U.S. patent application number 13/349385 was filed with the patent office on 2012-06-14 for treatment of diseases and disorders using self-renewing colony forming cells cultured and expanded in vitro.
This patent application is currently assigned to Garnet Biotherapeutics, Inc.. Invention is credited to Richard S. Gore, Baron Heimbach, Gene KOPEN, Vanessa Ragaglia, Joseph Wagner.
Application Number | 20120148544 13/349385 |
Document ID | / |
Family ID | 40156544 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148544 |
Kind Code |
A1 |
KOPEN; Gene ; et
al. |
June 14, 2012 |
Treatment of Diseases and Disorders Using Self-Renewing Colony
Forming Cells Cultured and Expanded In Vitro
Abstract
The present invention relates to methods and uses of cells for
the prevention and treatment of a wide variety of diseases and
disorders and the repair and regeneration of tissues and organs
using low passage and extensively passaged in vitro cultured,
self-renewing, colony forming somatic cells (CF-SC). For example,
adult bone marrow-derived somatic cells (ABM-SC), or compositions
produced by such cells, are useful alone or in combination with
other components for treating, for example, cardiovascular,
neurological, integumentary, dermatological, periodontal, and
immune mediated diseases, disorders, pathologies, and injuries.
Inventors: |
KOPEN; Gene; (Wynnewood,
PA) ; Wagner; Joseph; (West Chester, PA) ;
Ragaglia; Vanessa; (Newtown Square, PA) ; Heimbach;
Baron; (Philadelphia, PA) ; Gore; Richard S.;
(West Chester, PA) |
Assignee: |
Garnet Biotherapeutics,
Inc.
Malvern
PA
|
Family ID: |
40156544 |
Appl. No.: |
13/349385 |
Filed: |
January 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12140065 |
Jun 16, 2008 |
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13349385 |
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60929151 |
Jun 15, 2007 |
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60929152 |
Jun 15, 2007 |
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60955204 |
Aug 10, 2007 |
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60996093 |
Nov 1, 2007 |
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Current U.S.
Class: |
424/93.7 ;
435/373; 435/375 |
Current CPC
Class: |
A61K 35/28 20130101;
A61K 2035/122 20130101; A61P 5/00 20180101; A61P 3/10 20180101;
A61P 37/06 20180101; A61P 13/02 20180101; C12N 5/0669 20130101;
A61K 2035/124 20130101; A61P 1/16 20180101; A61P 17/00 20180101;
A61P 13/10 20180101; A61P 27/02 20180101; A61K 45/06 20130101; A61P
1/18 20180101; A61P 13/12 20180101; A61P 19/00 20180101; A61P 37/00
20180101; A61P 1/02 20180101; A61P 7/06 20180101; A61P 25/00
20180101; A61P 37/02 20180101; A61P 9/00 20180101; A61P 11/00
20180101; A61P 29/00 20180101; A61P 35/00 20180101; A61P 19/04
20180101; A61P 15/00 20180101; A61P 1/04 20180101; A61P 21/00
20180101; A61K 38/1722 20130101 |
Class at
Publication: |
424/93.7 ;
435/373; 435/375 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C12N 5/071 20100101 C12N005/071; C12N 5/078 20100101
C12N005/078; A61P 29/00 20060101 A61P029/00; A61P 37/00 20060101
A61P037/00 |
Claims
1. A method of administering a therapeutically useful amount of a
biological composition or compositions to an organ, tissue, or
subject, comprising administering to said organ, tissue, or subject
an isolated population of bone marrow-derived self-renewing
colony-forming somatic cells (CF-SC), wherein said CF-SC do not
have multipotent differentiation capacity, wherein said CF-SC have
a normal karyotype, wherein said CF-SC are non-immortalized,
wherein said CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and
.beta. (beta) 2-Microglobulin, and wherein said CF-SC do not
express CD10, CD34, CD45, CD62L, or CD106.
2. (canceled)
3. A method of administering a therapeutically useful amount of a
biological composition or compositions to an organ, tissue, or
subject, comprising administering to said organ, tissue, or subject
an isolated population of bone marrow-derived self-renewing
colony-forming somatic cells (CF-SC), wherein said CF-SC do not
have multipotent differentiation capacity, wherein said CF-SC have
a normal karyotype, wherein said CF-SC are non-immortalized, and
wherein said CF-SC are obtained from bone marrow by steps
comprising: i) incubating bone marrow cells under a low oxygen
condition such that said bone marrow cells when allowed to adhere
to a tissue culture-treated surface produce adherent colony forming
units; and, ii) passaging cells in said adherent colony forming
units at low cell seeding densities.
4. (canceled)
5. The method of claim 1, wherein the therapeutically useful amount
is an amount useful for, preventing tissue damage or for repairing,
treating, or promoting regeneration of damaged tissue.
6. (canceled)
7. The method of claim 3, wherein the therapeutically useful amount
is an amount useful for preventing tissue damage or for repairing,
treating, or promoting regeneration of damaged tissue in an organ,
tissue, or subject.
8. (canceled)
9. The method of claim 1, wherein the therapeutically useful amount
is an amount useful for treating or reducing inflammation, immune,
or autoimmune activity in an organ, tissue, or subject.
10. (canceled)
11. The method of claim 3, wherein the therapeutically useful
amount is an amount useful for treating or reducing inflammation,
immune, or autoimmune activity in an organ, tissue, or subject.
12. (canceled)
13. The method of claim 1, wherein said CF-SC further express one
or more molecules selected from the group consisting of: a) CD29;
b) CD59; c) CD147; d) CD166; and, e) telomerase and, wherein said
CF-SC further do not express one or more molecules selected from
the group consisting of: f) CD11c; g) CD14; h) CD33; i) CD62P; j)
CD80; k) STRO-1; l) HLA-Class-II; m) CD178. n) p53; and, o)
p21.
15. The method of claim 3, wherein said low oxygen condition is
selected from the group consisting of a) less than about 20%
oxygen; b) less than about 15% oxygen; c) less than about 10%
oxygen; d) less than about 5% oxygen; e) between about 1 to 10%
oxygen; f) between about 2 to 7% oxygen; g) between about 3 to 6%
oxygen; h) between about 4 to 6% oxygen; and, i) between about 4 to
5% oxygen.
16.-19. (canceled)
20. The method of claim 3, wherein said low cell seeding density is
selected from the group consisting of: a) less than about 2500
cells/cm.sup.2; b) less than about 1000 cells/cm.sup.2; and, c)
less than about 500 cells/cm.sup.2.
21.-26. (canceled)
27. The method of claim 1, wherein said CF-SC have unipotent
differentiation capacity.
28. (canceled)
29. The method of claim 1, wherein said CF-SC maintain an
approximately constant population doubling rate through multiple in
vitro cell doublings.
30. (canceled)
31. The method of claim 1, wherein said bone marrow is human bone
marrow.
32. The method of claim 1, wherein said cell population has
undergone a number of population doublings selected from the group
consisting of: a) at least about 15 population doublings; b) at
least about 20 population doublings; c) at least about 25
population doublings; d) at least about 30 population doublings; e)
at least about 35 population doublings; f) at least about 40
population doublings; g) at least about 45 population doublings;
and, h) at least about 50 population doublings.
33.-37. (canceled)
38. A composition comprising a pharmaceutically acceptable mixture
of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
39. (canceled)
40. A composition comprising a pharmaceutically acceptable mixture
of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
41.-69. (canceled)
70. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
71.-72. (canceled)
73. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
74.-94. (canceled)
95. The method of claim 3, wherein said CF-SC have unipotent
differentiation capacity.
96. The method of claim 3, wherein said CF-SC maintain an
approximately constant population doubling rate through multiple in
vitro cell doublings.
97. The method of claim 3, wherein said bone marrow is human bone
marrow.
98. The method of claim 3, wherein said cell population has
undergone a number of population doublings selected from the group
consisting of: a) at least about 15 population doublings; b) at
least about 20 population doublings; c) at least about 25
population doublings; d) at least about 30 population doublings; e)
at least about 35 population doublings; f) at least about 40
population doublings; g) at least about 45 population doublings;
and, h) at least about 50 population doublings.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/140,065 (filed Jun. 16, 2008), now pending; which
claims benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Patent Application Nos. 60/929,151 and 60/929,152 (each filed Jun.
15, 2007); U.S. Provisional Patent Application No. 60/955,204
(filed Aug. 10, 2007); and, U.S. Provisional Patent Application No.
60/996,093 (filed on Nov. 1, 2007). Each of the above-referenced
patent applications are hereby incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the generation
and use of in vitro cultured self-renewing colony forming somatic
cells (CF-SC), and compositions produced by such cells, for the
treatment of a variety of diseases and disorders. One example of
such CF-SC are adult human bone marrow-derived somatic cells
(hABM-SC).
[0003] The present invention also relates to manipulation of CF-SC
cell populations during cultivation to modulate (i.e., up- or
down-regulate) production of various soluble or secreted
compositions produced by in vitro cultured and expanded
self-renewing colony forming cells.
[0004] The field of the invention also relates to cell-based and
tissue-engineering therapies; particularly, methods of using and/or
administering CF-SC, or compositions produced by such cells,
including administration via incorporation in, or mixture with,
pharmaceutically acceptable carriers (such as a pharmaceutically
acceptable solution or a transient, permanent, or biodegradable
matrix).
Cell Based Therapies
[0005] There are two major options in using cell-based therapies to
manage and treat chronic and acute tissue damage in which the
overall objective is the functional and/or cosmetic restoration of
damaged tissue. These cell based therapy options include: 1) Cell
Replacement--Use of cells to replace damaged tissue by establishing
long-term engraftment; and 2) Supply Trophic Factors--Use of cells
and compositions produced by cells (e.g., growth factors) to
stimulate endogenous repair mechanisms through release of factors
delivered or produced by cells without long-term engraftment.
[0006] The present invention relates to use of cell based therapies
without relying on long-term cell engraftment. In particular, the
invention relates to use of cells, and compositions produced by
cells, in the treatment of various diseases and disorders;
particularly those involving tissues and organs with limited
self-renewal capability (such as, for example, neurological and
cardiac tissues and organs).
[0007] Cell-based therapeutic options in managing and treating
tissue damage also present the possibility for use of autologous or
allogeneic cells. Each of these have certain advantages and
disadvantages. Use of autologous cells involves the following
factors or parameters: [0008] Patient is the donor; [0009] Requires
manufacture of cell product on a patient-by-patient basis; [0010]
Variability in the identity, purity and potency of cell product;
and, [0011] Lag time between clinical decision to treat and
availability of cells for transplant.
[0012] In contrast, the use of allogeneic cells involves the
following factors or parameters: [0013] Donor is second party
(i.e., donor is not the patient); [0014] Risk associated with donor
variability; [0015] Multiple patients treatable per manufactured
batch of cell product; [0016] Increased consistency of identity,
purity and potency of cell product; and, [0017] Decreased lag time
between clinical decision to treat and availability of cell
product.
[0018] The present invention relates primarily to treatments
involving use of allogeneic cells. However, it would also be
equally possible to perform these same treatments using autologous
cells.
Organ and Tissue Repair
[0019] The regenerative potential of certain tissues in the
mammalian body has been known for centuries, for example tissues
like skin and bone are known to repair themselves after injury.
However, a number of conditions and diseases of the central nervous
system (i.e., brain and spinal cord), peripheral nervous system and
heart adversely affect humans because of the deficit of
regenerative capacity in the effected tissues. These conditions and
diseases include, for example, spinal cord injury, amyotrophic
lateral sclerosis (ALS), Parkinson's disease, stroke, Huntington's
disease, traumatic brain injury, brain tumors, Fabry Disease, heart
diseases (such as congestive heart failure and myocardial
infarction). Clinical management strategies, for example,
frequently focus on the prevention of further damage or injury
rather than replacement or repair of the damaged tissue (e.g.,
neurons, glial cells, cardiac muscle); include treatment with
exogenous steroids and synthetic, non-cellular pharmaceutical
drugs; and have varying degrees of success which may depend on the
continued administration of the steroid or synthetic drug.
[0020] For example, the majority of spinal cord injuries are
compression injuries with the remaining cases involving complete
transection of the spinal cord. Current therapeutic treatments for
spinal cord injury include the prevention of additional spinal cord
injury by physically stabilizing the spine through surgical and
non-surgical procedures and by inhibiting the inflammatory response
with steroidal therapy.
[0021] Additionally, aging is a major negative component to nearly
every common disease affecting mammals, and one of the principle
features of aging in a degeneration of many tissue including those
of skin, bone, eye, brain, liver, kidney, heart, vasculature,
muscle, et cetera. Furthermore, the already limited regenerative
capacity of certain tissues of the body is known to decline with
age, tissue maintenance and repair mechanisms in almost every
tissue decline over the course of life.
[0022] Thus, there is a need to develop new, improved and effective
methods of treatment for diseases and conditions, in particular,
neurological and cardiac diseases and age-related degenerative
conditions in humans.
Erythropoiesis
[0023] Hematopoietic cells in a healthy human or other mammal do
not ordinarily have a limited long-term self-renewal capability.
However, the potential for catastrophic loss of blood (or need
otherwise for a supplemental supply of blood) combined with limited
supplies of donor blood, entails that methods for enhancing,
maintaining, or generating red blood supplies in vitro are quite
desirable.
[0024] Blood is a highly specialized circulating tissue consisting
of several types of cells suspended in a fluid medium known as
plasma. The cellular constituents are: red blood cells
(erythrocytes), which carry respiratory gases and give it its red
color because they contain hemoglobin (an iron-containing protein
that binds oxygen in the lungs and transports it to tissues in the
body), white blood cells (leukocytes), which fight disease, and
platelets (thrombocytes), cell fragments which play an important
part in the clotting of the blood. Medical terms related to blood
often begin with hemo- or hemato- (BE: haemo- and haemato-) from
the Greek word "haima" for "blood." Blood cells are produced in the
bone marrow; in a process called hematopoiesis. Blood cells are
degraded by the spleen and liver. Healthy erythrocytes have a
plasma half-life of 120 days before they are systematically
replaced by new erythrocytes created by the process of
hematopoiesis. Blood transfusion is the most common therapeutic use
of blood. It is usually obtained from human donors. As there are
different blood types, and transfusion of the incorrect blood may
cause severe complications, crossmatching is done to ascertain the
correct type is transfused.
[0025] A shortage of blood donors and inadequate supplies of red
blood cells for transfusion is a common problem in treating
patients worldwide. Accordingly, there is a need for new, improved
and effective methods of increasing the availability of red blood
cells as this would provide a means for alleviating at least some
of the global shortages in red blood cell supplies.
Skin
[0026] The present invention relates in part to treatment of skin
wounds. There are currently available a number of different
treatments for wounds of the skin such as epidermal replacement
products, dermal replacement products, artificial skin products,
and wound dressings. Examples of some of these are described
briefly below.
Epidermal Replacement Products
[0027] According to the manufacturer, EPICEL.TM. (Genzyme Corp.,
Cambridge, Mass.) is composed of autologous epidermal cells skin
grown from biopsy of patients own skin for treatment of burns.
Cells are co-cultured with mouse feeder cell line into sheets of
autologous epidermis.
[0028] According to the manufacturer, MYSKIN.TM. (CellTran LTD,
Sheffield, S1 4DP United Kingdom) is a cultured autologous
epidermal substitute for the treatment of burns, ulcers and other
non-healing wounds. MYSKIN.TM. contains living cells expanded from
the tissue of individual patients. MYSKIN.TM. comprises a layer of
keratinocytes (epidermal cells) on an advanced polymer-like coating
which facilitates the transfer of cells into the wound where they
can initiate healing. MYSKIN.TM. uses a medical grade silicone
substrate layer to support cell delivery, wound coverage and allow
exudate management.
[0029] According to the manufacturer, EPIDEX.TM. (Modex
Therapeutics Ltd, Lausanne, Switzerland) is an autologous epidermal
skin equivalent that is grown directly from stem and pre-cursor
cells derived from hair taken directly from a patient in a
non-surgical procedure.
[0030] According to the manufacturer, CELLSPRAY.TM. (Clinical Cell
Culture Europe Ltd, Cambridge CB2 1NL, United Kingdom) is a
cultured epithelial autograft suspension that is sprayed onto
injured skin in order to provide a rapid epidermal cover, promote
healing and optimize scar quality.
Dermal Replacement Products
[0031] According to the manufacturer, INTEGRA.TM. Dermal
Regeneration Template (Integra LifeSciences Corporation,
Plainsboro, N.J.) is a bilayer membrane system for skin
replacement. The dermal replacement layer is made of a porous
matrix of fibers of cross-linked bovine tendon collagen and a
glycosaminoglycan (chondroitin-6-sulfate) that is manufactured with
a controlled porosity and defined degradation rate. The temporary
epidermal substitute layer is made of synthetic polysiloxane
polymer (silicone) and functions to control moisture loss from the
wound. The collagen dermal replacement layer serves as a matrix for
the infiltration of fibroblasts, macrophages, lymphocytes, and
capillaries derived from the wound bed.
[0032] According to the manufacturer, DERMAGRAFT.TM. (Advanced
Biohealing, La Jolla, Calif.) Allogeneic newborn fibroblasts grown
on a biodegradable mesh scaffold, indicated for full-thickness
diabetic ulcers.
[0033] According to the manufacturer, PERMACOL.TM. (Tissue Science
Laboratories, Inc. Andover, Mass. 01810) Permacol.TM. surgical
implant is collagen-derived from porcine dermis which, when
implanted in the human body, is non-allergenic and
long-lasting.
[0034] According to the manufacturer, TRANSCYTE.TM. (Advanced
Biohealing, La Jolla, Calif. 92037) TRANSCYTE.TM. is a human
foreskin-derived fibroblast temporary skin substitute (allogeneic).
The product consists of a polymer membrane and newborn human
fibroblast cells cultured under aseptic conditions in vitro on a
nylon mesh. Prior to cell growth, this nylon mesh is coated with
porcine dermal collagen and bonded to a polymer membrane
(silicone). This membrane provides a transparent synthetic
epidermis when the product is applied to the burn. The human
fibroblast-derived temporary skin substitute provides a temporary
protective barrier. TRANSCYTE.TM. is transparent and allows direct
visual monitoring of the wound bed.
[0035] According to the manufacturer, RENGRANEX.TM. Gel
(Ortho-McNeil Pharmaceutical, Inc..COPYRGT. ETHICON, INC.) is a
topical wound care product made of recombinant PDGF in a gel.
Artificial Skin Products (Epidermal and Dermal Combination
Products)
[0036] According to the manufacturer, PERMADERM.TM. (Cambrex Bio
Science Walkersville, Inc., Walkersville, Md.) PERMADERM.TM. is
constructed from autologous epidermal and dermal layers of the skin
and is indicated for the treatment of severe burns. The product is
reported to be pliable and to grow with the patient.
[0037] According to the manufacturer, ORCEL.TM. (Ortec
International, New York, N.Y.) Bilayered construct made from
allogeneic epidermal cells and fibroblasts cultured in bovine
collagen, indicated for split-thickness burns. The manufacturer
reports no evidence of product-derived DNA detectable in two human
patients treated with product at 2 or 3 weeks, respectively.
[0038] According to the manufacturer, APLIGRAF.TM. (Smith &
Nephew, London, WC2N 6LA United Kingdom) Allogeneic epidermal cells
and fibroblasts cultured in bovine collagen, indicated for venous
leg ulcers.
Wound Dressings
[0039] According to the manufacturer, 3M.TM. TEGADERM.TM.
Transparent Film Dressing (3M, St. Paul, Minn.) is a breathable
film that provides a bacterial and viral barrier to outside
contaminants.
[0040] According to the manufacturer, TISSEEL.TM. VH Fibrin Sealant
(Baxter, Deerfield, Ill.) is indicated for use as an adjunct to
hemostasis.
SUMMARY OF THE INVENTION
[0041] The present invention relates to the production and use of
stable cell populations and compositions produced thereby. The term
"stable cell population" as used herein means an isolated, in vitro
cultured, cell population that when introduced into a living
mammalian organism (such as a mouse, rat, human, dog, cow, etc.)
does not result in detectable production of cells which have
differentiated into a specialized cell type or cell types (such as
a chondrocyte, adipocyte, osteocyte, etc.) and wherein the cells in
the cell population express, or maintain the ability to express or
the ability to be induced to express, detectable levels of at least
one therapeutically useful composition (such as membrane bound or
soluble TNF-alpha receptor, IL-1R antagonists, IL-18 antagonists,
compositions shown in Table 1A, 1B, 1C, etc.).
[0042] Another characteristic of the stable cell populations of the
present invention is that the cells do not exhibit ectopic
differentiation. The term "ectopic" means "in the wrong place" or
"out of place". The term "ectopic" comes from the Greek "ektopis"
meaning "displacement" ("ek", out of+"topos", place=out of place).
For example, an ectopic kidney, is one that is not in the usual
location; or, an extrauterine pregnancy is an "ectopic pregnancy".
In the present context, an example of ectopic differentiation would
be cells that when introduced into cardiac tissue, produce bone
tissue-like calcifications and/or ossifications. This phenomenon
has been demonstrated to occur, for example, when mesenchymal stem
cells are injected into cardiac tissue. See, Breitbach et al.,
"Potential Risks of Bone Marrow Cell Transplantation Into Infarcted
Hearts", Blood, Vol. 110, No. 4 (August 2007).
[0043] The present invention relates to the generation and use of
expanded, in vitro cultured, self-renewing colony forming somatic
cells (hereinafter referred to as "CF-SC"), and products produced
by such cells, for the treatment of a variety of diseases and
disorders. Further, the present invention also relates to the
generation and use of extensively expanded, in vitro cultured,
self-renewing colony forming somatic cells (hereinafter referred to
as "exCF-SC"), and products produced by such cells, for the
treatment of a variety of diseases and disorders. ExCF-SC are
self-renewing colony forming somatic cells (CF-SC) which have
undergone at least about 30, at least about 40, or at least about
50 cell population doublings during in vitro cultivation. Hence,
self-renewing colony forming somatic cells which have been expanded
in vitro are hereinafter referred to as "CF-SC" (such that, unless
specified otherwise, this term encompasses both cell populations
which have undergone less than about 30 population doublings (e.g.,
less than about 5, less than about 10, less than about 15, less
than about 20, less than about 25 population doublings) and also
cell populations which have undergone more than about 30, more than
about 40, or more than about 50 populations doublings in vitro).
One particular example of CF-SC are adult human bone marrow-derived
somatic cells (hereinafter referred to as "ABM-SC"). Further, one
particular example of exCF-SC are adult human bone marrow-derived
somatic cells which have undergone at least about 30, at least
about 40, or at least about 50 cell population doublings during in
vitro cultivation (hereinafter referred to as "exABM-SC").
Accordingly, the term "ABM-SC", unless specified otherwise,
encompasses both ABM-SC cell populations which have undergone less
than about 30 population doublings (e.g., less than about 5, less
than about 10, less than about 15, less than about 20, less than
about 25 population doublings) and also ABM-SC cell populations
which have undergone more than about 30, more than about 40, or
more than about 50 populations doublings in vitro).
[0044] The term "extensively expanded" as used herein refers to
cell populations which have undergone at least about 30 or more
cell population doublings and wherein the cells are non-senescent,
are not immortalized, and continue to maintain the normal karyotype
found in the cell species of origin.
[0045] As used herein, the term "substantial capacity for
self-renewal" means having the ability to go through numerous
cycles of cell division resulting in the production of multiple
generations of cell progeny (thus, with each cell division, one
cell produces two "daughter cells" wherein at least one daughter
cell is capable of further cell division). One measure of
"substantial capacity for self-renewal" is indicated by the ability
of a cell population to undergo at least about 10, 15, 20, 25, 30,
35, 40, 45, 50 or more cell doublings. Another measure of
"substantial capacity for self-renewal" is indicated by maintenance
of the ability of a cell population to re-populate, or approach
confluence in, a tissue culture vessel after cell culture passaging
(when the same or similar culture conditions are maintained). Thus,
an example of "substantial capacity for self-renewal" is
demonstrated when a cell population continues to re-populate a
tissue culture vessel in a period of time of at least about 25%,
50%, 60%, 70%, 80%, 90%, 95% or 100% of the time required for such
re-population during early cell culture doublings (such as before a
cell population has undergone more than about 10 population
doublings). Another measure of "substantial capacity for
self-renewal" is maintenance of a consistent rate of population
doubling time or of a consistent and relatively rapid rate of
population doubling.
[0046] As used herein, the term "substantially no multipotent
differentiation capacity" means cell populations which cannot
differentiate into multiple different types of cells, either in
vitro or in vivo. An example of cells which do have substantial
multipotent differentiation capacity are hematopoietic stem cells
which can differentiate into red blood cells, T-cells, B-cells,
platelets, etc. either in vitro or in vivo. Another example of
cells which do have substantial multipotent differentiation
capacity are mesenchymal stem cells which can differentiate, for
example, into osteocytes (bone), adipocytes (fat), or chondrocytes
(cartilage). In contrast, cells in a cell population which have
"substantially no multipotent differentiation capacity" cannot
differentiate into multiple cell types in vitro or when introduced
into an organism or target tissue(s) in vivo. In a preferred
embodiment of the invention, a cell population with "substantially
no multipotent differentiation capacity" is one in which at least
about 80%, 90%, 95%, 98%, 99% or 100% of the cells in the cell
population cannot be induced to detectably differentiate in vitro
or in vivo into more than one cell type. A "unipotent" cell or
"unipotent progenitor cell" is an example of a cell which has
substantially no multipotent differentiation capacity.
[0047] As used herein, "stem cell" means a cell or cells possessing
the following two properties: 1) capacity for self-renewal, which
is the ability to go through numerous cycles of cell division while
maintaining the undifferentiated state; and, 2) differentiation
potency, which is the capacity to change into one or more kinds of
mature cell types and, upon such change, no longer undergoing
cycles of cell division (for example, capacity to change into an
osteocyte, adipocyte, chondrocyte, etc.). As used herein,
differentiation potency means the cells are either totipotent,
pluripotent, multipotent, or unipotent progenitor cells. A
"mesenchymal stem cell" is a stem cell of this same definition but
wherein said cell has been derived or obtained from mesenchyme
tissue (such as, for example, bone marrow, adipose or cartilage).
See, Horwitz et al., "Clarification of the nomenclature for MSC:
The International Society for Cellular Therapy position statement",
Cytotherapy, vol. 7, no. 5, pp. 393-395 (2005); and references
cited therein.
[0048] As used herein, "totipotent" means cells which can become
any type of cell as may be found during any stage of development in
the organism of the cells origin. Totipotent cells are typically
produced by the first few divisions of the fertilized egg (i.e.,
following fusion of an egg and sperm cell). Thus, totipotent cells
can differentiate into embryonic and extraembryonic cell types.
[0049] As used herein, "pluripotent" means cells which can
differentiate into cells derived from any of the three germ layers
(endoderm, mesoderm, ectoderm) found in the organism of the cells
origin.
[0050] As used herein, "multipotent" means cells which can produce
multiple types (i.e., more than one type) of differentiated cells.
A mesenchymal stem cell is an example of a multipotent cell.
[0051] As used herein, "unipotent" means cells which can produce
only one cell type. Unipotent cells have the property of
self-renewal, but can change into only one kind of mature cell
type.
[0052] As used herein, "normal karyotype" means having a genetic
composition comprising chromosomes of the number and of the
structure typically found in, and considered normal for, the
species from which the cells are derived.
[0053] As used herein, "connective tissue" is one of the four types
of tissue usually referenced in traditional classifications (the
others being epithelial, muscle, and nervous tissue). Connective
tissue is involved in organism and organ structure and support and
is usually derived from mesoderm. As used herein, "connective
tissue" includes those tissues sometimes referred to as "connective
tissue proper", "specialized connective tissues", and "embyronic
connective tissue".
[0054] "Connective tissue proper" includes areolar (or loose)
connective tissue, which holds organs and epithelia in place and
has a variety of proteinaceous fibres, including collagen and
elastin. Connective tissue proper also includes dense connective
tissue (or, fibrous connective tissue) which forms ligaments and
tendons.
[0055] "Specialized connective tissue" includes blood, bone,
cartilage, adipose and reticular connective tissue. Reticular
connective tissue is a network of reticular fibres (fine collagen,
type III) that form a soft skeleton to support the lymphoid organs
(lymph nodes, bone marrow, and spleen)
[0056] "Embryonic connective tissue" includes mesenchymal
connective tissue and mucous connective tissue. Mesenchyme (also
known as embryonic connective tissue) is the mass of tissue that
develops mainly from the mesoderm (the middle layer of the
trilaminar germ disc) of an embryo. Viscous in consistency,
mesenchyme contains collagen bundles and fibroblasts. Mesenchyme
later differentiates into blood vessels, blood-related organs, and
connective tissues. Mucous connective tissue (or mucous tissue) is
a type of connective tissue found during fetal development; it is
most easily found as a component of Wharton's jelly (a gelatinous
substance within the umbilical cord which serves to protect and
insulate cells in the umbilical cord).
[0057] As used herein, "immortalized" refers to a cell or cell line
which can undergo an indefinite number of cell doublings in vitro.
Immortalized cells acquire such ability through genetic changes
which eliminate or circumvent the natural limit on a cells ability
to continually divide. In contrast, "non-immortalized" cells are
eukaryotic cells which, when taken directly from the organism and
cultured in vitro (producing a "primary cell culture"), can undergo
a limited number of cell doublings before senescencing (losing
ability to divide) and dying. For example, primary cultures of most
types of mammalian, non-immortalized cells can usually undergo a
relatively defined but reproducibly limited range of cell doublings
(depending on the primary cell type) before differentiating,
senescing, or dying.
[0058] As used herein "long-term engraftment" means the detectable
presence of donor cells residing within (or as part of) target
tissue to which (or in which) said cells were delivered after more
than about 4 weeks from the time of administration. "More than
about 4 weeks" includes time periods of more than about 5, 6, 7, 8,
10, 12, 14, 16, 18, 20, and 24 weeks. "More than about 4 weeks"
also includes time periods of more than about 6 months, 8 months,
10 months, 12 months, 18 months, 24 months, 30 months, 36 months,
42 months and 48 months.
[0059] The present invention also relates to manipulation of CF-SC
and exCF-SC cell populations during cultivation to modulate (i.e.,
up- or down-regulate) production of various soluble or secreted
compositions produced by the in vitro cultured and expanded
self-renewing colony forming cells.
[0060] The present invention also relates to extensively expanded
cell populations which are characterized by loss of ability to
differentiate into bone cells (osteocytes). For example, the
present invention relates to extensively expanded cell populations
which are characterized by loss of ability to generate calcium
deposits when cultured under osteoinductive conditions, including
with or without cultivation in the presence of the supplemental
bone morphogen Noggin (see Example 16). (Mouse and Human Noggin:
See, e.g., the U.S. National Center for Biotechnology PubMed
Protein Database Accession Nos. NP.sub.--032737 and NP.sub.--005441
(respectively); see also e.g., Valenzuela, et al., "Identification
of mammalian noggin and its expression in the adult nervous
system", J. Neurosci. 15 (9), 6077-6084 (1995)).
[0061] The present invention also relates to extensively expanded
cell populations characterized by the loss of ability to
differentiate into bone cells and/or loss of ability to generate
calcium deposits (as described above), but wherein said cell
populations continue to secrete, or maintain the ability to secrete
or to be induced to secrete, at least one therapeutically useful
composition.
[0062] The present invention also relates to cell-based and
tissue-engineering therapies; particularly, methods of using and/or
administering CF-SC and exCF-SC, or compositions produced by such
cells, including administration via incorporation in, or mixture
with, pharmaceutically acceptable carriers (such as a
pharmaceutically acceptable solution or a transient, permanent, or
biodegradable matrix).
[0063] The present invention also relates to expanded (i.e., in
vitro cultured and passaged) and extensively expanded cell
populations which are preferably negative for expression of the
STRO-1 cell surface marker. See, e.g., Stewart et al., "STRO-1,
HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive
human marrow stromal cells and their more differentiated progeny: a
comparative investigation in vitro" Cell Tissue Res. 2003
September; 313(3):281-90; and, Dennis et al., "The STRO-1+ marrow
cell population is multipotential" Cells Tissues Organs. 2002;
170(2-3):73-82; and, Oyajobi et al., "Isolation and
characterization of human clonogenic osteoblast progenitors
immunoselected from fetal bone marrow stroma using STRO-1
monoclonal antibody", J Bone Miner Res. 1999 March;
14(3):351-61.
[0064] The present invention also relates to manufacture and use of
pharmaceutically acceptable compositions containing CF-SC and
exCF-SC (for example, ABM-SC and exABM-SC) with additional
structural and/or therapeutic components. As one example, CF-SC or
exCF-SC (for example, ABM-SC or exABM-SC) and collagen may be
combined in a pharmaceutically acceptable solution to generate
compositions in liquid, semi-solid, or solid-like forms (matrices)
for use, for example, in the treatment, repair, and regeneration of
skin disorders (e.g., skin wounds such as burns, abrasions,
lacerations, ulcers, infections).]
[0065] The present invention relates generally to use of
self-renewing cells, referred to herein as colony-forming somatic
cells (CF-SC) including extensively passaged colony-forming somatic
cells (exCF-SC). Examples of such cells are adult human bone
marrow-derived somatic cells (ABM-SC) including extensively
passaged adult human bone marrow-derived somatic cells (exABM-SC),
for use in treatment of various diseases and disorders;
particularly diseases and disorders involving ischemia, trauma,
and/or inflammation (such as, for example, heart failure due to
acute myocardial infarction (AMI) and stroke).
[0066] Self-renewing colony-forming somatic cells (CF-SC) such as
adult human bone marrow-derived somatic cells (ABM-SC) as used in
the present invention are prepared as described in U.S. Patent
Publication No. 20030059414 (U.S. application Ser. No. 09/960,244,
filed Sep. 21, 2001) and U.S. Patent Publication No. 20040058412
(U.S. application Ser. No. 10/251,685, filed Sep. 20, 2002). Each
of these patent applications are hereby incorporated by reference
in their entirety. In particular, CF-SC isolated from a source
population of cells (such as, for example, from bone marrow, fat,
skin, placenta, muscle, umbilical cord blood, or connective tissue)
are cultured under low oxygen conditions (e.g., less than
atmospheric) and passaged at low cell densities such that the CF-SC
maintain an essentially constant population doubling rate through
numerous population doublings. After expansion of the CF-SC to an
appropriate cell number, the CF-SC may be used to generate the
compositions of the present invention. For example, after expansion
of the CF-SC in vitro for at least about 30, at least about 40, or
at least about 50 cell population doublings exCF-SC may be used to
generate compositions of the present invention. In one embodiment
CF-SC and exCF-SC, as used in the present invention, are derived
from bone marrow (and are referred to herein as ABM-SC and
exABM-SC, respectively).
[0067] One embodiment of CF-SC and exCF-SC (such as for example,
ABM-SC and exABM-SC, respectively), as used in the present
invention, is an isolated cell population wherein the cells of the
cell population co-express CD49c and CD90 and wherein the cell
population maintains a doubling rate of less than about 30 hours
after at least about 30, at least about 40, or at least about 50
cell population doublings.
[0068] Another embodiment of CF-SC and exCF-SC (such as for
example, ABM-SC and exABM-SC, respectively), as used in the present
invention, is an isolated cell population wherein the cells of the
cell population co-express CD49c, CD90, and one or more cell
surface proteins selected from the group consisting of CD44, HLA
Class-1 antigen, and .beta. (beta) 2-Microglobulin, and wherein the
cell population maintains a doubling rate of less than about 30
hours after at least about 30, at least about 40, or at least about
50 cell population doublings.
[0069] Another embodiment of CF-SC and exCF-SC (such as for
example, ABM-SC and exABM-SC, respectively), as used in the present
invention, is an isolated cell population wherein the cells of the
cell population co-express CD49c and CD90, but are negative for
expression of cell surface protein CD 10, and wherein the cell
population maintains a doubling rate of less than about 30 hour
after at least about 30, at least about 40, or at least about 50
cell population doublings.
[0070] Another embodiment of CF-SC and exCF-SC (such as for
example, ABM-SC and exABM-SC, respectively), as used in the present
invention, is an isolated cell population wherein the cells of the
cell population co-express CD49c, CD90, and one or more cell
surface proteins selected from the group consisting of CD44, HLA
Class-1 antigen, and .beta. (beta) 2-Microglobulin, but are
negative for expression of cell surface protein CD10, and wherein
the cell population maintains a doubling rate of less than about 30
hours after at least about 30, at least about 40, or at least about
50 cell population doublings.
[0071] Another embodiment of CF-SC and exCF-SC (such as for
example, ABM-SC and exABM-SC, respectively), as used in the present
invention, is an isolated cell population wherein the cells of the
cell population express one or more proteins selected from the
group consisting of soluble proteins shown in Table 1A, 1B and 1C,
and wherein the cell population maintains a doubling rate of less
than about 30 hours after at least about 30, at least about 40, or
at least about 50 cell population doublings.
[0072] Damaged tissues and organs may result from, for example,
disease (e.g., heritable (genetic) or infectious diseases (such as
bacterial, viral, and fungal infections)), physical trauma (such as
burns, lacerations, abrasions, compression or invasive tissue and
organ injuries), ischemia, aging, toxic chemical exposure, ionizing
radiation, and dysregulation of the immune system (e.g., autoimmune
disorders).
[0073] The present invention encompasses the use of CF-SC and
exCF-SC (such as for example, ABM-SC and exABM-SC, respectively),
CF-SC and exCF-SC purified protein fractions, supernatants of CF-SC
and exCF-SC conditioned media, and fractions of cell-supernatants
derived from CF-SC and exCF-SC conditioned media. In one embodiment
of the invention, the above mentioned components may be combined
with, or introduced into, physiologically compatible biodegradable
matrices which contain additional components such as collagen
and/or fibrin (for example, purified natural or recombinant human,
bovine, or porcine collagen or fibrin), and/or polyglycolic acid
(PGA), and/or additional structural or therapeutic compounds.
Combination matrices such as these may be administered to the site
of tissue or organ damage to promote, enhance, and/or result in
repair and/or regeneration of the damaged tissue or organ.
[0074] Embodiments of the invention include use of CF-SC and
exCF-SC (such as for example, ABM-SC and exABM-SC, respectively),
incorporated into pharmaceutically acceptable compositions which
may be administered in a liquid, semi-solid, or solid-like state.
Embodiments of the invention may be administered by methods
routinely used by those skilled in the relevant art, such as for
example, by topical application, as spray-on or aerosolized
compositions, by injection, and implantation.
[0075] Use of CF-SC and exCF-SC (such as for example, ABM-SC and
exABM-SC, respectively), cells and compositions produced by these
cells as described in the present invention for tissue regenerative
therapies may provide a number of benefits compared to previously
described tissue regenerative therapies and products. For example,
use of the CF-SC and exCF-SC (such as for example, ABM-SC and
exABM-SC, respectively), exABM-SC cells and compositions produced
thereby provides a means of tissue regenerative therapy which may
exhibit reduced adverse immune responses (such as reduced
inflammation and T-cell activation; see e.g., Examples 3A, 3B, 5,
18, and 19. Moreover, since ABM-SC and exABM-SCs are
immunologically silent, subjects do not need to be HLA-matched or
pre-conditioned prior to treatment. See, Example 10, Part II; see
also, FIG. 17.
[0076] The present invention also relates to the use of CF-SC and
exCF-SC (such as expanded and extensively expanded adult human bone
marrow-derived somatic cells (human ABM-SC and exABM-SC,
respectively)), and the cell products generated by these cells, for
inducing, enhancing, and/or maintaining hematopoiesis (in
particular, for the in vitro generation and production of red blood
cells (erythrocytes) from hematopoietic progenitor cells in a
process called erythropoiesis). Thus, another embodiment of the
invention encompasses the use of such cells and/or compositions
produced by such cells, to induce, enhance, and/or maintain the
generation and production of red blood cells (erythrocytes).
[0077] Another example of the field of the invention relates to the
prevention and treatment of immune, autoimmune, and inflammatory
disorders via use of such cells, cell populations, and compositions
produced thereby.
[0078] In another example, the present invention provides
compositions and methods for repair and regeneration of wounds of
the skin (i.e., epidermis, dermis, hypodermis); including the
manufacture and use of liquid, semi-solid, and solid-like matrices
which incorporate CF-SC and exCF-SC (for example, human ABM-SC and
exABM-SC), or products generated by such cells, and additional
structural or therapeutic compounds.
Exemplary Results of Preclinical Studies
[0079] In vivo preclinical pharmacology studies have demonstrated
the beneficial effects of ABM-SC in treating myocardial infarction
and stroke. For example, in a study investigating the effects of
intra-cardiac injection of hABM-SC in a rat model of myocardial
infarction (in particular, to determine the efficacy of hABM-SC in
restoring cardiac function post-AMI (acute myocardial infarction)
and evaluate distribution and disposition of hABM-SC), it was shown
that hABM-SC produced a significant improvement in cardiac function
and significantly reduced fibrosis. Furthermore, the hABM-SC were
not observed to remain in the heart four weeks after cardiac
injection, nor in any of the peripheral organs examined eight weeks
after injection. Additionally, in a study investigating the safety
and efficacy of porcine and human ABM-SC in an AMI model in pigs
(in particular, to evaluate the feasibility, safety and efficacy of
percutaneous, NOGA.TM.-guided endomyocardial administration of
cells through a MYOSTAR.TM. catheter) it was demonstrated that this
particular delivery method was well-tolerated and led to
significant improvements in cardiac parameters. Likewise, in a
comparison of the method of delivery of hABM-SC and stroke recovery
(in particular, to determine efficacy of hABM-SC in promoting
neuromotor recovery from ischemic stroke) it was observed that I.V.
or intra-cerebral treatment resulted in significant improvements in
neuromotor activity.
DESCRIPTION OF THE DRAWINGS
[0080] FIG. 1 shows a 2-dimensional SDS PAGE separation (pH 3.5 to
10; 12% polyacrylamide) of proteins secreted by human adult bone
marrow-derived somatic cells (ABM-SC at about 27 population
doublings). Each spot on the gel represents a separate and distinct
protein, ranging in size from approximately 5-200 kilodaltons
(kDa). The X-axis shows proteins separated according to isoelectric
point (pH 3.5 to 10). The Y-axis shows proteins separated according
to molecular weight (via passage through 12% polyacrylamide).
[0081] FIG. 2 shows photomicrographs of PC-12 differentiation into
neurons using nerve growth factor (NGF) and conditioned media
derived from human exABM-SC (at about 43 population doublings). A)
RPMI-ITS medium only. B) RPMI-ITS supplemented with NGF. C)
RPMI-ITS supplemented with a 1:50 dilution of concentrated control
media and NGF. D) RPMI-ITS supplemented with a 1:50 dilution of
concentrated conditioned media derived from human ABM-SC and NGF.
Arrows indicate neurite outgrowth. Extent of neurite outgrowth in
panel D is significantly more robust than that of panel B and
C.
[0082] FIG. 3 is a graphical representation of inhibition of
mitogen-induced T cell proliferation using human ABM-SC. Lot
#RECB801 represents ABM-SC that have been sub-cultured to about 19
population doublings and Lot #RECB906 represents exABM-SC which
have been sub-cultured to about 43 population doublings. To
stimulate T cell proliferation, cultures were inoculated with 2.5
or 10 microg/mL Phytohaemagglutinin. Cells were then harvested
after 72 hrs later and stained with CD3-PC7 antibody. Human
mesenchymal stem cells were used as a positive control. (Human
mesenchymal stem cells were obtained from Cambrex Research
Bioproducts; now owned by Lonza Group Ltd., Basel,
Switzerland).
[0083] FIG. 4 shows photomicrographs of pig skin 7 days after
surgically-induced incisional wounding (left two panels, both
wounds from experimental group #4). A) Wound No. 3 treated with
allogeneic porcine ABM-SC (at about 28 population doublings) shows
complete wound closure with virtually no scar. B) In comparison,
Wound No. 4 treated with vehicle only reveals a visible scar. C)
The graph (right panel) represents histomorphometric scoring of
tissue sections from both treatment groups and shows a
statistically significant reduction (p=0.03) in the number of
histiocytes in the porcine ABM-SC treated wounds (statistical
significance determined using a two-tailed unpaired T-test);
compare, bars for "Histiocytes" PBSG versus pABM-SC treated.
[0084] FIG. 5 is a graphical representation (top panel) of the
extent of re-epithelialization across the incisional wounds 7 days
post-treatment. Wounds treated with porcine ABM-SC (at about 28
population doublings) had a thicker epidermis than those treated
with vehicle only. The photomicrograph in the lower left panel
shows (histologically) complete and anatomically correct repair of
the epidermis in the wounds treated with porcine ABM-SC. The
photomicrograph in the lower right panel shows (histologically)
porcine ABM-SC (arrow heads) which appear engrafted, at least
transiently, in the hypodermis at this 7 day time point.
[0085] FIG. 6 is a graphical representation of ABM-SC mediated
contraction of hydrated collagen gel lattices seeded 24 hours after
cell reconstitution. Human ABM-SC (at about 27 population
doublings) were reconstituted in liquid biodegradable
collagen-based media (at 1.8.times.10.sup.6 cells/mL) and then
stored for 24 hours at approximately 4-8.degree. C. The following
day the liquid cell suspension was placed into a culture dish to
form a semi-solid collagen lattice. The semi-solid collagen
lattices were maintained in a cell culture incubator to facilitate
contraction over the course of three days. Collagen lattices
prepared without cells did not contract, demonstrating that
contraction is dependent upon the presence of cells.
[0086] FIG. 7 is a graphical representation of ABM-SC mediated
contraction of hydrated collagen gel lattices seeded at different
cell concentrations utilizing exABM-SC at about 43 population
doublings. The data demonstrate that rate and absolute magnitude of
contraction is related to cell number. Heat inactivated cells do
not contract the gels, demonstrating that this activity is a
biophysical event.
[0087] FIG. 8 is a graphical representation of ABM-SC mediated
secretion of several cytokines and matrix proteases IL-6, VEGF,
Activin-A, MMP-1, and MMP-2) when cultured for 3 days in hydrated
collagen gel lattices utilizing exABM-SC at about 43 population
doublings.
[0088] FIG. 9 shows photomicrographs of human ABM-SC reconstituted
in biodegradable collagen-based media as a liquid (left panel) or a
semi-solid (right panel) (Utilizing exABM-SC at about 43 population
doublings). When reconstituted using this formulation, the cell
suspension can remain as a liquid at 4.degree. C. for more than 24
hrs. When placed in a culture dish and incubated at 37.degree. C.,
the cell suspension will solidify within 1-2 hours, giving rise to
a semi-solid structure than can be physically manipulated.
[0089] FIG. 10 shows photomicrographs of a solid-like neotissue
formed by culturing human ABM-SC (at about 43 population doublings)
reconstituted in the biodegradable collagen-based media for three
days. The upper left panel shows the pliability of the tissue when
stretched. The upper right panel shows the general texture of the
solid-like neotissue. The lower panel shows a histological section
of the tissue stained by Masson's Trichrome, demonstrating the rich
extracellular matrix synthesized by the ABM-SC. Control gels
constructed by the same method, but lacking cells, do not stain
blue by this method, demonstrating that the collagen and
glycosaminoglycan-rich matrix is produced by the cells.
[0090] FIG. 11 shows an example of the quantities of multiple
pro-regenerative cytokines secreted by human ABM-SC with and
without TNF-alpha stimulation. When sub-cultured, ABM-SC secrete
potentially therapeutic concentrations of several growth factors
and cytokines known to augment angiogenesis, inflammation and wound
healing. ABM-SC have been shown to consistently secrete several
cytokines and growth factors in vitro; including proangiogenic
factors (e.g., SDF-1 alpha, VEGF, ENA-78 and angiogenin),
immunomodulators (e.g., IL-6 and IL-8) and scar inhibitors/wound
healing modulators (e.g., MMP-1, MMP-2, MMP-13 and Activin-A).
Furthermore, the release of several of these factors is modulated
by tumor necrosis factor alpha (TNF-alpha), a known inflammatory
cytokine released during the course of acute tissue injury.
[0091] FIG. 12 shows a model injury-response cascade (inflammation,
regeneration, and fibrosis from injury through scar) and examples
of molecules that can play roles in inflammation, regeneration, and
fibrosis.
[0092] FIG. 13 shows an example of improved cardiac function
results in rats treated with human ABM-SC. Four weeks after
treatment, rats receiving ABM-SC demonstrated significantly higher
+dp/dt (peak positive rate of pressure change) values (A).
Expressing changes in cardiac function over the course of the study
by subtracting 0 week +dp/dt values from 4 week values ("delta
+dp/dt") demonstrated that while vehicle treated rats had decreases
in cardiac function over the course of the study (negative delta),
animals treated with either cell preparation showed significant
improvement in cardiac function (B). Compared to vehicle treated
rats, those receiving ABM-SC demonstrated significantly lower tau
values (C), suggesting increased left ventricular compliance. Tau
is the time constant of isovolumetric left ventricular pressure
decay. For peak negative rate of pressure change (-dp/dt),
expressing changes in cardiac function over the course of the study
by subtracting 0 week -dp/dt values from 4 week values ("delta
-dp/dt") demonstrated that while vehicle-treated rats had decreases
in cardiac function over the course of the study (negative delta),
animals treated with cell preparation showed significant
improvement in cardiac function (D). [*p<0.05, **p<0.01 by
ANOVA]
[0093] FIG. 14 shows reduction of fibrosis and enhanced
angiogenesis in a rat model myocardial infarct treated with
hABM-SC. Semi-quantitative scoring was used to evaluate changes in
infarct size in the hearts of rats receiving vehicle or ABM-SC
seven days after myocardial infarction. Histopathological analysis,
performed approximately 30 days after administration of ABM-SC,
indicated significant reduction in infarct size in rats receiving
hABM-SC compared to vehicle. According to a preset scale, rats
receiving hABM-SC had histological scores approximately two points
lower than vehicle controls. This figure shows an example of
typical infarct size reduction.
[0094] FIG. 15 shows results obtained from histological, performed
approximately 30 days after administration of ABM-SC, measurement
of changes in the heart structure of rats receiving vehicle or
ABM-SC seven days after myocardial infarction.
[0095] FIG. 16 shows that allogeneic human ABM-SC and exABM-SC
suppress mitogen-induced T-cell proliferation in one-way MLR (mixed
lymphocyte reaction) assay.
[0096] FIG. 17 shows that allogeneic porcine ABM-SC fail to illicit
T-cell mediated immune response in a 2-way MLR challenge
experiment. A Division Index was calculated for samples collected
at baseline and 3 or 30 days post-treatment and then challenged in
vitro with media, vehicle, pABM-SC or ConA. The average division
index from all animals at Day 3 or Day 30 for CD3+ cells which were
stimulated with ConA was significantly higher than the division
index for CD3+ cells from vehicle and pABM-SC treated animals at
both pre-treatment and necropsy (* p<0.05).
[0097] FIG. 18 shows the changes in cardiac fixed perfusion deficit
size in three patients by comparison of baseline (BL) measurements,
with measurements obtained 90 days post-treatment with hABM-SC.
[0098] FIG. 19 shows the changes in cardiac ejection fractions
measured in three patients by comparison of baseline (BL)
measurements with measurements obtained 90 days post-treatment with
hABM-SC.
[0099] FIG. 20 shows examples of quantities of erythropoietic
cytokines secreted in vitro by hABM-SC (i.e., IL-6, Activin-A,
VEGF, LIF, IGF-II, SDF-1 and SCF). ABM-SC lots were tested for
cytokine secretion using RAYBIO.TM. Human Cytokine Antibody Array
(RayBiotech, Inc.). Cells were first cultured in serum-free
Advanced DMEM (GIBCO.TM.) for three days to generate conditioned
medium (CM). The CM was then concentrated using CENTRICON.TM.
PLUS-20 Centrifugal Filter Units (Millipore) prior to analysis.
[0100] FIG. 21 demonstrates that exABM-SC reduce TNF-.alpha. levels
in vitro in a dose-dependent manner. Human exABM-SC (at about 43
population doublings) were tested for their ability to reduce
TNF-.alpha. levels when cultured at various seeding densities (e.g.
10,000 cells/cm.sup.2, 20,000 cells/cm.sup.2, and 40,000
cells/cm.sup.2). Cells were cultured for 3 days in serum-free
Advanced DMEM (GIBCO.TM.) either alone or supplemented with 10
ng/mL TNF-.alpha.. Heat inactivated cells were also included as a
negative control. Concentration of TNF is shown on the Y-axis.
(Y-axis represents concentration of substances in media which has
been concentrated 100.times.).
[0101] FIGS. 22A and 22B demonstrates that reduction of TNF-.alpha.
appears to be mediated by the secretion of sTNF-RI and sTNF-RII by
exABM-SC (at about 43 population doublings). Basal level expression
of sTNF-RI occurs in the absence of a pro-inflammatory inducer (A),
while sTNF-RII is detected at appreciable levels only when first
primed with TNF-.alpha. (B). These data reveal an inverse
relationship between the number of cells seeded and the levels of
both sTNF-RI and sTNF-RII detected, suggesting that the secreted
receptors themselves may be binding to and masking the TNF-.alpha..
(Y-axis represents concentration of substances in media which has
been concentrated 100.times.).
[0102] FIG. 23 demonstrates that secretion levels of IL-IRA (by
exABM-SC at about 43 population doublings) is dose-dependent. Basal
level expression of IL-IRA occurs in the absence of a
pro-inflammatory inducer, but when primed when TNF-.alpha., soluble
levels increase approximately 10-fold. (Y-axis represents
concentration of substances in media which has been concentrated
100.times.).
[0103] FIG. 24 shows expression of IL-1 receptor antagonist
(IL-1RA) and IL-18 binding protein (IL-18BP) by exABM-SC. Human
exABM-SC express basal levels of IL-1 receptor antagonist (IL-1RA;
FIG. 36A) and IL-18 binding protein (IL-18BP; FIG. 36B) even in the
absence of an inflammatory signal such as TNF-alpha.
[0104] FIGS. 25A, B, and C show that human ABM-SC reduce levels of
TNF-alpha (FIG. 25A) and IL-13 (FIG. 25B) while simultaneously
inducing elevated expression of IL-2 (FIG. 25C) in a Mixed PBMC
reaction assay. (R=Responder PBMC, Self=Mitomycin-C treated PBMC
isolated from same donor as Responder, Stim=Mitomycin-C treated
PBMC isolated from a different donor.)
[0105] FIG. 26 shows a graphical representation of inhibition of
mitogen-induced human peripheral blood mononuclear cell (PBMC)
proliferation using human ABM-SC. RECB801 represents a particular
lot of ABM-SC that have been sub-cultured to about 19 population
doublings and #RECB906 represents a particular lot of ABM-SC that
have been sub-cultured to about 43 population doublings. To
stimulate PBMC proliferation, cultures were inoculated with 2.5
microg/mL phytohaemagglutinin. After 56 hours in culture, cells
were pulsed with Thymidine-[Methyl-3H] and at 72 hours isotope
incorporation was quantitated (CPM). Human mesenchymal stem cells
were included as a positive control.
DETAILED DESCRIPTION OF THE INVENTION
[0106] Typically, a stem cell or other early-stage progenitor cells
lose plasticity because the cells have committed to a particular
differentiation pathway. At the biomolecular level, as this process
begins to occur the cell loses the ability to respond to certain
signaling molecules (e.g., mitogens and morphogens) which would
otherwise drive the cell to divide or become another cell type.
Thus, as a cell begins to differentiate, it leaves the cell cycle
(i.e., can no longer go through mitosis) and enters an irreversible
state called G0 wherein the cell can no longer divide. Entry into
G0 is also associated with replicative senescence (hallmarks of
which include increased expression of intracellular proteins p21
and p53). Thus, loss of plasticity (the ability to differentiate
into a variety of cell types) is typically considered a prelude to
cellular differentiation or cellular senescence. Furthermore, loss
of plasticity is also typically associated with the loss of a cells
capacity for continued self-renewal. In contrast, to this typical
and traditionally accepted scenario, an unexpected and surprising
result of the present invention is that the exCF-SC of the present
invention (e.g., exABM-SC) continue to self-renew (including
self-renewal at a relatively constant rate) despite loss of
plasticity. Accordingly, one embodiment of the present invention
are therapeutically useful "end-stage cells" with a continued
capacity for self-renewal (e.g., cells capable of continued
self-renewal and production of trophic support factors (or "trophic
support cells")). In another embodiment, the exCF-SC and exABM-SC
of the present invention do not express significant quantities of
p21 and/or p53, wherein a "significant quantity" of said molecules
is a quantity which is indicative of cell senescence (wherein
senescence may require sufficient expression levels of p21, p53,
and/or other cell cycle regulators).
[0107] Additionally, most experts in the field of the present
invention would expect a non-hematopoietic stromal-type cell that
has lost plasticity to have limited utility or capability of
generating or promoting regeneration of organs and tissues. Thus,
another surprising and unexpected result of the present invention,
is the ability to generate extensively passaged CF-SC (e.g.,
ABM-SC) which have lost plasticity yet retain the ability to
generate new tissue in vitro and to promote regeneration of tissue
in vivo.
[0108] The present invention is drawn, inter alia, to methods of
repairing, regenerating, and/or rejuvenating tissues using
self-renewing cells, referred to herein as colony-forming somatic
cells (CF-SC) (an example of which are adult human bone
marrow-derived somatic cells (ABM-SC)). Self-renewing
colony-forming somatic cells (CF-SC) such as adult human bone
marrow-derived somatic cells (ABM-SC) as used in the present
invention are prepared as described in U.S. Patent Publication No.
20030059414 (U.S. application Ser. No. 09/260,244, filed Sep. 21,
2001) and U.S. Patent Publication No. 20040058412 (U.S. application
Ser. No. 10/251,685, filed Sep. 20, 2002). Each of these patent
applications are hereby incorporated by reference in their
entirety. Also incorporated by reference herein are U.S.
Provisional Patent Applications 60/929,151 and 60/929,152 (each
filed on Jun. 15, 2007), U.S. Provisional Patent Application
60/955,204 (filed on Aug. 10, 2007), and U.S. Provisional Patent
Application 60/996,093 (filed on Nov. 1, 2007).
[0109] In particular, CF-SC isolated from a source population of
cells (such as, for example, from bone marrow (ABM-SC and
exABM-SC), fat, skin, placenta, muscle, umbilical cord blood, or
connective tissue), are permitted to adhere to a cell culture
surface in the presence of an appropriate media (such as for
example, but not limited to, Minimal Essential Medium-Alpha (e.g.,
available from HYCLONE.TM.) supplemented with 4 mM glutamine and
10% fetal bovine serum) and cultured under low oxygen conditions
(such as for example, but not limited to, O.sub.2 at about 2-5%,
CO.sub.2 at about 5%, balanced with nitrogen) and subsequently
passaged at low cell densities (such as at about 30-1000
cells/cm.sup.2) such that the CF-SC maintain an essentially
constant population doubling rate (such as for example, but not
limited to, a doubling rate of less than about 30 hours) through
numerous population doublings (such as for example, but not limited
to, going through 10, 15, 20, 25, 30, 35, 40, 45 and/or 50
population doublings).
[0110] Embodiments of the invention may be generated with CF-SC and
exCF-SC (for example, ABM-SC and exABM-SC) cultured under low
oxygen conditions wherein said O.sub.2 concentrations range from
about 1-20% (for example, wherein the O.sub.2 concentration is
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, or 20%), plus CO.sub.2 and balanced with nitrogen. For
example, ABM-SC may be cultured under low oxygen conditions wherein
said O.sub.2 concentrations are about 20%, less than about 20%,
about 15%, less than about 15%, about 10%, less than about 10%,
about 7%, less than about 7%, about 6%, less than about 6%, about
5%, less than about 5%, about 4%, less than about 4%, about 3%,
less than about 3%, about 2%, less than about 2%, about 1%; or,
wherein said low oxygen conditions are in a range from about 1% to
about 20%, about 1% to about 15%, about 1% to about 10%, about 1%
to about 5%, about 5% to about 20%, about 5% to about 15%, about 5%
to about 10%, about 10% to about 15%, about 10% to about 20%, about
2% to about 8%, about 2% to about 7%, about 2% to about 6%, about
2% to about 5%, about 2% to about 4%, about 2% to about 3%, about
3% to about 8%, about 3% to about 7%, about 3% to about 6%, about
3% to about 5%, about 3% to about 4%, about 4% to about 8%, about
4% to about 7%, about 4% to about 6%, about 4% to about 5%, about
5% to about 8%, about 5% to about 7%, about 5% to about 6%, or
about 5%.
[0111] Embodiments of the invention may be generated with CF-SC and
exCF-SC (for example, ABM-SC and exABM-SC) cultured under low
oxygen conditions wherein CO.sub.2 concentration range from about
1-15% (for example, wherein the CO.sub.2 concentration is about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%),
plus low O.sub.2 and balanced with nitrogen. Embodiments of the
invention may be generated with CF-SC and exCF-SC (for example,
ABM-SC and exABM-SC) passaged by seeding cells at low cell
densities, wherein said cell density ranges from about 1-2500
cells/cm.sup.2 (for example, wherein the cell density is about 1,
2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, 1500, 2000 or 2500 cells/cm.sup.2). For
example, ABM-SC may be passaged at seeding densities of less than
about 2500 cell/cm.sup.2, less than about 1000 cells/cm.sup.2, less
than about 500 cells/cm.sup.2, less than about 100 cells/cm.sup.2,
less than about 50 cells/cm.sup.2, less than about 30
cells/cm.sup.2, or less than about 10 cells/cm.sup.2. Embodiments
of the invention may be generated with CF-SC and exCF-SC (for
example, ABM-SC and exABM-SC) wherein the cell population doubling
rates are maintained in a range of less than about 24-96 hours (for
example, wherein the cell population doubling rate is maintained at
less than about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or
96 hours). Embodiments of the invention may be generated with CF-SC
and exCF-SC (for example, ABM-SC and exABM-SC) wherein the cell
population maintains an essentially constant doubling rate through
a range of population doublings such as in a range of about 5-50
population doublings (for example, wherein the population doubling
rate is maintained for about 5-10, 5-15, 5-20, 5-25, 5-30, 5-35,
5-40, 5-45, or 5-50 population doublings).
[0112] Embodiments of the invention include use of CF-SC and
exCF-SC (for example, ABM-SC and exABM-SC) incorporated into
pharmaceutically acceptable compositions which may be in a liquid,
semi-solid, or solid-like state. Use of the terms "liquid,
semi-solid, or solid-like state" is intended to indicate that the
pharmaceutically acceptable composition in which the cells are
contained can span a range of physical states from 1) a common
liquid state (such as in an ordinary physiological saline
solution); 2) to a wide-range of low-to-highly viscous states
including jelly-like, gelatinous, or viscoelastic states (wherein
the pharmaceutical composition contains from very high to very low
levels of extracellular water, for example, such that the
composition ranges in viscosity from a state where it "oozes"
slowly like oil or honey to increasingly gelatinous or viscoelastic
states which may be jelly-like, pliable, semi-elastic and/or
malleable; 3) to a solid-like state (having very low levels of
extracellular water) wherein the living cells within the matrix
have remodeled the milieu in which they were initially suspended
into a durable, non-gelatinous, but still pliable, semi-elastic,
and malleable matrix (which, for example, has some of the same
pliable, semi-elastic properties of mammalian skin); see, FIGS. 10A
and 10B.
[0113] (Note: Viscoelasticity, also known as anelasticity,
describes materials that exhibit both viscous and elastic
characteristics when undergoing plastic deformation. Viscous
materials, like honey, resist shear flow and strain linearly with
time when a stress is applied. Elastic materials strain
instantaneously when stretched and just as quickly return to their
original state once the stress is removed. Viscoelastic materials
have elements of both of these properties and, as such, exhibit
time dependent strain.)
[0114] Clinical administration of cells in liquid, semi-solid, and
solid-like vehicles will enable application of treatments that
shape to the contour of the wound bed, without trapping unwanted
exudate in the wound.
[0115] Combining soluble matrix components such as collagens or
fibrin with CF-SC and exCF-SC (for example, ABM-SC and exABM-SC)
induces the cell population to up-regulate expression of important
secreted proteins such as cytokines and matrix metalloproteinases.
Moreover, application of ABM-SC to surgically-induced wounds
appears to facilitate wound closure and prevent scarring thereby
resulting in minimal scarring (see, Example 7).
[0116] Additionally, the apparent immunomodulatory properties of
CF-SC and exCF-SC (such as, ABM-SC and exABM-SC) (see e.g., Example
4) make compositions and therapies incorporating these cells
attractive for the treatment of immunological disorders and
diseases involving the skin, such as for example, but not limited
to, chronic inflammatory dermatoses, psoriasis, lichen planus,
lupus erythematosus (LE), graft-versus-host disease (GVHD), and
drug eruptions (i.e., adverse cutaneous drug reactions).
[0117] Secreted proteins and cell-supernatant fractions from CF-SC
and exCF-SC (such as, ABM-SC and exABM-SC) conditioned media can be
manufactured from serum-free conditions, concentrated and prepared
in such manner as to make them suitable for in vivo use. When
prepared this way, conditioned serum-free media from ABM-SC has
been demonstrated to contain numerous pro-regenerative cytokines,
growth factors, and matrix proteases in therapeutically effective
concentrations (see, Table 1A, 1B and 1C). The complex mixture of
hundreds of soluble factors produced by ABM-SC can be distinguished
by 2D SDS PAGE (see, FIG. 1). Individual proteins and other
macromolecules can be excised from these gels and identified using
techniques routinely practiced in the art, such as, for example,
MALDI-TOF mass spectrometry (Matrix Assisted Laser Desorption
Ionisation-Time Of Flight spectrometry).
[0118] Utilizing the methods disclosed (as well as other separation
techniques such as chromatography or hollow fiber cell culture
systems), the desired proteins or cell supernatant fractions can be
isolated, dialyzed, lyophilized and stored as a solid, or
reconstituted in an appropriate vehicle for therapeutic
administration. In one embodiment, the proteins or cell-supernatant
fractions would be reconstituted in a semi-solid collagen or
fibrin-based vehicle, and applied topically to the wound bed.
[0119] In addition to products generated by CT-SC and exCF-SC (such
as, ABM-SC and exABM-SC), any number and type of pharmaceutically
acceptable small molecules to large macromolecular compounds
(including biologics such as lipids, proteins, and nucleic acids)
may be incorporated for administration with a pharmaceutically
acceptable carrier such as biodegradeable matrices in which CF-SC
and exCF-SC (such as, ABM-SC and exABM-SC), or products generated
by such cells, are contained. As a very small sampling, such
additional molecules may include small molecule pharmaceuticals
such as anti-inflammatories, antibiotics, vitamins, and minerals
(such as calcium) to name but a few categories, Likewise, a very
small sampling of biologics may include extracellular matrix
proteins, blood plasma coagulation proteins, antibodies, growth
factors, chemokines, cytokines, lipids (such as cardiolipin and
sphingomyelin), and nucleic acids (such as ribozymes, anti-sense
oligonucleotides, or cDNA expression constructs); including
therapeutically beneficial variants and derivatives of such
molecules such as various isoforms, fragments, and subunits, as
well as substitution, insertion, and deletion variants. These are
mentioned merely by way of example, as it can be appreciated by
those skilled in the art that, in combination with the teachings
provided herein, any number of additional structural or
therapeutically beneficial compounds could be included for
administration with a pharmaceutically acceptable carrier such as
biodegradeable matrices in which CF-SC and exCF-SC (such as, ABM-SC
and exABM-SC), or products generated by such cells, are
contained.
[0120] One embodiment of the invention encompasses a method of
stimulating wound closure in a diabetic patient, such as a diabetic
foot or venous leg ulcer, or a post-surgical wound. Stimulation of
wound closure may be promoted by treatment with a pharmaceutical
composition of CF-SC and exCF-SC (such as, ABM-SC and exABM-SC), or
products generated by such cells, combined with naturally occurring
extracellular matrix and/or blood plasma proteins components such
as, for example, purified natural or recombinant human, bovine,
porcine, or recombinant collagens, laminins, fibrinogen, and/or
thrombin. The pharmaceutical composition may be administered to a
mammal, including a human, at the site of tissue damage. In another
embodiment, a topically administered biodegradable matrix is formed
from a mixture of components such as purified natural or
recombinant collagen, fibrinogen, and/or thrombin, combined with
allogeneic CF-SC and exCF-SC (such as, ABM-SC and exABM-SC).
[0121] In another embodiment of the invention, a pharmaceutical
composition of allogeneic cells and matrix are cultured in vitro
for an extended period of time (such as, for example, but not
limited to 1 day to one month or longer), producing the de novo
formation of connective tissue. In another embodiment of the
invention, the biodegradable matrix is bovine collagen or
poylglycolic acid. In another embodiment, the pharmaceutical
composition is cultured in serum-free cell media under conditions
of reduced oxygen tension, for example but not limited to, oxygen
tension equivalent to about 4-5% O.sub.2, 5% CO.sub.2, and balanced
with nitrogen.
[0122] In one embodiment, the invention encompasses a method of
preparing a pharmaceutical composition comprising the steps:
[0123] (a) preparing a solution comprising soluble collagen,
serum-free cell culture media supplemented with glutamine, sodium
biocarbonate, and HEPES (optionally including supplementation with
insulin, transferrin, and/or selenium);
[0124] (b) re-suspending CF-SC or exCF-SC (for example, ABM-SC or
exABM-SC) in the solution; and,
[0125] (c) transferring the cell suspension to a tissue mold, or
equivalent thereof, to congeal at 37.degree. C., for example, when
placed in a cell culture incubator.
[0126] The above method of preparing a pharmaceutical composition
may additionally comprise the step of incubating the culture for an
extended period of time (such as, for example but not limited to,
1-3 days or longer) under low oxygen tension conditions equivalent
to about 4-5% O.sub.2, 5% CO.sub.2, and balanced with nitrogen.
[0127] In another embodiment, the invention encompasses a method of
preparing a pharmaceutical composition comprising the steps of:
[0128] a) preparing a solution comprising fibrinogen and
thrombin;
[0129] b) re-suspending CF-SC or exCF-SC (for example, ABM-SC or
exABM-SC) in the solution; and,
[0130] c) administering the re-suspended solution to an open
wound.
[0131] In another embodiment, the invention encompasses a method of
preparing a pharmaceutical composition comprising the steps of:
[0132] a) preparing a solution comprising soluble collagen,
serum-free cell culture media supplemented with glutamine, sodium
biocarbonate, and HEPES (optionally including supplementation with
insulin, transferrin, and/or selenium); and
[0133] b) mixing a fraction or fractions of cell-supernatant
derived from CF-SC or exCF-SC (for example, ABM-SC or exABM-SC)
into the solution; and,
[0134] c) transferring the solution to a tissue mold, or equivalent
thereof, to congeal at 37.degree. C., for example, when placed in a
cell culture incubator.
[0135] The above method of preparing a pharmaceutical composition
may additionally comprise the step of incubating the tissue mold,
or equivalent thereof, under atmospheric oxygen tension conditions
equivalent to about 18-21% O.sub.2 and 5% CO.sub.2
[0136] In another embodiment, the invention encompasses a method of
preparing a pharmaceutical composition comprising the steps of:
[0137] d) preparing a solution comprising fibrinogen and
thrombin;
[0138] e) mixing a fraction or fractions of cell-supernatant
derived from CF-SC or exCF-SC (for example, ABM-SC or exABM-SC)
into the solution; and,
[0139] f) administering the solution to an open wound.
[0140] In another embodiment, the present invention encompasses
tissue regeneration, particularly in the treatment of tissue damage
caused by: immune related disorders (such as autoimmune disorders);
inflammation (including both acute and chronic inflammatory
disorders); ischemia (such as myocardial infarction); traumatic
injury (such as burns, lacerations, and abrasions); infection (such
as bacterial, viral, and fangal infections); and, chronic cutaneous
wounds. The present invention encompasses treatment of a diversity
of damage and disorders, for example, but not limited to,
neurological damage and disorders of the central nervous system
(brain) and per'pheral nervous system (e.g., spinal cord) (for
example, such as may be caused by neurotrauma and neurodegenerative
diseases). Another embodiment of the invention encompasses
treatment of diseases and disorders requiring bone, connective
tissue, and cartilage regeneration, chronic and acute inflammatory
liver diseases, vascular insufficiency, and corneal and macular
degeneration. Another embodiment of the invention encompasses
treating cardiovascular and pulmonary damage and disorders (for
example, such as myocardial ischemia and repair and regeneration of
blood vessels). Another embodiment of the invention encompasses
treating damage and disorders of pancreatic and hepatic tissue as
well as other endocrine and exocrine glands. Another embodiment of
the invention encompasses treating damage and disorders of thymus
as well as other immune cell producing and harboring organs.
Another embodiment of the invention encompasses treating damage and
disorders of the genitourinary system (for example, such as the
ureter and bladder). Another embodiment of the invention
encompasses treating hernias and herniated tissues. Another
embodiment of the invention encompasses treatment, repair,
regeneration, and reconstruction of heart valves.
[0141] CF-SC and exCF-SC (such as, ABM-SC and exABM-SC) or protein
and cell-supernatant fractions derived from CF-SC and exCF-SC (such
as, ABM-SC and exABM-SC), can also be reconstituted in a solid-like
collagen-base device. When the cells are reconstituted in such
manner, the solid-like collagen matrix is remodeled over several
days, giving rise to a neotissue that has fabricated its own unique
matrix. Such CF-SC and exCF-SC (such as, ABM-SC and exABM-SC)
derived neotissues are pliable, suturable, and bioactive (see e.g.,
FIGS. 6, 7, and 10A-C). These structures could also be sterilized,
chemically cross-linked, freeze-dried, or further processed,
rendering the cells non-viable and incapable of further growth.
[0142] Such devices may be particularly beneficial in the treatment
of burns, including fall thickness burn wounds. To rebuild a
vascularized wound bed, patients with severe burns are often
treated with an artificial dermal replacement after surgical
resection of the dead tissue. After the wound bed has healed, these
patients are subsequently treated with artificial skin products or
applications of epithelial cells in an attempt to re-grow host
epidermis.
[0143] Compositions, such as descr'bed herein, when used in lieu of
a conventional artificial dermal products (e.g., DERMAGRAFT.TM.),
may increase the longevity of subsequently grafted allogeneic skin,
by inhibiting or reducing undesirable T-cell mediated immune
reactions (see, Example 5). By modulating T-cell mediated immune
responses, compositions of the present invention may permit
subsequent reapplication of the artificial skin for a durations
adequate to stimulate re-growth of the patients own skin.
[0144] The above-referenced ABM-SC have been shown to exhibit the
following properties:
In Vitro
[0145] Secretion of cytokines important in angiogenesis and tissue
repair. [0146] Release of factors for prevention and inhibition of
scarring and matrix turnover. [0147] Promotion of migration of
endothelial cells indicative of pro-angiogenic activity.
In Vivo
[0147] [0148] Significant improvement in outcomes in multiple
animal models of acute myocardial infarction (AMI) and stroke.
[0149] Effective and well-tolerated intracardiac or intracerebral
delivery of cells. [0150] Cells not detectable in tissues eight
weeks post-injection. [0151] No measurable immune response against
cells.
[0152] In one embodiment of the invention, a number of
pro-regenerative cellular factors secreted by CF-SC and exCF-SC
(such as, ABM-SC and exABM-SC) may be used in treatment, repair,
regeneration, and/or rejuvenation of damaged tissues and organs
(such as, for example, cardiac and neuronal organs and tissues
damaged by, for example, heart failure due to acute myocardial
infarction (AMI) or stroke). These include factors which can be
secreted by CF-SC such as ABM-SC as shown in FIG. 11. For example,
these factors include, but are not limited to, SDF-1 alpha, VEGF,
ENA-78, Angiogenin, BDNF, IL-6, IL-8, ALCAM, MMP-2, Activin, MMP-1,
MMP-13, MCP-1. See, FIG. 11. Additional factors, such as those
listed in Table 1A, 1B and 1C, may also be secreted by CF-SC and
exCF-SC (such as, ABM-SC and exABM-SC). See e.g., Table 1A, 1B and
1C.
[0153] Secretion of pro-regenerative factors by CF-SC and exCF-SC
(such as, ABM-SC and exABM-SC) may be enhanced or induced by
pre-treatment with stimulatory factors (such as, for example, tumor
necrosis factor-alpha (TNF-alpha)) to induce the production of
conditioned cell culture media or to prime the cells before
administration of cells to a patient.
[0154] Acute ischemia, trauma or inflammation lead to a
constellation of cellular and chemical events in the affected
organs and tissues. See e.g., FIG. 12. In the inflammation phase
there occurs a release of factors and an influx of cells to the
injured site. In the regeneration phase there occurs a recruitment
of circulating cells for the proper repair of functional tissue.
And, in the fibrosis phase, there occurs a deposition of fibrotic
scars which potentially compromise organ function. Moreover, a
variety of cytokines and other biological molecules play a
diversity of roles in each of these processes. See e.g., FIG.
12.
[0155] Use of CF-SC and exCF-SC (such as, ABM-SC and exABM-SC) in
the present invention includes methods of treating and preventing
inflammation, methods of stimulating organ and tissue regeneration
while reducing fibrosis (i.e., tissue scarf ng), and methods of
stimulating angiogenesis via compositions (e.g., cytokines,
proteases, extracellular matrix proteins, etc) produced by
stimulated or unstimulated CF-SC and exCF-SC (such as, ABM-SC and
exABM-SC).
[0156] In another embodiment, CF-SC and exCF-SC may inhibit the
biological process of fibrosis. Fibrosis is a natural byproduct of
wound healing, scarring, and inflammation in many human tissues.
Fibrosis, also known as fibrotic scarring, is a significant
impediment to regenerating tissue with optimal function, especially
in the heart and central nervous system (CNS), because scar tissue
displaces cells needed for optimal organ function. Treatment with
cells disclosed herein helps to prevent or reduce fibrosis and
thereby facilitates the healing of damaged tissue. The fibrosis may
be prevented by additive or synergistic effects of two or more
secreted proteins or cell produced compositions, including membrane
bound cell-surface molecules. Additionally matrix proteases induced
or produced by the administered CF-SC and exCF-SC (such as, ABM-SC
and exABM-SC) may play an important part in preventing
fibrosis.
[0157] In another exemplary use of the present invention,
angiogenesis, also known as neovascularization, is increased in a
desired tissue. Angiogenesis, or the formation of new blood
vessels, is a key component of regenerative medicine because newly
formed tissue must have a blood supply, and angiogenesis is crucial
if endothelial cells are lost during degenerative processes,
disease progression, or acute injuries for which the present
invention is a treatment. Hence, use of CF-SC and exCF-SC (for
example, ABM-SC and exABM-SC) or compositions produced by such
cells are useful in stimulating angiogenesis in target tissues and
organs (especially, for example, in damaged cardiac tissue).
Angiogenesis is an important component of tissue repair and can
operate in conjunction with fibrosis inhibition to optimize healing
of damaged tissues.
[0158] Another exemplary use of the present invention involves the
stimulation of regeneration or rejuvenation processes without the
engraftment of the administered cells. In vivo studies have shown
that long term cell engraftment or tissue-specific differentiation
of human ABM-SC or exABM-SC are generally not seen, suggesting that
the mechanism by which these cells incite tissue regeneration is
not through cell replacement, but instead through a host response
to the cells themselves and/or factors they produce. This is not
surprising, however, given that the role of ABM-SC in bone marrow
is to provide structural and trophic support. Hence, the present
invention includes treatment of damaged tissues and organs wherein
the administered CF-SC and exCF-SC (for example, ABM-SC and
exABM-SC) do not exhibit permanent or long-term tissue or organ
engraftment. Instead, the therapeutic CF-SC and exCF-SC (for
example, ABM-SC and exABM-SC) provide trophic support factors,
suppress cell-death, inhibit fibrosis, inhibit inflammation (e.g.,
immune cell inflammatory responses), promote extra-cellular matrix
remodeling, and/or stimulate angiogenesis without becoming part of
the repaired tissue at a significant or currently detectable
level.
[0159] A further example of the present invention teaches that
after a period of time, the administered cells are not detected
anywhere in the experimental animal, suggesting the administered
cells are completely cleared from the body. This suggests that
secreted factors play an essential role in the repair of damaged
tissue.
[0160] In yet another example of the present invention illustrating
its utility, the hABM-SCs disclosed herein come from one donor
source. As such, these cells will be allogeneic cell transplants in
patients which might suggest that these transplanted cells could
potentially stimulate an adverse immune response. However,
surprisingly, we find that transplanted allogeneic cells disclosed
herein actually can suppress mitogen induced T-cell proliferation
in vitro and avoid induction of a T-cell-dependent immune response
in vivo. A T-cell mediated immune response is a key factor in
immune processes that are detrimental to healing, regenerative, and
rejuvenation processes.
[0161] As used herein "an effective amount" is an amount sufficient
to produce detectable improvement in tissue, organ, or biological
system (e.g., immune system) performance, function, integrity,
structure, or composition wherein said improvement is indicative of
complete or partial amelioration, restoration, repair,
regeneration, or healing of the damaged tissue, organ or biological
system.
[0162] Table 1A, 1B and 1C shows an extensive list of cytokines,
growth factors, soluble receptors, and matrix proteases secreted by
human ABM-SC when sub-cultured in serum-free cell culture media.
Media Supernatant Concentrate #1=Advanced DMEM (Gibco.TM.)
supplemented with 4 mM L-glutamine. Media Supernatant Concentrate
#2 =RPMI-1640 containing 4 mM L-glutamine and HEPES (HyClone)
supplemented with Insulin-Transferrin-Selenium-A (Gibco.TM.).
[0163] The results demonstrate that numerous trophic factors and
soluble receptors important for tissue regeneration and modulation
of the immune system are produced by ABM-SC at therapeutically
relevant levels when cultured under these conditions. Notably,
earlier experiments demonstrated that supplementation of the base
culture medium with insulin, transferrin, and selenium was required
to achieve secreted protein levels such as those indicated in Table
1A, 1B and 1C.
Immune Disorders
[0164] Cells and compositions of the present invention may be used
to prevent, treat, and/or ameliorate, inter alia, immune,
autoimmune, and inflammatory diseases and disorders. Some examples
of such disorders are indicated below; these lists are exemplary
only and are not intended to be comprehensive with respect to all
immune, autoimmune, and inflammatory diseases and disorders; nor
should the following be construed as limiting with respect to
pathologies which may be treated with the cells and compositions of
the present invention.
[0165] Example of some diseases with a complete or partial
autoimmune etiology: Acute disseminated encephalomyelitis (ADEM),
Addison's disease, Ankylosing spondylitis, Antiphospholipid
antibody syndrome (APS), Aplastic anemia, Autoimmune hepatitis,
Autoimmune Oophoritis, Celiac disease, Crohn's disease, Diabetes
mellitus type 1, Gestational pemphigoid, Goodpasture's syndrome,
Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's
disease, Idiopathic thrombocytopenic purpura, Kawasaki's Disease,
Lupus erythematosus, Multiple sclerosis, Myasthenia gi avis,
Opsoclonus myoclonus syndrome (OMS), Optic neuritis, Ord's
thyroiditis, Pemphigus, Pernicious anaemia, Polyarthritis, Primary
biliary cirrhosis, Rheumatoid arthritis, Reiter's syndrome,
Sjogren's syndrome, Takayasu's arteritis, Temporal arteritis (also
known as "giant cell arteritis"), Warm autoimmune hemolytic anemia,
and Wegener's granulomatosis.
[0166] Examples of some diseases suspected of being linked to
autoimmunity: Alopecia universalis, Behcet's disease, Chagas'
disease, Chronic fatigue syndrome, Dysautonomia, Endometriosis,
Hidradenitis suppurativa, Interstitial cystitis, Lyme disease,
Morphea, Neuromyotonia, Narcolepsy, Psoriasis, Sarcoidosis,
Scleroderma, Ulcerative colitis, Vitiligo, and Vulvodynia.
[0167] Examples of some immune hypersensitivity diseases and
disorders: Allergic asthma, Allergic conjunctivitis, Allergic
rhinitis ("hay fever"), Anaphylaxis, Myasthenia gravis, Angioedema,
Arthus reaction, Atopic dermatitis (eczema), Autoimmune hemolytic
anemia, Autoimmune Pernicious anemia, Coeliac disease, Contact
dermatitis (poison ivy rash, Eosinophilia, Erythroblastosis
Fetalis, Farmer's Lung (Arthus-type reaction), for example),
Goodpasture's syndrome, Graves' disease, Graves' disease,
Hashimoto's thyroiditis, Hemolytic disease of the newborn, Immune
complex glomerulonephritis, Immune thrombocytopenia, Myasthenia
gravis, Pemphigus, Rheumatic fever, Rheumatoid arthritis, Serum
sickness, Subacute bacterial endocarditis, Symptoms of leprosy,
Symptoms of malaria, Symptoms of tuberculosis, Systemic lupus
erythematosus, Temporal arteritis, Transfusion reactions,
Transplant rejection, and Urticaria (hives).
[0168] Example of some inflammatory disorders: allergies,
ankylosing spondylitis, arthritis, asthma, autistic enterocolitis,
autoimmune diseases, Behcet's disease, chronic inflammation,
glomerulonephritis, inflammatory bowel disease (IBD), inflammatory
bowel diseases, pelvic inflammatory disease, psoriasis, psoriatic
arthritis, reperfusion injury, rheumatoid arthritis, transplant
rejection, and vasculitis.
[0169] Example of some immunodeficiency disorders: B cell
deficiencies (such as X-linked agammaglobulinemia and Selective
Immunoglobulin Deficiency), T cell deficiencies (such as DiGeorge's
syndrome (Thymic aplasia), Chronic mucocutaneous candidiasis,
Hyper-IgM syndrome and, Interleukin-12 receptor deficiency),
Combined T cell and B cell abnormalities (such as Severe Combined
Immunodeficiency Disease (SCID), Wiskott-Aldrich syndrome, and
Ataxia-telangiectasia), Complement Deficiencies (such as Hereditary
Angioedema or Hereditary angioneurotic edema and Paroxysmal
nocturnal hemoglobinuria), Phagocyte deficiencies (such as
Leukocyte adhesion deficiency, Chronic Granulomatous Disease (CGD),
Chediak-Higashi syndrome, Job's syndrome (Hyper-IgE syndrome),
Cyclic neutropenia, Myeloperoxidase deficiency, Glucose-6-phosphate
dehydrogenase deficiency, and Interferon-.gamma. deficiency), and
Common Variable Immunodeficiency (CVID), Vici syndrome, and
Acquired immune deficiency syndrome (AIDS).
Embodiments of the Invention
[0170] Particular embodiments of the invention include the
following:
[0171] A1. A method of administering a therapeutically useful
amount of a biological composition or compositions to a subject,
comprising administering to said subject an isolated population of
self-renewing colony forming cells, wherein the cells in said cell
population have substantially no multipotent differentiation
capacity, wherein said cells have a normal karyotype, and wherein
said cells are non-immortalized.
[0172] A2. A method of administering a therapeutically useful
amount of a biological composition or compositions to a subject,
comprising:
[0173] (i) isolating the biological composition or compositions
produced by an isolated population of self-renewing colony forming
cells; and,
[0174] (ii) administering said biological composition or
compositions to said
[0175] subject,
[0176] wherein the cells in said cell population have substantially
no multipotent differentiation capacity, wherein said cells have a
normal karyotype, and wherein said cells are non-immortalized.
[0177] A3. A method of repairing, treating, or promoting
regeneration of damaged tissue in a subject, comprising
administering to said subject an effective amount of an isolated
population of self-renewing colony forming cells, wherein the cells
in said cell population have substantially no multipotent
differentiation capacity, wherein said cells have a normal
karyotype, and wherein said cells are non-immortalized.
[0178] A4. A method of repairing, treating, or promoting
regeneration of damaged tissue in a subject, comprising:
[0179] (i) isolating the biological composition or compositions
produced by an isolated population of self-renewing colony forming
cells; and,
[0180] (ii) administering said biological composition or
compositions to said subject,
[0181] wherein the cells in said cell population have substantially
no multipotent differentiation capacity, wherein said cells have a
normal karyotype, and wherein said cells are non-immortalized.
[0182] A5. A method of treating or reducing inflammation, immune,
or autoimmune activity in a subject, comprising administering to
said subject an effective amount of an isolated population of
self-renewing colony forming cells, wherein the cells in said cell
population have substantially no multipotent differentiation
capacity, wherein said cells have a normal karyotype, and wherein
said cells are non-immortalized.
[0183] A6. A method of treating or reducing inflammation, immune,
or autoimmune activity in a subject, comprising:
[0184] (i) isolating the biological composition or compositions
produced by an isolated population of self-renewing colony forming
cells; and,
[0185] (ii) administering said biological composition or
compositions to said subject,
[0186] wherein the cells in said cell population have substantially
no multipotent differentiation capacity, wherein said cells have a
normal karyotype, and wherein said cells are non-immortalized.
[0187] A7. The method of any of embodiments A1 to A6, wherein prior
to administration, said cell population has been passaged in vitro
for a number of population doublings sufficient to cause the cells
in said population to lose multipotent differentiation
capacity.
[0188] A8. The method of any one of embodiments A1 to A7, wherein
said cell population has unipotent differentiation capacity.
[0189] A9. The method of any of embodiments A1 to A8, wherein said
cells have substantial capacity for self-renewal.
[0190] A10. The method of any of embodiments A1 to A9, wherein
prior to administration said cell population has been passaged in
vitro for a number of population doublings while retaining
substantial capacity for self-renewal.
[0191] A11. The method of any one of embodiments A1 to A10, wherein
the cells in said isolated cell population are not embryonic stem
cells.
[0192] A12. The method of any one of embodiments A1 to A11, wherein
the cells in said isolated cell population are not stem cells,
mesenchymal stem cells, hematopoietic stem cells, multipotent adult
progenitor cells (MAPCs), multipotent adult stem cells (MASCs), or
fibroblasts.
[0193] A13. The method of any one of embodiments A1 to A12, wherein
said cells do not differentiate into one or more cell types
selected from the group consisting of:
[0194] a) osteocytes; b) adipocytes; and, c) chondrocytes.
[0195] A14. The method of any one of embodiments A1 to A13, wherein
said cells do not deposit detectable levels of calcium following
treatment under osteoinductive conditions.
[0196] A15. The method of embodiment A14, wherein said
osteoinductive conditions include exposure to exogenously supplied
Noggin.
[0197] A16. The method of any one of embodiments A1 to A15, wherein
the cells in said isolated cell population are derived from
connective tissue.
[0198] A17. The method of any one of embodiments A1 to A16, wherein
the cells in said isolated cell population are stromal cells.
[0199] A18. The method of any one of embodiments A1 to A17, wherein
the cells in said isolated cell population co-express CD49c and
CD90.
[0200] A19. The method of any one of embodiments A1 to A18, wherein
the cell population maintains an approximately constant doubling
rate through multiple in vitro cell doublings,
[0201] A20. The method of any one of embodiments A1 to A19, wherein
said cells are negative for detectable expression of one or more
antigens selected from the group consisting of:
[0202] a) CD10; b) STRO-1; and, c) CD106/VCAM-1.
[0203] A21. The method of any one of embodiments A1 to A20, wherein
said cells are positive for detectable expression of one or more
antigens selected from the group consisting of:
[0204] a) CD44; b) HLA Class-1 antigen; and, c) 13 (beta)
2-Microglobulin,
[0205] A22. The method of any one of embodiments A1 to A21, wherein
said cells express or secrete detectable quantities of compositions
selected from the group consisting of:
[0206] a) TNF-RI; b) soluble TNF-RI; c) TNF-RII; d) soluble
TNF-RII; e) IL-1R antagonist;
[0207] and, f) IL-18 binding protein.
[0208] A23. The method of any one of embodiments A1 to A21, wherein
said cells express or secrete detectable quantities of compositions
selected from the group consisting of compositions shown in Table
1A, 1B and 1C.
[0209] A24. The method of any one of embodiments A1 to A23, wherein
the cells in said isolated cell population are initially isolated
from a tissue source selected from the group consisting of:
[0210] a) bone marrow; b) adipose tissue/fat; c) skin; d)
placental; e) umbilical cord; f) tendon; g) ligament; h) muscle
fascia; and, i) other connective tissues.
[0211] A25. The method of embodiment A24, wherein said tissue
source is human.
[0212] A26. The method of any one of embodiments A1 to A25, wherein
said cell population maintains an approximately constant doubling
rate through a number of in vitro cell doublings selected from the
group consisting of:
[0213] a) 1 to 5 cell doublings; b) 5 to 10 cell doublings; c) 10
to 20 cell doublings; d) 20 to 30 cell doublings; e) 30 to 40 cell
doublings; f) 40 to 50 cell doublings; g) 1 to 50 cell doublings;
h) 5 to 50 cell doublings; i) 10 to 50 cell doublings; j) 20 to 50
cell doublings; k) 30 to 50 cell doublings; 1) 1 to 10 cell
doublings; m) 1 to 20 cell doublings; n) 1 to 30 cell doublings; o)
1 to 40 cell doublings; p) 5 to 20 cell doublings; q) 5 to 30 cell
doublings; r) 5 to 40 cell doublings; s) 10 to 30 cell doublings;
t) 10 to 40 cell doublings; and, u) 20 to 40 cell doublings.
[0214] A27. The method of any one of embodiments A1 to A26, wherein
said cell population has undergone a number of population doublings
selected from the group consisting of:
[0215] a) at least about 10 population doublings; b) at least about
15 population doublings; c) at least about 20 population doublings;
d) at least about 25 population doublings; e) at least about 30
population doublings; 0 at least about 35 population doublings; g)
at least about 40 population doublings; h) at least about 45
population doublings; and, i) at least about 50 population
doublings.
[0216] A28. The method of any one of embodiments A1 to A27, wherein
said biological composition or compositions are bound in or to the
cell surface of said cell populations.
[0217] A29. The method of any one of embodiments A1 to A28, wherein
said biological composition or compositions are secreted into the
extracellular environment of said cell populations.
[0218] A30. The method of any one of embodiments A1 to A29, wherein
said biological composition or compositions are one or more
molecules selected from the group consisting of:
[0219] a) proteins; b) carbohydrates; c) lipids; d) fatty acids; e)
fatty acid derivatives; d) gases; and, e) nucleic acids.
[0220] A31. The method of embodiment A30, wherein said proteins are
selected from the group consisting of:
[0221] a) glycosylated proteins; b) cytokines; c) chemokines; d)
lymphokines; e) growth factors; f) trophic factors, g)
morphogenetic proteins; and, h) hormones.
[0222] A32. The method of embodiment A31, wherein said wherein said
biological composition or compositions bind to and inactivate, or
reduce, the biological activity of molecules selected from the
group consisting of:
[0223] a) fatty acids; b) fatty acid derivatives; c) receptor
molecules; d) cytokines; e) chemokines; f) lymphokines; g) growth
factors; h) trophic factors, i) morphogenetic proteins; and, j)
hormones.
[0224] A33. The method of embodiment A32, wherein said biological
composition or compositions are soluble receptors that bind cognate
ligands selected from the group consisting of:
[0225] a) fatty acids; b) fatty acid derivatives; c) receptor
molecules; d) cytokines; e) chemokines; f) lymphokines; g) growth
factors; h) trophic factors, i) morphogenetic proteins; and, j)
hormones.
[0226] A34. The method of any one of embodiments A1 to A33, wherein
said cells are induced to increase expression of one or more
biological compositions.
[0227] A35. The method of any one of embodiments A1 to A33, wherein
said cells are induced to express one or more biological
compositions.
[0228] A36. The method of any one of embodiments A1 to A29, wherein
said one or more biological compositions is/are selected from Table
1A, 1B and 1C.
[0229] A37. The method of any one of embodiments A1 to A29, wherein
said one or more biological compositions is selected from the group
consisting of:
[0230] a) TNF-RI; b) soluble TNF-RI; c) TNF-RII; d) soluble
TNF-RII; e) IL-1R antagonist; and, f) IL-18 binding protein.
[0231] A38. The method of any one of embodiments A1 to A37, wherein
the cells in said cell population do not exhibit long-term
engraftment in, or with, tissues or organs when administered to a
living mammalian organism.
[0232] A39. The method of any one of embodiments A1 to A38, wherein
the cells in said cell population maintain approximately constant
levels of production of one or more therapeutically useful
compositions in vivo.
[0233] A40. The method of embodiment A39, wherein said levels of
production are maintained for a measure of time selected from the
group consisting of:
[0234] a) at least about 24 hours; b) at least about 48 hours; c)
at least about 72 hours; d) at least about 4 days; e) at least
about 5 days; f) at least about 6 days; g) at least about 7 days;
h) at least about 2 weeks; i) at least about 3 weeks; j) at least
about 4 weeks; k) at least about 1 month; 1) at least about 2
months; m) at least about 3 months; n) at least about 6 months;
and, o) at least about 1 year.
[0235] A41. The method of any one of embodiments A1 to A40, wherein
said patient is human.
[0236] A42. The method of any one of embodiments A1 to A41, wherein
said method is used to treat a disease or disorder selected from
the group consisting of:
[0237] a) a neurological disease or disorder; b) a cardiac disease
or disorder; c) a skin disease or disorder; d) a skeletal muscle
disease or disorder; e) a respiratory disease or disorder; f) a
hepatic disease or disorder; g) a renal disease or disorder; h) a
genitourinary system disease or disorder; i) a bladder disease or
disorder; j) an endocrine disease or disorder; k) a hematopoietic
disease or disorder; 1) a pancreatic disease or disorder; m)
diabetes; n) an ocular disease or disorder; o) a retinal disease or
disorder; p) a gastrointestinal disease or disorder; q) a splenic
disease or disorder; r) an immunological disease or disorder; s) an
autoimmune disease or disorder; t) an inflammatory disease or
disorder; u) a hyperproliferative disease or disorder; and, v)
cancer.
[0238] A43. The method of any one of embodiments A1 to A42, wherein
said cells are genetically modified.
[0239] A44. The method of embodiment A43, wherein said cells are
genetically modified by introduction of a recombinant nucleic acid
molecule.
[0240] A45. A process for making an isolated cell population in any
one of embodiments A1 to A47, wherein said process comprises:
[0241] i) obtaining a source population of cells from an organism;
and,
[0242] ii) culturing said source population of cells in vitro.
[0243] B1. A composition comprising a pharmaceutically acceptable
mixture of self-renewing, colony-forming somatic cells (CF-SC), or
conditioned cell culture media derived from such cells, and
purified naturally occurring or isolated recombinant extracellular
matrix or blood plasma proteins.
[0244] B2. The composition of embodiment B1, wherein said CF-SC are
derived from bone marrow.
[0245] B3. The composition of embodiments B1 or B2, wherein said
CF-SC are derived from a human.
[0246] B4. The composition of any one of embodiments B1-B3, wherein
said CF-SC are derived from an adult mammal, including humans.
[0247] B5. The composition of any one of embodiments B1-B4, wherein
said CF-SC express one or more secreted proteins shown in Table 1A,
1B and 1C.
[0248] B6. The composition of any one of embodiment B1-B5, wherein
said extracellular matrix or blood plasma proteins comprise one or
more full-length or alternatively processed isoforms, proteolytic
fragments, or subunits of molecules selected from the group
consisting of:
[0249] a) collagen; b) elastin; c) fibronectin; d) laminin; e)
entactin (nidogen); f) hyaluronic acid; g) polyglycolic acid (PGA);
h) fibrinogen (Factor I); i) fibrin; j) prothrombin (Factor II); k)
thrombin; 1) anti-thrombin; m) Tissue factor Co-factor of VIIa
(Factor III); n) Protein C; o) Protein S; p) protein Z; q) Protein
Z-related protease inhibitor; r) heparin cofactor II; s) Factor V
(proaccelerin, labile factor); t) Factor-VII; u) Factor-VIII; v)
Factor-IX; w) Factor-X; x) Factor-XI; y) Factor-XII; z)
Factor-XIII; aa) von Willebrand factor; ab) prekallikrein; ac) high
molecular weight kininogen; ad) plasminogen; ae) plasmin; af)
tissue-plasminogen activator; ag) urokinase; ah) plasminogen
activator inhibitor-1; and, ai) plasminogen activator
inhibitor-2.
[0250] B7. The composition of any one of embodiments B1-B6, further
comprising purified naturally occurring or isolated recombinant
cytokines or chemokines.
[0251] B8. The composition of any one of embodiments B1-B7, wherein
said extracellular matrix, blood plasma proteins, cytokines, and/or
chemokines are derived from humans.
[0252] B9. The composition of any one of embodiments B1-B8, wherein
said pharmaceutically acceptable mixture forms a semi-solidified or
solidified matrix.
[0253] B10. A method of treating damaged tissue with the
composition of any one of embodiments B1-B8, wherein the
composition is a liquid.
[0254] B11. The method of embodiment B10, wherein the liquid is
applied by injection.
[0255] B12. A method of treating damaged tissue with the
composition of any one of embodiments 1-9, wherein the composition
is applied as a liquid but thereafter forms a semi-solidified or
solidified matrix.
[0256] B13. The method of embodiments B10-B12 wherein said tissue
is damaged as a result of a condition selected from the group
consisting of:
[0257] a) disease; b) physical trauma; c) ischemia; d) aging; e)
burn; f) bacterial infection; g) viral infection; h) fungal
infection; and, i) dysregulation of the immune system.
[0258] B14. The method of embodiment B13, wherein the damaged
tissue is skin.
[0259] B15. A method of using the composition of any one of
embodiments B1-B9 for facial skin rejuvenation.
[0260] B16. A method of using the composition of any one of
embodiments B1-B9, wherein said composition inhibits acute
inflammation.
[0261] C1. A method for treating, repairing, regenerating, or
healing a damaged organ or tissue comprising contacting said
damaged organ or tissue with an effective amount of self-renewing
colony forming somatic cells or compositions produced from such
cells so as to effect said treatment, repair, regeneration, or
healing of the damaged organ or tissue.
[0262] C2. The method of embodiment C1, wherein said damaged organ
or tissue is contacted with an effective amount of self-renewing
colony forming somatic cells or compositions produced from such
cells by means selected from the group consisting of:
[0263] a) injection into the damaged organ or tissue; b)
application onto the damaged organ or tissue; c) injection proximal
to the damaged organ or tissue; d) application proximal to the
damaged organ or tissue; and, e) intravenous administration.
[0264] C3. The method of embodiments C1 or C2, wherein the cells
are derived from bone marrow.
[0265] C4. The method of any one of embodiments C1-C3, wherein the
cells are human.
[0266] C5. The method of any one of embodiments C1-C4, wherein the
cells, or compositions produced by said cells, inhibit or reduce
adverse immune responses (such as cell-mediated autoimmunity),
fibrosis (scarring) and/or adverse tissue remodeling (for example,
ventricular remodeling).
[0267] C6. The method of any one of embodiments C1C5, wherein the
cells, or compositions produced by said cells, control inflammation
and/or inhibit acute inflammation.
[0268] C7. The method of any one of embodiments C1-C5, wherein the
cells, or compositions produced by said cells, stimulate or enhance
angiogenesis.
[0269] C8. The method of any one of embodiments C1-C5, wherein said
cells do not exhibit significant or detectable levels of permanent
or long-term engraftment into said damaged organs or tissues.
[0270] C9. The method of any one of embodiments C1-C8, wherein said
damaged organs are selected from the group consisting of heart,
brain, and spinal cord.
[0271] C10. The method of any one of embodiments C1-C8, wherein
said damaged tissue is selected from the group consisting of
cardiac tissue, neuronal tissue (including central and peripheral
nervous system tissue), and vascular tissue (including major and
minor arteries, veins, and capillaries).
[0272] D1. A composition comprising a pharmaceutically acceptable
mixture of self-renewing, colony-forming somatic cells (CF-SC), or
conditioned cell culture media derived from such cells, and
purified naturally occurring or isolated recombinant extracellular
matrix or blood plasma proteins.
[0273] D2. The composition of embodiment D1, wherein said CF-SC are
derived from bone marrow.
[0274] D3. The composition of embodiments D1 or D2, wherein said
CF-SC are derived from a human.
[0275] D4. The composition of any one of embodiments D1-D3, wherein
said CF-SC are derived from an adult mammal, including humans.
[0276] D5. The composition of any one of embodiments D1-D4, wherein
said CF-SC express one or more secreted proteins shown in Table 1A,
1B and 1C.
[0277] D6. The composition of any one of embodiment D1-D5, wherein
said extracellular matrix or blood plasma proteins comprise one or
more full-length or alternatively processed isoforms, proteolytic
fragments, or subunits of molecules selected from the group
consisting of:
[0278] a) collagen; b) elastin; c) fibronectin; d) laminin; e)
entactin (nidogen); f) hyaluronic acid; g) polyglycolic acid (PGA);
h) fibrinogen (Factor I); i) fibrin; j) prothrombin (Factor II); k)
thrombin; 1) anti-thrombin; m) Tissue factor Co-factor of VIIa
(Factor III); n) Protein C; o) Protein S; p) protein Z; q) Protein
Z-related protease inhibitor; r) heparin cofactor II; s) Factor V
(proaccelerin, labile factor); t) Factor-VII; u) Factor-VIII; v)
Factor-IX; w) Factor-X; x) Factor-XI; y) Factor-XII; z)
Factor-XIII; aa) von Willebrand factor; ab) prekallikrein; ac) high
molecular weight kininogen; ad) plasminogen; ae) plasmin; af)
tissue-plasminogen activator; ag) urokinase; ah) plasminogen
activator inhibitor-1; and, ai) plasminogen activator
inhibitor-2.
[0279] D7. The composition of any one of embodiments D1-D6, further
comprising purified naturally occurring or isolated recombinant
cytokines or chemokines.
[0280] D8. The composition of any one of embodiments D1-D7, wherein
said extracellular matrix, blood plasma proteins, cytokines, and/or
chemokines are derived from humans.
[0281] D9. The composition of any one of embodiments D1-D8, wherein
said pharmaceutically acceptable mixture forms a semi-solidified or
solidified matrix.
[0282] D10. A method of treating damaged tissue with the
composition of any one of embodiments D1-D8, wherein the
composition is a liquid.
[0283] D11. The method of embodiment D10, wherein the liquid is
applied by injection.
[0284] D12. A method of treating damaged tissue with the
composition of any one of embodiments D1-D9, wherein the
composition is applied as a liquid but thereafter forms a
semi-solidified or solidified matrix.
[0285] D13. The method of embodiments D10-D12 wherein said tissue
is damaged as a result of a condition selected from the group
consisting of:
[0286] a) disease; b) physical trauma; c) ischemia; d) aging; e)
burn; f) bacterial infection; g) viral infection; h) fungal
infection; and, i) dysregulation of the immune system.
[0287] D14. The method of embodiment D13, wherein the damaged
tissue is skin.
[0288] D15. A method of using the composition of any one of
embodiments D1-D9 for facial skin rejuvenation.
[0289] D16. A method of using the composition of any one of
embodiments D1-D9, wherein said composition inhibits acute
inflammation.
[0290] D17. A method for treating, repairing, regenerating, or
healing a damaged organ or tissue comprising contacting said
damaged organ or tissue with an effective amount of self-renewing
colony forming somatic cells or compositions produced from such
cells so as to effect said treatment, repair, regeneration, or
healing of the damaged organ or tissue.
[0291] D18. The method of embodiment D17, wherein said damaged
organ or tissue is contacted with an effective amount of
self-renewing colony forming somatic cells or compositions produced
from such cells by means selected from the group consisting of:
[0292] a) injection into the damaged organ or tissue; b)
application onto the damaged organ or tissue; c) injection proximal
to the damaged organ or tissue; d) application proximal to the
damaged organ or tissue; and, e) intravenous administration.
[0293] D19. The method of embodiments D17 or D18, wherein the cells
are derived from bone marrow.
[0294] D20. The method of any one of embodiments D17-D19, wherein
the cells are human.
[0295] D21. The method of any one of embodiments D17-D20, wherein
the cells, or compositions produced by said cells, inhibit or
reduce adverse immune responses (such as cell-mediated
autoimmunity), fibrosis (scarring) and/or adverse tissue remodeling
(for example, ventricular remodeling).
[0296] D22. The method of any one of embodiments D12-D21, wherein
the cells, or compositions produced by said cells, control
inflammation and/or inhibit acute inflammation.
[0297] D23. The method of any one of embodiments D17-D21, wherein
the cells, or compositions produced by said cells, stimulate or
enhance angiogenesis.
[0298] D24. The method of any one of embodiments D17-D21, wherein
said cells do not exhibit significant or detectable levels of
permanent or long-term engraftment into said damaged organs or
tissues.
[0299] D25. The method of any one of embodiments D17-D24, wherein
said damaged organs are selected from the group consisting of
heart, brain, and spinal cord.
[0300] D26. The method of any one of embodiments D17-D24, wherein
said damaged tissue is selected from the group consisting of
cardiac tissue, neuronal tissue (including central and peripheral
nervous system tissue), and vascular tissue (including major and
minor arteries, veins, and capillaries).
[0301] D27. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro.
[0302] D28. The method of embodiment D27, wherein said induction,
enhancement, or maintenance is achieved by co-cultivation of
hematopoietic precursor cells with self-renewing colony forming
cells.
[0303] D29. The method of embodiment D28, wherein said
self-renewing colony forming cells are human bone marrow-derived
somatic cells (hABM-SC).
[0304] D30. The method of embodiment D29, wherein said hABM-SC are
derived from an adult.
[0305] D31. The method of any one of embodiments D27-D29, wherein
said co-cultivation utilizes a semi-permeable barrier to maintain
separation of the hematopoietic precursor cells from the
self-renewing colony forming cells while allowing exchange of
compositions produced by said self-renewing colony forming cells
across said barrier.
[0306] D32. The method of embodiment D27, wherein said induction,
enhancement, or maintenance is achieved by co-cultivation of
hematopoietic precursor cells with isolated compositions produced
by self-renewing colony forming cells.
[0307] D33. The method of embodiment D32, wherein said
self-renewing colony forming cells are human bone marrow-derived
somatic cells (hABM-SC).
[0308] D34. The method of embodiment D33, wherein said hABM-SC are
derived from an adult.
[0309] D35. The method of any one of embodiments D32-D34, wherein
said isolated compositions are lyophilized.
[0310] D36. The method of any one of embodiments D32-D34, wherein
said isolated compositions are cryopreserved.
[0311] D37. The method of any one of embodiments D32-D34, wherein
said isolated compositions are mixed with one or more
pharmaceutically acceptable carriers.
[0312] D38. A method of producing, isolating, purifying, and/or
packaging cell-derived compositions and/or trophic factors.
[0313] D39. A method of producing conditioned media, wherein said
media contains sera or is sera-free media.
[0314] D40. A method of isolating and purifying fractions and/or
cell-derived compositions from conditioned media, wherein said
media contains sera or is sera-free media.
[0315] D41. A method of isolating, cryopreserving, and/or expanding
CD34+Cord Blood Cells (CBC).
[0316] D42. The method of embodiment D41, wherein said CBC are
expanded in suspension cultures.
[0317] D43. The method of embodiment D41, wherein said CBC are
expanded by co-culturing with a feeder layer of self-renewing
colony forming cells.
[0318] D44. The method of embodiment D43, wherein said
self-renewing colony forming cells are human bone marrow-derived
somatic cells (hABM-SC).
[0319] D45. A wash solution comprising Balanced Salt Solution with
dextrose (BSSD).
[0320] D46. The wash solution of embodiment D45 wherein said
dextrose is at a concentration of about 4.5% dextrose.
[0321] D47. The wash solution of embodiment D45 or D46, further
comprising human serum albumin.
[0322] D48. The wash solution of embodiment D47, wherein said human
serum albumin is at a concentration of about 5% human serum
album.
[0323] D49. A cryopreservation media comprising dimethyl sulfoxide
(DMSO) and human serum albumin in a Balanced Salt Solution.
[0324] D50. The cryopreservation media of embodiment D49, wherein
said DMSO concentration is about 5% and said HSA concentration is
about 5%.
[0325] E1. An isolated cell population derived from bone marrow,
wherein greater than about 91% of the cells of the cell population
co-express CD49c and CD90, and wherein the cell population has a
doubling rate of less than about 30 hours.
[0326] E2. The isolated cell population of embodiment E1, wherein
the cell population is derived from human bone marrow.
[0327] E3. The isolated cell population of embodiments E1 or E2,
wherein the cells of the cell population that co-express CD49c and
CD90 do not express CD34 and/or CD45.
[0328] E4. The isolated cell population according to any one of
embodiments E1, E2, or E3, wherein the cells of the cell population
that co-express CD49c and CD90 further express at least one
cardiac-related transcription factor selected from the group
consisting of GATA-4, Irx4, and NRx2.5.
[0329] E5. The isolated cell population according to any one of
embodiments E1, E2, or E3, wherein the cells of the cell population
that co-express CD49c and CD90 further express at least one trophic
factor selected from the group consisting of:
[0330] a) Brain-Derived Neurotrophic Factor (BDNF);
[0331] b) Cystatin-C;
[0332] c) Interleukin-6 (IL-6);
[0333] d) Interleukin-7 (IL-7);
[0334] e) Interleukin-11 (IL-11);
[0335] f) Nerve Growth Factor (NGF);
[0336] g) Neurotrophin-3 (NT-3);
[0337] h) Macrophage Chemoattractant Protein-1 (MCP-1);
[0338] i) Matrix Metalloproteinase-9 (MMP-9);
[0339] j) Stem Cell Factor (SCF); and,
[0340] k) Vascular Endothelial Growth Factor (VEGF).
[0341] E6. The isolated cell population according 10 any one of
embodiments E1, E2, or E3, wherein the cells of the cell population
that co-express CD49c and CD90 further express p21 or p53, and
wherein expression of p53 is a relative expression of up to about
3000 transcripts of p53 per 10.sup.6 transcripts of an 18s rRNA and
expression of p21 is a relative expression of up to about 20,000
transcripts of p21 per 10.sup.6 transcripts of an 18s rRNA.
[0342] E7. The isolated cell population according to any one of
embodiments E1, E2, or E3, wherein the isolated cell population has
been cultured in vitro through a number of population doublings
selected from the group consisting of:
[0343] a) at least about 15 population doublings;
[0344] b) at least about 20 population doublings;
[0345] c) at least about 25 population doublings;
[0346] d) at least about 30 population doublings;
[0347] e) at least about 35 population doublings; and,
[0348] at least about 40 population doublings.
[0349] E8. A method of making an isolated cell population derived
from bone marrow, wherein greater than about 91% of the cells of
the cell population co-express CD49c and CD90, and wherein the cell
population has a doubling rate of less than about 30 hours,
comprising the steps of:
[0350] a) culturing a source of the cell population under a low
oxygen condition or a low oxidative stress condition to produce an
adherent cell population; and,
[0351] b) culturing the adherent cell population at a seeding
density of less than about 2500 cells/cm.sup.2,
[0352] E9. The method of embodiment E8, wherein the cell population
is derived from human bone marrow.
[0353] E10. The method of embodiments E8 or E9, wherein the source
of the cell population in embodiment 8, part a) is cultured at an
initial seeding density selected from the group consisting of:
[0354] a) less than about 75000 cells/cm.sup.2; and,
[0355] b) less than about 50000 cells/cm.sup.2.
[0356] E11. The method of any one of embodiments E8 to E10, wherein
the adherent cell population in embodiment 8, part b) is cultured
at a seeding density selected from the group consisting of:
[0357] a) less than about 2500 cells/cm.sup.2;
[0358] b) less than about 1000 cells/cm.sup.2;
[0359] c) less than about 100 cells/cm.sup.2;
[0360] d) less than about 50 cells/cm.sup.2; and,
[0361] e) less than about 30 cells/cm.sup.2.
[0362] E12. The method of any one of embodiments E8 to E11, wherein
the low oxygen condition is selected from the group consisting
of:
[0363] a) between about 1 to 10% oxygen;
[0364] b) between about 2 to 7% oxygen;
[0365] d) less than about 20% oxygen;
[0366] c) less than about 15% oxygen;
[0367] d) less than about 10% oxygen;
[0368] e) less than about 5% oxygen; and,
[0369] f) about 5% oxygen.
[0370] E13. The method of any one of embodiments E8 to E12, further
including lysing the red blood cells in a source of the cell
population prior to culturing the source of the cell
population.
[0371] E14. The method of any one of embodiments E8 to E12, further
including selecting a fractionated source of the cell population by
passage through a density gradient prior to culturing the source of
the cell population.
[0372] E15. The method of any one of embodiments E8 to E14, wherein
the cells of the cell population that co-express CD49c and CD90, do
not express CD34 and/or CD45.
[0373] E16. The method of any one of embodiments E8 to E15, wherein
the cells of the cell population that co-express CD49c and CD90
further express at least one cardiac-related transcription factor
selected from the group consisting of GATA-4, Irx4, and NRx2.5.
[0374] E17. The method of any one of embodiments E8 to E15, wherein
the cells of the cell population that co-express CD49c and CD90
further express at least one trophic factor selected from the group
consisting of:
[0375] a) Brain-Derived Neurotrophic Factor (BDNF);
[0376] b) Cystatin-C;
[0377] c) Interleukin-6 (IL-6);
[0378] d) Interleukin-7 (IL-7);
[0379] e) Interleukin-11 (IL-11);
[0380] f) Nerve Growth Factor (NGF);
[0381] g) Neurotrophin-3 (NT-3);
[0382] h) Macrophage Chemoattractant Protein-1 (MCP-1);
[0383] i) Matrix Metalloproteinase-9 (MMP-9);
[0384] j) Stem Cell Factor (SCF); and,
[0385] k) Vascular Endothelial Growth Factor (VEGF).
[0386] E18. The method of any one of embodiments E8 to E15, wherein
the cells of the cell population that co-express CD49c and CD90
further express p21 or p53, and wherein expression of p53 is a
relative expression of up to about 3000 transcripts of p53 per
10.sup.6 transcripts of an 18s rRNA and expression of p21 is a
relative expression of up to about 20,000 transcripts of p21 per
10.sup.6 transcripts of an 18s rRNA.
[0387] E19. The method of any one of embodiments E8 to E15, wherein
the isolated cell population has been cultured in vitro through a
number of population doublings selected from the group consisting
of:
[0388] a) at least about 15 population doublings;
[0389] b) at least about 20 population doublings;
[0390] c) at least about 25 population doublings;
[0391] d) at least about 30 population doublings;
[0392] e) at least about 35 population doublings; and,
[0393] 1) at least about 40 population doublings.
[0394] E20. Use of an isolated cell population according to any one
of embodiments E1 to E7 in the manufacture of a medicament for
treating a human suffering, from a condition selected from the
group consisting Of:
[0395] a) a degenerative condition;
[0396] b) an acute injury condition;
[0397] c) a neurological condition; and,
[0398] d) a cardiac condition.
[0399] E21. Use of an isolated cell population according to any one
of embodiments E1 to E7 in the manufacture of a medicament for
treating a human suffering from a degenerative or acute injury
condition.
[0400] E22. An isolated cell population derived from bone marrow,
*herein greater than about 91% of the cells of the cell population
co-express CD49c and CD90, and wherein the cell population has a
doubling rate of less than about 30 hours under a low oxygen
condition.
[0401] E23. The isolated cell population of embodiment E22, wherein
the cell population is derived from human bone marrow.
[0402] E24. The isolated cell population of embodiments E22 or E23,
wherein the low oxygen condition is between about 1 to 10%
oxygen.
[0403] E25. The isolated cell population of embodiment E24, wherein
the low oxygen condition is about 5% oxygen.
[0404] E26. The isolated cell population of any one of embodiments
E22 to E25, wherein the cell population is cultured as an adherent
cell population at a seeding density of less than about 2500
cells/cm.sup.2.
[0405] E27. The isolated cell population of any one of embodiments
E22 to 1325, wherein the seeding; density is less than about 1000
cells/cm.sup.2.
[0406] E28. The isolated cell population of any one of embodiments
E22 to E25, wherein the seeding density is less than about 100
cells/cm.sup.2.]
[0407] E29. The isolated cell population of any one of embodiments
E22 to E25, wherein the seeding density is less than about 50
cells/cm.sup.2.
[0408] E30. The isolated cell population of any one of embodiments
E22 to E25, wherein the seeding density is less than about 30
cells/cm.sup.2.
[0409] E31. A method of making an isolated cell population, wherein
greater than about 91% of the cells of the cell population
co-express CD49c and CD90, and wherein the cell population has a
doubling rate of less than about 30 hours, comprising the steps
of:
[0410] a) aspirating bone marrow cells from a human;
[0411] b) lysing the red blood cell component of the bone marrow
aspirate;
[0412] c) seeding the non-lysed bone marrow cells in a tissue
culturing device;
[0413] d) allowing the non-lysed bone marrow cells to adhere to a
surface;
[0414] e) culturing the adherent cells under a 5% oxygen condition;
and
[0415] f) passaging the adherent cells at a seeding density of 30
cells/cm.sup.2.
[0416] E32. An isolated cell population obtainable by the method of
embodiment E31.
[0417] E33. An isolated cell population obtained by the method of
embodiment E31.
[0418] E34. A method of making an isolated cell population, wherein
greater than about 91% of the cells of the cell population
co-express CD49c and CD90, and wherein the cell population has a
doubling rate of less than about 30 hours after 30 cell doublings,
comprising the steps of: [0419] a) aspirating bone marrow cells
from a human; [0420] b) selecting a fractionated source of the cell
population by passage through a density gradient; [0421] c) seeding
the fractionated cells in a tissue culturing device; [0422] d)
allowing the fractionated cells to adhere to a surface; [0423] e)
culturing the adherent cells under a 5% oxygen condition; and
[0424] f) passaging the adherent cells at a seeding density of 30
cells/cm.sup.2.
[0425] E35. An isolated cell population obtainable by the method of
embodiment E34.
[0426] E36. An isolated cell population obtained by the method of
embodiment E34.
[0427] F1. A method of administering a therapeutically useful
amount of a biological composition or compositions to an organ,
tissue, or subject, comprising administering to said organ, tissue,
or subject an isolated population of bone marrow-derived
self-renewing colony-forming somatic cells (CF-SC), wherein said
CF-SC do not have multipotent differentiation capacity, wherein
said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD 10, CD34, CD45, CD62L, or CD 106.
[0428] F2. A method of administering a therapeutically useful
amount of a biological composition or compositions to an organ,
tissue, or subject, comprising: (a) isolating the biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC); and, (b) administering said biological composition or
compositions to said organ, tissue, or subject, wherein said CF-SC
do not have multipotent differentiation capacity, wherein said
CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
[0429] F3. A method of administering a therapeutically useful
amount of a biological composition or compositions to an organ,
tissue, or subject, comprising administering to said organ, tissue,
or subject an isolated population of bone marrow-derived
self-renewing colony-forming somatic cells (CF-SC), wherein said
CF-SC do not have multipotent differentiation capacity, wherein
said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0430] F4. A method of administering a therapeutically useful
amount of a biological composition or compositions to an organ,
tissue, or subject, comprising: (a) isolating the biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC); and, (b) administering said biological composition or
compositions to said organ, tissue, or subject, wherein said CF-SC
do not have multipotent differentiation capacity, wherein said
CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0431] F5. A method of preventing tissue damage or of repairing,
treating, or promoting regeneration of damaged tissue, comprising
administering to an organ, tissue, or subject an isolated
population of bone marrow-derived self-renewing colony-forming
cells somatic cells (CF-SC), wherein said CF-SC do not have
multipotent differentiation capacity, wherein said CF-SC have a
normal karyotype, wherein said CF-SC are non-immortalized, wherein
said CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and .beta.
(beta) 2-Microglobulin, and wherein said CF-SC do not express CD10,
CD34, CD45, CD62L, or CD106.
[0432] F6. A method of preventing tissue damage or of repairing,
treating, or promoting regeneration of damaged tissue in an organ,
tissue, or subject, comprising: (a) isolating the biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC); and, (b) administering said biological composition or
compositions to said organ, tissue, or subject, wherein said CF-SC
do not have multipotent differentiation capacity, wherein said
CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
[0433] F7. A method of preventing tissue damage or of repairing,
treating, or promoting regeneration of damaged tissue in an organ,
tissue, or subject, comprising administering to said organ, tissue,
or subject an isolated population of bone marrow-derived
self-renewing colony-forming cells somatic cells (CF-SC), wherein
said CF-SC do not have multipotent differentiation capacity,
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0434] F8. A method of preventing tissue damage or of repairing,
treating, or promoting regeneration of damaged tissue in an organ,
tissue, or subject, comprising: (a) isolating the biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC); and, (b) administering said biological composition or
compositions to said organ, tissue, or subject, wherein said CF-SC
do not have multipotent differentiation capacity, wherein said
CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0435] F9. A method of treating or reducing inflammation, immune,
or autoimmune activity in a organ, tissue, or subject, comprising
administering to said organ, tissue, or subject an isolated
population of bone marrow-derived self-renewing colony-forming
cells somatic cells (CF-SC), wherein said CF-SC do not have
multipotent differentiation capacity, wherein said CF-SC have a
normal karyotype, wherein said CF-SC are non-immortalized, wherein
said CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and p
(beta) 2-Microglobulin, and wherein said CF-SC do not express CD10,
CD34, CD45, CD62L, or CD106.
[0436] F10. A method of treating or reducing inflammation, immune,
or autoimmune activity in an organ, tissue, or subject, comprising:
(a) isolating the biological composition or compositions produced
by an isolated population of bone marrow-derived self-renewing
colony forming somatic cells (CF-SC); and, (b) administering said
biological composition or compositions to said organ, tissue, or
subject, wherein said CF-SC do not have multipotent differentiation
capacity, wherein said CF-SC have a normal karyotype, wherein said
CF-SC are non-immortalized, wherein said CF-SC express CD13, CD44,
CD49c, CD90, HLA Class-1 and 13 (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD 10, CD34, CD45, CD62L, or CD 106.
[0437] F11. A method of treating or reducing inflammation, immune,
or autoimmune activity in an organ, tissue, or subject, comprising
administering to said organ, tissue, or subject an isolated
population of bone marrow-derived self-renewing colony-forming
cells somatic cells (CF-SC), wherein said CF-SC do not have
multipotent differentiation capacity, wherein said CF-SC have a
normal karyotype, wherein said CF-SC are non-immortalized, and
wherein said CF-SC are obtained from bone marrow by steps
comprising: i) incubating bone marrow cells under a low oxygen
condition such that said bone marrow cells when allowed to adhere
to a tissue culture-treated surface produce adherent colony forming
units; and, ii) passaging cells in said adherent colony forming
units at low cell seeding densities.
[0438] F12. A method of treating or reducing inflammation, immune,
or autoimmune activity in a organ, tissue, or subject, comprising:
(a) isolating the biological composition or compositions produced
by an isolated population of bone marrow-derived self-renewing
colony forming somatic cells (CF-SC); and, (b) administering said
biological composition or compositions to said organ, tissue, or
subject, wherein said CF-SC do not have multipotent differentiation
capacity, wherein said CF-SC have a normal karyotype, wherein said
CF-SC are non-immortalized, and wherein said CF-SC are obtained
from bone marrow by steps comprising: i) incubating bone marrow
cells under a low oxygen condition such that said bone marrow cells
when allowed to adhere to a tissue culture-treated surface produce
adherent colony forming units; and, ii) passaging cells in said
adherent colony forming units at low cell seeding densities.
[0439] F13. The method of any one of embodiments F1, F2, F5, F6,
F9, or F10, wherein said CF-SC further express one or more
molecules selected from the group consisting of a) CD29; b) CD59;
c) CD147; d) CD166; and, e) telomerase and, wherein said CF-SC
further do not express one or more molecules selected from the
group consisting of: f) CD11c; g) CD14; h) CD33; i) CD62P; j) CD80;
k) STRO-1; l) HLA-Class-II; m) CD178. n) p53; and, o) p21.
[0440] F14. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low oxygen condition is about 5%
oxygen.
[0441] F15. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low oxygen condition is selected from the
group consisting of: a) less than about 20% oxygen; b) less than
about 15% oxygen; c) less than about 10% oxygen; d) less than about
5% oxygen; e) between about 1 to 10% oxygen; 1) between about 2 to
7% oxygen; g) between about 3 to 6% oxygen; h) between about 4 to
6% oxygen; and,
[0442] i) between about 4 to 5% oxygen.
[0443] F16. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low cell seeding density is less than
about 200 cells/cm2.
[0444] F17. Tne method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low cell seeding density is less than
about 100 cells/cm2.
[0445] F18. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low cell seeding density is less than
about 50 cells/cm2.
[0446] F19. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low cell seeding density is less than
about 30 cells/cm2.
[0447] F20. The method of any one of embodiments F3, F4, F7, F8,
F11 or F12, wherein said low cell seeding density is selected from
the group consisting of: a) less than about 2500 cells/cm2; b) less
than about 1000 cells/cm2; and, c) less than about 500
cells/cm2.
[0448] F21. The method of any one of embodiments F1 to F20, wherein
lack of said multipotent differentiation capacity comprises the
inability of said CF-SC to differentiate into osteocytes.
[0449] F22. The method of any one of embodiments F1 to F20, wherein
lack of said multipotent differentiation capacity comprises the
inability of said CF-SC to deposit detectable levels of calcium
following treatment of said CF-SC under osteoinductive
conditions.
[0450] F23. The method of embodiment F22, wherein said treatment
comprises exposure of said CF-SC to media containing one or more
components selected from the group consisting of: a) dexamethasone;
b) ascorbate; and, c) beta glycerophosphate.
[0451] F24. The method of embodiment F23, wherein said treatment
further comprises exposure to Noggin.
[0452] F25. The method of any one of embodiments F1 to F20, wherein
lack of said multipotent differentiation capacity comprises the
inability of said CF-SC to differentiate into chondrocytes.
[0453] F26. The method of any one of embodiments F1 to F20, wherein
lack of said multipotent differentiation capacity comprises the
inability of said CF-SC to differentiate into adipocytes.
[0454] F27. The method of any one of embodiments F1 to F20, wherein
said CF-SC have unipotent differentiation capacity.
[0455] F28. The method of any one of embodiments F1 to F27, wherein
said CF-SC have substantial capacity for self-renewal.
[0456] F29. The method of any of embodiments F1 to F28, wherein
said CF-SC maintain an approximately constant population doubling
rate through multiple in vitro cell doublings.
[0457] F30. The method of any one of embodiments F1 to F29, wherein
said CF-SC are not embryonic stem cells, hematopoietic stem cells,
mesenchymal stem cells, multipotent adult progenitor cells (MAPCs),
multipotent adult stem cells (MASCs), or fibroblasts.
[0458] F31. The method of any one of embodiments F1 to F30, wherein
said bone marrow is human bone marrow.
[0459] F32. The method of any one of embodiments F1 to F31, wherein
said cell population has undergone a number of population doublings
selected from the group consisting of: a) at least about 15
population doublings; b) at least about 20 population doublings; c)
at least about 25 population doublings; d) at least about 30
population doublings; e) at least about 35 population doublings; f)
at least about 40 population doublings; g) at least about 45
population doublings; and, h) at least about 50 population
doublings.
[0460] F33. The method of any one of embodiments F1 to F32, wherein
said organ, tissue, or subject is human.
[0461] F34. The method of any one of embodiments F1 to F33, wherein
said method is used to treat or prevent cell, organ, or tissue
damage, or diseases and disorders selected from the group
consisting of: a) neurological damage, disease or disorder; b)
cardiac damage, disease or disorder; c) skin damage, disease or
disorder; d) periodontal damage, disease or disorder; e)
maxillofacialary damage, disease or disorder; f) skeletal muscle
damage, disease or disorder; g) ligament damage, disease or
disorder; h) pulmonary damage, disease or disorder; i) hepatic
damage, disease or disorder; j) renal damage, disease or disorder;
k) genitourinary system damage, disease or disorder; 1) bladder
damage, disease or disorder; m) endocrine damage, disease or
disorder; n) hematopoietic damage, disease or disorder; o)
pancreatic damage, disease or disorder; p) diabetes; q) ocular
damage, disease or disorder; r) retinal damage, disease or
disorder; s) gastrointestinal disease or disorder; t) splenic
damage, disease or disorder; u) immunological damage, disease or
disorder; v) autoimmune damage, disease or disorder; w)
inflammatory damage, disease or disorder; x) hyperproliferative
damage, disease or disorder; and, y) cancer.
[0462] F35. The method of any one of embodiments F5 to F8, wherein
said damage is prevented in an organ or tissue during the course of
organ or tissue transplant.
[0463] F36. The method of any one of embodiments F1 to F35, wherein
said cells are genetically modified.
[0464] F37. The method of embodiment F36, wherein said cells are
genetically modified by introduction of a recombinant nucleic acid
molecule.
[0465] F38. A composition comprising a pharmaceutically acceptable
mixture of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
[0466] F39. A composition comprising a pharmaceutically acceptable
mixture of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and a biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC), wherein said CF-SC have a normal karyotype, wherein said
CF-SC are non-immortalized, wherein said CF-SC express CD13, CD44,
CD49c, CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and
wherein said CF-SC do not express CD10, CD34, CD45, CD62L, or
CD106.
[0467] F40. A composition comprising a pharmaceutically acceptable
mixture of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0468] F41. A composition comprising a pharmaceutically acceptable
mixture of purified naturally occurring or isolated recombinant
extracellular matrix or blood plasma proteins and a biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC), wherein said CF-SC have a normal karyotype, wherein said
CF-SC are non-immortalized, and wherein said CF-SC are obtained
from bone marrow by steps comprising: i) incubating bone marrow
cells under a low oxygen condition such that said bone marrow cells
when allowed to adhere to a tissue culture-treated surface produce
adherent colony forming units; and, ii) passaging cells in said
adherent colony forming units at low cell seeding densities.
[0469] F42. The composition of any one of embodiments F38 to F41,
wherein said extracellular matrix or blood plasma proteins comprise
one or more full-length or alternatively processed isoforms,
proteolytic fragments, or subunits of molecules selected from the
group consisting of: a) collagen; b) elastin; c) fibronectin; d)
laminin; e) entactin (nidogen); f) hyaluronic acid; g) polyglycolic
acid (PGA) h) fibrinogen (Factor I); i) fibrin; j) prothrombin
(Factor II); k) thrombin; 1) anti-thrombin; m) Tissue factor
Co-factor of VIIa (Factor III); n) Protein C; o) Protein S; p)
protein Z; q) Protein Z-related protease inhibitor; r) heparin
cofactor II; s) Factor V (proaccelerin, labile factor); t)
Factor-VII; u) Factor-VIII; v) Factor-IX; w) Factor-X; x)
Factor-XI; y) Factor-XII; z) Factor-XIII; aa) von Willebrand
factor; ab) prekallikrein; ac) high molecular weight kininogen; ad)
plasminogen; ae) plasmin; af) tissue-plasminogen activator; ag)
urokinase; ah) plasminogen activator inhibitor-1; and, ai)
plasminogen activator inhibitor-2.
[0470] F43. The composition of any one of embodiments F38 to F42,
further comprising purified naturally occurring or isolated
recombinant cytokines or chemokines.
[0471] F44. The composition of embodiment F38 or F39, wherein said
CF-SC further express one or more molecules selected from the group
consisting of: a) CD29; b) CD59; c) CD147; d) CD166; and, e)
telomerase and, wherein said CF-SC further do not express one or
more molecules selected from the group consisting of f) CD11c; g)
CD14; h) CD33; i) CD62P; j) CD80; k) STRO-1; 1) HLA-Class-II; m)
CD178; n) p53; and, o) p21.
[0472] F45. The composition of embodiment F40 or F41, wherein said
low oxygen condition is about 5% oxygen.
[0473] F46. The composition of embodiment F40 or F41, wherein said
low oxygen condition is selected from the group consisting of: a)
less than about 20% oxygen; b) less than about 15% oxygen; c) less
than about 10% oxygen; d) less than about 5% oxygen; e) between
about 1 to 10% oxygen; f) between about 2 to 7% oxygen; g) between
about 3 to 6% oxygen; h) between about 4 to 6% oxygen; and, i)
between about 4 to 5% oxygen.
[0474] F47. The composition of embodiment F40 or F41, wherein said
low cell seeding density is less than about 200 cells/cm2.
[0475] F48. The composition of embodiment F40 or F41, wherein said
low cell seeding density is less than about 100 cells/cm2.
[0476] F49. The composition of embodiment F40 or F41, wherein said
low cell seeding density is less than about 50 cells/cm2.
[0477] F50. The composition of embodiment F40 or F41, wherein said
low cell seeding density is less than about 30 cells/cm2.
[0478] F51. The composition of embodiment F40 or F41, wherein said
low cell seeding density is selected from the group consisting of:
a) less than about 2500 cells/cm2; b) less than about 1000
cells/cm2; and, c) less than about 500 cells/cm2.
[0479] F52. The composition of any one of embodiments F38 to F51,
wherein said CF-SC have unipotent differentiation capacity.
[0480] F53. The composition of any one of embodiments F38 to F52,
wherein said CF-SC have substantial capacity for self-renewal.
[0481] F54. The composition of any of embodiments F38 to F53,
wherein said CF-SC maintain an approximately constant population
doubling rate through multiple in vitro cell doublings.
[0482] F55. The composition of any one of embodiments F38 to F54,
wherein said CF-SC are not embryonic stem cells, hematopoietic stem
cells, mesenchymal stem cells, multipotent adult progenitor cells
(MAPCs), multipotent adult stem cells (MASCs), or fibroblasts.
[0483] F56. The composition of any one of embodiments F38 to F55,
wherein said bone marrow is human bone marrow.
[0484] F57. The composition of any one of embodiments F38 to F56,
wherein said cell population has undergone a number of population
doublings selected from the group consisting of: a) at least about
15 population doublings; b) at least about 20 population doublings;
c) at least about 25 population doublings; d) at least about 30
population doublings; e) at least about 35 population doublings; f)
at least about 40 population doublings; g) at least about 45
population doublings; and, h) at least about 50 population
doublings.
[0485] F58. The composition of any one of embodiments F38 to F57,
wherein said organ, tissue, or subject is human.
[0486] F59. The composition of any one of embodiments F38 to F58,
wherein said method is used to treat cell, organ, or tissue damage,
diseases and disorders selected from the group consisting of: a)
neurological damage, disease or disorder; b) cardiac damage,
disease or disorder; c) skin damage, disease or disorder; d)
periodontal damage, disease or disorder; e) maxillofacialary
damage, disease or disorder order; f) skeletal muscle damage,
disease or disorder; g) ligament damage, disease or disorder; h)
respiratory damage, disease or disorder; i) hepatic damage, disease
or disorder; j) renal damage, disease or disorder; k) genitourinary
system damage, disease or disorder; l) bladder damage, disease or
disorder; m) endocrine damage, disease or disorder; n)
hematopoietic damage, disease or disorder; o) pancreatic damage,
disease or disorder;
[0487] p) diabetes; q) ocular damage, disease or disorder; r)
retinal damage, disease or disorder; s) gastrointestinal disease or
disorder; t) splenic damage, disease or disorder;
[0488] u) immunological damage, disease or disorder; v) autoimmune
damage, disease or disorder; w) inflammatory damage, disease or
disorder; x) hyperproliferative damage, disease or disorder; and,
y) cancer.
[0489] F60. The composition of any one of embodiments F38 to F59,
wherein said cells are genetically modified.
[0490] F61. The composition of embodiment F60, wherein said cells
are genetically modified by introduction of a recombinant nucleic
acid molecule.
[0491] F62. The composition of any one of embodiments F38 to F61,
wherein said pharmaceutically acceptable mixture forms a
semi-solidified or solidified matrix.
[0492] F63. A method of treating damaged tissue with the
composition of any one of embodiments F38 to F62, wherein the
composition is a liquid.
[0493] F64. The method of embodiment F63, wherein the liquid is
applied by injection.
[0494] F65. A method of treating damaged tissue with the
composition of any one of embodiments F38 to F64, wherein the
composition is applied as a liquid but thereafter forms a
semi-solidified or solidified matrix.
[0495] F66. The method or composition of any one of embodiments F1
to F65, wherein said CF-SC or biological composition or
compositions produced by said CF-SC are administered by a means
selected from the group consisting of: a) injection into the
damaged organ or tissue; b) application onto the damaged organ or
tissue; c) injection proximal to the damaged organ or tissue; d)
application proximal to the damaged organ or tissue; e) intravenous
administration; and, f) organ or tissue perfusion.
[0496] F67. The method or composition of any one of embodiments F1
to F66, wherein said CF-SC, or biological composition or
compositions produced by said CF-SC, inhibit or reduce one or more
biological effects selected from the group consisting of: a)
adverse immune responses; b) fibrosis; c) adverse tissue
remodeling; d) organ or tissue ischemia; and, e) cell death.
[0497] F68. The method or composition of embodiment F67, wherein
said adverse immune responses are selected from the group
consisting of: a) autoimmune responses; b) inflammation; c) immune
cell proliferation; d) immune cell activation; e) immune cell
degranulation; and, f) T-cell mediated immune responses.
[0498] F69. The method or composition of any one of embodiments F1
to F68, wherein said CF-SC, or biological composition or
compositions produced by said CF-SC, stimulate or enhance
angiogenesis.
[0499] F70. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
[0500] F71. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with a biological
composition or compositions produced by bone marrow-derived
self-renewing colony-forming somatic cells (CF-SC), wherein said
CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,
CD90, HLA Class-1 and .beta. (beta) 2-Microglobulin, and wherein
said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.
[0501] F72. The method of embodiment F70 or F71, wherein said CF-SC
further express one or more molecules selected from the group
consisting of: a) CD29; b) CD59; c) CD147; d) CD166; and, e)
telomerase and, wherein said CF-SC further do not express one or
more molecules selected from the group consisting of: f) CD11c; g)
CD14; h) CD33; i) CD62P; j) CD80; k) STRO-1; l) HLA-Class-II; m)
CD178; n) p53; and, o) p21.
[0502] F73. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with bone
marrow-derived self-renewing colony-forming somatic cells (CF-SC),
wherein said CF-SC have a normal karyotype, wherein said CF-SC are
non-immortalized, and wherein said CF-SC are obtained from bone
marrow by steps comprising: i) incubating bone marrow cells under a
low oxygen condition such that said bone marrow cells when allowed
to adhere to a tissue culture-treated surface produce adherent
colony forming units; and, ii) passaging cells in said adherent
colony forming units at low cell seeding densities.
[0503] F74. A method of inducing, enhancing, and/or maintaining the
generation of new red blood cells in vitro or in vivo, wherein said
induction, enhancement, or maintenance is achieved by
co-cultivation of hematopoietic precursor cells with a biological
composition or compositions produced by an isolated population of
bone marrow-derived self-renewing colony forming somatic cells
(CF-SC), wherein said CF-SC have a normal karyotype, wherein said
CF-SC are non-immortalized, and wherein said CF-SC are obtained
from bone marrow by steps comprising: i) incubating bone marrow
cells under a low oxygen condition such that said bone marrow cells
when allowed to adhere to a tissue culture-treated surface produce
adherent colony forming units; and, ii) passaging cells in said
adherent colony forming units at low cell seeding densities.
[0504] F75. The method of embodiment F73 or F74, wherein said low
oxygen condition is about 5% oxygen.
[0505] F76. The method of embodiment F73 or F74, wherein said low
oxygen condition is selected from the group consisting of: a) less
than about 20% oxygen; b) less than about 15% oxygen; c) less than
about 10% oxygen; d) less than about 5% oxygen; e) between about 1
to 10% oxygen; f) between about 2 to 7% oxygen; g) between about 3
to 6% oxygen; h) between about 4 to 6% oxygen; and, i) between
about 4 to 5% oxygen.
[0506] F77. The method of embodiment F73 or F74, wherein said low
cell seeding density is less than about 200 cells/cm2
[0507] F78. The method of embodiment F73 or F74, wherein said low
cell seeding density is less than about 100 cells/cm2.
[0508] F79. The method of embodiment F73 of F74, wherein said low
cell seeding density is less than about 50 cells/cm2.
[0509] F80. The method of embodiment l'73 or F74, wherein said low
cell seeding density is less than about 30 cells/cm2.
[0510] F81. The method of embodiment F73 or F74, wherein said low
cell seeding density is selected from the group consisting of: a)
less than about 2500 cells/cm2; b) less than about 1000 cells/cm2;
and, c) less than about 500 cells/cm2.
[0511] F82. The method of any one of embodiments F70 to F81,
wherein said CF-SC have unipotent differentiation capacity.
[0512] F83. The method of any one of embodiments F70 to F82,
wherein said CF-SC have substantial capacity for self-renewal.
[0513] F84. The method of any one of embodiments F70 to F83,
wherein said CF-SC maintain an approximately constant population
doubling rate through multiple in vitro cell doublings.
[0514] F85. The method of any one of embodiments F70 to F84,
wherein said CF-SC are not embryonic stem cells, hematopoietic stem
cells, mesenchymal stem cells, multipotent adult progenitor cells
(MAPCs), multipotent adult stem cells (MASCs), or fibroblasts.
[0515] F86. The method of any one of embodiments F70 to F85,
wherein said bone marrow is human bone marrow.
[0516] F87. The method of any one of embodiments F70 to F86,
wherein said cell population has undergone a number of population
doublings selected from the group consisting of: a) at least about
15 population doublings; b) at least about 20 population doublings;
c) at least about 25 population doublings; d) at least about 30
population doublings; e) at least about 35 population doublings; f)
at least about 40 population doublings; g) at least about 45
population doublings; and, h) at least about 50 population
doublings.
[0517] F88. The method of any one of embodiments F70 to F87,
wherein said cell population is human.
[0518] F89. The method of any one of embodiments F9 to F12, wherein
said treatment or reduction of inflammation, immune, or autoimmune
activity results in one or more changes selected from the group
consisting, of: a) decreased focal or systemic levels of IL-13; b)
decreased focal or systemic levels of TNF-alpha; c) increased focal
or systemic levels of IL-2; d) decreased focal or systemic immune
cell proliferation; e) decreased focal or systemic immune cell
activation; and, f) decreased focal or systemic immune cell
degranulation.
[0519] F90. The method of embodiment F89, wherein said decreased
immune cell proliferation comprises inhibition of proliferation of
one or more cell types selected from the group consisting of: a)
monocytes; b) granulocytes; c) lymphocytes; and, d)
neutrophils.
[0520] F91. The method of embodiment F89, wherein said increased
focal or systemic levels of IL-2 supports maturation of T-cell
regulatory cells.
[0521] F92. The method or composition of any one of embodiments
F1-F91, wherein said biological composition or compositions are
lyophilized.
[0522] F93. The method or composition of any one of embodiments
F1-F91, wherein said biological composition or compositions are
cryopreserved.
[0523] F94. The method or composition of any one of embodiments
F1-F91, wherein said biological composition or compositions are
mixed with one or more pharmaceutically acceptable carriers.
EXAMPLES
Example 1
Bioactivity of Adult Bone Marrow-Derived Somatic Cells: Production
of Serum-Free Conditioned Media
[0524] Production of serum-free conditioned media was produced as
described below for use in assays, such as the solid-phase antibody
capture of secreted proteins (also as described below). Human
exABM-SC (Lot #RECB-819; at .about.43 population doublings) were
thawed and re-suspended in either Advanced DMEM (GIBCO.TM.; Catalog
#12491-015, Lot #1216032 (Invitrogen Corp., Carlsbad, Calif., USA))
supplemented with 4 mM L-glutamine (Catalog #SH30034.01. Lot
#134-7944, (HYCLONE.TM. Laboratories Inc., Logan, Utah, USA)) or
HyQ.RTM. RPMI-1640 (HYCLONE.TM. Catalog #SH30255.01, Lot #ARC25868)
containing 4 mM L-glutamine and supplemented with
Insulin-Transferrin-Selenium-A (ITS) (GIBCO.TM.; Catalog
#51300-044, Lot #1349264). Cell suspensions were then seeded in
T-225 cm.sup.2 CELLBIND.TM. (Corning Inc., NY, USA) culture flasks
(culture surfaces treated with a patented microwave plasma process;
see, U.S. Pat. No. 6,617,152) (n=3) at 20,000 cells/cm.sup.2 in 36
mL of media (n=3 per condition). Cultures were placed in a
37.degree. C. humidified trigas incubator (4% O.sub.2, 5% CO.sub.2,
balanced with nitrogen) for approximately 24 hours. Cultures were
then re-fed with fresh media on same day to remove non-adherent
debris and returned to the incubator. On day 3, cell culture media
were concentrated using 20 mL CENTRICON.TM. PLUS-20 Centrifugal
Filter Units (Millipore Corp., Billerica, Mass., USA), as per
manufacturer's instructions. Briefly, concentrators were
centrifuged for 45 minutes at 1140.times.G. Concentrated
supernatants were transferred to clean 2 mL cryovials and stored at
-80.degree. C. Fresh culture media were also concentrated as
described for use as a negative control. The cells were then
removed from the flasks using 0.25% porcine trypsin EDTA
(CELLGRO.TM.; Catalog #30-004-C1 (Mediatech Inc., Herndon, Va.,
USA)). Trypsin was then neutralized by adding back an equal volume
of cell culture media containing 10% fetal bovine serum. Cell count
and viability analysis was performed using a COULTER.TM. AcT 10
Series Analyzer (Beckman Coulter, Fullerton, Calif.) and trypan
blue exclusion assays, respectively.
[0525] To perform 2D SDS-PAGE, human ABM-SC (Lot #PCH627; at
.about.27 population doublings) were thawed and re-suspended in
either HyQ.RTM. Minimum Essential Medium (MEM), Alpha Modification
(HYCLONE.TM.; Catalog #SH30265.01, Lot #ASA28110) supplemented with
4 mM L-glutamine (HYCLONE.TM.; Catalog #SH30034.01, Lot #134-7944))
or RPMI1640 (HYCLONE.TM.; Catalog #SH30255.01) supplemented with 4
mM L-glutamine (HYCLONE.TM.; Catalog #SH30034.01, Lot #134-7944).
Cell suspensions were then seeded in T-225 cm.sup.2 CELLBIND.TM.
culture flasks (n 3) at 24-40,000 cells/cm.sup.2 in 36 mL of media
(n=3 per condition). Cultures were placed in a 37.degree. C.
humidified trigas incubator (4% O.sub.2, 5% CO.sub.2, balanced with
nitrogen) for approximately 24 hours. Cultures were re-fed with
fresh media on same day to remove non-adherent debris and then
returned to the incubator. The following day, conditioned media
were collected, pooled, and centrifuged at 1140.times.G for 15
minutes to remove cell debt's, and then transferred to sterile
centrifuge tubes for short-term storage at -80.degree. C.
Example 2
[0526] Two Dimensional (2-D) SDS PAGE Separation of Secreted
Factors (FIG. 1)
[0527] Frozen aliquots of conditioned media and control media
(samples) were shipped to Kendrick Labs, Inc. (Madison, Wis.) for
analysis. Prior to use, samples were thawed and warmed to room
temperature. Approximately 50 mL of each sample was lyophilized
then re-dissolved in 200 microL of SDS Boiling Buffer (5% sodium
dodecyl sulfate, 5% beta mercaptoethanol ethanol, 10% glycerol and
60 mM Tris, pH 6.8) and 2 mL of ultrapure water. The samples were
then dialyzed against 5 mM Tris, pH 7.0 for two days at 4.degree.
C. using 6-8,000 MWCO membranes. The final dialysis was performed
using water only. The samples were lyophilized once again,
re-dissolved in 200 microL of SDS Boiling Buffer, and heated in a
boiling water bath for 5 minutes before loading into the gels.
[0528] Two-dimensional gel electrophoresis was performed according
to the method of O'Farrell (O'Farrell, P. H., J. Biol. Chem. 250:
4007-4021, 1975) as follows: Isoelectric focusing was first carried
out in glass tubes of inner diameter 2.0 mm using 2.0% ampholines,
pH 3.5-10 (Amersham Biosciences, Piscataway, N.J.) for 20,000
volt-hrs. 50 ng of IEF internal standard (tropomyosin) was then
added to each sample. The tropomyosin standard is used as a
reference point on the gel, it migrates as a doublet with a lower
polypeptide spot of MW 33,000 and pI 5.2. The tube gel pH gradient
for this set of ampholines was determined using a surface pH
electrode.
[0529] After equilibration for 10 min in buffer 0 (10% glycerol, 50
mm dithiothreitol, 2.3% SDS, 0.0625 M tris, pH 6.8) each tube gel
was sealed to the top of a stacking gel that, itself, is placed on
top of a 12% acrylamide slab gel (1.0 mm thickness). SDS slab gel
electrophoresis was carried out for about 5 hours at 25 mA. The
following proteins (Sigma Chemical Co.) were added as molecular
weight standards to a single well in the agarose portion of the gel
(the agarose is cast between the tube gel to the slab gel): myosin
(220,000 daltons), phosphorylase A (94,000 daltons), catalase
(60,000 daltons), actin (43,000 daltons), carbonic anhydrase
(29,000 daltons), and lysozyme (14,000 daltons). Following
silver-staining the standards appear as bands on the basic edge of
the acrylamide slab gel (Oakley et al. Anal. Biochem. 105:361-363,
1980). The gel was then dried between two sheets of cellophane
paper with the acid end to the left (FIG. 1). If gels are intended
for use with mass spectroscopy analysis they are stained using the
silver stain method of O'Connell and Stults (O'Connell and Stults.
Electrophoresis. 18:349-359, 1997).
[0530] The results show that using the methods provided, human
ABM-SC can be cultured in the absence of animal serum to produce
conditioned media rich in secreted proteins, and that such proteins
can be individually identified and isolated. Conditioned media
produced in such can also be processed, alternatively, by
fractionating the expressed proteins based on a range of molecular
weights. Techniques for protein concentration and fractionation are
well-known and routinely used by those of ordinary skill in the
art. These techniques include techniques such as affinity
chromatography, hollow fiber filtration, 2D PAGE, and
low-absorption ultrafiltration.
Example 3A
[0531] Pro-Regenerative Cytokine Secretion by Human ABM-SC
[0532] Human ABM-SC were plated in triplicate at 6,000 viable
cells/cm.sup.2 in cell culture "T" flasks containing AFG 104 media.
After allowing cells to attach and equilibrate for 24 hours,
culture media was completely changed and flasks were incubated for
72 hours. Media was collected, centrifuged and stored at
-80.degree. C. until analysis for cytokines using commercially
available colorimetric ELISA assay kits. For analysis of secreted
cytokine release, sister flasks were treated with 10 mg/mL
TNF-alpha, added during the last 24 hours of the 72 hour
incubation. For each, lot three flasks of cells and supernatant
were prepared, processed and banked independently for the basal and
stimulated conditions, designated Basal Flask A, B and C or
Stimulated Flask A, B and C, respectively.
[0533] Results show that when sub-cultured, ABM-SC secrete
potentially therapeutic concentrations of several growth factors
and cytokines known to augment angiogenesis, inflammation and wound
healing. See, FIG. 11. Hence, ABM-SC have been shown to
consistently secrete several cytokines and growth factors in vitro;
including proangiogenic factors (e.g., SDF-1 alpha, VEGF, ENA-78
and angiogenin), immunomodulators (e.g., IL-6 and IL-8) and scar
inhibitors/wound healing modulators (e.g., MMP-1, MMP-2, MMP-13 and
Activin-A). Furthermore, the release of several of these factors is
modulated by tumor necrosis factor alpha (TNF-alpha), a known
inflammatory cytokine released during the course of acute tissue
injury.
Example 3B
[0534] Solid-Phase Capture and Identification of Secreted Factors
(Table 1A, 1B and 1C)
[0535] Conditioned media were screened for the presence of various
proteins such as cytokines, proteases, and soluble receptors by
solid phase antibody capture protein array, using RAYBIO.TM. Human
Cytokine Antibody Array (RayBiotech, Inc., Norcross, Ga., USA).
Briefly, frozen aliquots of conditioned media were thawed and
warmed to room temperature prior to use. Array membranes were
placed into the well of an eight-well tray (C series 1000). To each
well, 2 mL 1.times. Blocking Buffer (RayBiotech, Inc.) was added
and then incubated at room temperature for 30 min to block the
membranes. Blocking Buffer was then decanted from each container,
and the membranes were then incubated with conditioned media
(diluted 1:10 with Blocking Buffer) at room temperature for 1 hr.
Fresh cell culture media were used in place of PBS as negative
controls. Samples were then decanted from each container and washed
3 times with 2 mL of 1.times. Wash Buffer I (RayBiotech, Inc.) at
room temperature, while shaking for 5 min. Array membranes were
then placed into one well, with 1 mL biotin-conjugated secondary
antibody prepared in 1.times. Blocking Buffer, and incubated at
room temperature for 1 hr. Arrays were then washed several times
with Wash Buffer. 2 mL HRP-conjugated streptavidin diluted 1:1000
with 1.times. Blocking Buffer was added to each membrane and then
incubated at room temperature for 2 hrs. Membranes were then washed
several times with 1.times. Wash Buffer. Detection reagents for
chemiluminescence were prepared as per manufacturer's instructions
(RayBiotech, Inc.) and applied to each membrane and incubated at
room temperature for 2 minute. Membranes were then placed protein
side up on a plastic sheet. The opposite of the membrane was then
covered with another piece of plastic sheet. Air bubbles were
purged from the membranes by smoothing out the plastic. The
membranes were then expose to x-ray film (Kodak X-OMAT AR.TM. film)
and then processed using a film developer.
[0536] Table 1A, 1B and 1C shows an extensive list of cytokines,
growth factors, soluble receptors, and matrix proteases secreted by
human ABM-SC when sub-cultured in serum-free cell culture media.
Media Supernatant Concentrate #1=Advanced DMEM (Gibco.TM.)
supplemented with 4 mM L-glutamine. Media Supernatant Concentrate
#2=RPMI-1640 containing 4 mM L-glutamine and HEPES (HyClone)
supplemented with Insulin-Transferrin-Selenium-A (Gibco.TM.)
[0537] The results demonstrate that numerous trophic factors and
soluble receptors important for tissue regeneration and modulation
of the immune system are produced by ABM-SC when cultured under
these conditions. Notably, earlier experiments demonstrated that
supplementation of the base culture medium with insulin,
transferrin, and selenium was required to achieve secreted protein
levels such as those indicated in Table 1A, 1B and 1C. Protein
levels shown in Table 1A, 1B and 1C were assessed using a
RAYBIO.TM. Human Cytokine Antibody Array (RayBiotech, Inc.). Values
are expressed as mean optical densities (O.D.). (N=2 for test
samples. N=4 for controls.) Values reported with a (+) indicate
mean O.D. values for that particular analyte greater than two
standard deviations above the mean O.D. values for the respective
negative control. Values reported with a (-) represent mean O.D.
values for that particular analyte that are not greater than two
standard deviations above the mean O.D. values for the respective
negative control.
TABLE-US-00001 TABLE 1A Media Supernatent Media Supernatent
Cytokine Concentrate #1 Concentrate #2 POSITIVE CTL 11,020 (Mean
O.D.) 11,127 (Mean O.D.) NEG CTL (Background) 2,360.00 2,271.00
Angiogenin 5800.5 (+) 4651 (+) BDNF 5855.5 (+) 3587 (+) BLC 3852
(+) 3164.5 (+) BMP-4 3299 (+) 2610 (+) BMP-6 2359.5 (-) 2290.5 (-)
CK beta 8-1 2408.5 (-) 2426 (-) CNTF 2655.5 (+) 2663 (+) EGF 3932.5
(+) 2517 (+) Eotaxin 2527 (+) 2488 (+) Eotaxin-2 2467 (-) 2452.5
(+) Eotaxin-3 4564 (+) 4450 (+) FGF-6 2863.5 (+) 2883.5 (+) FGF-7
2328 (-) 2374.5 (-) Flt-3 Ligand 2661 (+) 2414.5 (-) Fractalkine
2432.5 (-) 2379.5 (-) GCP-2 2546.5 (+) 2270 (-) GDNF 2299.5 (-)
2208.5 (-) GM-CSF 2294 (-) 2129 (-) I-309 2431.5 (-) 2222 (-)
IFN-gamma 2807.5 (+) 2848.5 (+) IGFBP-1 3192 (+) 4528.5 (+) IGFBP-2
4813.5 (+) 4244 (+) IGFBP-4 4640 (+) 4222.5 (+) IGF-I 2206.5 (-)
2238 (-) IL-10 2225.5 (-) 2200.5 (-) IL-13 2582 (+) 2473 (+) IL-15
2472.5 (-) 2622.5 (+) IL-16 2339.5 (-) 2229.5 (-) IL-1alpha 2698.5
(+) 2571.5 (+) IL-1beta 2276 (-) 2253 (-) IL-1ra 2609 (+) 2505.5
(+) IL-2 2523.5 (+) 2381 (-) IL-3 2346 (-) 2270 (-) IL-4 2591 (+)
2402 (+) IL-5 3159 (+) 3808 (+) IL-6 45570 (+) 40260.5 (+) IL-7
7336.5 (+) 5805 (+) Leptin 4187 (+) 3733.5 (+) LIGHT 3689.5 (+)
3378.5 (+) MCP-1 9925.5 (+) 5561 (+) MCP-2 3117.5 (+) 2481.5 (+)
MCP-3 2532 (+) 2382 (-) MCP-4 2702.5 (+) 2694 (+) M-CSF 2387 (-)
2381.5 (-) MDC 2414.5 (-) 2510.5 (+) MIG 2344 (-) 2342.5 (-)
MIP-1-delta 2324 (-) 2259.5 (-) MIP-3-alpha 2323.5 (-) 2261.5 (-)
NAP-2 2517.5 (+) 2467.5 (+) NT-3 2973.5 (+) 3205.5 (+) PARC 2668
(+) 2630 (+) PDGF-BB 2580.5 (+) 2780 (+) RANTES 2803 (+) 2760 (+)
SCF 2765 (+) 2701.5 (+) SDF-1 3721 (+) 2562 (+) TARC 2488 (-) 2395
(-) TGF-beta 1 2381 (-) 2311 (-) TGF-beta 3 2422 (-) 2531 (+)
TNF-alpha 2243 (-) 2321 (-) TNF-beta 2355 (-) 2410.5 (-)
TABLE-US-00002 TABLE 1B Media Supernatent Media Supernatent
Cytokine Concentrate #1 Concentrate #2 POSITIVE CTL 12,318 (Mean
O.D.) 11,936 (Mean O.D.) NEG CTL 2,452.00 2,392.00 Acrp30 2539.5
(+) 2436.5 (-) AgRP 2670 (+) 2494 (-) Angiopoietin-2 3372 (+)
2656.5 (+) Amphiregulin 2692 (+) 2447 (-) axl 3398.5 (+) 3438.5 (+)
bFGF 2915 (+) 2901.5 (+) Beta-NGF 2573.5 (+) 2544 (+) BTC 2653.5
(+) 2554.5 (+) CCL28 2706.5 (+) 2553.5 (+) CTACK 3502 (+) 3217 (+)
dtk 2610.5 (+) 2512 (+) EGF-R 3057.5 (+) 2767.5 (+) ENA-78 2630.5
(+) 2503 (+) Fas/TNFRSF6 3312 (+) 3322.5 (+) FGF-4 2711 (+) 2650.5
(+) FGF-9 2770 (+) 2538.5 (+) G-CSF 3950.5 (+) 3951 (+) GITR ligand
2973.5 (+) 3107.5 (+) GITR 3198 (+) 2935 (+) GRO 29446.5 (+) 10214
(+) GRO-alpha 7351 (+) 3553.5 (+) HCC-4 3241 (+) 2720.5 (+) HGF
5535 (+) 3936.5 (+) ICAM-1 3043 (+) 2701.5 (+) ICAM-3 2621.5 (+)
2427 (-) IGF-BP-3 3392 (+) 3190.5 (+) IGF-BP-6 5858 (+) 6111 (+)
IGF-I SR 2737.5 (+) 2757 (+) IL-1 R4/ST2 3463.5 (+) 3235.5 (+) IL-1
RI 2522.5 (+) 2401 (-) IL11 2444.5 (-) 2273 (-) IL12-p40 2584 (+)
2536 (+) IL12-p70 2612 (+) 2618 (+) IL17 2610.5 (+) 2555.5 (+) IL-2
Ra 2491 (-) 2441.5 (-) IL-6 R 3202 (+) 2836 (+) IL8 24199.5 (+)
17594.5 (+) I-TAC 3898 (+) 3564 (+) Lymphotactin 3415.5 (+) 3166
(+) MIF 3743 (+) 3524 (+) MIP-1-alpha 2792 (+) 2747.5 (+)
MIP-1-beta 2638.5 (+) 2523 (+) MIP-3-beta 2495.5 (-) 2377 (+) MSP-a
2524.5 (+) 2394 (-) NT-4 2735 (+) 2635 (+) Osteoprotegerin 4183.5
(+) 3399 (+) Oncostatin M 2610 (+) 2508 (-) PlGF 2705 (+) 2493 (-)
sgp130 3232 (+) 2866.5 (+) sTNF RII 3124 (+) 3127 (+) sTNF-RI 9981
(+) 7929.5 (+) TECK 2887.5 (+) 2851 (+) TIMP-1 8718 (+) 9342.5 (+)
TIMP-2 11927 (+) 12602 (+) TPO 3712 (+) 3141.5 (+) TRAIL-R3 3129
(+) 3051 (+) TRAIL-R4 3417 (+) 3381 (+) uPAR 9557.5 (+) 8158.5 (+)
VEGF 8587.5 (+) 6851 (+) VEGF-D 3477 (+) 3190.5 (+)
TABLE-US-00003 TABLE 1C Media Supernatent Media Supernatent
Cytokine Concentrate #1 Concentrate #2 POS 16,092 (Mean O.D.)
15,396 (Mean O.D.) NEG 2,338 1,747 Avtivin A 23239.5 (+) 18339 (+)
ALCAM 14185.5 (+) 15463.5 (+) B7-1 (CD80) 2983.5 (+) 2222.5 (+)
BMP-5 2770.5 (+) 2011.5 (+) BMP-7 2564 (+) 1828 (-) Cardiotrophin-1
2816.5 (+) 2097 (+) CD14 3556 (+) 2334.5 (+) CXCL-16 4108.5 (+)
2559 (+) DR6 (TNFRSF21) 3477 (+) 2312 (+) Endoglin 3070 (+) 2135
(+) ErbB3 3366 (+) 2313.5 (+) E-Selectin 2846.5 (+) 1918 (+)
Fas-Ligand 3531.5 (+) 2943.5 (+) ICAM-2 3158.5 (+) 2155.5 (+)
IGF-II 3212 (+) 2395.5 (+) IL-1 R II 2855 (+) 1834 (-) IL-10 Rb
2780 (+) 1916 (+) IL-13 Ra2 2559.5 (+) 1693 (-) IL-18 BPa 2921 (+)
1881 (-) IL-18 Rb 3238.5 (+) 2387 (+) IL-2 Ra 3666 (+) 2316.5 (+)
IL-2 Rb 3001 (+) 2083.5 (+) IL-2 Rg 3121 (+) 2185.5 (+) IL-21R
3567.5 (+) 2534.5 (+) IL-5 Ra 3084.5 (+) 2237 (+) IL-9 3676 (+)
2324.5 (+) IP-10 3300.5 (+) 2262.5 (+) LAP 6202 (+) 5383.5 (+)
Leptin R 3487 (+) 2791 (+) LIF 3486.5 (+) 2400.5 (+) L-Selectin
3036.5 (+) 2160 (+) M-CSF R 3140 (+) 2330.5 (+) MMP-1 3469 (+) 2499
(+) MMP-13 3083.5 (+) 2316.5 (+) MMP-9 3058.5 (+) 2370 (+) MPIF-1
2974 (+) 2274.5 (+) NGF R 2887.5 (+) 2355 (+) PDGF-AA 4130 (+)
3423.5 (+) PDGF-AB 3191.5 (+) 2278.5 (+) PDGF Ra 4430 (+) 4027 (+)
PDGF Rb 3768 (+) 2784 (+) PECAM-1 4071.5 (+) 3450 (+) Prolactin
3199.5 (+) 2151 (+) SCF R 3431.5 (+) 2668.5 (+) SDF-1b 2268.5 (-)
2156 (+) Siglec-5 2691 (+) 2160.5 (+) TGF-a 3058.5 (+) 2388.5 (+)
TGF b2 3316 (+) 2583 (+) Tie-1 2883 (+) 3178 (+) Tie-2 3565 (+)
3802.5 (+) TIMP-4 6468 (+) 6248 (+) VE-Cadherin 3164.5 (+) 2428 (+)
VEGF R2 4030.5 (+) 3003 (+) VEGF R3 3200 (+) 2651.5 (+)
Example 4
Bioactivity of Adult Bone Marrow-Derived Somatic Cells: In Vitro
Neurogenesis Enhanced by Secreted Factors
[0538] A stock solution of collagen was first prepared by
re-suspending rat tail collagen (Sigma Chemical) in 0.1N acetic
acid at a final concentration of 3.0 mg/mL. The collagen-based
medium then was prepared as described by Bell et al., Proc. Natl.
Acad. Sci. USA, vol. 76, no. 3, pp. 1274-1278 (March 1979) with
minor modifications as described herein. Briefly, the collagen
medium was prepared by mixing the rat tail collagen solution with
DMEM 5.times. (JRH Biosciences) supplemented with 5 mM L-glutamine
(CELLGRO.TM.), Antibiotic-Antimycotic Solution (CELLGRO.TM.), and a
buffer solution (0.05N NaOH (Sigma Chemical), 2.2% NaHCO.sub.3
(Sigma Chemical), and 60 mM HEPES (JRH Biosciences) at a ratio of
4.7:2.0:3.3. Approximately 500 microL of the collagen cell
suspension was added to each well of a 24-well culture plate. The
24-well plates were then placed in a 37.degree. C. humidified
trigas incubator (4% O.sub.2, 5% CO.sub.2, balanced with nitrogen)
for 1 hour to permit the collagen solution to congeal. Frozen rat
PC-12 were thawed, washed in RPMI-1640 supplemented with 4 mM
L-glutamine and HEPES (HYCLONE.TM.) supplemented with
Insulin-Transferrin-Selenium-A (GIBCO.TM.) and centrifuged at
350.times.g for 5 minutes at 25.degree. C. Cell pellets were
re-suspended in same solution at a concentration of 75,000 viable
cells/mL, with and without 136 ng/mL rat beta-NGF (.beta.-NGF)
(Sigma Chemical), 1:50 dilution of unconditioned concentrated
RPMI-1640/ITS medium (used as a negative control), and a 1:50
dilution of conditioned concentrated
RPMI-1640/Insulin-Transferrin-Selenium-A (ITS) media (media was
conditioned as described in Example 1; conditioned and
unconditioned, negative control media were concentrated as
described in Example 1). Next, l mL of cell suspension was
dispensed evenly across the surface of each of 2 gels (1 mL gel)
for each cohort and then verified by phase contrast microscopy. The
plates were then placed in a 37.degree. C. humidified trigas
incubator (4% O.sub.2, 5% CO.sub.2, balanced with nitrogen). Spent
culture media was replaced every 3 days with fresh media. Images
were captured on Day 10. See, FIG. 2.
[0539] These results demonstrate that PC12 differentiation into
neurons by NGF is augmented dramatically when supplemented with
conditioned media produced by human ABM-SC. Interestingly, the
extent of neural differentiation, as assessed by the number of axon
and neurites in the culture, was not significant when conditioned
media was added alone. While some neurite outgrowth was observed in
the presence of NGF alone, supplementing the cultures with
conditioned media dramatically increased both the number and length
of neurites. Previous work in our lab showed that supplementing
RPMI culture media with insulin, transferrin, and selenium was
critical for neural differentiation of PC 12 under all standard
published experimental conditions tested. These data indicate that
media conditioned by human ABM-SC contain components which
supplement or induce neurite outgrowth over and above the levels
obtained with RPMI/ITS media alone or with RPMI/ITS media
containing NGF. See, FIG. 2.
Example 5
Bioactivity of Adult Bone Marrow-Derived Somatic Cells
Inhibition of Mitogen-Induced T Cell Proliferation In Vitro
[0540] Human ABM-SC (Lot #RECB801 at .about.18 population
doublings) and exABM-SC (RECB906 at .about.43 population
doublings), were plated in 75 cm.sup.2 flasks at a concentration of
6000 viable cells/cm.sup.2 in complete media (Minimal Essential
Medium-Alpha (HYCLONE.TM.) supplemented with 4 mM glutamine and 10%
sera-lot selected, gamma-irradiated, fetal bovine serum
(HYCLONE.TM.) and incubated at 37.degree. C. in a humidified trigas
incubator (4% O.sub.2, 5% CO.sub.2, balanced with nitrogen). After
24 hrs, spent media was aspirated and replaced with 15 mL fresh
media. Human mesenchymal stem cells (hMSC, Catalog #PT2501, Lot
#6F3837; obtained from Cambrex Research Bioproducts; now owned by
Lonza Group Ltd., Basel, Switzerland) were plated in 75 cm.sup.2
flasks at a concentration of 6000 viable cells/cm.sup.2 in 15 mL
Mesenchymal Stem Cell Growth Medium (MSCGM.TM.; Lonza Group Ltd.,
Basel, Switzerland) and incubated at 37.degree. C. in a humidified
incubator at atmospheric O.sub.2 and 5% CO.sub.2. After 24 hrs,
spent media was aspirated and replaced with 15 mL fresh MSCGM.TM..
Both human ABM-SC (hABM-SC) and hMSC were harvested after 96 hours
in culture. Harvested hABM-SC and hMSC were plated in 96-well round
bottom plates at a concentration of 25,000 viable cells/mL in
RPMI-complete media (HYCLONE.TM.) Human peripheral blood
mononuclear cells (PBMCs) were labeled in 1.25 microM
CarboxyFluoroscein Succinimidyl Ester (CFSE) and cultured at
250,000 cells/well in RPMI-complete media along with hMSC, Lot
#RECB801, Lot #RECB906 hABM-SC or alone. To stimulate T cell
proliferation, cultures were inoculated with 2.5 or 10 microg/mL
Phytohaemagglutinin (Sigma Chemical). Cells were then harvested 72
hrs later and stained with CD3-PC7 antibody (Beckman Coulter), as
per manufacturer's instructions, and analyzed on a Beckman FC 500
Cytometer, using FlowJo 8.0 software (Tree Star, Inc., Ashland,
Oreg.). Only CD3+ cells were analyzed for division index. See, FIG.
3.
[0541] These findings demonstrate that exABM-SC possess the
capacity to inhibit T cell activation and proliferation and,
therefore, may be useful as a therapeutic to suppress T
cell-mediated graft rejection, autoimmune disorders involving
dysregulation of T cells, or to induce a state of immune tolerance
to an otherwise immunogenic skin product. Thus, one could envision
the use of allogeneic human exABM-SC or compositions produced by
such cells, to treat burn patients awaiting surgical application of
an allogeneic skin product. In such an embodiment, treating an open
wound first with exABM-SC, or compositions produced by such cells,
may act not only to help rebuild the wound bed by inciting host
cells to migrate to the cite of injury, but also to provide an
environment permissive to long term engraftment of allogeneic skin
or skin substitutes.
Example 6
Reconstitution of Porcine ABM-SC in Aqueous Vehicle for In Vivo
Administration
[0542] Porcine ABM-SC were seeded at 60 cells/cm.sup.2, refed at
day 4, and grown for a total of 6 days. Cells were collected and
frozen until subsequent use. Frozen aliquots of porcine ABM-SC were
thawed, washed in DPBSG (Dulbecco's Phosphate Buffered Saline
(CELLGRO.TM.)) supplemented with 4.5% glucose) and centrifuged at
350.times.g for 5 minutes at 25.degree. C. Cell pellets were
re-suspended in DPBSG at a concentration of approximately
50,000/microL. Cell counts and viability assays were performed
using a COULTER.TM. AcT 10 Series Analyzer (Beckman Coulter,
Fullerton, Calif.) and by trypan blue exclusion, respectively. The
cell suspension was then loaded into a 1 cc tuberculin syringe.
Example 7
Bioactivity of Adult Bone Marrow-Derived Somatic Cells
Treatment of Incisional Wounds with Allogeneic Porcine ABM-SC
[0543] Two Yucatan swine, weighing between 57 kg and 78 kg were
anesthetized and prepared for aseptic surgery. Four incisional
wounds measur'ng approximately 50 mm in length were made with a
scalpel blade on both sides of two animals (Nos. 3 and 4) for a
total of eight wounds per animal along the paravertebral and
thoracic area skin. Bleeding was stopped by inserting sterile gauze
soaked with epinephrine into the lesion site. Gauze was then
removed after about 10-20 minutes and each wound was treated with a
single dose of porcine ABM-SC, divided into 12 separate injections
evenly spaced around the incision with an additional 10-300 microL
applied to the wound bed itself. Control wounds were injected
similarly with vehicle only (DPBSG). Wounds were then closed with
Steri-Strips.TM. (3M) and the animals were covered with protective
aluminum jackets. The jackets were checked several times each day
to ensure stable and proper position. The wound dressings were
monitored daily and changes photographed on days 0, 1, 3, 5, and 7.
Animals were euthanized on day 7 for histopathology. Formalin fixed
paraffin embedded tissue sections were prepared and stained by
H&E. Histomorphometric scoring was conducted by an expert
veterinary pathologist blinded to the treatment group.
[0544] Seven days following treatment of the wounds, lesions
treated with allogeneic porcine ABM-SC shown almost no signs of
visible scarring (FIG. 4) while those treated with vehicle
exhibited visible signs of scarring. Histomorphometric analysis of
the wounds showed a marked reduction in tissue macrophages
(histiocytes) in those treated with the ABM-SC, while no
significant difference was seen in any of the other histological
scores assessed.
[0545] When similar tissue sections were scored for the extent of
re-epithelialization (a crude indicator wound healing rate), those
treated with ABM-SC exhibited a marked increase in the amount of
epithelial cells repopulating the site of the incisions (FIG.
5).
Example 8
Bioactivity of Human ABM-SC in Collagen Vehicle for In Vivo
Administration as a Liquid, Semi-Solid, or Solid-like therapeutic
(FIG. 6-9)
[0546] When reconstituted in a collagen-based biodegradable vehicle
and stored at 4.degree. C., human ABM-SC (Lot #PCH610; .about.27
population doublings) retain high cell viability for at least 24
hours (as demonstrated by cell bioactivity in gel contraction
assays). Stored this way, the collagen solution will remain as a
liquid and will preserve the cells in a suspended state without
significant loss of viability (FIG. 6). Bioactivity of the cells
can then be assessed using an in vivo assay of wound repair. To
conduct this assay, a stock solution of collagen was first prepared
by re-suspending rat tail collagen (Sigma Chemical) in 0.1N acetic
acid at a final concentration of 3.0 mg/mL. The collagen medium was
prepared as described by Bell et al. (Proc. Natl. Acad. Sci. USA,
vol. 76, no. 3, pp. 1274-1278 (March 1979)) with minor
modifications as described herein. Briefly, the collagen medium was
prepared by mixing the rat tail collagen solution with DMEM
5.times. (JRH Biosciences) supplemented with 5 mM L-glutamine
(CELLGRO.TM.), Antibiotic-Antimycotic Solution (CELLGRO.TM.;
Catalog #30-004-C1), and a buffer solution (0.05N NaOH (Sigma
Chemical), 2.2% NaHCO.sub.3 (Sigma Chemical), and 60 mM HEPES (JRH
Biosciences) at a ratio of 4.7:2.0:3.3. Frozen human adult bone
marrow derived somatic cells (hABM-SC) were thawed, washed in DMEM
1.times. and centrifuged at 350.times.g for 5 minutes at 25.degree.
C. The cell pellets were re-suspended in DMEM 1.times. at
concentration of approximately 72,000 total cells/microL. Fifty
microliters of cell suspension was then added to 2 mL collagen
medium and gently triturated (i.e., gently pipetted up and down to
obtain a homogeneous suspension of cells in collagen medium),
yielding a final cell concentration of approximately 1,800
cells/microL. The cell suspension was then stored at approximately
4-8.degree. C. overnight. The following day, the liquid cell
suspension was transferred from the 15 mL conical tube and
dispensed into 24-well cell culture plates at approximately 500
microL/well. The plates were then placed in a 37.degree. C.
humidified trigas incubator (4% O.sub.2, 5% CO.sub.2, balanced with
nitrogen) for 1 hour to permit the collagen to solidify into a
semi-solid gel. The gels were then removed from the 24-well plates
using disposable sterile spatulas (VWR) and transferred to 12-well
culture plates. The gels were then floated in 1.0 mL DMEM 1.times.
per well. For negative controls, gels were prepared as described
but without cells. Three wells were seeded for each condition
(n=3).
[0547] To evaluate the extent to which gel contraction is
dose-dependent, a similar assay was conducted wherein human
exABM-SC (Lot#RECB819; at .about.43 population doublings) were
reconstituted in collagen solution at different cell concentrations
immediately after removal from cryostorage (FIG. 7). A stock
solution of collagen was first prepared by re-suspending rat tail
collagen (Sigma Chemical) in 0.1N acetic acid at a final
concentration of 3.0 mg/mL. The collagen medium then was prepared
as described by Bell et al. (1979) with minor modifications as
described herein. Briefly, the collagen medium was prepared by
mixing the rat tail collagen solution with DMEM 5.times. (JRH
Biosciences) supplemented with 5 mM L-glutamine (CELLGRO.TM.),
Antibiotic-Antimycotic Solution (Cellgro.TM.), and a buffer
solution (0.05N NaOH (Sigma Chemical), 2.2% NaHCO.sub.3 (Sigma
Chemical), and 60 mM HEPES (JRH Biosciences)) at a ratio of
4.7:2.0:3.3. Frozen human adult bone marrow derived somatic cells
(hABM-SC) were thawed, washed in DMEM 1.times. and centrifuged at
350.times.g for 5 minutes at 25.degree. C. The cell pellets were
re-suspended in DMEM 1.times. at concentration of approximately
40,000, 80,000 and 200,000 viable cells/microL. Fifty microliters
of each cell suspension was added to 2 mL collagen medium and
gently triturated. Approximately 500 microL of the collagen cell
suspension was added to each well of a 24-well culture plate. The
plates were then placed in a humidified 37.degree. C. trigas
incubator (4% O.sub.2, 5% CO.sub.2 balanced with nitrogen) for 1
hour to permit the collagen solution to solidify. The gels were
then removed from the plates using disposable sterile spatulas
(VWR) and transferred to 12-well culture plates. The gels were
floated in 1.0 mL DMEM 1.times. per well.
[0548] As a negative control, gels were prepared as described above
using the highest concentration of hABM-SC (5.times.10.sup.6/mL)
except that the cells were heat-inactivated (to eliminate
biological activity). Heat-inactivated cells were first prepared by
heating the initial cell suspension in DMEM 1.times. medium to
70.degree. C. in a heat block containing water (heat transfer) for
40 minutes. Three wells were seeded for each condition (n=3).
[0549] To determine the extent to which the gels contracted over
time, the percentage initial or starting surface area was
calculated from digital images captured at 0, 24, 48 and 72 hours
using a flatbed scanner. From each image, the diameter of the gel
was measure both horizontally and vertically and then averaged.
Results demonstrate that both the rate and extent of gel
contraction was effected in a dose dependent manner (FIG. 7).
[0550] To determine the levels of certain secreted proteins
produced from the human ABM-SC in these semi-solid gels,
enzyme-linked immunosorbant assay (ELISA) was performed (on day 3
of culture) on conditioned cell culture supernatants collected from
the liquid media surrounding the gels (FIG. 8). Supernatants were
transferred to sterile 15 mL conical tubes and centrifuged at
1140.times.g for 15 minutes to remove cell debris. Supernatants
were then transferred to 2 mL cryovials and transferred to
-80.degree. C. for short-term storage. On the day of assay,
supernatants were thawed and equilibrated to room temperature
before use. ELISA analysis was performed to detect IL-6, VEGF,
Activin-A, pro-MMP-1, and MMP-2 ELISA (conducted as per
manufacturer's instructions; all kits were purchased from R&D
Systems, Inc. (Minneapolis, Minn., USA)). Results demonstrate that
therapeutically relevant levels of trophic factors can be produced
by these semi-solid neotissues and that these levels can be
controlled by adjusting cell concentration. Of the trophic factors
measured, detectable levels were not seen in cultures containing
heat inactivated cells only. Statistical comparisons between assay
conditions were determined by One-way ANOVA (*** p<0.001).
[0551] Human ABM-SC can also be reconstituted in a collagen
solution to construct a large-format semi-solid structure that
could be used as topical therapeutic (FIG. 9). To construct such a
structure, a stock solution of collagen was first prepared by
re-suspending rat tail collagen (Sigma Chemical) in 0.1N acetic
acid at a final concentration of 3.0 mg/mL. The aqueous collagen
medium was prepared by mixing the rat tail collagen solution with
DMEM 5.times. (JRH Biosciences) supplemented with 5 mM L-glutamine
(CELLGRO.TM.), Antibiotic-Antimycotic Solution (CELLGRO.TM.), and a
buffer solution (0.286N NaOH (Sigma Chemical), 1.1% NaHCO.sub.3
(Sigma Chemical), and 100 mM HEPES (JRH Biosciences) at a ratio of
6:2:2. Frozen hABM-SC were thawed and washed in 1.times.DMEM and
then centrifuged at 350.times.g for 5 minutes at 25.degree. C. The
cell pellet was re-suspended in 1.times.DMEM at a concentration of
approximately 90,000 cells/microL. Approximately 1.1 mL of cell
suspension was then added to 20 mL collagen medium and gently
triturated to achieve a final cell concentration of
5.times.10.sup.6 cells/mL. The final concentration of collagen was
1.8 mg/mL. The cell suspension was then dispensed into a 10 cm
Petri dish (forming dish). The effective dose of cells in the
collagen solution dispensed was approximately 100.times.10.sup.6
viable cells. The 10 cm forming dish containing the cell suspension
was then placed in a humidified 37.degree. C. incubator (5%
CO.sub.2) for 1 hour to permit the collagen solution to solidify.
The semi-solid gel was then carefully removed from the 10 cm
forming dish and transferred to a 15 cm Petri dish (culture dish)
and photographed.
[0552] To construct a solid-like neotissue derived from human
ABM-SC and collagen, the semi-solid structure described above can
be placed back into a 37.degree. C. humidified cell culture
incubator (5% CO.sub.2) for an additional 2 days (FIG. 10). To form
a solid-like neotissue, a semi-solid gel prepared as described
above, with the exception that the final collagen solution was 1.4
mg/mL (instead of 1.8 mg/mL), was carefully dislodged from the
edges of the 10 cm forming dish and floated in approximately 82 mL
1.times.DMEM containing Antibiotic-Antimycotic Solution
(CELLGRO.TM.) in a 15 cm culture dish. The semi-solid gel was then
transferred to a 37.degree. C. humidified incubator (5% CO.sub.2)
for an additional 48 hrs to facilitate remodeling of the matrix
into a solid-like tissue structure, free of the starting collagen
substrate. The solid-like neotissue was then removed from the 15 cm
culture dish and photographed (FIGS. 10A and 10B). Histological
analysis of the neotissue by Masson's Trichrome stain demonstrates
that the matrix is rich in newly synthesize human collagens and
proteoglycans (FIG. 10C). Control collagen gels do not stain by
this method. Collagens and proteoglycans stain blue.
[0553] The results of these studies indicate that frozen stocks of
ABM-SC can be dispensed upon thaw and reconstituted in a liquid
collagen-based medium that could be used therapeutically as a
liquid suspension, semi-solid construct, or solid-like neotissue.
When prepared in such a way and stored at approximately
4-10.degree. C., the cell suspension will remain as liquid while
maintaining satisfactory cell viability for greater than 24 hours.
Employing such a method to formulate ABM-SC for clinical
application then would provide considerable latitude to the
clinician administering the cells. The suspension could be
administered as a liquid injectable or, alternatively, could be
applied topically to a wound bed. In the latter case, the liquid
cell suspension would be anticipated to mold to the contour of the
wound and then congeal into a semi-solid structure (for example,
when warmed to .about.37 degrees C.). Alternatively, the suspension
could be used in such a way as to manufacture semi-solid constructs
or solid-like neotissues.
[0554] These data also show that when prepared by the methods, the
resulting compositions each possess bioactivity important for
mediating repair of various types of wounds, particularly those
involving the skin.
[0555] ExCF-SC (for example, exABM-SC), or compositions produced by
such cells, prepared in a liquid collagen-based medium could
therefore be used topically to treat open wounds or as an
injectable alternative to dermal fillers for facial
rejuvenation.
[0556] In the semi-solid form, exCF-SC (for example, exABM-SC) or
compositions produced by such cells, cold be used topically to
treat severe burn patients that have had damaged full-thickness
skin removed surgically, thereby acting as a dermal
replacement.
[0557] Solid neotissues produced by exCF-SC (for example, exABM-SC)
could be used surgically as an alternative to human cadaveric skin
(ALLODERM.TM.), porcine skin (PERMACOL.TM.) and other
animal-derived constructs (INTEGRA.TM.). Moreover, these data also
show that the potency of each of these various constructs can be
controlled by altering dose of cells or compositions produced by
the cells.
Example 9
Improvement of Cardiac Function in Rats Treated with hABM-SC
[0558] Administration of human ABM-SC to animals following
myocardial infarct demonstrates that CF-SC (such as ABM-SC) improve
cardiac function and enhance repair of cardiac tissue damage by
stimulating angiogenesis and reducing fibrosis. See, FIG. 15. A rat
model for acute myocardial infarction was utilized by occluding a
coronary artery thereby creating a cardiac lesion (i.e., damaged
region of heart). Lesioned rats were injected intercardially with
either hABM-SC or vehicle.
[0559] Heart Function Methods: Sprague-Dawley rats of both sexes
(age approx. 3 months) received experimentally-induced myocardial
infarction via the placement of a permanent silk ligature around
the left-anterior descending (LAD) coronary artery via a midline
sternotomy. Five days after this procedure, the rats were begun on
a standard regimen of Cyclosporine A treatment that lasted for the
duration of the study. On day 7-8 following infarction, rats were
anesthetized, intubated and an intercostal incision was made to
expose the apex of the heart. An ultrasonic Millar catheter was
then inserted through the ventricular wall, and pressure over time
measurements were recorded for a period of approximately 30-60
seconds. This model of infarct production and pressure/time
measurements of cardiac function is a standard, well characterized
model by which the effects of cellular therapies on cardiac
function can be assessed (See e.g., Muller-Ehmsen, et al.,
Circulation., 105(14):1720-6 (2002)).
[0560] The test composition was delivered using a 100 microL
Hamilton syringe fitted with a 30 gauge, low dead-space needle.
Five separate injections of 20 microL were performed over the
course of 2-3 minutes. Four injections were performed at equal
distances around the visualized infarct, while the fifth was placed
directly into the center of the infarcted region as determined by
area of discoloration. After injection, the incision was sutured
closed, the pneumothorax was reduced, and the animals were weaned
from the respirator and extubated. Four weeks after injection (5
weeks post-infarction), animals were reanesthetized, the heart was
exposed through a midline sternotomy, and a Millar catheter was
inserted. DpAlt measurements were taken as described above, after
which the rats were euthanized via exsanguination.
[0561] Heart Function Results (FIG. 13): Four weeks after
treatment, rats receiving ABM-SC demonstrated significantly higher
+dp/dt (peak positive rate of pressure change) values (A).
Expressing changes in cardiac function over the course of the study
by subtracting 0 week +dp/dt values from 4 week values ("delta
+dp/dt") demonstrated that while vehicle treated rats had decreases
in cardiac function over the course of the study (negative delta),
animals treated with either cell preparation showed significant
improvement in cardiac function (B). Compared to vehicle treated
rats, those receiving ABM-SC demonstrated significantly lower tau
values (C), suggesting increased left ventricular compliance. Tau
is the time constant of isovolumetric left ventricular pressure
decay. For peak negative rate of pressure change (-dp/dt),
expressing changes in cardiac function over the course of the study
by subtracting 0 week -dp/dt values from 4 week values ("delta
-dp/dt") demonstrated that while vehicle-treated rats had decreases
in cardiac function over the course of the study (negative delta),
animals treated with cell preparation showed significant
improvement in cardiac function (D). [*p<0.05, **p<0.01 by
ANOVA]
[0562] Heart Structure Methods: Sprague-Dawley rats received
experimentally-induced myocardial infarction via the placement of a
permanent silk ligature around the left-anterior descending (LAD)
coronary artery. Animals received a standard regimen of
Cyclosporine A treatment (10 mg/kg s.c. daily) that lasted for the
duration of the study.
[0563] On day 7-8 following infarction, rats were anesthetized,
intubated and an intercostal incision was made to expose the apex
of the heart. Cardiac function was accesses after which the test
article was delivered using a 100 microL Hamilton syringe fitted
with a 30 gauge, low dead-space needle. Five separate injections of
20 microL were performed over the course of 2-3 minutes. Four
injections were performed at equal distances around the visualized
infarct, while the fifth was placed directly into the center of the
infracted region as determined by area of discoloration. After
injection, the incision was sutured shut, the pneumothorax was
reduced, and the animals were weaned from the respirator and
extubated. Four weeks after injection (5 weeks post-infarction),
animals were reanesthetized, the heart was exposed through a
midline sternotomy, and cardiac function accessed. After functional
measures were completed rats were euthanized via exsanguination.
Rats were first deeply anesthetized using a mixture of ketamine (75
mg/kg) and medetomidine (0.5 mg/kg). The thoracic cavity was then
surgically exposed and the heart dissected and immersion fixed in
10% neutral buffered formalin. Hearts were then grossly sectioned
into three pieces, oriented into embedding molds, and processed for
paraffin embedding. Heart tissues were then sectioned at 6 .mu.m
and stained by Hemotoxylin & Eosin (H&E) or Masson's
Trichrome. At least six sections from every heart were also stained
with hemotoxylin/eosin and Trichrome respectively. Specifically,
trichrome staining allows for the visualization of collagen (blue)
versus muscle tissue (red). Since collagen indicates the presence
of scar tissue (absence of regeneration), the ratios of collagen to
normal cardiac muscle were determined. A semiquantitative scoring
scale was devised, with 0 as no detectable collagen and 5 as
maximal/severe. Stained sections were then sent to a board
certified pathologist for histomorphometric scoring.
[0564] Each slide contained three cross-sections of the heart,
demonstrating a cross-sectional view of both ventricles from the
mid-ventricular area (1) distal 1/3 of the ventricle (2), and apex
of the ventricle (3). For histomorphometric analyses, the following
grading scheme was used:
[0565] Location of tissue damage: Left ventricle (LV), Right
ventricle (LV), Both ventricles (By).
[0566] Percent of affected ventricle damaged (size of injury):
Given in percent (0-100%)
[0567] Thickness score of experimentally damaged area of ventricle:
Given a grade of 1-4 based on estimated thickness in millimeters.
Compared with known landmarks in the tissue sections (e.g. average
erythrocyte is 7 microns in diameter; average myocardial muscle
bundle is 30 microns in diameter). Grade 1 (less than 0.5 mm);
Grade 2 (0.5 mm to 1 mm); Grade 3 (1 mm to 1.5 mm); Grade 4 (1.5
mm+).
[0568] Neovascularization in area of tissue damage: (Grade of 0 to
4, from normal (0) to neovascularization throughout the entire area
of initial tissue damage (4).
[0569] Initial vascular damage: Includes degeneration/necrosis of
pre-existing blood vessels, with thrombosis and/or inflammation
resulting from removal of remaining vascular debr's, expressed as a
grade of 0 to 4, with 0 being no vascular damage present, and 4
being vascular damage throughout the affected area.
[0570] Extent of fibrosis within the area of tissue damage:
Expressed as a grade of 0 to 4, from no fibrosis (0) to (4) in 20%
graduating levels of fibrosis and scarring of the initial area of
damage caused by the infarction procedure. For example, fibrosis of
20% of the ventricle would be assigned a grade of (1), and fibrosis
of 40% of the ventricle would be assigned a grade of (2), 60% would
receive a (3), and above 60% would receive a (4).
[0571] Heart Structure Results: Rats were subsequently sacrificed
and cardiac tissue was sectioned and stained. A board certified
veterinary pathologist performed semiquantitative scoring (FIG. 15)
to evaluate changes in infarct size in the hearts of rats receiving
vehicle or ABMSC seven days after myocardial infarction.
Histopathological analysis indicated significant reduction in
infarct size in rats receiving hABM-SC compared to vehicle.
According to a preset scale, rats receiving hABM-SC had
histological scores approximately two points lower than vehicle
controls. FIG. 14 shows an example of typical infarct size
reduction. Histopathological analysis determined that hABM-SC
reduced fibrosis and increased vascularization in the infarct zone
(FIG. 15), consistent with pro-regenerative activity. Thus, it was
observed that rats treated with hABM-SC showed dramatic improvement
of cardiac tissue structure. See, FIGS. 14 and 15.
Example 10
Adult Bone Marrow-Derived Somatic Cells Suppress Immune Mediated
Responses
[0572] Part I: Suppression of Mitogen-Induced T-Cell Proliferation
in One-Way MLR (Mixed Lymphocyte Reaction) Assay.
[0573] Methods: Human ABM-SC and exABM-SC (Lot #RECB801 and
RECB906, respectively), were plated in 75 cm.sup.2 flasks at a
concentration of 6000 viable cells/cm.sup.2 in 15 mL complete media
such as Advanced DMEM (GIBCO.TM.; Catalog #12491-015, Lot #1216032
(Invitrogen Corp., Carlsbad, Calif., USA)) supplemented with 4 mM
L-glutamine (Catalog #SH30034.01. Lot #134-7944, (HYCLONE.TM.
Laboratories Inc., Logan, Utah, USA)) or HyQ.RTM. RPMI-1640
(HYCLONE.TM. Catalog #SH30255.01, Lot #ARC25868) containing 4 mM
L-glutamine and supplemented with Insulin-Transferrin-Selenium-A
(ITS) (GIBCO.TM.; Catalog #51300-044, Lot #1349264) and incubated
at 37.degree. C. in a humidified trigas incubator (4% O.sub.2, 5%
CO.sub.2, balanced with Nitrogen). After 24 hrs, spent media was
aspirated and replaced with 15 mL fresh media. Human mesenchymal
stem cells (hMSC) (Lonza BioScience, formerly Cambrex Bioscience,
Catalog #PT2501, Lot #6F3837) were plated in 75 cm.sup.2 flasks at
a concentration of 6000 viable cells/cm.sup.2 in 15 mL MSCGM.TM.
(Lonza BioScience) and incubated at 37.degree. C. in a humidified
incubator at atmospheric O.sub.2 and 5% CO.sub.2. After 24 hrs,
spent media was aspirated and replaced with 15 mL fresh MSCGM.TM..
Both hABM-SC and hMSC were harvested after 96 hours in culture.
Harvested hABM-SC and hMSC were plated in 96-well round bottom
plates at a concentration of 25,000 viable cells/mL in
RPMI-complete media (Hyclone). Human peripheral blood mononuclear
cells (PBMCs) were labeled in 1.25 uM CarboxyFluoroscein
Succinimidyl Ester (CFSE) and cultured at 250,000 cells/well in
RPMI-complete media along with hMSC, RECB801, RECB906 or alone. To
stimulate T cell proliferation, cultures were inoculated with 2.5
or 10 micrograms/mL Phytohaemagglutinin (Sigma Chemical). Cells
were then harvested after 72 hrs later and stained with CD3-PC7
antibody (Beckman Coulter), as per manufacturer's instructions, and
analyzed on a Beckman FC 500 Cytometer, using Flow Jo software.
Only CD3+ cells were gated analyzed for division index.
[0574] Results: Allogeneic human ABM-SC and exABM-SC suppress
mitogen-induced T-cell proliferation in one-way MLR assay. See,
FIG. 16.
[0575] Part II: Allogeneic Porcine ABM-SC Fail to Illicit T-Cell
Mediated Immune Response in 2-Way MLR Challenge Assay.
[0576] Methods: Porcine whole blood was collected for immunoassays
on Day 0 (prior to treatment) and at necropsy (Day 3 or Day 30
post-treatment) for cellular immune response analysis. PBMC from
each animal were cultured with pABM-SC, the mitogen ConA, or media
alone. Samples were analyzed by flow cytometry and the amount of
proliferation calculated using FlowJo software.
[0577] Whole blood samples were diluted 1:1 with DPBS (Dulbecco's
PBS)-2% Porcine Serum. Diluted blood was overlayed on Ficoll (2:1
ratio diluted blood to Ficoll) and centrifuged at 350.times.G for
30 minutes, with centrifugation cycle ending with zero braking. The
resulting top layer was aspirated. The middle layers, which contain
the desired mononuclear cells, were pooled for each sample, and
washed in 3.times. with DPBS-2% Porcine Serum. After washing, the
pellet was resuspended in 20 mL ACK lysis buffer and incubated for
3 minutes, to remove residual red blood cells, then centrifuged for
5 minutes, at 250.times.G. The pellets were washed in 20 mL DPBS-2%
Porcine Serum and resuspended in 5 mL RPMI Complete media
(RPMI-1640, 10% Porcine serum, 2 mM L-glut, 20 mM HEPES, 0.1 mM
NEAA, 1.times. Penn-Strep). Cells were frozen at a concentration of
20.times.10.sup.6 cells/mL by centrifugation, and resuspension in
ice cold freeze media (10% DMSO in Porcine Serum) and immediately
added to 2 mL cryovials and placed into a cryorate freezer. (freeze
rate=-25.degree. C./min to -40.degree. C., +15.degree. C./min to
-12.0.degree. C., -1.degree. C./min to -40.degree. C., -10.degree.
C./min to -120.degree. C.). Cells were stored in 2 mL aliquots per
vial in vapor phase of liquid nitrogen until use.
[0578] On day 0, pABM-SC were plated in 96 well culture dishes at a
concentration of 10,000 cells/well in AFG-104 media according to
study template for each test condition. Plates were incubated
overnight at 37.degree. C. in a humidified incubator with low
O.sub.2 (4-5%), .about.5% CO.sub.2 balanced with nitrogen.
[0579] The following day, PBMC were labeled with CFSE
(carboxy-fluorescein diacetate, succinimidyl ester). In short,
thawed vials of PBMC in 37.degree. C. water bath, washed with 10 mL
RPMI-Complete media centrifuged cells at 300.times.G, and
resuspended in DPBS. Cell concentrations were adjusted to
10.times.10.sup.6 cells/mL and incubated with CFSE at a final
concentration of 0.625 mM for 5 minutes. Cells were immediately
washed in 40 mL ice cold DPBS/5% porcine serum and centrifuged 10
minutes at 300.times.G. Cells were again washed in 25 mL DPBS/5%
porcine serum and centrifuged as before. Cells were washed a third
and final time in 10 mL RPMI-complete media. Cell concentrations
were adjusted to a final concentration of 5.times.10.sup.6
cells/mL. Labeled PBMC were added to the assay plate according to
study template as follows: AFG-104 media was aspirated and replaced
with 100 microL RPMI-Complete media. 100 microL of RPMI-complete
media was added to non-stimulated wells, 100 microL media with 20
microg/mL ConA in RPMI-Complete media was added to stimulated
wells, and 4.5% Glucose-RPMI-Complete media to vehicle cells.
500,000 labeled PBMC were plated per well in 96 well plates
according to study template. Plates were incubated for 5 days at
37.degree. C., atmospheric O.sub.2 (high O.sub.2), with humidity,
5% CO.sub.2 no nitrogen. All conditions were completed in
triplicate for each blood sample received. Vehicle stimulation was
completed for a subset of blood samples, but was not significantly
different than media alone. After 5 days of co-culture, cells were
harvested for flow cytometry by transferring cells from 96 well
plate to a flow tube. Indirect staining was conducted in accordance
with standard protocols. The primary antibody used was Biotin
Conjugated Mouse anti-Pig CD3 Monoclonal antibody; followed by
exposure to Streptavidin-PE-Cy7 secondary reagent. Cells were
resuspended in 200 microL flow wash buffer and analyzed on a
Coulter FC500 device.
[0580] Results: A Division Index was calculated for samples
collected at baseline and at 3 or 30 days post-treatment and then
challenged in vitro with media, vehicle, pABM-SC or ConA. The
average division index from all animals at Day 3 or Day 30 for CD3+
cells stimulated with ConA was significantly higher than the
division index for CD3+ cells from vehicle and pABM-SC treated
animals at pre-treatment and at necropsy (*p<0.05). See, FIG.
17.
Example 11
Clinical Development
[0581] A Phase 1, open label, dose escalation study to evaluate the
safety of a single escalating dose of hABM-SC administered by
endomyocardial injection to cohorts of adults 30-60 days following
initial acute myocardial infarction has been undertaken. The
primary objective of this study was investigate the safety and
feasibility of single escalating doses of hABM-SC delivered via
multiple endomyocardial injections using the MYOSTAR.TM. catheter,
guided by the NOGA.TM. or NOGA XP.TM. electromechanical cardiac
mapping system. A secondary objective was to investigate the
preliminary efficacy of single escalating doses of hABM-SC,
measured by left ventricular volume, dimension, myocardial
infarction size and voltage.
[0582] The study protocol provides that test subjects are to be
followed for 12 months with frequent monitoring for safety.
Efficacy assessments are to be performed at 90 day and six month
follow-up visits. The intended study population is 30 to 75 year
old consenting adults with an acute myocardial infarction (AMI)
within the previous 30 days who have been successfully treated with
percutaneous revascularization restoring TIMI II or higher flow,
with a left ventricular ejection fraction of greater than or equal
to 30% as measured by myocardial perfusion imaging (SPECT).
[0583] Inclusion and Exclusion Criteria: Inclusion criteria for the
study comprises: (1) 30-75 years of age (inclusive); (2) 30-60 days
since AMI (defined as the most recent MI causing a doubling in
cardiac-specific troponin I (cTnI) enzyme concentrations relative
to normal levels in addition to ECG changes consistent with MI with
confirmation by myocardial perfusion imaging [SPECT]); (3)
successful percutaneous revascularization of restoring TIMI II or
higher flow to infarcted area; (4) negative pregnancy test
(serum_hCG) in women of childbearing potential (within 24 hours
prior to dosing); (5) left ventricular ejection fraction
(LVEF)>30% as measured by myocardial perfusion imaging (SPECT);
(6) cardiac enzyme tests (CPK, CPK MB, cTnI) within the normal
range at baseline; (7) must be ambulatory.
[0584] Exclusion criteria for the study comprises: (1) significant
coronary artery stenosis that may require percutaneous or surgical
revascularization within six months of enrollment; (2) left
ventricular (LV) thrombus (mobile or mural); (3) high grade
atrioventricular block (AVB); (4) frequent, recurrent, sustained
(>30 seconds) or non-sustained ventricular tachycardia >48
hours after AMI; (5) clinically significant electrocardiographic
abnormalities that may interfere with subject safety during the
intracardiac mapping and injection procedure; (6) atrial
fibrillation with uncontrolled heart rate; (7) severe valvular
disease (e.g., aortic stenosis, mitral stenosis, severe valvular
insufficiency requiring valve replacement); (8) history of heart
valve replacement; (9) idiopathic cardiomyopathy; (10) severe
peripheral vascular disease; (11) liver enzymes (Aspartate
aminotransferase [AST]/alanine aminotransferase [ALT])>3 times
upper limit of normal (ULN); (12) serum creatinine >2.0 mg/dL;
(13) history of active cancer within the preceding three years
(with exception of basal cell carcinoma); (14) previous bone marrow
transplant; (15) known human immunodeficiency virus (HIV)
infection; (16) evidence of concurrent infection or sepsis on chest
X-ray (CXR) or blood culture; (17) participation in an experimental
clinical trial within 30 days prior to enrollment; (18) alcohol or
recreational drug abuse within six months prior to enrollment; (19)
major surgical procedure or major trauma within the 14 days prior
to enrollment; (20) known autoimmune disease (e.g., systemic lupus
erythematosus [SLE], multiple sclerosis); (21) clinically
significant elevations in prothrombin (PT) or partial
thromboplastin time (PTT) relative to laboratory norms; (22)
thrombocytopenia (platelet count <50,000/mm.sup.3); (23)
inadequately controlled diabetes mellitus type I or type II,
defined as a change in anti-diabetic medication regimen within the
prior 3 months or HbA1c >7.0%; (24) uncontrolled hypertension
defined as systolic blood pressure (SBP)>180 mmHg and/or
diastolic blood pressure (DBP)>100 mmHg; (25) use of ionotrophic
drugs>24 hours post AMI; (26) other co-morbid conditions such as
hemodynamic instability, unstable arrythmias, and intubation,
which, in the opinion of the principal investigator, may place
subjects at undue risk or interfere with the objectives of the
study; (27) any other major illness, which, in the opinion of the
principal investigator, may interfere with the subject's ability to
comply with the protocol, compromise subject safety, or interfere
with the interpretation of the study results; and, (28)
contraindication (either allergy or impaired renal function) to
injection with contrast media for adequate CT scan evaluations.
[0585] Study Drug Dosage and Administration: Subjects in the same
cohort will be dosed no closer than three days apart, and dosing of
successive cohorts will be separated by approximately four weeks,
following review of at least three weeks of safety data on all
subjects in the previous cohort.
TABLE-US-00004 Cohorts Dose Cohort 1 30 .times. 10.sup.6 cells
Cohort 2 100 .times. 10.sup.6 cells Cohort 3 300 .times. 10.sup.6
cells Cohort 4 300 .times. 10.sup.6 cells or MTD
[0586] On the day of dosing, after baseline evaluations are
complete and immediately following percutaneous ventricular mapping
with the NOGA.TM. or NOGA XP.TM. electromechanical mapping system
(Biosense Webster, Diamond Bar, Calif.), multiple sequential
injections of hABM-SC will be delivered directly into the
myocardium from a percutaneous, LV approach using a MYOSTAR.TM.
catheter.
[0587] Study Procedures: Potential subjects will be consented and
screened within 21 days prior to planned hABM-SC administration,
which must occur within 30-60 days following AMI. Screening
procedures to deter-nine eligibility also will be used as baseline
values, unless hospital SOPs require additional tests (i.e.,
immediately prior to catheterization). Baseline testing for
treatment efficacy is to consist of a Six Minute Walk Test, NYHA
classification, blood analysis for B-type natriuretic peptide (BNP)
concentration, echocardiography, right and left cardiac
catheterization, myocardial perfusion imaging (SPECT), and NOGA.TM.
or NOGA XP.TM. electromechanical mapping. On the day of admission,
additional baseline blood testing (including pregnancy testing
[serum_hCG] for female subjects of childbearing potential) will be
done, and eligibility will be verified. On the day of dosing (Study
Day 0), subjects will undergo NOGA.TM. or NOGA XP.TM.
electromechanical cardiac mapping and a MYOSTAR.TM. catheter will
be placed into the left ventricle. The dose of hABM-SC will be
administered via multiple sequential endomyocardial injections into
the damaged (defined by NOGA.TM. or NOGA XP.TM.) myocardial tissue.
After administration of hABM-SC, echocardiography will be performed
to detect possible transmural perforation, and the subject will be
admitted directly to the intensive care unit (ICU) for a minimum of
twenty-four hours of observation with continuous cardiac telemetric
monitoring. Stable subjects without complications will be
discharged from the ICU to a step down unit (with cardiac
monitoring) and then discharged to home no sooner than 72 hours
after the dosing procedure. Subjects with complications will remain
in the ICU under optimal medical management until stable and
appropriate for discharge to the step down unit. Safety evaluations
will be performed 7, 14, 21, 60 and 90 days and at six and twelve
months following administration of hABM-SC. Efficacy evaluations
will be performed at 90 days and six months after the procedure,
and will include left ventricular volume, dimension, size of
myocardial infarction and voltage, measured respectively by
contrast enhanced echocardiography, myocardial reperfusion and
viability analysis (SPECT), right and left cardiac catheterization
(90 days only), six minute walk test, NYHA classification, and
NOGA.TM. or NOGA XP.TM. electromechanical mapping (90 days
only).
[0588] Safety endpoints in the study will comprise: (1) adverse
events as detailed in the study protocol; (2) clinically
significant changes from baseline in blood or blood components
including CBC, CMP, CPK/CPK MB, cTnl, PT/PTT, and HLA antibody
analysis; (3) clinically significant changes from baseline in
cardiac electrical activity as assessed by electrocardiogram (ECG)
or cardiac telemetry; (4) clinically significant changes from
baseline in cardiac electrical activity as assessed by 24 hour
Holter monitoring; (5) perioperative myocardial perforation as
assessed by echocardiogram (post procedure); and, (6) clinically
significant changes from baseline in physical and mental status as
assessed by physical examination including a focused neurological
examination. If signs and symptoms consistent with cerebrovascular
accident (stroke) are observed, a neurological consult will be
obtained for further evaluation
[0589] Efficacy Endpoints: Efficacy endpoints to be monitored
comprise: (1) end systolic and/or end diastolic volume compared to
baseline, as measured by myocardial perfusion imaging (SPECT); (2)
myocardial infarction size compared to baseline as measured by
myocardial perfusion imaging (SPECT); (3) end systolic and/or end
diastolic dimensions compared to baseline as measured by contrast
enhanced 2-D echocardiography; (4) action potential voltage
amplitude in the area of hABM-SC injected myocardium as compared to
baseline and historical controls (provided by the core laboratory)
as measured by NOGA.TM. or NOGA XP.TM. electromechanical mapping;
(5) cardiac output and pressure gradients compared to baseline as
determined by right and left cardiac catheterization; (6) quality
of life compared to baseline as assessed by the Six Minute Walk
Test; and, (7) functional cardiovascular disease class (NYHA
functional classification scheme) compared to baseline as assessed
by the physician performing scheduled physical examinations.
[0590] Endomyocardial Delivery of hABM-SC: hABM-SC will be
delivered to the myocardium via direct catheter-guided injection
from within the ventricular chamber. Endomyocardial delivery of
hABM-SC will be accomplished with the aid of the NOGA.TM. Cardiac
Navigation System (one of the most advanced systems for three
dimensional visualization of the physical, mechanical and
electrical properties of intact myocardium in vivo; from
Biosense-Webster, Diamond Bar, Calif.). The actual injection will
be performed with the Cordis MYOSTAR.TM./catheter. The NOGA.TM.
system allows for real time viewing of left ventricular heart
function, detection of heart tissue damage, observation and
placement of the catheter tip. Given the tenuous cardiac condition
of patients post-AMI, a relatively non-invasive delivery system
(compared to open heart or direct intracardiac delivery), i.e. the
MYOSTAR.TM. Injection Catheter used in conjunction with the
NOGA.TM. mapping system, was selected for administration of
hABM-SC.
[0591] Preliminary Results: Preliminary results for 5 patients have
been obtained. The first 3 patients comprised the initial dose
group (30 million cells), while the last two patients received the
second escalating dose (100 million cells). Overall, hABM-SC was
well tolerated in all patients, with some trends to improvement in
cardiac function noted in several patients. More detailed results
are discussed below.
[0592] Safety Findings: No evidence of allogeneic immune response
(as measured by pre- and post-treatment antibody profiling) was
found in any patients.
[0593] Cardiac Functional Assessments: NOGA Electromechanical
Mapping: Functional mapping was performed at time of treatment and
at 90 days after cell treatment. Representative unipolar voltage
maps were obtained from the second patient in the first dose
cohort. A clear voltage deficit could be seen in the area of
infarct (data not shown). Fifteen cell injections were performed at
the margin of the infarct using unipolar voltage as a guide. At 90
days follow up, a clear improvement in unipolar voltage could be
seen, with near normal voltages prevailing in the infarct zone.
Similar degrees of improvement in voltage were noted in patients 1,
3 and 4 (data not shown).
[0594] Myocardial Perfusion Imaging (SPECT): Perfusion imaging was
performed at baseline, 90 days and 6 months after cell treatment
according to previously published methods.
[0595] All images were digitally captured and analyzed. Ejection
fraction, perfusion deficit size, and ventricular volumes were
derived from this analysis, under basal and adenosine-stress
conditions, along with a 24 hour-washout rescan. Results for each
patient at each time point are discussed below.
[0596] Perfusion Deficit: In general, perfusion deficit sizes,
which are thought to represent overall infarct sizes, either
decreased or remained unchanged over the six months of follow up
for treated patients. Two patients demonstrated reductions in
deficit deemed "clinically significant" meaning the deficits
resolved to less than 4-5% of the total ventricular wall. In both
of these cases, the areas of improvement corresponded to areas of
voltage improvement as measured by NOGA mapping. Although NOGA
mapping is considered investigational, this data supports validity
of the hypothesis that unipolar voltage may be a surrogate for
infarct size measurement.
[0597] Ejection Fraction (EF): In general, ejection fraction in
study patients either improved or remained relatively unchanged.
One patient experienced a significant drop in overall EF (63% to
50% over six months), but this patient experienced a serious
adverse event during the course of cell treatment which renders it
questionable whether or not a complete dose of cells was actually
administered to the endomyocardium. Two patients demonstrated
increases in EF well above the expected for this patient group. The
lack of placebo controls precludes any conclusions as to the
mechanism of this improvement.
[0598] End-Diastolic Volume (EDV): EDV was measure at baseline and
at 90 days and 6 months following treatment. In general, EDV
remained unchanged in all patients over the 6 month follow up
period, suggesting no significant remodeling occurred in these
patients.
[0599] FIG. 18 shows the changes in cardiac fixed perfusion deficit
size in three patients by comparison of a baseline (BL)
measurements with measurements obtained 90 days post-treatment with
hABM-SC. FIG. 19 shows the changes in cardiac ejection fractions
measured in three patients by comparison of a baseline (BL)
measurements with measurements obtained 90 days post-treatment with
hABM-SC.
Example 12
Human ABM-SC and Compositions Derived Thereby for the Production of
Red Blood Cells In Vitro
[0600] It is well known that the bone marrow microenvironment
provides the requisite combination of matrix molecules, growth
factors and cytokines necessary to support and modulate
hematopoiesis (Dexter at al. 1981). Most, if not all, of the
trophic factors known to drive hematopoietic cell self-renewal and
lineage restricted differentiation derive from the mesenchymal
support cells (Quesenberry et al. 1985). Roecklein and Torok-Storb
(1995) showed that even within a relatively pure population of
these cells, sub-populations can be isolated that differentially
support hematopoiesis. Unlike the immortalized clones described in
these previous publications, the hABM-SC utilized as described
herein represent a pure population of CD45 negative, CD90/CD49c
co-positive non-hematopoietic support cells that secrete many
factors important for inducing and maintaining erythropoiesis
including, but not limited to, IL-6 (Ullrich et al. 1989), LIF
(Cory et al. 1991), SDF-1 (Hodohara et al. 2000), SCF (Dai et al.
1991), Activin-A (Shao et al. 1992), VEGF and IGF-II (Miharada et
al. 2006) (FIG. 20).
[0601] To generate red blood cells from a starting population of
hematopoietic precursors (e.g. embryonic stem cells (ES),
hematopoietic stem cells (HSC), cord blood cells (CBC) or committed
erythroblast precursors (BFU-E)), human ABM-SC and/or compositions
produced by such cells can be utilized to induce, enhance, and/or
maintain erythropoiesis by delivering a "cocktail" of
erythropoietic factors necessary for, or to supplement, growth and
differentiation of hematopoietic precursors into erythroblasts.
See, FIG. 20.
Example 13
Production, Isolation, Purification, and Packaging of Cell-Derived
Compositions and Trophic Factors
[0602] A two-step, downstream bioprocess has been developed to
manufacture, collect and purify compositions such as secreted
growth factors, cytokines, soluble receptors and other
macromolecules produced by human ABM-SC and exABM-SC. This cocktail
of secreted cell compositions, produced as such in the
stoichiometric ratios created by the cells, has tremendous
potential as a pro-regenerative therapeutic, cell culture reagent
and/or research tool for studying in vitro cell and tissue
regeneration. Such compositions can also be used as an alternative
to the cells themselves to support the growth and
lineage-appropriate differentiation of starting erythroid
progenitor cell populations in suspension cultures.
Production of Sera-Free Conditioned Media
[0603] Cryopreserved human ABM-SC (Lot no. P25-T2S1F1-5) are thawed
and re-suspended in one liter of Advanced DMEM (GIBCO, catalog
#12491-015, lot 284174 (Invitrogen Corp., Carlsbad, Calif., USA))
supplemented with 4 mM L-glutamine (HYCLONE Laboratories Inc.,
Logan, Utah, USA catalog #SH30255.01). Cells are seeded in a
Corning.RTM. CellBind.RTM. polystyrene CellSTACK.RTM. ten chamber
(catalog number 3312, (Corning Inc., NY, USA)) at a density of
20,000 to 25,000 cells per cm.sup.2, One port of the CellSTACK.RTM.
ten chamber unit is fitted with a CellSTACK.RTM. Culture chamber
filling accessory (Corning.RTM. Catalog number 3333, (Corning Inc.,
NY, USA)) while the other port is fitted with a CellSTACK.RTM.
Culture chamber filling accessory 37 mm, 0.1 .mu.M filter
(Corning.RTM. Catalog number 3284, (Corning Inc., NY, USA)).
[0604] Cultures are placed in a 37.degree. C..+-.1.degree. C.
incubator and aerated with a blood gas mixture (5.+-.0.25%
CO.sub.2, 4.+-.0.25% O.sub.2, balance Nitrogen (GTS, Allentown,
Pa.)) for 5.+-.0.5 hrs. After 24.+-.2 hrs post seeding, the media
is removed, replaced with 1 liter of fresh media and aerated as
previously described. Approximately, 72.+-.2 hours later the
sera-free conditioned media is aseptically removed from the
CellSTACK.RTM. ten chamber unit within a biological safety cabinet
and transferred to a one liter PETG bottle. The sera free
conditioned media is subsequently processed by tangential flow
filtration.
Isolation and Purification of Sera-Free Conditioned Media
[0605] Tangential flow filtration (TFF) is performed on a reservoir
of sera free conditioned media, recovered from a CellSTACK.RTM. ten
chamber unit, as described above. A polysulfone hollow fiber with a
molecular weight cut-off of 100 kilodaltons (kD) (Catalog number
M1ABS-360-01P (Spectrum Laboratories, Inc., Rancho Dominguez,
Calif., USA)) is employed. The reservoir of sera free conditioned
(the retentate) is re-circulated through the lumen of the hollow
fiber tangential to the face of the lumen. Molecules with a
molecular weight of 100 kD or less pass through the lumen into a 2
liter PETG bottle; this fraction is called the permeate or
filtrate. The retentate is continually re-circulated until the
volume is reduced to approximately less than 50 mL. The retentate
is subsequently discarded and the permeate is retained for further
processing. The resulting permeate (approximately 1 liter) is a
clear, sera-free solution containing small molecular weight
molecules free of cellular debris and larger macromolecules, herein
referred to as Fraction #1.
[0606] Fraction #1 is subsequently subjected to additional TFF
using a polysulfone hollow fiber with a molecular weight cut off of
10 kilodaltons (kD) (Catalog number M11S-360-01P (Spectrum
Laboratories, Inc., Rancho Dominguez, Calif., USA)). Fraction #1 is
subsequently used as the retentate and re-circulated through the
lumen of the hollow fiber, tangential to the face of the lumen.
Smaller molecules <10 kD (i.e. ammonia, lactic acid etc.) are
allowed to pass through the lumen. After the volume of the
retentate is reduced to 100 mL, diafiltration of the solution is
begun. One liter of alpha-MEM without phenol red (HYCLONE, catalog
number RR11236.01 (HYCLONE Laboratories Inc., Logan, Utah, USA)) is
added to the retentate reservoir at the same rate that the permeate
is pumped out; thus maintaining the volume of the reservoir
constant. The resulting retentate contains small only small
molecules ranging in molecular weight from 10 kD to 100 kD; herein
referred to as Fraction #2.
[0607] Fraction #2 can be further processed by subjecting it to
additional TFF using a polysulfone hollow fiber with a molecular
weight cut off of 50 kilodaltons (kD) (Catalog number M15S-360-01 P
(Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA)).
Fraction #2 is thus re-circulated through the lumen of the hollow
fiber, tangential to the face of the lumen. Smaller molecules
.ltoreq.50 kD are passed through the lumen. Both processing streams
are retained as product. The resulting permeate/filtrate is
composed primarily of molecules 10 kD to 50 kD (Fraction #3), while
the retentate comprises macromolecules in the range of 50 kD to 100
kD (Fraction #4).
[0608] Each of the resulting fractions is frozen in 60 mL PETG
bottles (Catalog number 2019-0060, Nalgene Nunc International
Rochester N.Y.).
[0609] Such Isolated protein fractions can subsequently be
subjected to further aseptic downstream processing and packaging,
wherein such compositions can be dialyzed, lyophilized, and
reconstituted into a dry, biocompatible matrix, such as
LYOSPHERES.TM. (manufactured by BIOLYPH.TM., Hopkins, Minn.,
USA).
Example 14
Isolation, Cryopreservation, and Expansion of CD34+ Cord Blood
Cells (CBC)
[0610] Large scale production of lineage-committed erythroid cells
(CFU-E or Reticulocytes) can be manufactured from a starting
population of stem cells or erythroblast precursors (e.g. cord
blood cells, embryonic stem cells, hematopoietic stem cells and
BFU-E) employing the methods described below.
[0611] Umbilical cord blood from healthy full-term newborns is
collected in heparinized blood collection bags. A clean nucleated
cell preparation is made by adding ammonium chloride lysis solution
to cord blood, then centrifuging the mixture at 300.times.g for 15
minutes at room temperature. The supernatant is aspirated from the
cell pellet, and the cell pellet is washed in BSSD with 5% human
serum albumin (wash solution). The cells are centrifuged again at
300.times.g for 15 minutes at room temperature and the wash
solution is removed from the cell pellet by aspiration. CD34+cells
are separated by magnetic cell sorting using MASC LS-columns
(MACS.RTM.; Miltenyi Biotech, Gladbach, Germany) using established
protocols. The CD34+ CBCs are subsequently re-suspended in CSM-55
at approximately 2 million cells/mL and cryopreserved using a
controlled-rate freezer.
[0612] BSSD (Balanced Salt Solution with 4.5% Dextrose) is prepared
as follows:
[0613] To Balanced Salt Solution, Sterile Irrigating Solution (BSS;
Baxter, Deerfield, Ill., USA) add 450.+-.0.5 grams Dextrose (EMD
Life Sciences, Gibbstown N.J. USA), QS to a final volume of 10.0
Liters with BSS.
CSM-55 (Cryogenic Storage Media 5% DMSO, 5% HSA) is Prepared as
follows:
[0614] In a 2 liter bottle combine 1.4 liters of BSSD with 400 mLs
of 25% HSA (25% solution human serum albumin from ZLB Behring,
Ill., USA) and 200 mLs of 50% DMSO (50% dimethyl sulfoxide from
Edwards Lifesciences Irvine Calif. USA).
Wash Solution is Prepared with 400 mLs of BSSD plus 100 mLs of 25%
HSA.
CD34+ CBC Expansion in Suspension Cultures
[0615] The cells are subsequently re-suspended in StemSpan.RTM.
H300 (StemCell Technology) supplemented with 1.0 U/mL recombinant
human EPO(R&D Systems, Cat #287-TC), 10 LYOSPHERES.TM./L, and
inoculated into a disposable HYCLONE.TM., perfusion BIOPROCESS
CONTAINER.TM. (bioreactor) or equivalent, at a cell concentration
of 1.0.times.10.sup.6/mL. Cultures are maintained at 37.degree. C.
with 5% CO.sub.2, 4% O.sub.2, and balanced with Nitrogen, for 3
weeks using continuous flow of fresh culture media. On day 14,
cultures are supplemented with the glucocorticoid antagonist
Mifepristone to accelerate enucleation, as described by Miharada et
al. 2006. Continuous flow of fresh culture media is maintained at a
fixed rate under these conditions until harvest on day 21.
CBC Expansion on a Human ABM-SC Feeder Layer
[0616] Cryopreserved human ABM-SC are thawed and re-suspended in
Advanced RPMI Media 1640 (INVITROGEN.TM.) supplemented with 1.0
U/mL recombinant human EPO (R&D Systems, Cat #287-TC), 4 mM
L-Glutamine, 10% lot selected, gamma-irradiated fetal bovine serum
(Hyclone), and seeded at a density of 10,000 cells/cm.sup.2 in 40
layer cell culture factories (Corning) and maintained at 37.degree.
C. under 5% CO.sub.2, 4% O.sub.2, and balanced with Nitrogen at
37.degree. C. On day 5, one-half volume of spent media is removed
from the cultures and replenished by adding back one-half volume of
fresh media along with 1.0.times.10.sup.6 CBC/mL. Discontinuous
flow (on-off-on) of fresh culture media is subsequently engaged to
enable the media conditions to cycle between fresh (on) to
conditioned (off), and back to fresh media again (on). On day 14,
cultures are supplemented with the glucocorticoid antagonist
Mifepristone to accelerate enucleation, as described by above.
Co-cultures are maintained under these conditions until harvest on
day 21.
Example 15
ABM-SC Secrete Scavenger Receptors and Antagonists and Reduce Tumor
Necrosis Factor-Alpha Levels in a Dose Dependent Manner
[0617] Background: Embodiments of the present invention include
methods and compositions for treating, reducing, or preventing
adverse immune activity (such as inflammation or autoimmune
activity) in a subject by delivering therapeutically effective
amounts of exABM-SC or compositions produced by exABM-SC.
Embodiments of the invention include utilization of exABM-SC, or
compositions produced thereby, relying on the naturally occurring
or basal level production of secreted compositions in vitro.
Alternatively, embodiments of the invention also include
utilization of exABM-SC, or compositions produced thereby, by
manipulating the exABM-SC to modulate (up- or down-regulate) the
quantity and kind of compositions produced (for example, by
administration of pro-inflammatory factors such as TNF-alpha).
[0618] For example, it has now been found that exABM-SC produce at
least one scavenger receptor for the cytokine Tumor Necrosis
Factor-alpha (TNF-.alpha.), and at least one antagonist of the
Interleukin-1 Receptor (IL-1R), and at least one binding protein
(antagonist) of cytokine Interleukin-18 (IL-18). Accordingly,
embodiments of the invention include methods and compositions for
use and administration of stable cell populations (such as
exABM-SC) that consistently secrete therapeutically useful proteins
in their native form.
[0619] The term "stable cell population" as used herein means an
isolated, in vitro cultured, cell population that when introduced
into a living mammalian organism (such as a mouse, rat, human, dog,
cow, etc.) does not result in detectable production of cells which
have differentiated into a new cell type or cell types (such as a
neuron(s), cardiomyocyte(s), osteocyte(s), hepatocyte(s), etc.) and
wherein the cells in the cell population continue to secrete, or
maintain the ability to secrete or the ability to be induced to
secrete, detectable levels of at least one therapeutically useful
composition (such as soluble TNF-alpha receptor, IL-1R antagonists,
IL-18 antagonists, compositions shown in Table 1A, 1B and 1C,
etc.).
[0620] For purposes of the present invention, "scavenger receptor"
is intended to mean any soluble or secreted receptor (whether
membrane bound or free in the extracellular milieu) capable of
binding to and neutralizing its cognate ligand.
[0621] In addition to the pro-inflammatory factors listed above, in
view of the present disclosure it is also understood that cell
populations of the present invention may be treated with any
number, variety, combination, and/or varying concentrations of
factors now known or subsequently discovered or identified in order
to manipulate the concentration and kind of compositions produced
by cell populations of the present invention. For example, the cell
populations of the invention may preferably be treated with factors
such as: IL-1alpha, IL-1beta, IL-2, IL-12, IL-15, IL-18, IL-23,
TNF-alpha, TNF-beta, and Leptin. This brief list of preferred
factors, however, is not intended nor should it be construed as
limiting with respect to the number of different compositions that
can be used to treat cell populations of the present invention, nor
are these compositions limited to proteins, as is it is also
appreciated that many other types of compounds could also be used
to manipulate the cell populations of the present invention
(including, by way of brief examples, other biological
macromolecules such as nucleic acids, lipids, carbohydrates, etc.
and small molecules and chemicals such as dimethylsulfoxide (DMSO)
and nitrous oxide (NO), etc).
[0622] Methods: Production of serum-free conditioned media was
produced as described below for use in enzyme-linked immunosorbant
assays (ELISA) (also described below). Cryopreserved human exABM-SC
(Lot #MFG-05-15; at .about.43 population doublings) were thawed and
re-suspended in Advanced DMEM (GIBCO.TM.; Catalog #12491-015, Lot
#1216032 (Invitrogen Corp., Carlsbad, Calif. USA)) supplemented
with 4 mM L-glutamine (Catalog #SH30034.01. Lot #134-7944,
(HYCLONE.COPYRGT. Laboratories Inc., Logan, Utah, USA)) with and
without 10 ng/mL TNF-.alpha.. Cell suspensions were then seeded in
T-225 cm.sup.2 CELLBIND.TM. (Corning Inc., NY, USA) culture flasks
(culture surfaces treated with a patented microwave plasma process;
see, U.S. Pat. No. 6,617,152) (n=3) at 10,000, 20,000, 40,000
cells/cm.sup.2 in 36 mL of media (n=3 per condition).
Heat-inactivated cells seeded at 40,000 cells/cm.sup.2 were used as
a negative control. Cells were heat-inactivated by transferring an
aliquot to a sterile tube and incubating it for .about.40 minutes
in a 70.degree. C. heat block containing water (for efficient heat
transfer). Cultures were placed in a 37.degree. C. humidified
trigas incubator (4% O.sub.2, 5% CO.sub.2, balanced with nitrogen)
for approximately 24 hours. Cultures were then re-fed with fresh
media on same day to remove non-adherent debris and returned to the
incubator. On day 3, cell culture media was concentrated using 20
mL CENTRICON.TM. PLUS-20 Centrifugal Filter Units (Millipore Corp.,
Billerica, Mass., USA), as per manufacturer's instructions.
Briefly, concentrators were centrifuged for 45 minutes at
1140.times.G. Concentrated supernatants (100.times. final
concentration) were transferred to clean 2 mL cryovials and stored
at -80.degree. C. until later use.
[0623] To determine the levels of certain secreted proteins
produced from the human ABM-SC in these adherent cultures,
enzyme-linked immunosorbant assays (ELISA) were performed on day 3,
100.times. concentrated, conditioned cell culture supernatants
collected as described above. On the day of assay, supernatants
were thawed and equilibrated to room temperature before use. ELISA
analysis was performed to detect TNF-.alpha., soluble TNF-RI
(sTNF-RI), soluble TNF-RII (sTNF-RII), IL-1 receptor antagonist
(IL-IRA) and IL-2 receptor alpha (conducted as per manufacturer's
instructions; all kits were purchased from R&D Systems, Inc.
(Minneapolis, Minn., USA)).
[0624] The results demonstrate that therapeutically relevant levels
of secreted scavenger receptors (e.g. sINT-RI) and receptor
antagonists (e.g. IL-IRA) are produced by these adherent cultures
and that these levels can be controlled by adjusting cell
concentration or dose (FIG. 21-23), Importantly, these data also
demonstrate that the cells respond to the inflammatory milieu in
which they are placed. For example, following treatment with the
potent inflammatory cytokine TNF-alpha, the cells up-regulate
secretion of sTNF-RII (FIG. 22B) and IL-IRA (FIG. 23).
Interestingly, in these sample cultures, the levels of TNF-alpha
were significantly reduced with each increase in cell seeding
density (FIG. 21), suggesting that the TNF-alpha itself was
sequestered in some way by either the ABM-SCs or factors that they
secrete.
[0625] It is well established that both sTNF-RI and sTNF-RII can
bind and neutralize the biological activity of TNF-alpha. Since the
measurable levels of both forms of the TNF receptor, as well as
TNF-alpha itself, are each reduced significantly with each increase
in cell seeding density, it is likely that the ABM-SC derived
sTNF-RI and sTNF-RII are binding to and masking TNF-alpha in this
assay system.
[0626] Of the soluble receptors and receptor antagonists measured,
detectable levels were not seen in cultures containing
heat-inactivated cells only. Statistical comparisons between assay
conditions were determined by one-way ANOVA.
Example 16
Osteogenesis Induction Assay
Human ABM-SC Cells do not Exhibit a Bone Differentiation
Characteristic In Vitro when Cell Populations Expanded Beyond
Approximately 25 Population Doublings are Exposed to Standard
Osteoinductive Conditions Or when Cell Populations Expanded Beyond
Approximately 30 Population Doublings are Exposed to Enhanced
Osteoinductive Conditions
[0627] Methods: Human ABM-SC and exABM-SC were seeded at 3100
cells/cm.sup.2 in 6-well culture dishes (Corning, Catalog #3516)
with 2.4 mL Mesenchymal Stem Cell Basal Medium (MSCBM.TM.; Lonza,
Catalog #PT-3238) supplemented with MSCGM.TM. SingleQuot Kit
(Lonza, Catalog #PT-4105) per well, hereafter referred to as
Mesenchymal Stem Cell Growth Medium (MSCGM.TM.). Approximately four
hours later, the MSCGM.TM. was changed to the appropriate test
conditions. Negative control wells were those re-fed with either
MSCGM.TM. alone, or MSCGM.TM. supplemented with 5 ng/mL recombinant
mouse Noggin/Fc Chimer (R&D Systems, Catalog #719-NG). The test
wells were those treated with either Osteogenesis Induction Medium
(OIM; Lonza Catalog #PT-3924 and #PT-4120) alone (standard
osteoinductive conditions) or OIM supplemented with 5 ng/mL
recombinant mouse Noggin/Fc Chimer (enhanced osteoinductive
conditions). Cultures were then maintained in a humidified CO.sub.2
incubator at 37.degree. C. and re-fed with fresh medium every 3-4
days for 2 weeks. After 14 days, cultures were processed for
calcium determination using the Calcium Liquicolor kit (Stanbio,
Catalog #0150-250), as per manufacturer's instructions. Plates were
read at 550 nm using a SpectraMax Plus.sup.384 microplate
reader.
[0628] Results: Human ABM-SC and exABM-SC derived from research lot
#MCB 109 were cultured under standard osteoinductive conditions
(OIM only) or under enhanced osteoinductive conditions (OIM and the
morphogen Noggin; OIM+Noggin). Negative control cultures were
maintained in either growth media alone (MSCGM.TM.) or MSCGM.TM.
supplemented with Noggin (MSCGM.TM.+Noggin).
[0629] ABM-SC at about 16 population doublings exhibited a calcium
deposition increase of approximately 6-fold when the OIM media was
supplemented with Noggin (i.e., ABM-SC at about 16 population
doublings deposited .about.5 micrograms calcium/well under OIM
conditions and .about.30 micrograms/well under OIM+Noggin
conditions). ABM-SC lost the capacity to deposit detectable levels
of calcium beyond about 16 population doublings under standard OIM
conditions, however, this could be reversed by supplementing with
Noggin (i.e., exABM-SC at about 25 population doublings deposited
no detectable calcium under OIM conditions whereas these same cells
deposited .about.5 micrograms calcium/well under OIM+Noggin
conditions). In contrast, beyond about 30 population doublings
(e.g., at about 35 and 43 populations doublings) exABM-SC did not
deposit detectable levels of calcium under any of the conditions
tested (standard or enhanced OIM).
Example 17
Expression of IL-1 receptor antagonist (IL-MA) and IL-18 binding
protein (IL-18 BP) by ABM-SC
[0630] Methods: Human ABM-SC which had undergone about 43 cell
population doublings (lot #P17-T2S1F1-5) were thawed and seeded in
AFG growth medium supplemented with Brefeldin A at 3 micrograms/mL
(1.times.) and placed in a humidified 5% CO.sub.2 incubator at
37.degree. C. for 24 hours. Cultured cells were then removed from
the culture flasks using porcine trypsin, washed and prepared for
flow cytometry, as per CALTAG FIX & PERM.RTM. staining protocol
(CALTAG LABORATORIES; now part of Invitrogen Corp. (Carlsbad,
Calif., USA). Cells were stained with either FITC conjugated mouse
anti-human IL-1 Receptor Antagonist (IL-1R.sup.A; eBioscience,
Catalog #11-7015, clone CRM17) antibody neat or unlabeled rabbit
anti-IL-18 Binding Protein (IL-18BP; Epitomics, Catalog #1893-1,
clone EP1088Y) at a 1:10 dilution, both for 45 minutes at room
temperature. FITC-rabbit FITC-labeled goat anti-rabbit antibody was
then used to detect the IL-18BP. Isotype matched controls were
included as a negative control (Beckman Coulter).
[0631] Results: Human exABM-SC express basal levels of IL-1
receptor antagonist (IL-1RA; FIG. 24A) and IL-18 binding protein
(IL-18BP; FIG. 24B) even in the absence of an inflammatory signal
such as TNF-alpha.
Example 18
Human ABM-SC Reduce Expression of TNF-Alpha and IL-13 while
Simultaneously Increasing Expression of IL-2
[0632] Methods: Human peripheral blood mononuclear cells (PBMC)
were co-cultured in RPMI-1640 containing 5% Human Sera Albumin, 10
mM HEPES, 2 mM glutamine, 0.05 mM 2-mercaptoethanol, 100 U/mL
penicillin, and 100 microg/mL streptomycin, in a 24 well plate with
either 1) Mitomycin-C treated PBMC from same donor (Responder+Self)
or 2) Mitomycin-C treated PBMC derived from a different donor
(Responder+Stimulator). PBMC from each source were each seeded at
4.times.10.sup.5 cells/well. For each condition, cultures were
supplemented with or without human ABM-SC at a seeding density of
40,000 cells/well. Cultures were maintained in a humidified 5%
CO.sub.2 incubator at 37.degree. C. for 7 days to condition the
media. Conditioned cell culture supernatants were collected and
analyzed for the presence of the various cytokines using the
SEARCHLIGHT.TM. 9-Plex assay (Pierce Protein Research Products,
Thermo Fisher Scientific Inc., Rockford, Ill.). Statistical
analysis was performed by one-way ANOVA (analysis of variance).
[0633] Results: Co-culture of allogeneic PBMC
(Responders+Stimulators) resulted in a marked increase in the
levels of TNF-alpha and IL-13, as would be expected for a mixed
PBMC reaction. When challenged with human ABM-SC, however, both
IL-13 and TNF-alpha were significantly reduced (P<0.001),
suggesting that ABM-SC could be utilized therapeutically to treat
chronic inflammatory disorders or graft rejection by reducing focal
or serum levels of inflammatory mediators. See, FIGS. 25A, B, and
C.
[0634] Notably, ABM-SC induced elevated expression of IL-2 in both
autologous (Responders +Self) and allogeneic (Responders
+Stimulators) mixed PBMC cultures (P<0.001) while simultaneously
suppressing PBMC proliferation. While this result appears somewhat
paradoxical given the importance of IL-2 in promoting T cell
proliferation, recently it has been shown in mice that disruption
of the IL-2 pathway results in lymphoid hyperplasia and
autoimmunity rather than immune deficiency, suggesting that the
major physiological role of IL-2 may be to limit or regulate,
rather than enhance T cell responses (Nelson, "IL-2, Regulatory
T-Cells, and Tolerance," Jour. Immunol. 172: 3983-3988 (2004)).
Additionally, it is now known that IL-2 is also critical for
promoting self-tolerance by suppressing T cell responses in vivo
and that the mechanism by which this occurs is through the
expansion and maturation of CD4+/CD25+ regulatory T cells. It is,
therefore, contemplated that ABM-SC could be employed
therapeutically to induce T-cell tolerance by indirectly supporting
the maturation of T regulatory cells through the induced
up-regulation of IL-2.
Example 19
Human ABM-SC Inhibit Mitogen-Induced Peripheral Blood Mononuclear
Cell Proliferation
[0635] Methods: Human adult bone-marrow derived somatic cells
(ABM-SC) were cultured in vitro for 96 hours in a humidified
incubator under 5% CO.sub.2 then passaged onto 96-well round bottom
plates at a concentration of 25,000 viable cells/mL in
RPMI-complete media (HYCLON.TM.). Human peripheral blood
mononuclear cells (PBMC) were cultured either separately at 250,000
cells/mL in RPMI-complete media, or with ABM-SC Lots RECB801
(sub-cultured to about 19 population doublings) or RECB906
(sub-cultured to about 43 population doublings). To stimulate PBMC
proliferation, cultures were inoculated with 2.5 microg/mL
phytohaemagglutinin (Sigma Chemical Co.). After 56 hours in
culture, cells were pulsed with Thymidine-[Methyl-3H] (Perking
Elmer, 1 microCi/well). Cells were harvested at 72 hours using a
Filtermaster harvester onto glass filters. Filters were read in
Omnifilter platers using an NXT TopCount Scintillation counter.
Human mesenchymal stem cells were included as a positive control.
(Human mesenchymal stem cells were obtained from Cambrex Research
Bioproducts; now owned by Lonza Group, Ltd, Basel, Switzerland).
Statistical analysis was performed by one-way ANOVA (analysis of
variance).
[0636] Results: PBMC-induced proliferation was significantly
reduced when challenged with either lot of ABM-SC (P<0.001).
See, FIG. 26. Mesenchymal stem cells (MSC) were included as a
positive control. These data suggest that ABM-SC not only inhibit
mitogen-induced proliferation of the total PBMC preparation, but
that the presence of ABM-SC in this assay system does not induce
proliferation of various cell subpopulations within the preparation
(e.g., monocytes, granulocytes, lymophocytes).
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