U.S. patent application number 13/958706 was filed with the patent office on 2018-09-13 for adherent cells from adipose or placenta tissues and use thereof in therapy.
This patent application is currently assigned to Pluristem Ltd.. The applicant listed for this patent is Pluristem Ltd.. Invention is credited to Zami Aberman, Nirit Drori-Carmi, Moran Meiron, Rachel Ofir, Amir Toren.
Application Number | 20180256648 13/958706 |
Document ID | / |
Family ID | 40118825 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180256648 |
Kind Code |
A9 |
Meiron; Moran ; et
al. |
September 13, 2018 |
ADHERENT CELLS FROM ADIPOSE OR PLACENTA TISSUES AND USE THEREOF IN
THERAPY
Abstract
A method of treating ischemia in a subject in need thereof is
disclosed. The method comprising administering to the subject a
therapeutically effective amount of adherent cells of a tissue
selected from the group consisting of a placenta and an adipose
tissue, thereby treating the ischemia in the subject. A method of
treating a medical condition requiring connective tissue
regeneration and/or repair is also disclosed.
Inventors: |
Meiron; Moran;
(Zikhron-Yaakov, IL) ; Toren; Amir;
(Zikhron-Yaakov, IL) ; Ofir; Rachel; (Mitzpe Adi,
IL) ; Aberman; Zami; (Tel-Mond, IL) ;
Drori-Carmi; Nirit; (Doar-Na Hof HaCarmel, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pluristem Ltd. |
Haifa |
|
IL |
|
|
Assignee: |
Pluristem Ltd.
Haifa
IL
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130323213 A1 |
December 5, 2013 |
|
|
Family ID: |
40118825 |
Appl. No.: |
13/958706 |
Filed: |
August 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12678756 |
Mar 18, 2010 |
8529888 |
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PCT/IL2008/001185 |
Sep 2, 2008 |
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13958706 |
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60960184 |
Sep 19, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/35 20130101;
A61K 35/28 20130101; A61P 37/06 20180101; A61P 19/00 20180101; A61P
19/08 20180101; A61P 35/00 20180101; A61P 13/12 20180101; A61P
19/02 20180101; A61P 9/10 20180101; A61K 35/36 20130101; A61P 19/10
20180101; A61P 43/00 20180101; A61P 19/04 20180101; A61P 7/02
20180101; A61K 35/50 20130101; A61P 17/02 20180101; A61P 9/00
20180101 |
International
Class: |
A61K 35/50 20060101
A61K035/50; A61K 35/12 20060101 A61K035/12 |
Claims
1. A method of treating ischemia in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of adherent cells of a tissue selected from the
group consisting of a placenta and an adipose tissue, thereby
treating the ischemia in the subject.
2-30. (canceled)
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The invention relates to methods of treating diseases using
adherent cells from adipose or placenta tissues, more specifically,
to methods of treating ischemia and/or medical conditions requiring
connective tissue regeneration and/or repair using the adherent
cells.
[0002] In the developing medical world a growing need exists for
large amounts of adult stem cells for the purpose of cell
engraftment and tissue engineering. In addition, adult stem cell
therapy is continuously developing for treating and curing various
conditions such as hematopoietic disorders, heart disease,
Parkinson's disease, Alzheimer's disease, stroke, burns, muscular
dystrophy, autoimmune disorders, diabetes and arthritis.
[0003] In recent years, considerable activity has focused on the
therapeutic potential of mesenchymal stromal cells (MSCs) for
various medical applications including tissue repair of damaged
organs such as the brain, heart, bone and liver and in support of
bone marrow transplantations (BMT). MSCs, a heterogeneous
population of cells obtained from e.g. bone marrow, adipose tissue,
placenta, and blood, is capable of differentiating into different
types of mesenchymal mature cells (e.g. reticular endothelial
cells, fibroblasts, adipocytes, osteogenic precursor cells)
depending upon influences from various bioactive factors.
Accordingly, MSCs have been widely studied in regenerative medicine
as the foundation to build new tissues such as bone, cartilage and
fat for the repair of injury or replacement of pathologic tissues
and as treatment for genetic and acquired diseases [Fibbe and
Noort, Ann N Y Acad Sci (2003) 996: 235-44; Horwitz et al.,
Cytotherapy (2005) 7(5): 393-5; Zimmet and Hare, Basic Res Cardiol
(2005) 100(6): 471-81]. Furthermore, the multipotent ability of
MSCs, their easy isolation and culture, as well as their high ex
vivo expansion potential make them an attractive therapeutic tool
[Fibbe and Noort, supra; Minguell et al. Exp Biol Med (Maywood)
(2001) 226(6): 507-20].
[0004] Placental derived MSCs exhibit many markers common to MSCs
isolated from other tissues, e.g. CD105, CD73, CD90 and CD29, and
the lack of expression of hematopoietic, endothelial and
trophoblastic-specific cell markers. Adipogenic, osteogenic, and
neurogenic differentiation have been achieved after culturing
placental derived MSCs under appropriate conditions [Yen et al.,
Stem Cells (2005) 23(1): 3-9]. Furthermore, MSCs isolated from
placenta and cultured in vitro have been demonstrated to be immune
privileged in a similar fashion as MSCs. Thus, the placenta
provides an ethically non-controversial and easily accessible
source of MSCs for experimental and clinical applications [Zhang et
al., Exp Hematol (2004) 32(7): 657-64].
[0005] The present inventors have previously devised three
dimensional (3D) culturing conditions suitable for expansion of
placental derived MSCs (PCT Application No. IL2007/000380) fully
incorporated herein by reference in its entirety.
[0006] Leading clinical uses of MSCs are summarized infra.
[0007] Ischemia
[0008] Peripheral Arterial Disease (PAD)
[0009] Peripheral arterial disease (PAD) is a chronic disease that
progressively restricts blood flow in the limbs that can lead to
serious medical complications. This disease is often associated
with other clinical conditions, including hypertension,
cardiovascular disease, hyperlipidemia, diabetes, obesity and
stroke. Critical Limb Ischemia (CLI) is used to describe patients
with chronic ischemia induced pain, ulcers, tissue loss or gangrene
in the limb. CLI represents the end stage of PAD patients who need
comprehensive treatment by a vascular surgery or vascular
specialist. In contrast to coronary and cerebral artery disease,
peripheral arterial disease (PAD) remains an under-appreciated
condition that despite being serious and extremely prevalent is
rarely diagnosed and even less frequently treated. Consequently,
CLI often leads to amputation or death and mortality rates in PAD
patients exceed that of patients with myocardial infarction and
stroke.
[0010] In attempts to treat ischemic conditions, various adult stem
cells have been used. Thus, co-culturing of adipose tissue derived
stromal cells (ADSC) and endothelial cells (EC) resulted in a
significant increase in EC viability, migration and tube formation
mainly through secretion of VEGF and HGF. Four weeks after
transplantation of the stromal cells into the ischemic mouse hind
limb the angiogenic scores were improved [Nakagami et al., J
Atheroscler Thromb (2006) 13(2): 77-81]. Moon et al. [Cell Physiol
Biochem. (2006) 17: 279-90] have tested the ability of adipose
tissue-derived progenitor cells (ADSC) to treat limb ischemia in
immunodeficient mice and demonstrated a significant increase in the
laser Doppler perfusion index in ADSC-transplanted group.
[0011] In addition, when umbilical cord blood (UCB)-derived
mesenchymal stem cells were transplanted into four men with
Buerger's disease who had already received medical treatment and
surgical therapies, ischemic rest pain, suddenly disappeared from
their affected extremities [Kim et al., Stem Cells (2006) 24(6):
1620-6]. Moreover, transplantation of human mesenchymal stem cells
isolated from fetal membranes of term placenta (FMhMSC) into
infarcted rat hearts was associated with increased capillary
density, normalization of left ventricular function, and
significant decrease in scar tissue, which was enhanced when the
stem cells were preconditioned with a mixed ester of hyaluronan
with butyric and retinoic acid [Ventura et al., (2007) J. Biol.
Chem., 282: 14243-52].
[0012] Stroke
[0013] Stroke is one of the leading causes of death around the
world, causing approximately 9% of all deaths and consuming about
2-4% of total health-care costs. Although there has been a constant
reduction in stroke mortality in developed countries, probably due
improved control of stroke risk factors (especially high blood
pressure, diabetes and cigarette smoking), stroke still leads to
permanent damage (e.g. tissue damage, neurological damage).
[0014] New treatment regimens for stroke include stem cell therapy.
Transplantation of stem cells or progenitors into the injured site,
either locally or via intravenous routes, to replace nonfunctional
cells, enhance proliferation and/or differentiation of endogenous
stem or progenitor cells and supply necessary immune modulators has
been contemplated and stand as the major cell-based strategy.
Potential sources of stem/progenitor cells for stroke include fetal
neural stem cells, embryonic stem cells, neuroteratocarcinoma
cells, umbilical cord blood-derived non-hematopoietic stem cells,
bone marrow-derived stem cells and placental-derived mesenchymal
stem cells [Andres et al., Neurosurg Focus (2008) 24(3-4):
E16].
[0015] In a recent study, Koh et. al. [Koh et al., Brain Res.
(2008)] examined the neuroprotective effects and mechanisms of
implanted human umbilical cord-derived mesenchymal stem cells
(hUC-MSCs) in an ischemic stroke rat model. Twenty days after the
induction of in-vitro neuronal differentiation, hUC-MSCs displayed
morphological features of neurons and expressed neuronal cell
markers and neuronal factors (e.g. glial cell line-derived
neurotrophic factor, brain-derived neurotrophic factor).
Furthermore, in-vivo implantation of the hUC-MSCs into the damaged
hemisphere of immunosuppressed ischemic stroke rats improved
neurobehavioral function and reduced infarct volume relative to
control rats. Three weeks after implantation, hUC-MSCs were present
in the damaged hemisphere and expressed neuron-specific markers,
yet these cells did not become functionally active neuronal
cells.
[0016] Orthopedic Applications
[0017] Various conditions and pathologies require connective tissue
(e.g., bone, tendon and ligament) regeneration and/or repair. These
include, for example, bone fractures, burns, burn wound, deep
wound, degenerated bone, various cancers associated with connective
tissue loss (e.g. bone cancer, osteosarcoma, bone metastases), and
articular cartilage defect.
[0018] The use of autologous BM-MSCs to enhance bone healing has
been described for veterinary and human orthopedic applications and
include percutaneous injection of bone marrow for ligament healing
(Carstanjen et al., 2006), treatment of bone defects by autografts
or allografts of bone marrow in orthopedic clinic (Horwitz et al.,
1999, Horwitz et al., 2002), regeneration of critical-sized bone
defect in dogs using allogeneic [Arinzeh T L, et al., J Bone Joint
Surg Am. 2003, 85-A(10):1927-35] or autologous [Bruder S P, et al.,
J Bone Joint Surg Am. 1998 July; 80(7):985-96] bone marrow-MSCs
loaded onto ceramic cylinder consisting of
hydroxyapatite-tricalcium phosphate, or in rabbit using allogeneic
peripheral blood derived MSCs (Chao et al., 2(006.), and extensive
bone formation using MSCs implantation in baboon (Livingston et al.
2003).
[0019] Within the equine orthopedic field, mesenchymal stem cells
of BM and adipose sources have been used experimentally for
surgical treatment of subchondral-bone cysts, bone fracture repair
[Kraus and Kirker-Head,Vet Surg (2006) 35(3): 232-42] and cartilage
repair [Brehm et al., Osteoarthritis Cartilage (2006) 14(12):
1214-26; Wilke et al., J Orthop Res (2007) 25(7): 913-25] and
clinically in the treatment of overstrain induced injuries of
tendons in horses. Furthermore, different therapeutic approaches
have been used to promote suspensory ligament healing in horses
(Herthel, 2001). Herthel (2001) have demonstrated a novel
biological approach to facilitate suspensory ligament healing that
involves the intra lesional injection of autologous stem cells and
associated bone marrow components to stimulate natural ligament
regeneration.
[0020] Rabbit models for injured tendons showed that MSC-treated
tissues were stronger and stiffer than natural repaired tissues
(Gordon et al., 2005). In addition, seeding of cultured MSCs into a
tendon gap resulted in significantly improved repair biomechanics
(Young et al., 1998, Osiris Therapeutics, www.osiris.com).
[0021] Osiris Chondrogen (adult Mesenchymal Stem Cells) is being
tested in patients in order to evaluate safety and efficacy. In MSC
treated animals, surgically removed meniscal tissue was
regenerated, the cartilage surface was protected, and lessened
joint damage was observed in comparison to control animals. These
benefits persisted in animal models at least through one year
(Osiris Therapeutics, www.osiris.com).
SUMMARY OF THE INVENTION
[0022] According to an aspect of some embodiments of the present
invention there is provided a method of treating ischemia in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of adherent cells of a
tissue selected from the group consisting of a placenta and an
adipose tissue, thereby treating the ischemia in the subject.
[0023] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition requiring connective tissue regeneration and/or repair in
a subject in need thereof, the method comprising administering to
the subject a therapeutically effective amount of adherent cells of
a tissue selected from the group consisting of a placenta and an
adipose tissue, thereby treating the medical condition requiring
connective tissue regeneration and/or repair in the subject.
[0024] According to an aspect of some embodiments of the present
invention there is provided a use of adherent cells from a tissue
selected from the group consisting of a placenta and an adipose
tissue for the manufacture of a medicament identified for treating
ischemia.
[0025] According to an aspect of some embodiments of the present
invention there is provided a use of adherent cells from a tissue
selected from the group consisting of a placenta and an adipose
tissue for the manufacture of a medicament identified for treating
a medical condition requiring connective tissue regeneration and/or
repair.
[0026] According to an aspect of some embodiments of the present
invention there is provided an article of manufacture comprising a
packaging material which comprises a label for use in treating
ischemia, the packaging material packaging a pharmaceutically
effective amount of adherent cells of a tissue selected from the
group consisting of a placenta and an adipose tissue.
[0027] According to an aspect of some embodiments of the present
invention there is provided an article of manufacture comprising a
packaging material which comprises a label for use in treating a
medical condition requiring connective tissue regeneration and/or
repair, the packaging material packaging a pharmaceutically
effective amount of adherent cells of a tissue selected from the
group consisting of a placenta and an adipose tissue.
[0028] According to some embodiments of the invention, the adherent
cells are capable of suppressing immune reaction in the
subject.
[0029] According to some embodiments of the invention, at least 10%
of the adherent cells are at a proliferative phase.
[0030] According to some embodiments of the invention, the ischemia
is peripheral arterial disease (PAD).
[0031] According to some embodiments of the invention, the
peripheral arterial disease (PAD) is critical limb ischemia
(CLI).
[0032] According to some embodiments of the invention, the ischemia
comprises ischemia of the central nervous system (CNS).
[0033] According to some embodiments of the invention, the ischemia
is selected from the group consisting of peripheral arterial
disease, ischemic vascular disease, ischemic heart disease,
ischemic brain disease, ischemic renal disease and ischemic
placenta.
[0034] According to some embodiments of the invention, the adherent
cells are obtained from a three-dimensional (3D) culture.
[0035] According to some embodiments of the invention, the
three-dimensional (3D) culture comprises a 3D bioreactor.
[0036] According to some embodiments of the invention, the
culturing of the cells in the 3D culture is effected under
perfusion.
[0037] According to some embodiments of the invention, the
culturing conditions of the three-dimensional culture comprise an
adherent material selected from the group consisting of a polyester
and a polypropylene.
[0038] According to some embodiments of the invention, the
culturing of the cells is effected for at least 3 days.
[0039] According to some embodiments of the invention, the
culturing of the cells is effected until at least 10% of the cells
are proliferating.
[0040] According to some embodiments of the invention, the adherent
cells comprise a positive marker expression selected from the group
consisting of CD73, CD90, CD29 and CD105.
[0041] According to some embodiments of the invention, the adherent
cells comprise a negative marker expression selected from the group
consisting of CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34
and CD79.
[0042] According to some embodiments of the invention, the adherent
cells comprise an expression profile essentially as described
herein.
[0043] According to some embodiments of the invention, the adherent
cells comprise cells comprising a stromal stem cell phenotype.
[0044] According to some embodiments of the invention, the stromal
stem cell phenotype comprises T cell suppression activity.
[0045] According to some embodiments of the invention, the
connective tissue comprises tendon, bone and/or ligament.
[0046] According to some embodiments of the invention, the medical
condition requiring connective tissue regeneration and/or repair is
selected from the group consisting of bone fracture, bone cancer,
burn wound, articular cartilage defect and deep wound.
[0047] According to some embodiments of the invention, the medical
condition is selected from the group consisting of a
subchondral-bone cyst, a bone fracture, an osteoporosis, an
osteoarthritis, a degenerated bone, a bone cancer, a cartilage
damage, an articular cartilage defect, a degenerative disc disease,
an osteogenesis imperfecta (OI), a burn, a burn wound, a deep
wound, a delayed wound-healing, an injured tendon and an injured
ligament.
[0048] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. In case of
conflict, the patent specification, including definitions, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the embodiments of the invention only,
and are presented in the cause of providing what is believed to be
the most useful and readily understood description of the
principles and conceptual aspects of the invention. In this regard,
no attempt is made to show structural details of the invention in
more detail than is necessary for a fundamental understanding of
the invention, the description taken with the drawings making
apparent to those skilled in the art how the several forms of the
invention may be embodied in practice.
[0050] In the drawings:
[0051] FIGS. 1A-G depict the bone-like microenvironment created in
the bioreactor system containing 3-D carriers. FIGS. 1A-B are
electron micrographs depicting the comparison of natural bone (FIG.
1A) and the structure of the PluriX.TM. 3D carrier 7 days after
seeding adherent cells, imitating the bone micro-environment (FIG.
1B). FIGS. 1C-F are electron micrographs depicting the PluriX.TM.
3D matrix seeded with adherent cell, produced from bone marrow, 20
days (FIGS. 1C-D, magnified X 150 and 250 respectively) and 40 days
(FIGS. 1E-F, magnified X 350 and 500 respectively) after seeding.
FIG. 1G is a diagram of the Plurix 3D plug flow bioreactor with
separate parts defined by numbers: Culture medium reservoir (1),
gas mixture supply (2), filter (3), injection point (4), column in
which the 3D carriers are placed (5) flow monitor (6), flow valve
(6a), separating container (7), cell growth analyzers (8);
peristaltic pump (9), sampling point (10), dissolved O.sub.2
measurement electrode (11), pH measurement electrode (12), control
system (13), fresh growth media (14), used growth media (15).
[0052] FIG. 2 is a graph depicting different production lots of
adherent cells (Lots 5-8) originating from placenta, grown in 3D
growth conditions within the bioreactor systems. Adherent cells
(2.times.10.sup.6) were seeded in the bioreactor at a density of
10000-15000 cells/a carrier. Following a 12 day culture 3D-adherent
cells reached a density of between 150,000-250,000 cells/carrier or
22.5-37.5.times.10.sup.6 in a bioreactor containing 150
carriers.
[0053] FIGS. 3A-B are bar graphs depicting difference in expression
levels of expressed membrane markers in placenta derived
3D-adherent cell (dark purple) as compared to membrane markers in
placenta cells cultured in conventional 2D culture conditions
(light purple). Adherent cells were grown for 4-6 weeks in flasks
(2D) or for 2-3 weeks in the bioreactor system, on polystyrene
carriers (3D). Following harvesting from either flasks or carriers,
cells were incubated and bound to a panel of monoclonal antibodies
(MAb), which recognize membrane markers characteristic of adherent
cells (FIG. 3A), or hematopoietic cells (FIG. 3B). Note the
significantly higher expression of MSC membrane markers in 2D
cultured cells as shown for CD90. CD105, CD73 and CD29 membrane
markers, compared to MSC membrane markers expressed in 3D-cultured
adherent cells, especially CD105 which showed 56% expression in 3D
cultured cells vs. 87% in the 2D cultured cells (FIG. 3A). Adherent
cells of both 2D and 3D cultures, did not express any hematopoietic
membrane markers (FIG. 3B).
[0054] FIGS. 4A-D are bar graphs depicting a comparison of protein
levels in adherent cells produced from the placenta cultured under
2D and 3D Conditions or conditioned media of same. FIGS. 4A-C
depict levels of Flt-3 ligand (FIG. 4A), IL-6 (FIG. 4B) and SCF
(FIG. 4C) in pg/ml, normalized for 1.times.10.sup.6 cells/ml, as
analyzed by ELISA, in the conditioned media of 2D and 3D cultured
adherent cells. Results represent one of three independent
experiments. FIG. 4D shows the expression levels of different
cellular proteins, as analyzed by mass spectrometry with iTRAQ
reagents labeled protein samples compared therebetween. Protein
samples were taken from adherent cells grown under 2D (white bars)
and 3D (grey bars) conditions. The figure represents one of two
replica experiments. Note the difference in expression level of
some of the proteins in cells and conditioned media of 2D and 3D
culture conditions.
[0055] FIGS. 5A-D are micrographs depicting in vitro
differentiation capability of placenta derived 3D-adherent cell to
osteoblasts. Human placenta derived adherent cell were cultured in
an osteogenic induction medium (DMEM containing 10% FCS, 100 nM
dexamethasone, 0.05 mM ascorbic acid 2-phosphate, 10 mM
B-glycerophosphate) for a period of 3 weeks. FIGS. 5A-B show cells
expressing calcified matrix, as indicated by Alizzarin Red S
staining. FIGS. 5C-D show control cells, which were not treated
with osteogenic induction medium and maintained a fibroblast like
phenotype and demonstrating no mineralization.
[0056] FIG. 6 is a graph depicting percentage of human CD45+ cells
detected in bone marrow (BM) of NOD-SCID mice, treated with
chemotherapy (25 mg/kg busulfan intraperitoneal injections for two
consecutive weeks) 3.5 weeks following transplantation. CD34+ cells
(100,000) purified from mononuclear cord blood derived cells, were
transplanted alone (5 mice, a) or co-transplanted with
0.5.times.10.sup.6 placenta derived adherent cells cultured in 2D
conditions (2D-adherent cell; 2 mice, b), or placenta derived
adherent cells cultured in 3D conditions (3D-adherent cell), in the
pluriX.TM. bioreactor (5 mice, c). BM was then collected from mice
femurs and tibias. Human cells in the BM were detected by flow
cytometry. The percentage of CD45 expressing human cells was
determined by incubating cells with anti-human CD45-FITC. Note the
higher percentage of human cells (hCD45+) in the bone marrow of
mice co-transplanted with 2D-adherent cell (b) as well as with
3D-adherent cell (c) in comparison to the percentage of human cells
in the mice treated with HSCs alone (a). The higher engraftment
seen in mice treated with 3D-adherent cell cultured cells in
comparison to mice treated with 2D-adherent cell cultured cells
indicates a higher therapeutic advantage unique to 3D cultured
adherent cells.
[0057] FIGS. 7A-B are FACS analyses of human graft CD45+ cells in
mice transplanted with CD34+ cells only (FIG. 7A) in comparison to
CD34+ cells together with adipose tissue derived adherent cells
(FIG. 7B). Note the significantly higher percentage of human
hematopoietic population (hCD45+) (7A--29%) in a mouse
co-transplanted with adipose tissue derived adherent cell in
comparison to a mouse treated with human CD34+ alone (7B--12%).
[0058] FIG. 5A is a bar graph depicting a mixed lymphocyte reaction
conducted between human cord blood mononuclear cells (CB), and
equal amounts of irradiated (3000 Rad) cord blood cells (iCB),
human peripheral blood derived monocytes (PBMC), 2D cultured (2D)
or 3D cultured (3D) placental derived adherent cells, or a
combination of PBMC and 2D and 3D cultured placental derived
adherent cells (PBMC+2D and PBMC+3D). Size of CB cell population is
represented by the .sup.3H-thymidine uptake (measured in CPM) which
was measured during the last 18 hours of culturing. Elevation in
stimulated CB cell proliferation indicates an immune response of a
higher level. Note the lower level of immune response exhibited by
cells incubated with adherent cells, and, in particular, the
reduction of CB immune response to PBMCs when co-incubated with
adherent cells. Three replicates were made of each reaction.
[0059] FIG. 8B is a flow chart depicting production of 3D adherent
cell from placentas by Celligen.TM. (designated PLX-C cells).
[0060] FIG. 8C is a diagram of a Celligen.TM. bioreactor vessel and
ports adapted from The New Brunswick Scientific web site.
[0061] FIGS. 9A-B depict cell cycle analysis of 3D adherent cells
manufacture by Plurix (designated PLX, FIG. 9B) and by Celligen
(designated PLX-C, FIG. 9A). Cells were fixed in 70% EtOH O.N,
centrifuged and re-suspended in a Propidium Iodide (PI) solution
and then analyzed by FACS.
[0062] FIGS. 10A-C depict expression of fibroblast-typical markers
but not expression of endothelial typical markers on PLX-C. FIG.
10A depicts negative expression of the endothelial marker CD31;
FIG. 10B depicts negative expression of the endothelial marker KDR;
and FIG. 10C depicts positive expression of the human fibroblast
marker (D7-FIB). Of note, the red histograms for Isotype IgG1
(FITC) represent the negative control while the blue histograms
represents the positively stained cells.
[0063] FIGS. 11A-D depict expression of stimulatory and
co-stimulatory molecules on PLX-C cells. FIG. 11A depicts PLX-C
expression of CD80; FIG. 11B depicts PLX-C expression of CD86; FIG.
11C depicts PLX-C expression of CD40; and FIG. 11D depicts PLX-C
expression of HLA-A/B/C. Negative controls were prepared with
relevant isotype fluorescence molecules. Of note, red histograms
indicate PLX-C marker-expressing population of cells, blue
histograms indicate bone marrow (BM) marker-expressing population
of cells, and green histograms indicate mononuclear cell (MNC)
marker expressing population of cells.
[0064] FIGS. 12A-B depict inhibition of lymphocyte proliferation by
PLX-C. FIG. 12A depicts MLR tests performed with 2.times.10.sup.5
peripheral blood (PB) derived MNC (donor A) stimulated with equal
amount of irradiated (3000 Rad) PB derived MNCs (donor B) followed
by addition of increasing amounts of PLX-C cells to the cultures.
Three replicates of each group were seeded in 96-well plates.
Proliferation rate was measured by [.sup.3H-]thymidine
incorporation: FIG. 12B depict peripheral blood (PB) derived MNCs
stimulated with ConA (1.5 mg/ml). Increasing amounts of PLX-C cells
were added to the cultures. Three replicates of each group were
seeded in 96-well plates. Proliferation rate was measured by
[.sup.3H]thymidine incorporation;
[0065] FIGS. 13A-C depict PLX-C regulation of pro-inflammatory and
anti-inflammatory cytokine secretion following co-culture with
peripheral blood cells. FIGS. 13A-B depict secretion of IFN.gamma.
(FIG. 13A) and TNF.alpha. (FIG. 13B) following co-culture of human
derived MNCs (isolated from peripheral blood) stimulated with ConA
with PLX-C; FIG. 13C depicts secretion of IFN-.gamma., TNF.alpha.
and IL-10 following co-culture of human derived MNCs (isolated from
peripheral blood) stimulated with LPS with PLX-C. Supernatants were
collected and subjected to cytokines analysis using ELISA.
[0066] FIG. 14 depicts the luciferase expression vector used to
infect PLX-C cells. Expression vector Lv33 from OmicsLink was used
herein. The Luciferase gene was cloned into the ORF.
[0067] FIG. 15 depicts high luciferase expression by infected PLX-C
cells. Cells were infected with the luciferase expression vector
and visualized by the IVIS system 48 hours post infection. Of note,
cells exhibited high levels of luciferase expression.
[0068] FIGS. 16A-D depict injection of 2.times.10.sup.6 luciferase
expressing PLX-C cells into SCID/Beige mice. One mouse was injected
IM and one IV. The injected mice were monitored using the IVIS
system in order to asses the in vivo biodistribution of PLX-C. IVIS
results of days 1 (FIG. 16A), day 4 (FIG. 16B), day 6 (FIG. 16C)
and day 22 (FIG. 16D) are presented.
[0069] FIG. 17 is a graph depicting increased perfusion in hip and
foot of mice treated with the adherent cells of the invention
(designated PLX-C). The figure depicts the median of the percent of
perfusion in the mouse hip and foot. Blood flows on hip and foot
was measured using a non contact laser Doppler from both sides on
days 0, 6, 9, 14 and 21 post operation (shown are measurements on
day 21). Results are expressed as the ratio of the blood flow in
the ischemic limb to that in the normal limb during the
experiment.
[0070] FIG. 18 is a graph depicting the in vivo assessment of limb
function and ischemic damage. Semiquantitative assessment of
impaired use of the ischemic limb was performed serially using the
following score system: 3=dragging of foot, 2=no dragging but no
plantar flexion, 1=plantar flexion, and 0=flexing the toes to
resist gentle traction of the tail.
[0071] FIGS. 19A-C depict increased capillary density after PLX-C
treatment. FIG. 19A depicts capillary density in mice treated with
PBS; FIG. 19B depicts capillary density in mice treated with PLX-C
cells; FIG. 19C is a bar graph depicting the number of capillaries
per muscle cells. Of note, increase capillary density was noted in
PLX-C treated mice but not in control mice, following induced limb
ischemia demonstrated by specific capillary staining.
[0072] FIGS. 20A-B depict reduced oxidative stress and endothelial
inflammation following PLX-C administration. FIG. 20A is a bar
graph depicting oxidative stress (Nitrotyrosin staining); and FIG.
20B is a bar graph depicting endothelial inflammation (VCAM
evaluation). Of note, reduced oxidative stress and endothelial
inflammation is noted in mice treated with PLX-C.
DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION
[0073] The invention is of, in some embodiments, methods of
increasing angiogenesis in a tissue and treating ischemia or
medical conditions requiring connective tissue regeneration and/or
repair using adherent cells of placenta or adipose tissues.
[0074] The principles and operation of the invention may be better
understood with reference to the drawings and accompanying
descriptions.
[0075] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0076] While reducing the invention to practice, the present
inventors have uncovered that adherent cells from a placenta or an
adipose tissue are highly efficient in increasing angiogenesis in a
tissue and in treating ischemia and medical conditions requiring
connective tissue regeneration and/or repair.
[0077] As is illustrated herein below and in Example 1.-8 of the
Examples section which follows, the present inventors were able to
expand adipose and placenta-derived adherent cells which comprise
stromal stem cells properties. Cells expanded accordingly were
found viable, following cryo-preservation, as evidenced by
adherence and re-population assays (see Example 1). Flow cytometry
analysis of placenta-derived adherent cells uncovered a distinct
marker expression pattern (see FIGS. 3A-B). As is further shown in
Example 6 of the Examples section which follows, implantation of
placental derived adherent cells significantly induced blood flow
in the hip and foot (FIG. 17) of mice subjected to artery ligation
(the ischemic hind limb model), significantly improved limb
function (FIG. 18), increased capillary density (FIGS. 19A-C) and
reduced oxidative stress and endothelial inflammation (FIGS.
20A-B).
[0078] Thus, according to one aspect of the invention, there is
provided a method of increasing angiogenesis in a tissue. The
method is effected by contacting the tissue with adherent cells of
a tissue selected from the group consisting of a placenta and an
adipose tissue, thereby increasing the angiogenesis in the
tissue.
[0079] As used herein the phrase "increasing angiogenesis in a
tissue" refers to increasing (inducing, upregulating) the process
of generating new capillary blood vessels in a tissue.
[0080] As used herein the phrase "adherent cells" refers to a
homogeneous or heterogeneous population of cells which are
anchorage dependent, i.e., require attachment to a surface in order
to grow in vitro.
[0081] As used herein the phrase "adipose tissue" refers to a
connective tissue which comprises fat cells (adipocytes).
[0082] As used herein the term "placenta tissue" refers to any
portion of the mammalian female organ which lines the uterine wall
and during pregnancy envelopes the fetus, to which it is attached
by the umbilical cord. Following birth, the placenta is expelled
(and is referred to as a post partum placenta). In an exemplary
embodiment, placenta refers to whole placenta.
[0083] Placenta or adipose tissue derived adherent cells can be
propagated using two dimensional or three dimensional culturing
conditions.
[0084] Conditions for propagating adherent cells in 2D culture are
further described hereinbelow and in the examples section which
follows.
[0085] As used herein the phrase "three dimensional culture" refers
to a culture in which the cells are disposed to conditions which
are compatible with cell growth while allowing the cells to grow in
more than one layer. It is well appreciated that the in situ
environment of a cell in a living organism (or a tissue) is in a
three dimensional architecture. Cells are surrounded by other
cells. They are held in a complex network of extra cellular matrix
nanoscale fibers that allows the establishment of various local
microenvironments. Their extra cellular ligands mediate not only
the attachment to the basal membrane but also access to a variety
of vascular and lymphatic vessels. Oxygen, hormones and nutrients
are ferried to cells and waste products are carried away. The
conditions in the three dimensional culture of the invention are
designed to mimic such an environment as is further exemplified
below.
[0086] It will be appreciated that the conditions of the
three-dimensional culture are such that enable expansion of the
adherent cells.
[0087] As used herein the terms "expanding" and "expansion" refer
to substantially differentiation-less maintenance of the cells and
ultimately cell growth, i.e., increase of a cell population (e.g.,
at least 2 fold) without differentiation accompanying such
increase.
[0088] As used herein the terms "maintaining" and "maintenance"
refer to substantially differentiation-less cell renewal, i.e.,
substantially stationary cell population without differentiation
accompanying such stationarity.
[0089] As mentioned, the adherent cells of this aspect of the
invention are retrieved from an adipose or placental tissue.
[0090] Placental cells may be obtained from a full-term or pre-term
placenta. Placenta is preferably collected once it has been ex
blooded. The placenta is preferably perfused for a period of time
sufficient to remove residual cells. The term "perfuse" or
"perfusion" used herein refers to the act of pouring or passaging a
fluid over or through an organ or tissue. The placental tissue may
be from any mammal; for example, the placental tissue is human. A
convenient source of placental tissue is from a post partum
placenta (e.g., 1-6 hours), however, the source of placental tissue
or cells or the method of isolation of placental tissue is not
critical to the invention.
[0091] Placenta derived adherent cells may be obtained from both
fetal (i.e., amnion or inner parts of the placenta, see Example 1)
and maternal (i.e., decidua basalis, and decidua parietalis) parts
of the placenta. Tissue specimens are washed in a physiological
buffer [e.g., phosphate-buffered saline (PBS) or Hank's buffer].
Single-cell suspensions are made by treating the tissue with a
digestive enzyme (see below) or/and mincing and flushing the tissue
parts through a nylon filter or by gentle pipetting (Falcon,
Becton, Dickinson, San Jose, Calif.) with washing medium.
[0092] Adipose tissue derived adherent cells may be isolated by a
variety of methods known to those skilled in the art. For example,
such methods are described in U.S. Pat. No. 6,153,432. The adipose
tissue may be derived from omental/visceral, mammary, gonadal, or
other adipose tissue sites. One source of adipose tissue is omental
adipose. In humans, the adipose is typically isolated by
liposuction.
[0093] Isolated adherent cells from adipose tissue may be derived
by treating the tissue with a digestive enzyme such as collagenase,
trypsin and/or dispase; and/or effective concentrations of
hyaluronidase or DNAse; and ethylenediaminetetra-acetic acid
(EDTA); at temperatures between 25-50.degree. C., for periods of
between 10 minutes to 3 hours. The cells may then be passed through
a nylon or cheesecloth mesh filter of between 20 microns to 1 mm.
The cells are then subjected to differential centrifugation
directly in media or over a Ficoll or Percoll or other particulate
gradient. Cells are centrifuged at speeds of between 100 to
3000.times.g for periods of between 1 minutes to 1 hour at
temperatures of between 4-50.degree. C. (see U.S. Pat. No.
7,078,230).
[0094] In addition to placenta or adipose tissue derived adherent
cells, the invention also envisages the use of adherent cells from
other cell sources which are characterized by stromal stem cell
phenotype (as will be further described herein below). Tissue
sources from which adherent cells can be retrieved include, but are
not limited to, cold blood, scalp, hair follicles [e.g. as
described in Us Pat. App. 20060172304], testicles [e.g., as
described in Guan K., et al., Nature. 2006 Apr. 27; 440(7088):l
199-203], human olfactory mucosa [e.g., as described in Marshall,
Conn., et al., Histol Histopathol. 2006 June; 21(6):633-43],
embryonic yolk sac [e.g., as described in Geijsen N, Nature. 2004
Jan. 8; 427(6970):148-54] and amniotic fluid [Pieternella et al.
(2004) Stem Cells 22:1338-1345], all of which are known to include
mesenchymal stem cells. Adherent cells from these tissue sources
can be isolated by culturing the cells on an adherent surface, thus
isolating adherent cells from other cells in the initial
population.
[0095] Regardless of the origin (e.g., placenta or adipose tissue),
cell retrieval is preferably effected under sterile conditions.
Once isolated cells are obtained, they are allowed to adhere to an
adherent material (e.g., configured as a surface) to thereby
isolate adherent cells. Culturing may proceed under 2D conditions
as described in Example 4 of the Examples section and cells may be
further transferred to 3D conditions
[0096] As used herein "an adherent material" refers to a synthetic,
naturally occurring or a combination of same of a non-cytotoxic
(i.e., biologically compatible) material having a chemical
structure (e.g., charged surface exposed groups) which may retain
the cells on a surface.
[0097] Examples of adherent materials which may be used in
accordance with this aspect of the invention include, but are not
limited to, a polyester, a polypropylene, a polyalkylene, a
polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a
polysulfone, a cellulose acetate, a glass fiber, a ceramic
particle, a matrigel, an extra cellular matrix component (e.g.,
fibronectin, chondronectin, laminin), a collagen, a poly L lactic
acid and an inert metal fiber.
[0098] Further steps of purification or enrichment for stromal stem
cells may be effected using methods which are well known in the art
(such as by FACS using stromal stern cell marker expression, as
further described herein below).
[0099] Non-limiting examples of base media useful in culturing
according to the invention include Minimum Essential Medium Eagle,
ADC-1, LPM (Bovine Serum Albumin-free), F10(HAM), F12 (HAM), DCCM1,
DCCM2. RPMI 1640, BGJ Medium (with and without Fitton-Jackson
Modification), Basal Medium Eagle (BME-with the addition of Earle's
salt base), Dulbecco's Modified Eagle Medium (DMEM-without serum),
Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM),
Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E-with
Earle's sale base), Medium M199 (M199H-with Hank's salt base),
Minimum Essential Medium Eagle (MEM-E-with Earle's salt base),
Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and
Minimum Essential Medium Eagle (MEM-NAA with non essential amino
acids), among numerous others, including medium 199, CMRL 1415,
CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145,
Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB
501, MCDB 401, MCDB 411, MDBC 153. A preferred medium for use in
the invention is DMEM. These and other useful media are available
from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet
HaEmek, Israel, among others. A number of these media are
summarized in Methods in Enzymology, Volume LVIII, "Cell Culture",
pp. 62 72, edited by William B. Jakoby and Ira H. Pastan, published
by Academic Press, Inc.
[0100] The medium may be supplemented such as with serum such as
fetal serum of bovine or other species, and optionally or
alternatively, growth factors, vitamins (e.g. ascorbic acid),
cytokines, salts (e.g. B-glycerophosphate), steroids (e.g.
dexamethasone) and hormones e.g., growth hormone, erythropoietin,
thrombopoietin, interleukin 3, interleukin 6, interleukin 7,
macrophage colony stimulating factor, c-kit ligand/stem cell
factor, osteoprotegerin ligand, insulin, insulin like growth
factors, epidermal growth factor, fibroblast growth factor, nerve
growth factor, cilary neurotrophic factor, platelet derived growth
factor, and bone morphogenetic protein at concentrations of between
picogram/ml to milligram/ml levels.
[0101] It is further recognized that additional components may be
added to the culture medium. Such components may be antibiotics,
antimycotics, albumin, amino acids, and other components known to
the art for the culture of cells. Additionally, components may be
added to enhance the differentiation process when needed (see
further below).
[0102] It will be appreciated that in case the adherent cells of
the invention are administered to a human subject, the cells and
the culture medium (e.g., with the above described medium
additives) should be substantially xeno-free, i.e., devoid of any
animal contaminants e.g., mycoplasma. For example, the culture
medium can be supplemented with a serum-replacement, human serum
and/or synthetic or recombinantly produced factors.
[0103] As mentioned, once adherent cells are at hand they may be
passaged to two dimensional or three dimensional settings (see
Examples 1 and 4 of the Examples section which follows). It will be
appreciated though, that the cells may be transferred to a
3D-configured matrix immediately after isolation or alternatively,
may be passaged to three dimensional settings following two
dimensional conditions (as mentioned hereinabove).
[0104] Thus, the adherent material of this aspect of the invention
is configured for 3D culturing thereby providing a growth matrix
that substantially increases the available attachment surface for
the adherence of the cells so as to mimic the infrastructure of the
tissue (e.g., placenta).
[0105] For high scale production, culturing can be effected in a 3D
bioreactor.
[0106] Examples of such bioreactors include, but are not limited
to, a plug flow bioreactor, a continuous stirred tank bioreactor, a
stationary-bed bioreactor, a CelliGen Plus.RTM. bioreactor system
(New Brunswick Scientific (NBS) or a BIOFLO 310 bioreactor system
(New Brunswick Scientific (NBS).
[0107] As shown Example 4 of the Examples section, the Celligen
bioreactor is capable of 3D expansion of adherent cells under
controlled conditions (e.g. pH, temperature and oxygen levels) and
with constant cell growth medium perfusion. Furthermore, the cell
cultures can be directly monitored for concentration levels of
glucose, lactate, glutamine, glutamate and ammonium. The glucose
consumption rate and the lactate formation rate of the adherent
cells enable to measure cell growth rate and to determine the
harvest time.
[0108] Other 3D bioreactors that can be used with the invention
include, but are not limited to, a continuous stirred tank
bioreactor, where a culture medium is continuously fed into the
bioreactor and a product is continuously drawn out, to maintain a
time-constant steady state within the reactor. A stirred tank
bioreactor with a fibrous bed basket is available for example at
New Brunswick Scientific Co., Edison, N.J.), A stationary-bed
bioreactor, an air-lift bioreactor, where air is typically fed into
the bottom of a central draught tube flowing up while forming
bubbles, and disengaging exhaust gas at the top of the column], a
cell seeding perfusion bioreactor with Polyactive foams [as
described in Wendt. D. et al., Biotechnol Bioeng 84: 205-214,
(2003)]tubular poly-L-lactic acid (PLLA) porous scaffolds in a
Radial-flow perfusion bioreactor [as described in Kitagawa et al.,
Biotechnology and Bioengineering 93(5): 947-954 (2006). Other
bioreactors which can be used in accordance with the invention are
described in U.S. Pat. Nos. 6,277,151, 6,197,575, 6,139,578,
6,132,463, 5,902,741 and 5,629,186.
[0109] Cell seeding is preferably effected 100,000-1,500,000
cells/mm at seeding. In an exemplary embodiment a total of
150.+-.30.times.10.sup.6 cells are seeded, 3-5.times.10.sup.6
cell/gr carrier are seeded, or 0.015-0.1.times.10.sup.6 cell/ml are
seeded.
[0110] Cells can be harvested when at least about 10% of cells are
proliferating while avoiding uncontrolled differentiation and
senescence.
[0111] Culturing is effected for at least about 2 days, 3 days, 4
days, 5 days, 10 days, 20 days, a month or even more. It will be
appreciated that culturing in a bioreactor may prolong this period.
Culturing of the adherent cells in the 3D culture can be effected
under a continuous flow of a culture medium. Passaging may also be
effected to increase cell number. It will be appreciated that
culture medium may be changed in order to prolong and improve
culturing conditions.
[0112] Adherent cells of some embodiments of the present invention
comprise at least about 10%, 28%, 30%, 50%, 80% or more
proliferative cells (as can be assayed by FACS monitoring S and
G2/M phases).
[0113] Adherent cells of some embodiments of the invention may
comprise at least one "stromal stem cell phenotype".
[0114] As used herein "a stromal stem cell phenotype" refers to a
structural or functional phenotype typical of a bone-marrow derived
stromal (i.e., mesenchymal) stem cell
[0115] As used herein the phrase "stem cell" refers to a cell which
is not terminally differentiated.
[0116] Thus for example, the cells may have a spindle shape.
Alternatively or additionally the cells may express a marker or a
collection of markers (e.g. surface marker) typical to stromal stem
cells. Examples of stromal stem cell surface markers (positive and
negative) include but are not limited to CD105+, CD29+, CD44+,
CD73+, CD90+, CD3-, CD4-, CD34-, CD45-, CD80-, CD19-, CD5-, CD20-,
CD11B-, CD14-, CDI9-, CD79-, HLA-DR-, and FMC7-. Other stromal stem
cell markers include but are not limited to tyrosine hydroxylase,
nestin and H-NF.
[0117] Adherent cells of placenta tissue generated according to the
present teachings have a gene expression profile essentially as
described in Example 4 of the Examples section which follows.
[0118] Examples of functional phenotypes typical of stromal stem
cells include, but are not limited to, T cell suppression activity
(don't stimulate T cells and conversely suppress same),
hematopoietic stem cell support activity, as well as any of
adipogenic, hepatogenic, osteogenic and neurogenic
differentiation.
[0119] Any of these structural or functional features can be used
to qualify the cells of the invention (see Examples 4 of the
Examples section which follows).
[0120] Populations of cells generated according to the present
teachings are characterized by a unique protein expression profile
as is shown in Example 1 of the Examples section. Thus for example,
adherent cells of placenta or adipose tissue generated according to
the present teachings are capable of expressing and/or secreting
high levels of selected factors. For example, such cells express or
secrete SCF, Flt-3, H2A histone family (H2AF) or Aldehyde
dehydrogenase X (ALDH X) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
even 12 fold higher than that expressed or secreted by adherent
cells of placenta or adipose tissue grown in a 2D culture.
Additionally or alternatively, population of cells of the invention
secrete or express IL-6, eukaryotic translation elongation factor 2
(EEEF2), reticulocalbin 3, EF-hand calcium binding domain (RCN2) or
calponin 1 basic smooth muscle (CNN1) at a level least 2, 3 or 5
fold higher than that expressed or secreted by adherent cells of
placenta or adipose tissue grown in a 2D culture. Additionally or
alternatively, population of cells of the invention are
characterized by lower level of expression of various other
proteins as compared to 2D cultured cells. Thus for example,
secrete or express less than 0.6, 0.5, 0.25 or 0.125 of the
expression level of heterogeneous nuclear ribonucleoprotein H1
(Hnrph1). CD44 antigen isoform 2 precursor, 3 phosphoadenosine 5
phosphosulfate synthase 2 isoform a (Papss2) or ribosomal protein
L7a (rpL7a) expressed or secreted by adherent cells of placenta or
adipose tissue grown in a 2D culture.
[0121] As is shown in Examples 3-4 of the Examples section which
follows, the adherent cells, and particularly 3D-adherent cells,
were found to suppress the immune reaction of human cord blood
mononuclear cells in a mixed lymphocyte reaction (MLR) assay, thus
exhibit biological activities which may be preferentially used in
the clinic (e.g., T cell suppression activity, hematopoietic stem
cell support activity).
[0122] According to one embodiment of the invention, the adherent
cells of the invention are capable of suppressing immune reaction
in a subject.
[0123] As used herein the phrase "suppressing immune reaction in a
subject" refers to decreasing or inhibiting the immune reaction
occurring in a subject in response to an antigen (e.g., a foreign
cell or a portion thereof). The immune response which can be
suppressed by the adherent cells include the humoral immune
responses, and cellular immune responses, which involve specific
recognition of pathogen antigens via antibodies and T-lymphocytes
(proliferation of T cells), respectively.
[0124] According to one embodiment of the invention, the adherent
cells of the invention are characterized by a higher
immunosuppressive activity than that of adherent cells of the
placenta or the adipose tissue grown in a two-dimensional (2D)
culture.
[0125] According to one embodiment of the invention, the
immunosuppressive activity comprises reduction in T cell
proliferation.
[0126] As mentioned hereinabove and described in Example 6 of the
Examples section which follows, the adherent cells of the invention
induced angiogenesis in vivo (e.g., blood flow in the hip and leg),
significantly improved limb function of animals subjected to
arterial ligation, increased capillary density and reduced
oxidative stress and endothelial inflammation. Furthermore, as
described in detail in Example 7 of the Examples section which
follows, the adherent cells of the invention significantly improved
recovery from stroke in a rat model.
[0127] Thus, according to another aspect of the invention, there is
provided a method of treating ischemia in a subject in need
thereof. The method is effected by administering to the subject a
therapeutically effective amount of the adherent cells of the
invention, thereby treating the ischemia in the subject.
[0128] The term "ischemia" as used herein refers to any pathology
(disease, condition, syndrome or disorder) characterized by or
associated with insufficient angiogenesis. Examples include, but
are not limited to, a peripheral arterial disease (PAD) such as
limb ischemia and critical limb ischemia (CLI), ischemic heart
disease, ischemic brain disease (e.g. stroke), delayed
wound-healing, delayed ulcer healing, reproduction associated
disorders, arteriosclerosis, ischemic vascular disease, ischemic
heart disease, myocardial ischemia, coronary artery disease (CAD),
atherosclerotic cardiovascular disease, left main coronary artery
disease, arterial occlusive disease, peripheral ischemia,
peripheral vascular disease, vascular disease of the kidney,
peripheral arterial disease, limb ischemia, lower extremity
ischemia, cerebral ischemia, cerebro vascular disease, retinopathy,
retinal repair, remodeling disorder, von Hippel-Lindau syndrome,
hereditary hemorrhagic telengiectasiaischemic vascular disease,
Buerger's disease, ischemic renal disease and ischemic
placenta.
[0129] As used herein the term "treating" refers to inhibiting or
arresting the development of a pathology (e.g., ischemia) and/or
causing the reduction, remission, or regression of a pathology.
Those of skill in the art will understand that various
methodologies and assays can be used to assess the development of a
pathology, and similarly, various methodologies and assays may be
used to assess the reduction, remission or regression of a
pathology. The term "treating" may also refer to alleviating or
diminishing a symptom associated with the pathology.
[0130] As used herein the phrase "subject in need thereof" refers
to any subject (e.g., mammal), such as a human subject who is
diagnosed with or suffers from the pathology.
[0131] As mentioned hereinabove and described in Example 8 of the
Examples section which follows, the present inventors have found
that the adherent cells of the invention are capable of connective
tissue regeneration and/or repair.
[0132] Thus, according to yet an additional aspect of the
invention, there is provided a method of treating a medical
condition requiring connective tissue regeneration and/or repair in
a subject in need thereof. The method is effected by administering
to the subject a therapeutically effective amount of the adherent
cells of the invention.
[0133] The phrase "connective tissue" refers to a supporting
framework tissue comprising strands of collagen, elastic fibers
(e.g., between and around muscle and blood vessels) and simple
cells. Examples of connective tissues include, but are not limited
to dense connective tissue (e.g., ligament, tendon, periodontal
ligament), areolar connective tissue (e.g., with proteinaceous
fibers such as collagen and elastin), reticular connective tissue,
adipose tissue, blood, bone, cartilage, skin, intervertebral disc,
dental pulp, dentin, gingival, extracellular matrix (ECM)-forming
cells, loose connective tissue and smooth muscle cells.
[0134] As used herein the phrase "medical condition requiring
connective tissue regeneration and/or repair" refers to any
pathology characterized by connective tissue damage (i.e.,
non-functioning tissue, cancerous or pre-cancerous tissue, broken
tissue, fractured tissue, fibrotic tissue, or ischemic tissue) or
loss (e.g., following a trauma, an infectious disease, a genetic
disease, and the like). Non-limiting examples of such pathologies
include, bone fracture, bone cancer (e.g., osteosarcoma, bone
cancer metastasis), burn wound, articular cartilage defect and deep
wound.
[0135] The phrase "administering to the subject" refers to the
introduction of the cells of the invention to target tissue. The
cells can be derived from the recipient or from an allogeneic or
xenogeneic donor. This phrase also encompasses "transplantation",
"cell replacement" or "grafting" of the cells of the invention into
the subject.
[0136] The subject may be any mammal in need of connective tissue
regeneration and/or repair including e.g. human or domesticated
animals including, but not limited to, horses (i.e. equine),
cattle, goat, sheep, pig, dog, cat, camel, alpaca, llama and
yak.
[0137] According to an embodiment of the present teachings, the
adherent cells of the present invention may be used to treat
conditions including subchondral-bone cysts, bone fractures,
osteoporosis, osteoarthritis, degenerated bone, various cancers
associated with connective tissue loss (e.g., bone cancer,
osteosarcoma, bone metastases), cartilage damage, articular
cartilage defect, degenerative disc disease, osteogenesis
imperfecta (OI), burns, burn wounds, deep wounds, delayed
wound-healing, injured ligaments and injured tendons e.g.
overstrain-induced injuries of tendons in horses and other subjects
in need thereof (as stated above).
[0138] Cells which may be administered in accordance with this
aspect of the invention include the above-described adherent cells
which may be cultured in three-dimensional or two dimensional
settings as well as mesenchymal and-non mesenchymal partially or
terminally differentiated derivatives of same.
[0139] Methods of deriving lineage specific cells from the stromal
stem cells of the invention are well known in the art. See for
example, U.S. Pat. Nos. 5,486,359, 5,942,225, 5,736,396, 5,908,784
and 5,902,741.
[0140] The cells may be naive or genetically modified such as to
derive a lineage of interest (see U.S. Pat. Appl. No.
20030219423).
[0141] The cells may be of autologous or non-autologous source
(i.e., allogeneic or xenogeneic) of fresh or frozen (e.g.,
cryo-preserved) preparations.
[0142] Depending on the medical condition, the subject may be
administered with additional chemical drugs (e.g.,
immunomodulatory, chemotherapy etc.) or cells.
[0143] Since non-autologous cells may induce an immune reaction
when administered to the body several approaches have been
developed to reduce the likelihood of rejection of non-autologous
cells. These include either suppressing the recipient immune system
or encapsulating the non-autologous cells in immunoisolating,
semipermeable membranes before transplantation.
[0144] Encapsulation techniques are generally classified as
microencapsulation, involving small spherical vehicles and
macroencapsulation, involving larger flat-sheet and hollow-fiber
membranes (Uludag, H. et al. Technology of mammalian cell
encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
[0145] Methods of preparing microcapsules are known in the arts and
include for example those disclosed by Lu M Z, et al., Cell
encapsulation with alginate and
alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol
Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for
microencapsulation of enzymes, cells and genetically engineered
microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et
al., A novel cell encapsulation method using photosensitive
poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul.
2000, 17: 245-51.
[0146] For example, microcapsules are prepared by complexing
modified collagen with a ter-polymer shell of 2-hydroxyethyl
methylacrylate (HEMA), methacrylic acid (MAA) and methyl
methacrylate (MMA), resulting in a capsule thickness of 2-5 .mu.m.
Such microcapsules can be further encapsulated with additional 2-5
.mu.m ter-polymer shells in order to impart a negatively charged
smooth surface and to minimize plasma protein absorption (Chia, S.
M. et al. Multi-layered microcapsules for cell encapsulation
Biomaterials. 2002 23: 849-56).
[0147] Other microcapsules are based on alginate, a marine
polysaccharide (Sambanis, A. Encapsulated islets in diabetes
treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its
derivatives. For example, microcapsules can be prepared by the
polyelectrolyte complexation between the polyanions sodium alginate
and sodium cellulose sulphate with the polycation
poly(methylene-co-guanidine) hydrochloride in the presence of
calcium chloride.
[0148] It will be appreciated that cell encapsulation is improved
when smaller capsules are used. Thus, the quality control,
mechanical stability, diffusion properties, and in vitro activities
of encapsulated cells improved when the capsule size was reduced
from 1 mm to 400 .mu.m (Canaple L. et al., Improving cell
encapsulation through size control. J Biomater Sci Polym Ed. 2002;
13:783-96). Moreover, nanoporous biocapsules with well-controlled
pore size as small as 7 nm, tailored surface chemistries and
precise microarchitectures were found to successfully immunoisolate
microenvironments for cells (Williams D. Small is beautiful:
microparticle and nanoparticle technology in medical devices. Med
Device Technol. 1999, 10: 6-9: Desai, T. A. Microfabrication
technology for pancreatic cell encapsulation. Expert Opin Biol
Ther. 2002, 2: 633-46).
[0149] Examples of immunosuppressive agents include, but are not
limited to, methotrexate, cyclophosphamide, cyclosporine,
cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine
(sulphasalazopyrine), gold salts, D-penicillamine, leflunomide,
azathioprine, anakinra, infliximab (REMICADE), etanercept,
TNF.alpha, blockers, a biological agent that targets an
inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug
(NSAIDs). Examples of NSAIDs include, but are not limited to acetyl
salicylic acid, choline magnesium salicylate, diflunisal, magnesium
salicylate, salsalate, sodium salicylate, diclofenac, etodolac,
fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac,
meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam,
sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and
tramadol.
[0150] In any of the methods described herein, the cells can be
administered either per se or, preferably as a part of a
pharmaceutical composition that further comprises a
pharmaceutically acceptable carrier.
[0151] As used herein a "pharmaceutical composition" refers to a
preparation of the adherent cells of the invention (i.e., adherent
cells of a tissue selected from the group consisting of placenta
and adipose tissue, which are obtained from a three-dimensional
culture), with other chemical components such as pharmaceutically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of the cells to a
subject.
[0152] Hereinafter, the term "pharmaceutically acceptable carrier"
refers to a carrier or a diluent that does not cause significant
irritation to a subject and does not abrogate the biological
activity and properties of the administered compound. Examples,
without limitations, of carriers are propylene glycol, saline,
emulsions and mixtures of organic solvents with water.
[0153] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of a compound. Examples, without limitation, of
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0154] According to a preferred embodiment of the invention, the
pharmaceutical carrier is an aqueous solution of saline.
[0155] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0156] One may administer the pharmaceutical composition in a
systemic manner (as detailed hereinabove). Alternatively, one may
administer the pharmaceutical composition locally, for example, via
injection of the pharmaceutical composition directly into a tissue
region of a patient.
[0157] Pharmaceutical compositions of the invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0158] Pharmaceutical compositions for use in accordance with the
invention thus may be formulated in conventional manner using one
or more physiologically acceptable carriers comprising excipients
and auxiliaries, which facilitate processing of the active
ingredients into preparations which, can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0159] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, physiological salt buffer, or freezing medium
containing cryopreservents. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0160] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. Preferably, a dose
is formulated in an animal model to achieve a desired concentration
or titer. Such information can be used to more accurately determine
useful doses in humans.
[0161] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals.
[0162] The data obtained from these in vitro and cell culture
assays and animal studies can be used in formulating a range of
dosage for use in human. The dosage may vary depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition,
(see e.g., Fingl, et al. 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1). For example, Parkinson's patient can be
monitored symptomatically for improved motor functions indicating
positive response to treatment.
[0163] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer.
[0164] Dosage amount and interval may be adjusted individually to
levels of the active ingredient which are sufficient to effectively
regulate the neurotransmitter synthesis by the implanted cells.
Dosages necessary to achieve the desired effect will depend on
individual characteristics and route of administration. Detection
assays can be used to determine plasma concentrations.
[0165] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or diminution of the disease state is
achieved.
[0166] The amount of a composition to be administered will, of
course, be dependent on the individual being treated, the severity
of the affliction, the manner of administration, the judgment of
the prescribing physician, etc. The dosage and timing of
administration will be responsive to a careful and continuous
monitoring of the individual changing condition. For example, a
treated Parkinson's patient will be administered with an amount of
cells which is sufficient to alleviate the symptoms of the disease,
based on the monitoring indications.
[0167] Models for ligament injury include, but are not limited to,
rabbit model of anterior cruciate ligament reconstruction using
mesenchymal stem cells [Jit-Kheng et al., Arthroscopy (2004) 20(9):
899-910], goat model for use of long-term bioresorbable scaffolds
for anterior cruciate ligament repair [Altman et al. J Am Acad
Orthop Surg. (2008) 16(4):177-187]. Models for tendon repair
include, but are not limited to, adult New Zealand White rabbit
model for autologous mesenchymal stem cell-mediated repair of
tendon [Awad et al., Tissue Eng. (1999) 5(3):267-77]. Models for
bone repair were described in e.g. Stem Cells in Endocrinology,
Humana Press (2005) 183-206, describing the manipulation of
mesenchymal stem cells for bone repair.
[0168] Following transplantation, the cells of the invention
preferably survive in the diseased area for a period of time (e.g.
about 1 month), such that a therapeutic effect is observed.
[0169] Compositions including the preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0170] Compositions of the invention may, if desired, be presented
in a pack or dispenser device, such as an FDA approved kit, which
may contain one or more unit dosage forms containing the active
ingredient. The pack may, for example, comprise metal or plastic
foil, such as a blister pack. The pack or dispenser device may be
accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0171] The adherent cells of the invention can be suitably
formulated as pharmaceutical compositions which can be suitably
packaged as an article of manufacture. Such an article of
manufacture comprises a packaging material which comprises a label
for use in increasing angiogenesis in a tissue, treating ischemia
and/or treating a pathology requiring connective tissue
regeneration and/or repair, wherein the packaging material
packaging the adherent cells of the invention.
[0172] It will be appreciated that the adherent cells of the
present invention are capable of inducing immunosuppression and/or
tolerance in a subject. Thus, the adherent cells may be used to
treat any condition in need of immunosuppression and/or tolerance.
Such conditions included, but are not limited to, autoimmune
diseases and inflammatory diseases (including acute and chronic
inflammatory diseases) including, but are not limited to,
cardiovascular diseases, rheumatoid diseases, glandular diseases,
gastrointestinal diseases, cutaneous diseases, hepatic diseases,
neurological diseases, muscular diseases, nephric diseases,
diseases related to reproduction, connective tissue diseases and
systemic diseases.
[0173] Examples of autoimmune cardiovascular diseases include, but
are not limited to atherosclerosis (Matsuura E. et al., Lupus.
1998; 7 Suppl 2:S135), myocardial infarction (Vaurala O. Lupus.
1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7
Suppl 2:S107-9), Wegener's granulotmatosis, Takayasu's arteritis,
Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000
Aug. 25; 112 (15-16):660), anti-factor VIII autoinmmune disease
(Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26
(2):157), necrotizing small vessel vasculitis, microscopic
polyangiitis, Churg and Strauss syndrome, pauci-immune focal
necrotizing and crescentic glomerulonephritis (Noel L. H. Ann Med
Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome
(Flamholz R. et al., J Clin Apheresis 1999; 14 (4):171),
antibody-induced heart failure (Wallukat G. et al., Am J Cardiol.
1999 Jun. 17; 83 (12A):75H), thrombocytopenic purpura (Moccia F.
Ann Ital Med. Int. 1999 April-June; 14 (2): 114; Semple J W. et
al., Blood 1996 May 15; 87 (10):4245), autoimmune hemolytic anemia
(Efremov D G. et al., Leuk Lymphoma 1998 January; 28 (3-4):285;
Sallah S. et al. Ann Hematol 1997 March; 74 (3):139), cardiac
autoimmunity in Chagas' disease (Cunha-Neto E. et al., J Clin
Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyte
autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11
(1):9).
[0174] Examples of autoimmune rheumatoid diseases include, but are
not limited to rheumatoid arthritis (Krenn V. et al., Histol
Histopathol 2000 July; 15 (3):791; Tisch R, McDevitt H O. Proc Natl
Acad Sci units S A 1994 Jan. 18; 91 (2):437) and ankylosing
spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3):
189).
[0175] Examples of autoimmune glandular diseases include, but are
not limited to, pancreatic disease, Type I diabetes, thyroid
disease, Graves' disease, thyroiditis, spontaneous autoimmune
thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian
autoimmunity, autoimmune anti-sperm infertility, autoimmune
prostatitis and Type I autoimmune polyglandular syndrome. Diseases
include, but are not limited to autoimmune diseases of the
pancreas, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev.
Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 October; 34
Suppl:S125), autoimmune thyroid diseases. Graves' disease (Orgiazzi
J. Endocrinol Metab Clin North Am 2000 June; 29 (2):339; Sakata S.
et al., Mol Cell Endocrinol 1993 March; 92 (1):77), spontaneous
autoimmune thyroiditis (Braley-Mullen H. and Yu S. J Immunol 2000
Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,
Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema
(Mitsuma T. Nippon Rinsho. 1999 August; 57 (8):1759), ovarian
autoimmunity (Garza K M. et al., J Reprod Immunol 1998 February; 37
(2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am
J Reprod Immunol. 2000 March; 43 (3):134), autoimmune prostatitis
(Alexander R B. et al., Urology 1997 December; 50 (6):893) and Type
I autoimmnune polyglandular syndrome (Hara T. et al. Blood. 1991
Mar. 1; 77 (5):1127).
[0176] Examples of autoimmune gastrointestinal diseases include,
but are not limited to, chronic inflammatory intestinal diseases
(Garcia Herola A. et ail., Gastroenterol Hepatol. 2000 January; 23
(1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000
Jan. 16; 138 (2):122), colitis, ileitis and Crohn's disease.
[0177] Examples of autoimmune cutaneous diseases include, but are
not limited to, autoimmune bullous skin diseases, such as, but are
not limited to, pemphigus vulgaris, bullous pemphigoid and
pemphigus foliaceus.
[0178] Examples of autoimmune hepatic diseases include, but are not
limited to, hepatitis, autoimmune chronic active hepatitis (Franco
A. et al., Clin Immunol Immunopathol 1990 March; 54 (3):382).
primary biliary cirrhosis (Jones D E. Clin Sci (Colch) 1996
November; 91 (5):551; Strassburg C P. et al., Eur J Gastroenterol
Hepatol. 1999 June; 11 (6):595) and autoimmune hepatitis (Manns M
P. J Hepatol 2000 August; 33 (2):326).
[0179] Examples of autoimmune neurological diseases include, but
are not limited to, multiple sclerosis (Cross A H. et al., J
Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron
L. et al. J Neural Transm Suppl. 1997; 49:77), myasthenia gravis
(Infante A J. And Kraig E. Int Rev Immunol 1999; 18 (1-2):83:
Oshima M. et al., Eur J Immunol 1990 December; 20 (12):2563),
neuropathies, motor neuropathies (Kornberg A J. J Clin Neurosci.
2000 May; 7 (3):191); Guillain-Barre syndrome and autoimmune
neuropathies (Kusunoki S. Am J Med. Sci. 2000 April; 319 (4):234),
myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med
Sci. 2000 April; 319 (4):204); paraneoplastic neurological
diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and
stiff-man syndrome (Hiemstra H S. et al. Proc Natl Acad Sci units S
A 2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man
syndrome, progressive cerebellar atrophies, encephalitis,
Rasmussen's encephalitis, amyotrophic lateral sclerosis. Sydeham
chorea, Gilles de la Tourette syndrome and autoimmune
polyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol
(Paris) 2000 January; 156 (1):23); dysimmune neuropathies
(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol
Suppl 1999; 50:419); acquired neuormyotonia, arthrogryposis
multiplex congenita (Vincent A. et al., Ann N Y Acad. Sci. 1998 May
13; 841:482), neuritis, optic neuritis (Soderstrom M. et al., J
Neurol Neurosurg Psychiatry 1994 May; 57 (5):544) and
neurodegenerative diseases.
[0180] Examples of autoimmune muscular diseases include, but are
not limited to, myositis, autoimmune myositis and primary Sjogren's
syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 September;
123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al.
Biomed Pharmacother 1999 June; 53 (5-6):234).
[0181] Examples of autoimmune nephric diseases include, but am not
limited to, nephritis and autoimmune interstitial nephritis (Kelly
C J. J Am Soc Nephrol 1990 August; 1 (2):140).
[0182] Examples of autoimmune diseases related to reproduction
include, but are not limited to repeated fetal loss (Tincani A. et
al. Lupus 1998; 7 Suppl 2:S107-9).
[0183] Examples of autoimmune connective tissue diseases include,
but are not limited to ear diseases, autoimmune ear diseases (Yoo T
J. et al., Cell Immunol 1994 August; 157 (1):249) and autoimmune
diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997
Dec. 29; 830:266).
[0184] Examples of autoimmune systemic diseases include, but are
not limited to, systemic lupus erythematosus (Erikson J. et al.,
Immunol Res 1998; 17 (1-2):49) and systemic sclerosis (Renaudineau
Y. et al., Clin Diagn Lab Immunol. 1999 March; 6 (2):156); Chan O
T. et al. Immunol Rev 1999 June; 169:107).
[0185] Furthermore, the adherent cells may be used to treat
diseases associated with transplantation of a graft including, but
are not limited to, graft rejection, chronic graft rejection,
subacute graft rejection, hyperacute graft rejection, acute graft
rejection and graft versus host disease.
[0186] As used herein the term "about" refers to .+-.10%.
[0187] Additional objects, advantages, and novel features of the
invention will become apparent to one ordinarily skilled in the art
upon examination of the following examples, which are not intended
to be limiting. Additionally, each of the various embodiments and
aspects of the invention as delineated hereinabove and as claimed
in the claims section below finds experimental support in the
following examples.
EXAMPLES
[0188] Reference is now made to the following examples, which
together with the above descriptions illustrate the invention in a
non-limiting fashion.
[0189] Generally, the nomenclature used herein and the laboratory
procedures utilized in the invention include molecular,
biochemical, microbiological andrecombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985): "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I. ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal. B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press: "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Production and Culturing of Adherent Cells from Bone Marrow,
Placenta and Adipose Tissues
[0190] Adherent cells were cultured in a bioreactor system
containing 3D carriers to produce 3D-adherent cells, characterized
by a specific cell marker expression profile. Growth efficiency was
tested through cell count. The differentiation capacity of these
cells was tested by culturing in a differentiation medium.
[0191] Materials and Experimental Procedures
[0192] Bone Marrow Adherent Cells
[0193] Bone marrow (BM) adherent cells were obtained from aspirated
sterna marrow of hematologically healthy donors undergoing
open-heart surgery or BM biopsy. Marrow aspirates were diluted
3-fold in Hank's Balanced Salts Solution (HBSS; GIBCO
BRL/Invitrogen, Gaithersburg Md.) and subjected to Ficoll-Hypaque
(Robbins Scientific Corp. Sunnyvale, Calif.) density gradient
centrifugation. Thereafter, marrow mononuclear cells (<1.077
gm/cm.sup.3) were collected, washed 3 times in HBSS and resuspended
in growth media [DMEM (Biological Industries, Beit Ha'emek, Israel)
supplemented with 10% FCS (GIBCO BRL), 10.sup.-4 M mercaptoethanol
(Merck, White House Station, N.J.), Pen-Strep-Nystatin mixture (100
U/ml:100 .mu.g/ml:1.25 un/ml; Beit Ha'Emek), 2 mM L-glutamine (Beit
Ha'Emek)]. Cells from individual donors were incubated separately
in tissue culture flasks (Corning, Acton, M A) at 37.degree. C. (5%
CO.sub.2) with weekly change of culture media. Cells were split
every 3-4 days using 0.25% trypsin-EDTA (Beit Ha'Emek). Following
2-40 passages, when reaching 60-80% confluence, cells were
collected for analysis or for culturing in bioreactors.
[0194] Placenta Derived Adherent Cells
[0195] Inner parts of a full-term delivery placenta (Bnei Zion
medical center, 1Haifa, Israel) were cut under sterile conditions,
washed 3 times with Hank's Buffer and incubated for 3 hours at
37.degree. C. with 0.1% Collagenase (1 mg/ml tissue: Sigma-Aldrich,
St. Lewis, Mo.). Using gentle pipetting, suspended cells were then
washed with DMEM supplemented with 10% FCS, Pen-Strep-Nystatin
mixture (100 LU/ml:100 .mu.g/ml:1.25 un/ml) and 2 mM L-glutamine,
seeded in 75 cm.sup.2 flasks and incubated at 37.degree. C. in a
tissue culture incubator under humidified condition with 5%
CO.sub.2. Thereafter, cells were allowed to adhere to a plastic
surface for 72 hours after which the media was changed every 3-4
days. When reaching 60-80% confluence (usually 10-12 days), cells
were detached from the growth flask using 0.25% trypsin-EDTA and
seeded into new flasks. Cultured cells were thereafter collected
for analysis or for culturing in bioreactots.
[0196] Adipose Derived Adherent Cells
[0197] Adherent cells were obtained from human adipose tissue of
liposuction procedures (Rambam Haifa, Israel). Adipose tissue was
washed extensively with equal volumes of PBS and digested at
37.degree. C. for 30 minutes with collagenase (20 mg/ml). Cells
were then washed with DMEM containing 10% FCS, Pen-Strep-Nystatin
mixture (100 U/ml:100 .mu.g/ml:1.25 un/ml) and L-Glutamin and
centrifuged at 1200 rpm for 10 minutes at room temperature (RT),
resuspended with lysing solution (1:10; Biological Industries. Beit
Ha'emek, Israel, in order to discard red-blood cells) centrifuged
and resuspended with DMEM containing 10% FCS, Pen-Strep-Nystatin
mixture (100 U/ml:100 .mu.g/ml:1.25 un/ml) and L-Glutamin. Washed
cells were then seeded in a sterile tissue culture medium flask at
3-10.times.10.sup.7 cells/flask. At the next day cells were washed
with PBS to remove residual RBC and dead cells. The cells were kept
at 37.degree. C. in a tissue culture incubator under humidified
condition with 5% CO.sub.2. The medium was changed every 3 to 4
days. At 60-80% confluence, the cells were detached from the growth
flask using 0.25% trypsin-EDTA and seeded into new flasks.
Following 2-40 passages, when cells reached 60-80% confluence,
cells were collected for analysis or for culturing in
bioreactors.
[0198] PluriX.TM. Plug Flow Bioreactor
[0199] The PluriX.TM. Plug Flow bioreactor (Pluristem, Haifa,
Israel; as illustrated in FIG. 1G, see also U.S. Pat. No.
6,911,201), was loaded with 1-100 ml packed 3D porrosive carriers
(4 mm in diameter) made of a non woven fabric matrix of polyester.
These carriers enable the propagation of large cell numbers in a
relatively small volume. Glassware was designed and manufactured by
Pluristem (Pluristem, Haifa, Israel). The bioreactor was maintained
in an incubator of 37.degree. C., with flow rate regulated and
monitored by a valve (6a in FIG. 1G), and peristaltic pump (9 in
FIG. 1G). The bioreactor contains a sampling and injection point (4
in FIG. 10). allowing the sequential seeding of cells. Culture
medium was supplied at pH 6.7-7.4 from a reservoir (1 in FIG. 1G).
The reservoir was supplied by a filtered gas mixture (2, 3 in FIG.
1G), containing air/CO.sub.2/O.sub.2 at differing proportions,
depending on cell density in the bioreactor. The O.sub.2 proportion
was suited to the level of dissolved O.sub.2 at the bioreactor
exit, determined by a monitor (6 in FIG. 1G). The gas mixture was
supplied to the reservoir via silicone tubes or diffuser (Degania
Bet, Emek Hayarden, Israel). The culture medium was passed through
a separating container (7 in FIG. 1G) which enables collection of
circulating, nonadherent cells. Circulation of the medium was
obtained by a peristaltic pump (9 in FIG. 1G). The bioreactor was
further equipped with an additional sampling point (10 in FIG. 1G)
and containers for continuous medium exchange.
[0200] Production of 3D-Adherent Cells
[0201] Non-confluent primary human adherent 2D cell cultures, grown
as described above, were trypsinized. washed, resuspended in DMEM
supplemented with 10% FBS, Pen-Strep-Nystatin mixture (100 U/ml:100
ug/ml:1.25 un/ml) and 2 mM L-glutamine. and seeded
(10.sup.3-10.sup.5 cells/ml) via an injection point onto the 3D
carriers in a sterile Plug Flow bioreactor (see FIG. 1G). Prior to
inoculation, bioreactor was filled with PBS-Ca-Mg (Biological
Industries, Beit Ha'emek, Israel), autoclaved (120.degree. C., 30
min) and washed with Dulbecco's growth medium containing 10%
heat-inactivated fetal calf serum and a Pen-Strep-Nystatin mixture
(100 U/ml:100 ug/ml:1.25 un/ml). Flow was kept at a rate of 0.1-5
ml/min. Seeding process involved cease of circulation for 2-48 hrs,
thereby allowing the cells to settle on the carriers. Bioreactor
was kept under controlled temperature (37.degree. C.) and pH
conditions (pH=6.7-7.4); using an incubator supplied with sterile
air and CO.sub.2 as needed. Growth medium was replaced 2-3 times a
week. Circulation medium was replaced with fresh DMEM media, every
4 hr to 7 days. At a density of 1.times.10.sup.6-1.times.10.sup.7
cells/ml (following 12-40 days of growth), total medium volume was
removed from the bioreactor and bioreactor and carriers were washed
3-5 times with PBS. 3D-adherent cells were then detached from the
carriers with Trypsin-EDTA; (Biological Industries, Beit Ha'emek,
Israel; 3-15 minutes with gentle agitation, 1-5 times), and were
thereafter resuspended in DMEM and cryopreserved.
[0202] 3D-Adherent Cell Quality Biological Assays
[0203] Cryopreserved 3D-adherent cells were thawed and counted. For
cell viability evaluation. 2.times.10.sup.5 cells were seeded in a
150 cm.sup.2 tissue culture flask and their adherence capability
and repopulation was evaluated within 7 days following seeding.
Thereafter, the 3D-adherent cell membrane marker phenotype was
analyzed using fluorescence monoclonal antibodies flow-cytometer
(Beckman Coulter, Fullerton, Calif.).
[0204] Comparison Between the Cell Membrane Marker Profile of 31)
and 21) Cultured Adherent Cells Using Flow Cytometry Assays
[0205] 100,000-200,000 adherent cells from 2D cultures and 3D flow
system cultures were suspended in 0.1 ml of culture medium in a 5
ml tube and incubated (4.degree. C., 30 minutes, dark conditions)
with saturating concentrations of each of the following MAbs:
FITC-conjugated anti-human CD90 (Chemicon International Inc.
Temecula, Calif.), PE conjugated anti human CD73 (Bactlab
Diagnostic, Ceasarea, Israel), PE conjugated anti human CD105
(eBioscience. San Diego, Calif.), FITC conjugated anti human CD29
(eBioscience, San Diego, Calif.), Cy7-PE conjugated anti-human CD45
(eBiosience), PE-conjugated anti-human CD19 (IQProducts, Groningen.
The Netherlands), PE conjugated anti human CD14 MAb (IQProducts),
FITC conjugated anti human CD11b (IQProducts) and PE conjugated
anti human CD34 (IQProducts) or with FITC conjugated anti human
HLA-DR MAb (IQProducts). Following incubation the cells were washed
twice in ice-cold PBS containing 1% heat-inactivated FCS,
resuspended in 500 d formaldehyde 0.5% and analyzed using the
FC-500 flow-cytometer (Beckman Coulter, Fullerton, Calif.).
[0206] Comparison Between the Protein Profile of 3D1) and 2D
Cultured Adherent Cells Using Mass Spectrometry Analysis
[0207] 2D and 3D derived culturing procedures adherent cells were
produced from the placenta as described above. Briefly, the 2D
cultures were produced by culturing 0.3-0.75.times.10.sup.6 cells
in 175 cm.sup.2 flasks for 4 days under humidified 5% CO.sub.2
atmosphere at 37.degree. C., until reaching 60-80% confluence. The
3D cultures were produced by seeding 2-10.times.10.sup.6 cells/gram
in a bioreactor containing 2000 carriers, and culturing for 18
days. Following harvesting, cells were washed (X 3) to remove all
the serum, pelleted and frozen. Proteins were isolated from pellets
[using Tri Reagent kit (Sigma, Saint Louis, USA) and digested with
trypsin and labeled with iTRAQ reagent (Applied Biosciences, Foster
City, Calif.)], according to the manufacturers protocol. Briefly,
iTRAQ reagents are non-polymeric, isobaric tagging reagents.
Peptides within each sample are labeled with one of four isobaric,
isotope-coded tags via their N-terminal and/or lysine side chains.
The four labeled samples are mixed and peptides are analyzed with
mass spectrometry. Upon peptide fragmentation, each tag releases a
distinct mass reporter ion; the ratio of the four reporters
therefore gives relative abundances of the given peptide in a
sample. (information at:
www.docs.appliedbiosystems.com/pebiodocs/00113379.pdf).
[0208] Proteomics analysis of 2D culture versus 3D culture of
placenta derived adherent cells was performed in the Smoler
proteomic center (department of Biology, Technion, Haifa, Israel)
using LC-MS/MS on QTOF-Premier (Waters, San Francisco, Calif.),
with identification and analysis done by Pep-Miner software [Beer,
I., et al., Proteomics, 4, 950-60 (2004)] against the human part of
the nr database. The proteins analyzed were: heterogeneous nuclear
ribonucleoprotein H1 (Hnrph1 GenBank Accession No. NP_005511), H2A
histone family (H2AF, GenBank Accession No. NP_034566.1),
eukaryotic translation elongation factor 2 (EEEF2, GenBank
Accession No. NP_031933.1), reticulocalbin 3, EF-hand calcium
binding domain (RCN2, GenBank Accession No. NP_065701), CD44
antigen isoform 2 precursor (GenBank Accession No. NP_001001389,
calponin 1 basic smooth muscle (CNN1, GenBank Accession No.
NP_001290), 3 phosphoadenosine 5 phosphosulfate synthase 2 isoform
a (Papss2, GenBank Accession No. NP_004661), ribosomal protein L7a
(rpL7a, GenBank Accession No. NP_000963) and Aldehyde dehydrogenase
X (ALDH X, GenBank Accession No. P47738). Every experiment was done
twice. Because of the nature of the analysis, every protein was
analyzed according to the number of peptides of which appeared in a
sample (2-20 appearances of a protein in each analysis)
[0209] Comparison Between Secreted Proteins in 3D and 2D Cultured
Adherent Cells Using ELISA
[0210] 2D and 3D derived culturing procedures adherent cells
produced from the placenta, were produced as described above, with
3D cultures for the duration of 24 days. Conditioned media were
thereafter collected and analyzed for Flt-3 ligand, IL-6,
Thrombopoietin (TPO) and stem cell factor (SCF). using ELISA
(R&D Systems, Minneapolis, Minn.), in three independent
experiments. Results were normalized for 1.times.10.sup.6
cells/ml.
[0211] Osteoblast Differentiating Medium
[0212] Osteogenic differentiation was assessed by culturing of
cells in an osteoblast differentiating medium consisting DMEM
supplemented with 10% FCS, 100 nM dexamethasone, 0.05 mM ascotbic
acid 2-phosphate, 10 mM B-glycerophosphate, for a period of 3
weeks. Calcified matrix was indicated by Alizzarin Red S staining
and Alkaline phosphatase was detected by Alkaline phosphatase assay
kit (all reagents from Sigma-Aldrich, St. Lewis, Mo.).
[0213] Experimental Results
[0214] The PluriX.TM. Bioreactor System Creates a
Physiological-Like Microenvironment.
[0215] In order to render efficient culture conditions for adherent
cells, a physiological-like environment (depicted in FIG. 1A) was
created artificially, using the PluriX Bioreactor (Pluristem,
Haifa, Israel; carrier is illustrated in FIG. 1G and shown before
seeding in FIG. 1B). As is shown in FIGS. 1C-F, bone marrow
produced 3D-adherent cells were cultured successfully and expanded
on the 3D matrix, 20 days (FIGS. 1B-C, magnified X 150 and 250
respectively) and 40 days (FIGS. 1C-D, magnified X 350 and 500
respectively) following seeding.
[0216] Cells Grown in the PluriX Bioreactor System were
Significantly Expanded
[0217] Different production lots of placenta derived 3D-adherent
cells were grown in the PluriX bioreactor systems. The seeding
density was 13,300 cells/carrier (to a total of 2.times.10.sup.6
cells). Fourteen days following seeding, cell density multiplied by
15 fold, reaching approximately 200,000 cells/carrier (FIG. 2), or
30.times.10.sup.6 in a bioreactor of 150 carriers. In a different
experiment, cells were seeded into the bioreactor at density of
1.5.times.10.sup.4 cells/ml and 30 days following seeding the
carriers contained an over 50-fold higher cell number, i.e. approx.
0.5.times.10.sup.6 cells/carrier, or 0.5.times.10.sup.7 cells/ml.
The cellular density on the carriers at various levels of the
growth column was consistent, indicating a homogenous transfer of
oxygen and nutrients to the cells. The 3D culture system was thus
proven to provide supporting conditions for the growth and
prolonged maintenance of high-density mesenchymal cells cultures,
which can be grown efficiently to an amount sufficient for the
purpose of supporting engraftment and successful
transplantation.
[0218] 3D-Adherent Cells Show Unique Membrane Marker
Characteristics
[0219] In order to define the difference in the secretion profile
of soluble molecules and protein production, effected by the bone
environment mimicking 3D culturing procedure, FACs analysis was
effected. As is shown in FIG. 3A, FACS analysis of cell markers
depict that 3D-adherent cells display a different marker expression
pattern than adherent cells grown in 2D conditions. 2D cultured
cells expressed significantly higher levels of positive membrane
markers CD90, CD105, CD73 and CD29 membrane markers as compared to
3D cultured cells. For example, CDI05 showed a 56% expression in 3D
cultured cells vs. 87% in 2D cultured cells. Adherent cells of both
2D and 3D placenta cultures. did not express any hematopoietic
membrane markers (FIG. 3B).
[0220] 3D-Adherent Cells Show a Unique Profile of Soluble
Factors
[0221] The hematopoietic niche includes supporter cells that
produce an abundance of cytokines, chemokines and growth factors.
In order to further define the difference between 2D and 3D
cultured adherent cells, the profile of the four main hematopoietic
secreted proteins in the conditioned media of 2D and 3D adherent
cell cultures was effected by ELISA. FIGS. 4A-C show that cells
grown in 3D conditions produced condition media with higher levels
of Flt-3 ligand (FIG. 4A), IL-60 (FIG. 4B), and SCF (FIG. 4C),
while low levels of IL-6, and close to zero level of Flt-3 ligand
and SCF, were detected in the condition media of 2D cultures.
Production of Trombopoietin (TPO) was very low and equal in both
cultures.
[0222] 3D-Adherent Cells Show a Unique Protein Profile in Mass
Spectrometry Analysis
[0223] In order to further define the difference between 2D and 3D
cultured adherent cells, the protein profile of these cells was
analyzed by mass spectrometry. FIG. 4D shows that 2D and 3D
cultured adherent cells show a remarkably different protein
expression profile. As is shown in Table I below, 3D cultured cells
show a much higher expression level of H2AF and ALDH X (more than 9
and 12 fold higher, respectively) and a higher level of the
proteins EEEF2. RCN2 and CNN1 (ca. 3, 2.5 and 2 fold,
respectively). In addition, 3D cultured cells show ca. half the
expression levels of the proteins Hnrph1 and CD44 antigen isoform 2
precursor and ca. a third of the expression levels of Papss2 and
rpL7a.
TABLE-US-00001 TABLE 1 Protein level (relative to iTRAQ reporter
group) 2D cultured 3D cultured adherent cells adherent cells
protein Av SD Av SD Hnrph1 1.434493 0.260914 0.684687 0.197928 H2AF
0.203687 0.288058 1.999877 0.965915 EEEF2 0.253409 0.130064
0.799276 0.243066 RCN2 0.54 0.25 1.34 0.26 CD44 antigen 1.68 0.19
0.73 0.17 isoform 2 precursor CNN1 0.77 0.15 1.55 0.17 Papss2
1.48352 0.314467 0.45627 0.137353 rpL7a 1.22 0.24 0.43 0.05 ALDH X
0.15847 0.22411 1.986711 0.212851
[0224] 3D-Adherent Cells have the Capacity to Differentiate into
Osteoblasts
[0225] In order to further characterize 3D-adherent cells, cells
were cultured in an osteoblast differentiating medium for a period
of 3 weeks. Thereafter, calcium precipitation was effected.
Differentiated cells were shown to produce calcium (depicted in red
in FIGS. 5A-B) whereas control cells maintained a fibroblast like
phenotype and demonstrated no mineralization (FIGS. 5C-D). These
results show that placenta derived 3D-adherent cells have the
capacity to differentiate in vitro to osteoblasts cells.
Example 2
Assessment of the Ability of Placenta Derived 3D-Adherent Cells to
Improve HSC Engraftment
[0226] 3D-adherent cell's support of HSC engraftment was evaluated
by the level of human hematopoietic cells (hCD45+) detected in sub
lethally irradiated or chemotherapy pretreated immune deficient
NOD-SCID mice.
[0227] Materials and Experimental Procedures
[0228] Isolation of CD34+ Cells
[0229] Umbilical cord blood samples were taken under sterile
conditions during delivery (Bnei Zion Medical Center, Haifa,
Israel) and mononuclear cells were fractionated using Lymphoprep
(Axis-Shield PoC As, Oslo, Norway) density gradient centrifugation
and were cryopreserved. Thawed mononuclear cells were washed and
incubated with anti-CD34 antibodies and isolated using midi MACS
(Miltenyl Biotech, Bergish Gladbach, Germany). Cells from more than
one sample were pooled for achieving the desired amount
(50,000-100,000 cells).
[0230] Detection of Transplanted Cells in Irradiated Mice
[0231] Seven week old male and female NOD-SCID mice
(NOD-CB17-Prkdcscid/J; Harlan/Weizmann Inst., Rehovot Israel) were
maintained in sterile open system cages, given sterile diets and
autoclaved acidic water. The mice were sub lethally irradiated (350
cGy), and thereafter (48 hr post irradiation) transplanted with
50,000-100,000 hCD34.sup.+ cells, with or without additional
adherent cells (0.5.times.10.sup.6-1.times.10.sup.6) derived from
placenta or adipose tissue (3-7 mice in each group), by intravenous
injection to a lateral tail vein. Four to six weeks following
transplantation the mice were sacrificed by dislocation and BM was
collected by flushing both femurs and tibias with FACS buffer (50
ml PBS, 5 ml FBS, 0.5 ml sodium azid 5%). Human cells in the mice
BM were detected by flow cytometry, and the percentage of the human
and murine CD45 hematopoietic cell marker expressing cells in the
treated NOD-SCID mice was effected by incubating cells with
anti-human CD45-FITC (IQ Products, Groningen, The Netherlands). The
lowest threshold for unequivocal human engraftment was designated
at 0.5%.
[0232] Detection of Transplanted Cells in Mice Treated with
Chemotherapy
[0233] 6.5 week old male NOD.SCID mice (NOD.CB17/JhkiHsd-scid;
Harlan, Rehovot Israel), maintained as described hereinabove for
irradiated mice, were injected intraperitoneally with Busulfan (25
mg/kg--for 2 consecutive days). Two days following the second
Busulfan injection, mice were injected with CD34+ cells alone, or
together with 0.5.times.10.sup.6 adherent cells, produced from the
placenta. 3.5 weeks following transplantation, mice were
sacrificed, and the presence of human hematopoietic cells was
determined as described hereinabove for irradiated mice.
[0234] Experimental Results
[0235] 3D-Adherent Cells Improved Engraftment of HSC in Irradiated
Mice
[0236] Human CD34+ hematopoietic cells and 3D-adherent cells
derived from placenta or adipose tissues were co-transplanted in
irradiated NOD-SCID mice. Engraftment efficiency was evaluated 4
weeks following co-transplantation, and compared to mice
transplanted with HSC alone. As is shown in Table 2,
co-transplantation of 3D-adherent cells and UCB CD34+ cells
resulted in considerably higher engraftment rates and higher levels
of human cells in the BM of recipient mice compared to mice treated
with UCB CD34+ cells alone.
TABLE-US-00002 TABLE 2 Average Transplanted cells h-CD45 STDEV CD34
3.8 7.9 CD34 + 3D-adherent cells from placenta 5.1 12.2 CD34 +
3D-adherent cells from adipose 8.7 9.6
[0237] 3D-Adherent Cells Improved Engraftment of HSC in Mice
Treated with Chemotherapy
[0238] Human CD34+ hematopoietic cells were co-transplanted with
500,000-2D-adherent cells or 3D-adherent cells derived from
placenta. into NOD-SCID mice pretreated with chemotherapy.
Engraftment efficiency was evaluated 3.5 weeks following
co-transplantation, and compared to mice transplanted with HSC
alone. As is shown in Table 3 and FIG. 6, co-transplantation of
adherent cells and UCB CD34+ cells resulted in higher engraftment
levels in the BM of the recipient mice compared to UCB CD34+ cells
alone. Moreover, as is shown in Table 3, the average level of
engraftment was higher in mice co-transplanted with placenta
derived adherent cells grown in the PluriX bioreactor system
(3D-adherent cells) than in the mice co-transplanted with cells
from the same donor, grown in the conventional static 2D culture
conditions (flask).
TABLE-US-00003 TABLE 3 Average Transplanted cells h-CD45 STDEV CD34
0.9 1.1 CD34 + conventional 2D cultures from placenta 3.5 0.2 CD34
+ 3D-adherent cell from placenta 6.0 7.9
[0239] FACS analysis results shown in FIGS. 7A-B demonstrate the
advantage of co-transplanting adherent cells with hHSCs (FIG. 7B),
and the ability of adherent cells to improve the recovery of the
hematopoietic system following HSC transplantation.
[0240] Taken together, these results show that adherent cells may
serve as supportive cells to improve hematopoietic recovery
following HSCs transplantation (autologous or allogenic). The
ability of the 3D-adherent cells to enhance hematopoietic stem
and/or progenitor cell engraftment following HSCs transplantation
may result from the 3D-adherent cell ability to secrete HSC
supporting cytokines that may improve the homing, self-renewal and
proliferation ability of the transplanted cells, or from the
ability of those cells to rebuild the damaged hematopoietic
microenvironment needed for the homing and proliferation of the
transplantable HSCs
Example 3
The Suppression of Lymphocyte Response by 2D and 3D Cultured
Adherent Cells
[0241] Adherent cells, and particularly 3D-adherent cells, were
found to suppress the immune reaction of human cord blood
mononuclear cells in an MLR assay
[0242] Materials and Experimental Procedures
[0243] Mixed Lymphocyte Reaction (MLR) Assay
[0244] The immunosuppressive and immunoprivilaged properties of 2D
and 3D derived culturing procedures adherent cells produced from
the placenta, were effected by the MLR assay, which measures
histocompatibility at the HLA locus, as effected by the
proliferation rate of incompatible lymphocytes in mixed culturing
of responsive (proliferating) and stimulating (unproliferative)
cells. Human cord blood (CB) mononuclear cells (2.times.10.sup.5)
were used as responsive cells and were stimulated by being
co-cultured with equal amounts (10.sup.5) of irradiated (3000 Rad)
human peripheral blood derived Monocytes (PBMC), or with 2D or 3D
cultured adherent cells, produced from the placenta, or a
combination of adherent cells and PBMCs. Each assay was replicated
three times. Cells were co-cultured for 4 days in RPMI 1640 medium
(containing 20% FBS under humidified 5% CO.sub.2 atmosphere at
37.degree. C.), in a 96-well plate. Plates were pulsed with 1 .mu.C
.sup.3H-thymidine during the last 18 hours of culturing. Cells were
then harvested over fiberglass filter and thymidine uptake was
quantified with a scintillation counter.
[0245] Experimental Results
[0246] FIG. 8A shows the immune response of CB cells as represented
by the elevated proliferation of these cells when stimulated with
PBMCs, which, without being bound by theory. is probably associated
with T cell proliferation in response to HLA incompatibility.
However, a considerably lower level of immune response was
exhibited by these cells when incubated with the adherent cells of
the invention. Moreover, the CB immune response to PBMCs was
substantially reduced when co-incubated with these adherent cells.
Thus, in a similar manner to MSCs, adherent cells were found to
have the potential ability to reduce T cell proliferation of donor
cells. typical of GvHD. Although both cultures, 2D and 3D, reduced
the immune response of the lymphocytes, and in line with the other
advantages of 3D-adherent cells described hereinabove, the 3D
adherent cells were more immunosuppressive.
Example 4
3D Adherent Cell Manufactured by Plurix Compared to 3D Adherent
Cells Manufactured by Celligen
[0247] In order to provide large scale 3D adherent cells, a new
manufacturing system was utilized referred to herein as
Celligen.
[0248] Materials and Experimental Methods
[0249] PluriX.TM. Plug Flow Bioreactor
[0250] As described in Example 1, hereinabove.
[0251] Production of 3D-Adherent Cells by Plurix (PLX Cells)
[0252] As described in Example 1, hereinabove.
[0253] Celligen.TM. Plug Flow Bioreactor
[0254] The production of adherent cells by Celligen.TM. (PLX-C
cells) is composed of several major steps as illustrated in FIG.
8B. The process starts by collection of a placenta from a planned
cesarean delivery at term.
[0255] Adherent cells are then isolated from whole placentas, grown
in tissue culture flasks (2D cultures), harvested and stored in
liquid nitrogen as 2D-Cell Stock (2DCS), the appropriate amount of
2DCS are thawed, washed and seeded onto carriers in bioreactors for
further expansion as 3D-culture. After 1-3 weeks of growth in the
bioreactors, cells are harvested and cryopreserved in gas phase of
liquid nitrogen as PLX-C.
[0256] Receipt of Human Tissue
[0257] All placentas obtained were received from the maternity ward
under approval of the Helsinki Committee of the medical facility.
Accordingly, all placenta donors signed an informed consent and
Donor Screening and Donor Testing was performed (IPC1). Immediately
after taking the placenta from the donor (during the caesarean
procedure). it was placed in a sterile plastic bag and then in a
Styrofoam box with ice packs. The placenta was delivered and
immediately placed in a quarantine area until released to use by
Quality Control (QC) and Quality Assurance (QA). All the following
production steps were performed in a quarantine, clean room
facility until QC approval of mycoplasma test results arrived and
the cells were release for 2D cell growth.
[0258] Recovery and Processing of Adherent Cells
[0259] To initiate the process, the whole placenta was cut into
pieces under aseptic conditions under laminar flow hood, washed
with Hank's buffer solution and incubated for 3 hours at 37.degree.
C. with 0.1% Collagenase (1 mg Collagenase/ml tissue). 2D cell
medium (2D-Medium comprising DMEM supplemented with 10% FBS,
fungizone 0.25 .mu.g/ml and gentamycine 50 .mu.g/ml) was added and
the digested tissue was roughly filtered through a sterile metal
strainer, collected in a sterile beaker and centrifuged (10
minutes, 1200 RPM, 4.degree. C. Using gentle pipeting, suspended
cells were then washed with 2D-Medium supplemented with
antibiotics, seeded in 80 cm.sup.2 flasks and incubated at
37.degree. C. in a tissue culture incubator under humidified
condition supplemented with 5% CO.sub.2. Following 2-3 days, in
which the cells were allowed to adhere to the flask surface, they
were washed with PBS and 2D-Medium was added.
[0260] Two Dimensional (2D) Cell Growth
[0261] Prior to the first passage, growth medium samples of 10% of
the total flask number in quarantine was pooled and taken for
mycoplasma testing (IPC2). If cells were found to be negative for
Mycoplasma (EZ-PCR Mycoplasma kit, Biological Industries, Israel),
cells were released from quarantine. After 1-2 additional passages,
cells were transferred to the 2D production clean room (2DP). Once
in Room 2DP, culture was continued for another 3-5 passages. IPC-3
sample was taken for immune phenotype after passage 4. Throughout
the process, cultures were grown in 2D-Medium without antibiotics
in a tissue culture incubator under humidified conditions with 5%
CO2 at 37.degree. C. After a total of 6-8 passages (9-16 cell
doublings), cells were collected and cryopreserved as the 2D-Cell
Stock (2DCS).
[0262] The first passage was usually carried out after 10-15 days.
Beginning at passage 2 and continuing until passage 6-8, cells were
passaged when the culture reached 70-80% confluence, usually after
3-5 days (1.5-2 doublings). The cells were detached from the flasks
using 0.25% trypsin-EDTA (4 minutes at 37.degree. C.) and seeded in
a culture density of 3.+-.0.2.times.10.sup.3 cells/cm.sup.2. The
size of the tissue culture flasks raised as the passages proceed.
The culturing process started in 80 cm.sup.2 tissue culture flask,
continued in 175 cm.sup.2, then in 500 cm.sup.2 (Triple flask) and
finally the cells were seeded into Cell Factory 10 tray (6320
cm.sup.2).
[0263] Prior to cryopreservation, at the end of 2DCS growth period,
the growth medium was collected and the sample was prepared to be
sent to an approved GLP laboratory for Mycoplasma test (IPC 4).
[0264] Cryopreservation Procedure for 2D-Cell-Stock Product
[0265] For 2DCS cryopreservation, 2D-cultured cells were collected
under aseptic conditions using 0.25% trypsin-EDTA. The cells were
centrifuged (1200 RPM, 10', 4.degree. C.), counted and re-suspended
in 2D-Medium.
[0266] For freezing, cell suspensions were diluted 1:1 with
2D-Freezing Mixture (final concentrations was 10% DMSO, 40% FBS and
50% 2D-Medium). Approximately 1.5-2.5.times.10.sup.9 cells were
manufactured from one placenta, 4 ml of the cells were stored at a
final concentration of 10.times.10/ml in 5 ml cryopreservation
polypropylene vials. The vials were labeled and transferred to a
controlled rate freezer for a graduated temperature reducing
process (1.degree. C./min), after which they were transferred to
storage in gas-phase of a liquid nitrogen freezer located in the
Cold Storage Room. This material was referred to as the 2D-Cell
Stock (2DCS) batch.
[0267] Initiation of the Three Dimensional (3D) Culture
Procedures
[0268] To begin 3D culture, an appropriate amount
(150.+-.30.times.10.sup.6) of cells from 2DCS were thawed in the
2DP room and washed with 3D-Medium (DMEM with 10% FBS and 20 Mm
Hepes) to remove DMSO prior to seeding in the prepared-in-advanced
bioreactor systems. The content of each 2DCS vial was pipetted and
diluted 1:9 with pre-warmed (37.degree. C.) 3D-Medium. The cells
were centrifuged (1200 RPM, 10', 4.degree. C.) and re-suspended
again in 50-100 ml pre-warmed (37.degree. C.) 3D-Medium in a 250 ml
sterile bottle. A sample was taken and cells were counted using a
Trypan Blue stain in order to determine cell number and viability.
The cell suspension was transferred under a laminar flow hood into
a 0.5 L seeding bottle. From the seeding bottle the cell suspension
was transferred via sterile tubing to the bioreactor by
gravitation.
[0269] Production of 3D-Adherent Cells in the Celligen Bioreactor
(PLX-C) Bioreactor Description
[0270] 3D growth phase was performed using an automatic CelliGen
Plus.RTM. or BIOFLO 310 bioreactor system [(New Brunswick
Scientific (NBS)] depicted in FIG. 8C. The bioreactor system was
used for cultivation of cell culture, in which conditions were
suitable for high cell concentrations. The cultivation process was
carried out using a bioreactor in a perfusion mode. The lab scale
bioreactor was constructed of two main systems--the control system
and the bioreactor itself (vessel and accessories). The parameters
of the process were monitored and controlled by a control console
which included connectors for probes, motor and pumps, control
loops for Dissolved Oxygen (DO). pH, perfusion and agitation (with
a motor), a gases control system, water circulation and heating
system for temperature control and an operator interface. The
controlled process parameters (such as temperature, pH, DO etc.)
could be displayed on the operator interface and monitored by a
designated controller.
[0271] Cell Culture Growth Procedure in the Bioreactors
[0272] As noted in the section hereinabove,
150.+-.30.times.10.sup.6 cells from the cryopreserved 2DCS were
thawed, washed and seeded in a sterile bioreactor. The bioreactor
contained 30-50 gr caniers (FibraCel.RTM. disks, NBS), made of
Polyester and Polypropylene and 1.5.+-.0.1 L 3D-Medium. The growth
medium in the bioreactor was kept at the following conditions:
37.degree. C., 70% Dissolved Oxygen (DO) and pH 7.3. Filtered gases
(Air, CO.sub.2, N.sub.2 and O.sub.2) were supplied as determined by
the control system in order to keep the DO value at 70% and the pH
value at 7.3. For the first 24 hours, the medium was agitated at 50
Rounds Per Minutes (RPM) and increased up to 200 RPM by day 2. For
the first 2-3 days, the cells were grown in a batch mode. Perfusion
was initiated when the medium glucose concentration decreased below
550 mg/liter. The medium was pumped from the feeding container to
the bioreactor using sterile silicone tubing. All tubing
connections were performed under laminar flow using sterile
connectors. The perfusion was adjusted on a daily basis in order to
keep the glucose concentration constant at approximately 550.+-.50
mg/liter. A sample of the growth medium was taken every 1-2 days
for glucose, lactate, glutamine, glutamate and ammonium
concentration determination (BioProfile 400 analyzer, Nova
Biomedical). The glucose consumption rate and the lactate formation
rate of the cell culture enabled to measure cell growth rate. These
parameters were used to determine the harvest time based on
accumulated experimental data.
[0273] Harvest of the 3D Grown PLX-X Cells from the Bioreactor
[0274] The cell harvest process started at the end of the growth
phase (4-10 days). Two samples of the growth medium were collected.
One sample was prepared to be sent to an approved GLP laboratory
for Mycoplasma testing according to USP and Eu standards, and the
other one was transferred to a controlled rate freezer for a
graduated temperature reducing process (1.degree. C./min), after
which they were transferred to storage in gas-phase of a liquid
nitrogen freezer located in the Cold Storage Room, in case a repeat
Mycoplasma testing was needed. These medium samples were considered
as part of the Mycoplasma testing of the final product and the
results were considered as part of the criteria for product
release.
[0275] The 3D-grown culture was harvested in the Class-100 laminar
area in room 3DP as follows:
[0276] The bioreactor vessel was emptied using gravitation via
tubing to a waste container. The vessel was opened, by removing the
head plate, and the carriers were aseptically transferred, using
sterile forceps, from the basket to the upper basket net (see FIG.
5C). The bioreactor vessel was then closed and refilled with 1.5 L
pre-warmed PBS (37.degree. C.). The agitation speed was increased
to 150 RPM for 2 minutes. The PBS was drained via tubing by
pressure or gravity to the waste bottle. The washing procedure was
repeated twice.
[0277] In order to release the cells from the carriers, 1.5 L
pre-warmed to 37.degree. C. Trypsin-EDTA (Trypsin 0.25%, EDTA 1 mM)
was added to the bioreactor vessel and carriers were agitated for 5
minutes in 150 RPM, 37.degree. C. Cell suspension was collected to
a 5 L sterile container containing 250 ml FBS. Cell suspension was
divided to 4 500 ml sterile centrifuge tubes and a Mycoplasma test
sample was withdrawn. Closed centrifuge tubes were transferred
through the 3DP active pass-through into the class 10,000 filling
room (FR1) in which the cells were aseptically filled and
cryopreserved as PLX-C.
[0278] Cell Cycle Analysis
[0279] PLX-C cells obtained by Celligen and PLX cells obtained by
Plurix were fixed with 70% EtOH O.N. centrifuged and re-suspended
in a Propidium Iodide (PI) solution containing 2 .mu.g/ml PI
(Sigma), 0.2 mg/ml Rnase A (Sigma) and 0.1% (v/v) Triton (Sigma)
for 30 minutes. Cell cycle was analyzed by FACS.
[0280] Gene Expression Array (Microarray)
[0281] Adherent cells were obtained from human full term placentas
and were expanded Plurix or by Celligen. Three different batches of
cells were obtained from each of the expansion methods for further
examination.
[0282] RNA was extracted from the cells (Qiagen-Rneasy micro kit)
and applied to an Affymetrix whole genome expression array. The
chip used GeneChip.RTM. Human Exon 1.0 ST Array (Affymetrix, Santa
Clara, Calif., USA).
[0283] FACS Analysis of Membrane Markers
[0284] Cells were stained with monoclonal antibodies as previously
described. In short, 400,000-600,000 cells were suspended in 0.1 ml
flow cytometer buffer in a 5 ml test tube and incubated for 15
minutes at room temperature (RT), in the dark, with each of the
following monoclonal antibodies (MAbs): FITC-conjugated anti-human
CD29 MAb (eBioscience), PE conjugated anti human CD73 MAb (Becton
Dickinson), PE conjugated anti human CD105 MAb (eBioscience), PE
conjugated anti human CD90 MAb (Becton Dickinson), FITC-conjugated
anti-human CD45 MAb (IQProducts), PE-conjugated anti-human CDI9 MAb
(IQProducts), PE conjugated anti human CD14 MAb (IQProducts), FITC
conjugated anti human HLA-DR MAb (IQProduct), PE conjugated anti
human CD34 MAb (IQProducts), FITC conjugated anti human CD31 MAb
(eBioscience), FITC conjugated anti human KDR MAb (R&D
systems), anti human fibroblasts marker (D7-FIB) MAb(ACRIS),
FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugated
anti-human CD86 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD),
FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITC
conjugated (IQ Products), Isotype IgG1 PE conjugated (IQ
Products).
[0285] Cells were washed twice with flow cytometer buffer,
resuspended in 500 .mu.l flow cytometer buffer and analyzed by flow
cytometry using FC-500 Flow Cytometer (Beckman Coulter). Negative
controls were prepared with relevant isotype fluorescence
molecules.
[0286] Mixed Lymphocyte Reaction (MLR)
[0287] 2.times.10.sup.5 peripheral blood (PB) derived MNC (from
donor A) were stimulated with equal amount of irradiated (3000 Rad)
PB derived MNCs (from donor B). Increasing amounts of PLX-Cs were
added to the cultures. Three replicates of each group were seeded
in 96-well plates. Cells were cultured in RPMI 1640 medium
containing 20% FBS. Plates were pulsed with 1
.mu.C-.sup.3H-thymidine during the last 18 hrs of the 5-day
culturing. Cells were harvested over a fiberglass filter and
thymidine uptake was quantified with scintillation counter.
[0288] For CFSE staining, PB-MNC cells were stained for CFSE
(Molecular Probes) for proliferation measurement before culturing.
Cells were collected after 5 days and the intensity of CFSE
staining was detected by Flow Cytometry.
[0289] ELISA
[0290] ELISA was carried out as was previously described. In short,
MNCs (isolated from peripheral blood) were stimulated with 5
.mu.g/ml ConA (Sigma), 0.5 .mu.g/ml LPS (SIGMA), or 10 .mu.g/ml PHA
(SIGMA) in the presence of PLX-C under humidified 5% CO2 atmosphere
at 37.degree. C. Supernatants were collected and subjected to
cytokine analysis using ELISA kits for IFN.gamma. (DIACLONE),
TNF.alpha. (DIACLONE) and IL-10 (DIACLONE).
[0291] Experimental Results
[0292] The changes in manufacturing with Celligen as compared to
Plurix resulted in several major differences (summarized in Table
4, below).
TABLE-US-00004 TABLE 4 Comparison between Plurix system and
Celligen system Cell growth Teachings of according to the present
Parameter Example 1 invention Improvement Working 280 1500 Scale up
of volume (ml) the process. Higher production level in the present
teachings (2-8 population doubling) Weight of 1.4 30 Scale up of
carrier (gr) the process Bed Conic, 50 ml Cylinder Present
configuration column Packed bed teachings - Better flow of medium
and nutrients. Teachings of Example 1 - Inefficient flow due to
narrow outlet form of the conic structure Better homogeneity of
medium flow. Channeling in the teachings of Example 1. Cell 3
.times. 10.sup.6 cell/ 5 .times. 10.sup.6 cell/ Better cell to
concentration gr carrier gr carrier cell interaction at seeding in
the present (cell/gr teachings carrier) Cell 0.015 .times. 10.sup.6
0.1 .times. 10.sup.6 Better cell to concentration cell/ml cell/ml
cell interaction at seeding in the present (cell/ml) teachings
Seeding Seeding at Seeding at the Teachings of procedure low medium
final working Example 1 - volume for volume while Heterogenic 24 h
followed agitating distribution of by addition of the cell medium
to culture inside final working the carrier bed. volume
Insufficient medium volume in the first 24 h of the run. Leading to
unsuitable working conditions (acidic environment) Production 14-21
4-10 Better product phase days days quality. duration Efficient
harvest process. Better yield. Lower cost process in the present
teachings Mode of Repeated Perfusion Present operation batch - mode
- rate teachings - medium was adjusted Moderate change twice
according to changes of the a week the glucose conditions
concentration regarding (the medium medium was changed composition
at glucose throughout concentration the run of 550 .+-. 50
Continuous mg/L) removal of toxic agents produced by the cells. In
batch mode - lower concentration of essential nutrients (limiting
factors) Less cell debris Harvest Harvesting in Harvesting Present
procedure 50 ml tubes inside the teachings - Trypsinization
bioreactor More efficient 3 cycles Trypsinization process 1 cycle
Harvest is carried out in a close system. 1 trypsinization cycle -
better quality of the cells. Agitation medium Cell lift Present
Circulation impeller teachings - between Medium is reservoir
flowing container to through the the column packed bed - using
Better supply peristaltic of nutrients pump and oxygen to the
culture. Homogeneity of the medium Improves other control loops
(temp., DO, pH) Temperature The On-line direct Present control
production control. teachings - was carried Heat transfer More
accurate out inside an via water measurement incubator. jacket. of
the culture Indirect temperature. temperature Quick control (of the
response. incubator Short time to chamber). reach set Heat transfer
point. via air interface Temperature Manually. On-line direct
Present monitoring Indirect water monitoring. teachings -
temperature Better monitoring. monitoring and control of the
process. Quick response to malfunctions. DO None On-line Present
monitoring monitoring teachings - Better monitoring and control of
the process. Quick response to malfunctions DO None. On-line direct
Present control Introduction control of a teachings - of air only
specific set Better point using control of Air, O.sub.2 and DO
level. N.sub.2. Better maintenance of a specified working
conditions pH Only visual On-line Present monitoring monitoring
Control and teachings - and control (Phenol red as monitoring
Better part of the control of medium) pH level. Better maintenance
of a specified working conditions Aeration Sparge only Overlay
Teachings of (sparge as an Example 1 - option) Aeration by sparge
creates foam that might damage the cells.
[0293] The changes in the manufacturing process resulted in changes
in characteristics of the obtained 3D adherent cells. These
differences are summarized below.
[0294] Cell Cycle Analysis of PLX Manufactured by Plurix Compared
to PLX-C Manufactured by Celligen
[0295] PLX-C cells obtained by Celligen were compared to PLX cells
obtained by Plurix in order to examine the distribution of the
cells between the different phases of the cell cycle. As is clear
from FIGS. 9A-B, PLX-C cells expanded by Celligen exhibited typical
proliferating profile (distribution of cells between the different
phases of cell cycle). Specifically, 28% of cells were in S and
G2/M phases (FIG. 9A). These results indicated that cells were
harvested during proliferation and that the Celligen bioreactor
conditions supported cell growth.
[0296] Microarray Comparison Between Plurix and Celligen Obtained
Cells
[0297] Gene expression arrays enabled to simultaneously monitor
genome-wide expression profiles of adherent cells derived from
human full term placentas expanded by Plurix (PLX) or by Celligen
(PLX-C). These results enabled to asses the molecular mechanism
underlying phenotypic variation between cells obtained by these
different growth methods (see Table 5, below).
TABLE-US-00005 TABLE 5 Gene expression in Celligen compared to
Plurix cells Celligen vs. Plurix p-value Gene (fold change) (treat)
interferon-induced protein with tetratricopeptide repeats 17.52
0.0401812 aldehyde dehydrogenase 1 family, member A1 16.76
0.00145807 leukocyte-derived arginine aminopeptidase 13.99 3.88E-06
keratin 27 pseudogene 27 12.25 0.000224998 similar to Keratin, type
I cytoskeletal 18 (Cytokerati 11.83 0.000304949 G protein-coupled
receptor, family C, group 5, member A 10.35 3.39E-05 integrin,
alpha 6 9.84 0.0411667 G protein-coupled receptor 126 8.73
0.00197635 coagulation factor III (thromboplastin, tissue factor)
7.36 0.012192 Rho GDP dissociation inhibitor (GDI) beta 7.36
0.00200066 signal peptide, CUB domain, EGF-like 3 7.20 0.0255115
interferon-induced protein with tetratricopeptide repeats 7.09
0.0139777 dickkopf homolog 1 (Xenopus laevis) 7.06 3.06E-07 NAD(P)H
dehydrogenase, quinone 1 6.63 0.000282423 keratin 18 6.46
0.000514523 opioid growth factor receptor-like 1 5.96 0.00114551
mal, T-cell differentiation protein-like 5.95 0.00664216
neurofilament, medium polypeptide 150 kDa 5.86 0.0190611 DEP domain
containing 1 5.82 0.000370513 cathepsin C 5.72 0.00532262 WAS 5.47
0.00178153 serpin peptidase inhibitor, clade B (ovalbumin), member
5.44 0.0190218 solute carrier family 7, (cationic amino acid
transporte 5.33 0.00688017 interferon-induced protein with
tetratricopeptide repea 5.18 0.00357376 NUF2, NDC80 kinetochore
complex component, homolog (S. cere 5.05 0.00276524 SHC SH2-domain
binding protein 1 4.95 0.00430878 thioredoxin reductase 1 4.86
0.000197486 lung cancer metastasis-associated protein 4.85
0.00148024 Rho GTPase activating protein 29 4.85 0.0466211 cell
division cycle 20 homolog (S. cerevisiae) 4.80 0.00514206 family
with sequence similarity 111, member B 4.63 0.000125819 PDZ binding
kinase 4.54 0.00784983 establishment of cohesion 1 homolog 2 (S.
cerevisiae) 4.53 0.000773033 guanylate binding protein 4 4.47
0.000215944 lipase A, lysosomal acid, cholesterol esterase (Wolman
dise 4.42 0.0167385 kinesin family member 20A 4.39 0.00582352
KIAA0101 4.28 0.0105909 cyclin-dependent kinase inhibitor 3
(CDK2-associated dual 4.25 0.000732492 thymidylate synthetase 4.23
0.00685584 chromosome 13 open reading frame 3 4.18 0.000548296
aurora kinase A 4.16 0.00632571 nei endonuclease VIII-like 3 (E.
coli) 4.14 0.00115606 centrosomal protein 55 kDa 4.13 0.0021952
oxidized low density lipoprotein (lectin-like) receptor 1 4.11
0.0205198 denticleless homolog (Drosophila) 4.05 0.00141153
anillin, actin binding protein 4.01 0.010923 ribonucleotide
reductase M2 polypeptide 3.98 0.00834059 ankyrin repeat domain 1
(cardiac muscle) 3.93 0.00911953 transcription factor 19 (SC1) 3.89
0.00109627 keratin 18 3.89 0.000112551 non-SMC condensin I complex,
subunit G 3.88 0.00537097 cyclin E2 3.87 0.000203389 trypsinogen C
3.86 0.00416276 small nucleolar RNA, C 3.81 0.0334484 tight
junction protein 2 (zona occludens 2) 3.81 0.00012562 kinesin
family member 18A 3.78 0.00134108 kinesin family member 2C 3.77
0.0059888 shugoshin-like 1 (S. pombe) 3.76 0.00101318 polo-like
kinase 1 (Drosophila) 3.75 0.0140309 thymidine kinase 1, soluble
3.73 0.00124134 transcription factor 19 (SC1) 3.73 0.00124327
transcription factor 19 (SC1) 3.73 0.00124327 claspin homolog
(Xenopus laevis) 3.71 0.00683624 GINS complex subunit 1 (Psf1
homolog) 3.69 0.00104515 microsomal glutathione S-transferase 1
3.67 0.041701 arylacetamide deacetylase-like 1 3.67 0.000902645
SPC25, NDC80 kinetochore complex component, homolog (S. ce 3.65
0.00568662 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of
VLA-4 3.62 0.0158411 catenin (cadherin-associated protein),
alpha-like 1 3.57 7.46E-05 discs, large homolog 7 (Drosophila) 3.56
0.0317074 v-myb myeloblastosis viral oncogene homolog (avian)-lik
3.55 0.0043878 serglycin 3.54 0.0443487 centromere protein N 3.53
0.000540143 cyclin A2 3.53 0.00965934 heat shock 22 kDa protein 8
3.52 0.0219583 sema domain, immunoglobulin domain (Ig), short basic
doma 3.49 0.008548 Rho GTPase activating protein 11A 3.49
0.00834174 Fanconi anemia, complementation group I 3.43 0.00464532
BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast 3.42
0.0108258 ovary-specific acidic protein 3.42 0.00334641 cholinergic
receptor, muscarinic 2 3.41 0.0320078 cell division cycle 2, G1 to
S and G2 to M 3.41 0.0017111 protein regulator of cytokinesis 1
3.39 0.0325664 minichromosome maintenance complex component 5 3.38
0.00475504 sperm associated antigen 5 3.37 0.00906321 maternal
embryonic leucine zipper kinase 3.34 0.00908391 small nucleolar
RNA, C 3.33 0.0298703 carnitine palmitoyltransferase 1A (liver)
3.33 0.00170894 similar to Ubiquitin-conjugating enzyme E2S (Ubiqui
3.33 0.000415822 kinesin family member 11 3.33 0.00915145 NIMA
(never in mitosis gene a)-related kinase 7 3.33 0.00159114 ADAM
metallopeptidase with thrombospondin type 1 motif, 3.32 0.0102751
transforming, acidic coiled-coil containing protein 3 3.31
0.0014577 cyclin B1 3.29 0.0103092 MAD2 mitotic arrest
deficient-like 1 (yeast) 3.28 0.00488102 dihydrofolate reductase
3.28 0.00178879 NIPA-like domain containing 3 3.27 0.00164708 cell
division cycle associated 2 3.26 0.0122226 apolipoprotein B mRNA
editing enzyme, catalytic polypep 3.26 0.00308692 cyclin B2 3.25
0.016544 endonuclease domain containing 1 3.24 0.000429245
dihydrofolate reductase pseudogene 3.23 0.00141306 ATPase, Na+ 3.23
0.000381464 replication factor C (activator 1) 3, 38 kDa 3.23
0.00109668 WD repeat domain 76 3.22 0.0023531 pleckstrin 2 3.17
0.0304429 Rac GTPase activating protein 1 3.17 0.00381613 PHD
finger protein 19 3.17 0.000177604 deleted in lymphocytic leukemia,
2 3.15 0.0109528 centromere protein I 3.15 0.0106816 BRCA1
associated RING domain 1 3.14 0.000540414 regulator of G-protein
signalling 4 3.13 0.00781061 STAM binding protein-like 1 3.11
0.0181743 sulfiredoxin 1 homolog (S. cerevisiae) 3.10 5.14E-05
chromosome 15 open reading frame 23 3.08 0.000147331 TTK protein
kinase 3.08 0.0112171 non-SMC condensin II complex, subunit G2 3.08
0.0130322 villin 2 (ezrin) 3.07 0.0131934 stomatin 3.06 0.00387095
protein tyrosine phosphatase-like A domain containing 3.06
0.0419644 serpin peptidase inhibitor, clade B (ovalbumin), member
3.05 0.0030439 kinesin family member 4A 3.05 0.0114203 hypothetical
protein DKFZp762E1312 3.05 0.00726778 ubiquitin-conjugating enzyme
E2S 3.04 0.00118205 hydroxysteroid dehydrogenase like 2 3.03
3.71E-05 ATPase family, AAA domain containing 2 3.01 0.00415258
TPX2, microtubule-associated, homolog (Xenopus laevis) 3.00
0.0253137 histone cluster 1, H4d 3.00 0.030183 kinesin family
member 23 2.99 0.00790585 heat shock 70 kDa protein 2 2.99
0.0215102 origin recognition complex, subunit 1-like (yeast) 2.99
0.00207753 dihydrofolate reductase 2.98 0.00307793
hyaluronan-mediated motility receptor (RHAMM) 2.97 0.00467816
3'-phosphoadenosine 5'-phosphosulfate synthase 2 2.97 1.43E-05
glycerol-3-phosphate dehydrogenase 2 (mitochondrial) 2.95
0.00211969 nucleolar and spindle associated protein 1 2.95
0.00520875 diaphanous homolog 3 (Drosophila) 2.95 0.00107709
kinesin family member 14 2.94 0.00947901 histone cluster 1, H1b
2.93 0.0470898 guanine nucleotide binding protein (G protein),
alpha inhi 2.92 0.00184597 minichromosome maintenance complex
component 8 2.92 0.000841489 cancer susceptibility candidate 5 2.92
0.0330594 leukotriene B4 12-hydroxydehydrogenase 2.92 0.000685452
glutamate-cysteine ligase, modifier subunit 2.91 0.00378868
forkhead box M1 2.91 0.0203154 adipose differentiation-related
protein 2.90 0.000331751 membrane bound O-acyltransferase domain
containing 1 2.90 0.01185 ubiquitin-conjugating enzyme E2T
(putative) 2.90 0.00741886 cell division cycle associated 3 2.89
0.006289 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3
2.88 0.00574148 coagulation factor XIII, B polypeptide 2.88
0.0294465 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)
2.87 0.000854739 ATP-binding cassette, sub-family C (CFTR 2.87
0.00382491 family with sequence similarity 29, member A 2.85
0.00111165 SH2 domain containing 4A 2.84 0.0323646 membrane
protein, palmitoylated 1, 55 kDa 2.84 0.000396285 CDC28 protein
kinase regulatory subunit 1B 2.84 0.0107391 PSMC3 interacting
protein 2.84 0.00766442 elastin microfibril interfacer 2 2.84
0.0192072 topoisomerase (DNA) II alpha 170 kDa 2.83 0.0321109
transmembrane protein 106C 2.82 0.000214223 histone cluster 1, H3b
2.80 0.0304598 chromosome 18 open reading frame 24 2.80 0.00347442
epidermal growth factor receptor pathway substrate 8 2.79 0.0194949
high-mobility group nucleosomal binding domain 2 2.78 0.0030536 SCL
2.78 0.00390288 hect domain and RLD 4 2.78 0.00679184 ASF1
anti-silencing function 1 homolog B (S. cerevisiae) 2.77 0.00543408
thyroid hormone receptor interactor 13 2.76 0.0118319 cell division
cycle associated 8 2.75 0.00619878 kinesin family member C1 2.74
0.00821937 high-mobility group nucleosomal binding domain 2 2.73
0.00384071 ornithine decarboxylase 1 2.73 0.00144868 v-myb
myeloblastosis viral oncogene homolog (avian)-like 2 2.71
0.00989416 KIT ligand 2.70 0.00641955 dual-specificity
tyrosine-(Y)-phosphorylation regulated ki 2.70 0.0234606
intraflagellar transport 80 homolog (Chlamydomonas) 2.70 0.0247286
transmembrane protein 48 2.69 0.00458248 EBNA1 binding protein 2
2.69 0.00296292 ZW10 interactor 2.69 1.88E-05 exonuclease 1 2.68
0.00739393 transketolase (Wernicke-Korsakoff syndrome) 2.68
1.92E-05 somatostatin receptor 1 2.68 0.0144901 isocitrate
dehydrogenase 3 (NAD+) alpha 2.67 0.00297129 cytoskeleton
associated protein 2 2.67 0.0030499 minichromosome maintenance
complex component 4 2.67 0.00342054 inhibitor of DNA binding 1,
dominant negative helix-loop-hel 2.66 0.036485 CDC28 protein kinase
regulatory subunit 1B 2.66 0.0145263 keratin 18 2.66 8.40E-05 CD97
molecule 2.66 0.00994045 chromosome 6 open reading frame 173 2.64
0.00222408 BTB (POZ) domain containing 3 2.62 0.0166824 deafness,
autosomal dominant 5 2.62 0.00235481 KIAA0286 protein 2.62
0.00130563 Fanconi anemia, complementation group D2 2.61 0.0281405
polo-like kinase 4 (Drosophila) 2.60 0.00209633 ribonucleotide
reductase M1 polypeptide 2.60 0.000170076 malic enzyme 1,
NADP(+)-dependent, cytosolic 2.59 0.0435444 non-SMC condensin I
complex, subunit H 2.59 0.0216752 S100 calcium binding protein A3
2.58 0.0324073 ubiquitin-conjugating enzyme E2L 3 2.57 0.00343347
BUB1 budding uninhibited by benzimidazoles 1 homolog beta 2.56
0.0166047 glycerol kinase 2.55 2.66E-05 TAF9B RNA polymerase II,
TATA box binding protein 2.54 0.0170365 (TBP)-as TAF9B RNA
polymerase II, TATA box binding protein 2.54 0.0170365 (TBP)-as
histone cluster 1, H2bg 2.52 0.000180822 high-mobility group box 2
2.52 0.0196872 NIMA (never in mitosis gene a)-related kinase 2 2.50
0.00289469 proline rich 11 2.50 0.0357125 myopalladin 2.49
0.0255088 brix domain containing 1 2.49 0.00471977 cell division
cycle associated 5 2.49 0.01021 fucosidase, alpha-L- 2, plasma 2.49
0.00540929 cyclin-dependent kinase 2 2.49 0.00250724 lamin B
receptor 2.49 0.000151784 hypoxanthine phosphoribosyltransferase 1
(Lesch-Nyhan synd 2.49 0.000634057 tripartite motif-containing 25
2.47 0.0456344 proteasome (prosome, macropain) subunit, beta type,
9 (lar 2.46 0.0202595 proteasome (prosome, macropain) subunit, beta
type, 9 (lar 2.46 0.0202595 proteasome (prosome, macropain)
subunit, beta type, 9 (lar 2.46 0.0202595 sphingomyelin synthase 2
2.46 0.0020701 transmembrane protein 62 2.45 0.00761064
glucose-6-phosphate dehydrogenase 2.44 0.00278311 PHD finger
protein 1 2.44 0.010191 retinoblastoma-like 1 (p107) 2.44
0.00319946 KIAA1524 2.43 0.0380688 ST6
(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 2.43 0.00830766
cofilin 2 (muscle) 2.43 0.0459235 hypothetical protein LOC201725
2.42 0.000313319 cell division cycle 25 homolog A (S. pombe) 2.42
0.000341692 breast cancer 1, early onset 2.41 0.0180553
transaldolase 1 2.41 0.00199537
mRNA turnover 4 homolog (S. cerevisiae) 2.41 0.00373104
glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N- 2.41
0.0197148 cysteine rich transmembrane BMP regulator 1
(chordin-like) 2.41 0.0267286 tissue factor pathway inhibitor
(lipoprotein-associated 2.40 0.0356227 chromosome 16 open reading
frame 59 2.40 0.00185191 glycogenin 1 2.39 0.0224317 transmembrane
protein 154 2.39 0.0045589 tubulointerstitial nephritis
antigen-like 1 2.39 0.00510812 CTP synthase 2.38 8.80E-05
phenylalanyl-tRNA synthetase, beta subunit 2.38 0.000245973
geminin, DNA replication inhibitor 2.38 0.00167629 lamin B1 2.37
0.0477748 SPC24, NDC80 kinetochore complex component, homolog (S.
ce 2.36 0.00287227 glutathione reductase 2.36 0.00353875 ribosomal
protein L22-like 1 2.36 0.00335381 fumarylacetoacetate hydrolase
(fumarylacetoacetase) 2.36 3.88E-05 small nucleolar RNA, C 2.35
0.0188991 family with sequence similarity 64, member A 2.35
0.0019785 epithelial cell transforming sequence 2 oncogene 2.35
0.000571152 polymerase (DNA directed), epsilon 2 (p59 subunit) 2.34
0.00479612 glycerol kinase 2.34 3.37E-06 glutathione S-transferase
M2 (muscle) 2.33 0.0402076 elongation factor, RNA polymerase II, 2
2.33 0.0130017 thioredoxin 2.33 0.009636 polymerase (DNA directed),
alpha 2 (70 kD subunit) 2.32 0.0033903 breast cancer 2, early onset
2.32 0.00586847 CDC45 cell division cycle 45-like (S. cerevisiae)
2.32 0.00735977 H2A histone family, member Z 2.32 0.0129697
transporter 1, ATP-binding cassette, sub-family B (MDR 2.31
0.0164234 transporter 1, ATP-binding cassette, sub-family B (MDR
2.31 0.0164234 transporter 1, ATP-binding cassette, sub-family B
(MDR 2.31 0.0164234 nucleolar complex associated 3 homolog (S.
cerevisiae) 2.30 0.000373346 ATPase, Ca++ transporting, plasma
membrane 4 2.30 0.023011 minichromosome maintenance complex
component 7 2.30 0.0457691 TIMELESS interacting protein 2.29
0.00771062 von Hippel-Lindau binding protein 1 2.28 0.00329061
ras-related C3 botulinum toxin substrate 2 (rho family, sma 2.28
0.0292466 thymopoietin 2.28 0.0223176 peptidylprolyl isomerase F
(cyclophilin F) 2.28 0.00093846 activated leukocyte cell adhesion
molecule 2.27 0.00242163 polycomb group ring finger 5 2.27
0.000294142 Ran GTPase activating protein 1 2.27 9.68E-05
replication factor C (activator 1) 4, 37 kDa 2.26 0.00164152
tubulin, beta 2C 2.26 0.000346744 minichromosome maintenance
complex component 10 2.26 0.0037925 H2B histone family, member S
2.25 0.000885505 gamma-glutamyl hydrolase (conjugase,
folylpolygammaglutamyl 2.25 0.0195219 transcription termination
factor, RNA polymerase II 2.25 0.000393489 polymerase (DNA
directed), delta 2, regulatory subunit 50k 2.25 0.0123823
transporter 1, ATP-binding cassette, sub-family B (MDR 2.25
0.00859077 transporter 1, ATP-binding cassette, sub-family B (MDR
2.25 0.00859077 transporter 1, ATP-binding cassette, sub-family B
(MDR 2.25 0.00859077 histone cluster 1, H2bf 2.25 0.0124279
eukaryotic translation initiation factor 1A, X-linked 2.24
0.00330183 phosphoglucomutase 2 2.24 0.00818204 peroxisomal
D3,D2-enoyl-CoA isomerase 2.24 0.00148722 interferon-induced
protein with tetratricopeptide repeats 2.24 0.0177928 G-2 and
S-phase expressed 1 2.23 0.0241887 minichromosome maintenance
complex component 2 2.23 0.0021347 family with sequence similarity
72, member A 2.23 0.00143248 RMI1, RecQ mediated genome instability
1, homolog (S. cerev 2.23 0.00294705 FLJ20105 protein 2.23
0.0127979 multiple coagulation factor deficiency 2 2.22 0.0116892
phytoceramidase, alkaline 2.22 0.0157729 coiled-coil domain
containing 68 2.22 0.00227586 dedicator of cytokinesis 11 2.21
0.00697577 platelet-derived growth factor alpha polypeptide 2.21
0.00176418 N-acylsphingosine amidohydrolase (non-lysosomal cerami
2.20 0.00728536 S-phase kinase-associated protein 2 (p45) 2.20
0.00230153 polymerase (RNA) III (DNA directed) polypeptide G (32
kD) 2.20 0.0298794 ADP-ribosylation factor-like 6 interacting
protein 1 2.20 0.00139745 histone cluster 1, H2bh 2.19 0.0377748
origin recognition complex, subunit 5-like (yeast) 2.19 0.049697
CDC28 protein kinase regulatory subunit 2 2.19 0.0128024 histone
cluster 1, H4c 2.19 0.0112695 hypothetical protein LOC729012 2.19
0.000446087 DEAD (Asp-Glu-Ala-Asp) box polypeptide 39 2.19
0.000340561 chromatin assembly factor 1, subunit B (p60) 2.18
0.0119687 MLF1 interacting protein 2.18 0.0177203 microtubule
associated serine 2.18 0.00536974 MHC class I polypeptide-related
sequence B 2.18 0.0165406 shugoshin-like 2 (S. pombe) 2.18
0.000852557 COP9 constitutive photomorphogenic homolog subunit 6
(Arab 2.18 0.000793512 methylenetetrahydrofolate dehydrogenase
(NADP+ 2.18 0.00119726 dependent) chromosome 6 open reading frame
167 2.18 0.0011095 pituitary tumor-transforming 1 2.17 0.0485166
ribonuclease H2, subunit A 2.17 0.00669936 X-ray repair
complementing defective repair in Chinese ham 2.16 0.0369865
membrane protein, palmitoylated 5 (MAGUK p55 subfamily memb 2.16
0.00211873 karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
2.16 0.000650645 pleckstrin homology domain containing, family A
(phosphoi 2.15 0.0256434 ribosomal protein L39-like 2.15 0.00429384
karyopherin alpha 2 (RAG cohort 1, importin alpha 1) 2.15
0.000700649 amyloid beta (A4) precursor protein-binding, family B,
m 2.15 0.00201004 minichromosome maintenance complex component 3
2.14 0.0018389 histone cluster 1, H2ai 2.14 0.0129155 chromosome 13
open reading frame 34 2.14 0.000702936 RAD18 homolog (S.
cerevisiae) 2.14 0.0016685 WD repeat and HMG-box DNA binding
protein 1 2.13 0.0034833 sulfide quinone reductase-like (yeast)
2.13 0.0473641 chromosome 16 open reading frame 63 2.12 0.000804179
M-phase phosphoprotein 1 2.12 0.0271814 minichromosome maintenance
complex component 6 2.12 0.0161279 homeobox A9 2.11 0.00520942
fibroblast growth factor 9 (glia-activating factor) 2.10 0.0475844
cell division cycle 25 homolog C (S. pombe) 2.10 0.0169914
chromosome 9 open reading frame 64 2.10 0.0265979 U2AF homology
motif (UHM) kinase 1 2.09 0.0255167 replication factor C (activator
1) 2, 40 kDa 2.09 0.00768959 hypothetical protein LOC440894 2.09
0.0103358 small nuclear ribonucleoprotein D1 polypeptide 16 kDa
2.09 0.0334665 CSE1 chromosome segregation 1-like (yeast) 2.09
0.0013662 phosphatidylinositol glycan anchor biosynthesis, class W
2.09 0.0151967 centromere protein O 2.09 0.00397056 family with
sequence similarity 20, member B 2.09 0.00460031 hypothetical
protein FLJ40869 2.09 0.00444509 guanine nucleotide binding protein
(G protein), gamma 11 2.08 0.00140559 calcyclin binding protein
2.08 0.00524566 ATP-binding cassette, sub-family E (OABP), member 1
2.08 0.00454751 CD44 molecule (Indian blood group) 2.08 0.000651436
exosome component 8 2.08 0.00132017 family with sequence similarity
102, member B 2.08 0.025743 histone cluster 2, H3d 2.07 0.0102932
family with sequence similarity 33, member A 2.07 0.000318673
Fanconi anemia, complementation group B 2.07 0.000255109 kinesin
family member 22 2.07 0.0192406 histone cluster 1, H2ai 2.07
0.0161621 vaccinia related kinase 1 2.06 0.0233182 integrator
complex subunit 7 2.06 0.000841371 flap structure-specific
endonuclease 1 2.06 0.006882 hypothetical protein FLJ25416 2.06
0.000177531 ecotropic viral integration site 2B 2.06 0.0171408
retinitis pigmentosa 2 (X-linked recessive) 2.05 0.0264185
centromere protein L 2.05 0.000880856 cofactor required for Sp1
transcriptional activation, subu 2.04 0.00141809 chromosome 20 open
reading frame 121 2.04 0.0146323 family with sequence similarity
72, member A 2.04 0.00162905 family with sequence similarity 72,
member A 2.04 0.00165234 eukaryotic translation initiation factor
1A, X-linked 2.04 0.00520549 elongation factor, RNA polymerase II,
2 2.03 0.0458007 ATPase, Na+ 2.03 0.0189108 histone cluster 1, H3a
2.03 0.0244273 brix domain containing 1 2.03 0.00981178 sushi
domain containing 1 2.03 0.0258164 ectonucleoside triphosphate
diphosphohydrolase 6 (putativ 2.03 0.00423628 fructosamine 3 kinase
2.03 0.00470972 Bloom syndrome 2.02 0.0209259 tubulin, alpha 1c
2.01 0.00862586 E2F transcription factor 2 2.01 0.0496479 exosome
component 2 2.01 0.00649147 kinesin family member 22 2.01 0.0242075
LTV1 homolog (S. cerevisiae) 2.01 0.00812652 dihydrolipoamide
S-acetyltransferase (E2 component of pyruv 2.01 0.00179011 v-ral
simian leukemia viral oncogene homolog B (ras related 2.01 0.012225
ring finger and WD repeat domain 3 2.01 0.0013797 annexin A1 2.01
0.0173578 elaC homolog 2 (E. coli) 2.00 0.00266504 aldehyde
dehydrogenase 9 family, member A1 2.00 0.00911609 tubulin, alpha 4a
2.00 0.0435427 nuclear pore complex interacting protein -2.00
0.00111223 oculomedin -2.01 0.00778869 similar to
PI-3-kinase-related kinase SMG-1 -2.01 0.0356628 golgi autoantigen,
golgin subfamily a-like pseudogene -2.01 0.00770626 spectrin repeat
containing, nuclear envelope 1 -2.01 0.00438469 nuclear pore
complex interacting protein -2.01 0.00117582 sushi, nidogen and
EGF-like domains 1 -2.01 0.00161129 integrin, alpha V (vitronectin
receptor, alpha polypeptide -2.02 0.00252702 cyclin-dependent
kinase inhibitor 2B (p15, inhibits CDK4) -2.04 0.0150268 lysyl
oxidase-like 4 -2.04 0.0120148 nuclear pore complex interacting
protein -2.04 0.000213956 calcium -2.04 0.00657494 calsyntenin 3
-2.04 0.00300887 cell adhesion molecule 1 -2.05 0.0261129 solute
carrier family 22 (organic cation transporter), -2.05 0.0137275 RUN
and FYVE domain containing 3 -2.05 0.00387265 glucosidase, alpha;
acid (Pompe disease, glycogen storage di -2.05 0.000418401 nuclear
pore complex interacting protein -2.05 0.00988632 proline-rich
nuclear receptor coactivator 1 -2.06 0.0039587 membrane
metallo-endopeptidase -2.06 0.0152684 PHD finger protein 21A -2.06
0.00980401 Rho GTPase-activating protein -2.06 0.00705186 homeobox
B6 -2.06 0.00301714 nuclear pore complex interacting protein -2.07
0.00032839 phospholipase A2 receptor 1, 180 kDa -2.07 0.00069343
nuclear pore complex interacting protein -2.08 0.000352007 slit
homolog 3 (Drosophila) -2.08 0.02844 nuclear pore complex
interacting protein -2.09 0.000414309 cyclin-dependent kinase 6
-2.09 0.0456892 dynamin 1 -2.09 0.00139674 jumonji, AT rich
interactive domain 1B -2.09 0.00861002 calcium binding and
coiled-coil domain 1 -2.09 0.00370041 insulin-like growth factor 1
receptor -2.09 0.00114467 nuclear pore complex interacting protein
-2.10 0.000377834 CD82 molecule -2.10 0.0175517 bromodomain
adjacent to zinc finger domain, 2B -2.10 9.88E-05 -- -2.10
0.00666187 synaptotagmin XI -2.11 0.0129428 KIAA1546 -2.11
0.000255634 jun B proto-oncogene -2.12 0.0120169 CXXC finger 6
-2.12 0.0277527 nuclear pore complex interacting protein -2.14
0.00282604 Cdon homolog (mouse) -2.15 0.0350357 B-cell CLL -2.15
0.00343507 nuclear pore complex interacting protein -2.15
0.00263888 v-abl Abelson murine leukemia viral oncogene homolog 1
-2.16 0.0136688 nuclear pore complex interacting protein -2.16
0.00583397 FAT tumor suppressor homolog 1 (Drosophila) -2.18
0.0158766 transformer-2 alpha -2.18 0.012256 chimerin (chimaerin) 1
-2.18 0.0287031 milk fat globule-EGF factor 8 protein -2.18
0.000987073 vitamin D (1,25-dihydroxyvitamin D3) receptor -2.19
0.000192208 neuroblastoma, suppression of tumorigenicity 1 -2.20
0.00090639 jumonji domain containing 1A -2.20 0.0188513 WNK lysine
deficient protein kinase 1 -2.21 1.57E-05 protocadherin beta 14
-2.21 0.0103892 cortactin binding protein 2 -2.21 2.28E-05 WW
domain containing transcription regulator 1 -2.22 0.0379899 cyclin
L1 -2.22 0.00831474 nuclear factor of activated T-cells,
cytoplasmic, calcine -2.22 0.00786451 pellino homolog 1
(Drosophila) -2.23 0.00939357 golgi autoantigen, golgin subfamily
a-like pseudogene -2.24 0.00603583 chromosome 7 open reading frame
10 -2.26 0.00738442 golgi autoantigen, golgin subfamily a-like
pseudogene -2.27 0.00320764 small Cajal body-specific RNA 17 -2.27
0.0301336 latent transforming growth factor beta binding protein 2
-2.29 4.08E-05 golgi autoantigen, golgin subfamily a, 8A -2.29
0.0111179 inhibin, beta A (activin A, activin AB alpha polypeptide)
-2.29 0.00877271 solute carrier family 41, member 2 -2.30
0.00453672 forkhead box P1 -2.30 0.0463138 matrix metallopeptidase
14 (membrane-inserted) -2.31 1.93E-05 transcription factor 4 -2.31
0.0367869 jun oncogene -2.32 7.21E-05 neuroepithelial cell
transforming gene 1 -2.33 0.0109689 asporin -2.33 0.000659873 v-fos
FBJ murine osteosarcoma viral oncogene homolog -2.35 0.0138624
ephrin-B2 -2.36 0.00611474 WD repeat and SOCS box-containing 1
-2.36 0.0387851 similar to dJ402H5.2 (novel protein similar to wo
-2.36 0.00621503
PX domain containing serine -2.38 0.000927628 collagen, type VII,
alpha 1 (epidermolysis bullosa, dystr -2.38 0.00109233 AE binding
protein 1 -2.39 0.000105628 peroxidasin homolog (Drosophila) -2.40
0.00219049 calcium channel, voltage-dependent, L type, alpha 1C sub
-2.41 0.0189661 Prader-Willi syndrome chromosome region 1 -2.45
0.0415526 midline 1 (Opitz -2.45 0.00130803 nuclear pore complex
interacting protein -2.45 0.00354416 chromosome 1 open reading
frame 54 -2.47 0.0186089 transmembrane protein 16A -2.48 0.0481085
basic helix-loop-helix domain containing, class B, 2 -2.49
0.00270257 nuclear pore complex interacting protein -2.50
0.00316496 runt-related transcription factor 1 (acute myeloid
leukemi -2.50 0.000607387 zinc finger protein 292 -2.50 0.029832
fibronectin leucine rich transmembrane protein 2 -2.51 0.0135122
nuclear pore complex interacting protein -2.51 0.00283418 potassium
voltage-gated channel, subfamily G, member 1 -2.54 0.0244306
interleukin 19 -2.54 0.0310328 transforming growth factor, beta 3
-2.54 0.0287865 dihydropyrimidinase-like 3 -2.55 0.0165203 golgi
autoantigen, golgin subfamily a, 8B -2.56 0.0121417 hypothetical
protein PRO2012 -2.57 0.00756704 SATB homeobox 2 -2.57 0.039781
t-complex 11 (mouse)-like 2 -2.57 0.0324227 ring finger protein 122
-2.57 0.0236621 chromosome 8 open reading frame 57 -2.59 0.00261522
ADAM metallopeptidase with thrombospondin type 1 motif, -2.60
0.0113968 sushi, von Willebrand factor type A, EGF and pentraxin
dom -2.63 2.23E-05 ST6 beta-galactosamide
alpha-2,6-sialyltranferase 2 -2.64 0.0216987 sortilin-related VPS10
domain containing receptor 2 -2.65 0.00936311 protocadherin beta 9
-2.66 0.0285124 chromosome 5 open reading frame 13 -2.67 0.00410172
Enah -2.68 0.0077547 pyridoxal-dependent decarboxylase domain
containing 2 -2.69 0.00683647 similar to nuclear pore complex
interacting protein -2.70 0.0187322 nuclear pore complex
interacting protein -2.70 0.00368967 transmembrane protein 119
-2.70 0.00801387 chromosome 14 open reading frame 37 -2.70
0.0182453 sushi-repeat-containing protein, X-linked 2 -2.71
0.0253856 PDZ domain containing RING finger 3 -2.71 0.00931014
collagen, type XII, alpha 1 -2.72 0.000204664 matrix-remodelling
associated 5 -2.72 0.000317637 collagen, type V, alpha 1 -2.72
0.0166427 dystrophin related protein 2 -2.72 0.0137557 ATP-binding
cassette, sub-family A (ABC1), member 1 -2.73 0.00131361 trophinin
-2.77 0.00298044 cornichon homolog 3 (Drosophila) -2.78 0.0261738
formin binding protein 1-like -2.78 0.00290401 brain and acute
leukemia, cytoplasmic -2.78 0.0476919 protein tyrosine phosphatase,
receptor type, U -2.80 0.0270428 hypothetical protein MGC24103
-2.82 0.0346673 interferon induced with helicase C domain 1 -2.83
0.0024839 phospholipid transfer protein -2.84 0.00999206 immediate
early response 3 -2.87 0.0152127 immediate early response 3 -2.87
0.0152127 ADAM metallopeptidase domain 12 (meltrin alpha) -2.87
0.000870288 synaptic vesicle glycoprotein 2A -2.88 0.00704212
chromosome 9 open reading frame 3 -2.88 0.00410177 thioredoxin
interacting protein -2.90 0.0135494 early growth response 1 -2.93
0.000425035 small nucleolar RNA, C -2.94 0.00666866 small nucleolar
RNA, C -2.95 0.00765575 immediate early response 3 -2.99 0.0167309
low density lipoprotein-related protein 1 (alpha-2-macroglo -2.99
4.26E-05 bicaudal C homolog 1 (Drosophila) -2.99 0.0347162 homeobox
B2 -3.03 0.00665994 small nucleolar RNA, C -3.10 0.0274043 small
nucleolar RNA, C -3.10 0.0274043 matrix metallopeptidase 2
(gelatinase A, 72 kDa gelatinase, -3.13 5.59E-05 KIAA1641 -3.14
0.00659194 collagen, type VI, alpha 3 -3.14 2.09E-06 homeobox A2
-3.15 0.0435423 SH3 and PX domains 2B -3.15 0.0244357 collagen,
type VI, alpha 2 -3.16 0.0149554 chromosome 9 open reading frame 3
-3.21 0.0233723 small nucleolar RNA, C -3.24 0.0104491 small
nucleolar RNA, C -3.24 0.0104491 -- -3.27 0.00488845
UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylga -3.35
0.00964109 cholesterol 25-hydroxylase -3.38 0.0445558 KIAA1641
-3.40 0.013175 ring finger protein 144 -3.40 0.0135334 versican
-3.41 0.023885 angiopoietin-like 2 -3.42 0.0245161 KIAA1641 -3.44
0.0170531 FBJ murine osteosarcoma viral oncogene homolog B -3.54
0.00025573 similar to RIKEN cDNA 1110018M03 -3.59 0.00516476 early
growth response 2 (Krox-20 homolog, Drosophila) -3.62 0.00821813
dachsous 1 (Drosophila) -3.63 0.00697244 kinesin family member 26B
-3.64 0.00363199 distal-less homeobox 5 -3.66 0.000640157 similar
to Protein KIAA0220 -3.69 0.0302619 insulin-like growth factor 1
receptor -3.71 3.42E-05 protein tyrosine phosphatase, receptor
type, N -3.77 0.0294569 KIAA1641 -3.85 0.0191782
sushi-repeat-containing protein, X-linked -3.85 0.00370941
microfibrillar-associated protein 2 -3.91 0.0152901 complement
component 1, s subcomponent -3.97 0.0395863 CD24 molecule -3.99
0.0340122 homeobox B3 -4.02 0.0354368 trichorhinophalangeal
syndrome I -4.02 0.00557712 Kallmann syndrome 1 sequence -4.04
0.000548703 leucine rich repeat containing 17 -4.09 0.0263961
plexin domain containing 2 -4.32 0.031799 PTK7 protein tyrosine
kinase 7 -4.42 0.000116114 supervillin -4.43 0.0412717 zinc finger
protein 521 -4.58 0.00668815 calbindin 2, 29 kDa (calretinin) -4.77
0.0290743 ras homolog gene family, member J -4.79 0.00197982
integrin, alpha 11 -4.80 0.000390317 odz, odd Oz -5.05 0.00172671
F-box protein 32 -5.52 0.0212957 raftlin family member 2 -5.72
0.0260454 clusterin -5.74 0.0303973 neurotrimin -5.79 3.78E-06 WNT1
inducible signaling pathway protein 1 -5.86 0.000672342
insulin-like growth factor binding protein 5 -6.34 0.011614
sulfatase 2 -6.34 5.88E-05 microfibrillar-associated protein 4
-6.93 0.00155578 junctional adhesion molecule 2 -7.07 0.0306758
fibronectin type III domain containing 1 -7.29 0.0334696
sarcoglycan, delta (35 kDa dystrophin-associated glycoprotei -7.37
0.000881984 hephaestin -7.53 0.0123141 serpin peptidase inhibitor,
clade F (alpha-2 antiplasmi -7.66 0.00362941 cystatin SN -7.96
0.0496433 hemicentin 1 -8.18 0.0461603 tenascin C (hexabrachion)
-8.32 8.26E-05 biglycan -8.62 0.00161284 transmembrane, prostate
androgen induced RNA -11.20 0.000100935 carboxypeptidase E -11.22
0.00738131
[0298] Expression of Cellular Markers on PLX-C Cells
[0299] The surface antigens expressed by PLX-C were examined using
monoclonal antibodies. Results indicated that PLX-C cells were
characterized by the positive markers: CD73, CD29 and CD105 and the
negative markers: CD34, CD45, CD19, CD14 and HLA-DR (data not
shown). The immune phenotype test specifications were set as:
.gtoreq.90% for all positive markers and .ltoreq.3% for all
negative markers.
[0300] Furthermore, as shown in FIGS. 10A-B, PLX-C cultures did not
express endothelial markers as shown by negative staining for the
two endothelial markers CD31 and KDR. However, PLX-C expression of
a fibroblast-typical marker was evident (expression of D7-fib, FIG.
10C).
[0301] Immunogenecity and Immunomodulatory Properties of PLX-C
cells
[0302] As PLX-C is comprised of adherent cells derived from
placenta, it is expected to express HLA type 1, which is expressed
by all cells of the body and is known to induce an alloreactive
immune response. HLA type II and other co-stimulatory molecules are
typically expressed only on the surface of Antigen Presenting Cells
(APCs).
[0303] In order to examine the immunogenicity of the obtained PLX-C
cells, the expression of co-stimulatory molecules on the surface of
these cell membranes were performed. FACS analysis demonstrated the
absence of CD80, CD86 and CD40 on the PLX-C cell membranes (FIGS.
11A-C). Moreover, PLX-C expressed low levels HLA class I as
detected by staining for HLA A/B/C (FIG. 11D). The expression of
stimulatory and co-stimulatory molecules was similar to bone marrow
(BM) derived MSCs (as shown in FIGS. 11A-D).
[0304] To further investigate the immunogenecity as well as the
immunomodulation properties of PLX-C cells, Mix Lymphocyte Reaction
(MLR) tests were performed. As shown in FIG. 12A-B, PLX-C cells
both escape allorecognition, and reduce T cell response, as
measured by Thymidine incorporation. Furthermore, the reduction in
lymphocytes proliferation (evaluated by CPM measurement) was higher
as the number of PLX-C cells increased (in a dose dependent
manner). PLX-C also reduced lymphocyte proliferation following
mitogenic stimuli, such as Concavalin A (Con A, FIG. 12B) and
Phytohemagglutinin (PHA), and non-specific stimulation by anti-CD3,
anti-CD28 (data not shown).
[0305] In order to investigate the mechanism of action by which
PLX-C immunomodulate lymphocyte proliferation, and to see if this
action is mediated via cell to cell interaction or cytokines
secretion, PB derived Mononuclear cells (MNCs) were stimulated by
PHA using the transwell method (which prevents cell to cell contact
but enables the diffusion of cytokines between the two
compartments). Results showed that the inhibition of proliferation
maintained even when cell to cell contact was inhibited (data not
shown).
[0306] Cytokines Secretion
[0307] As depicted hereinabove, PLX-C reduce the proliferation rate
of lymphocytes, probably through soluble factors. Further
investigation of the cytokines secreted by lymphocytes in response
to PLX-C was performed to elucidate the mechanism of action of
PLX-C. As depicted in FIGS. 13A-B, culturing of mononuclear cells
with PLX-C slightly reduces the secretion of the pro-inflammatory
cytokine INF.gamma. and dramatically reduces the secretion of
TNF.alpha. (even in the presence of low amounts of PLX-C). In
addition, following lipopolysaccharide (LPS) stimulation, PB
derived MNCs secretion of IL-10 increased in the presence of PLX-C,
while the secretion level of TNF.alpha. decreased, in a dose
dependent manner (FIG. 13C).
Example 5
Biodistribution of PLX-C
Materials and Experimental Methods
[0308] Transfection of PLX-C Cells with Luciferase Expression
Vector
[0309] PLX-C cells were stably infected with a lentiviral construct
expressing the luciferase gene under the CMV promoter (FIG.
14).
[0310] Production of Infecting Virus
[0311] 293TN producer cells were grown in DMEM medium (Gibco)
supplemented with serum and antibiotics for 2-3 days (50-70%
confluency) prior to transfection. A mixture of 10 .mu.g of the
packaging plasmid and 2 .mu.g of expression construct and 20 .mu.l
of Plus.TM. Reagent (Invitrogen) were added to 400 .mu.l of DMEM
without supplements. The mixture was incubated for 15 min at room
temperature (RT) and Lipofectamine.TM. (30 .mu.l dilutes in 400
.mu.l of DMEM were added). The mixture was incubated at RT for 15
min. 293TN cells were washed and transferred to 2% serum media and
transfection mixture was added. Cells were incubated in CO.sub.2
incubator at 37.degree. C. over night and medium was collected
24-60 hrs post infection. Peak virus production was achieved after
48 hrs. Medium was collected, and centrifuged at 3000 rpm at room
temperature for 5 minutes to pellet cell debris. Following
centrifugation, the supernatant was filtered through Millex-HV 0.45
.mu.m PVDF filters (Millipore, Cat. #SLHVR25LS).
[0312] Infection of PLX-C
[0313] PLX-C cells were seeded in a 24-well plate at a density of
0.6-1.times.10.sup.5 cells per well in complete medium 24 hours
prior to viral infection. After 24 hrs, 0.5 ml of virus suspension
(diluted in complete medium with Polybrene at a final concentration
of 5-8 .mu.g/ml) was added. Cells were incubated for 24 hrs, then
medium was replaced by complete DMEM medium and cells were
incubated at 37.degree. C. with 5% CO.sub.2 overnight. At day 4,
the culture reached confluency and was split by 1:3 to 1:5, cells
were allowed to grow for 48 hours in complete DMEM then cells were
analyzed for Luciferase expression.
[0314] Efficiency rates of infection were close to 100%. Evaluation
of luminescence in living cells and in living mice was performed
using the IVIS Lumina Imaging system, which included a highly
sensitive CCD camera that captured the luciferase luminescence
signal.
[0315] Two weeks post infection 2.times.10.sup.6 cells were
injected IM or IV into SCID/Beige, NOD/SCID, SCID and Balb/C mice.
Injected cells were monitored using the described IVIS system.
[0316] Experimental Results
[0317] As evident from the results, CXL cells continued to divide
following infection, and expression levels of luciferase in the
growing cells remained strong and stable (FIG. 15).
[0318] Once PLX-C cells were injected into Balb/C mice, the
biodistribution pattern was examined. As evident from the results,
cells disappeared 72 hrs post IM injection (data not shown).
However, PLX-C cells retained constant high levels of luciferase
expression, in vitro, for over three weeks (data not shown).
[0319] As shown in FIGS. 16A-D, cells injected IM into SCID/Beige
mice immunodeficient mice retained up to 5 days at the site of
injection and were not observed thereafter. CXL cells injected IV
into SCID/Beige mice migrated after 24 hrs to the lungs, then to
the site of injection (presumably homing to site of injury).
Afterwards cells disappear gradually and were not observed after
3-4 weeks.
Example 6
Adherent Cells are Capable of Treating Limb Ischemia In Vivo
[0320] To determine whether implantation of placental derived
adherent cells can reduce ischemic damage and improve clinical and
motor functions, the hind limb ischemia model was used, as
follows.
[0321] Materials and Experimental Methods
[0322] Hind Limb Ischemia Model
[0323] Hind limb ischemia was induced in 20 Male Balb/c mice, which
are not immuno-deficient. at the age of 8-10 weeks, body weight
approximately 25 g.+-.20%. Animals handling was according to the
National Institute of Health (NIH) and the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Animals were housed under standard laboratory conditions. Animals
were kept in a climate controlled environment. Temperatures range
was between 20-24. .degree. C. and relative humidity (RH) was
between 30-70% with 12 hours light and 12 hours dark cycle.
[0324] Animals were randomized using a computer generated
randomization program "Research Randomizer" and divided into 2
groups of 10 animals. One group received intramuscular (IM)
injection of 1.times.10.sup.6 placental derived adherent cells
(PLX-C) cells and the other group served as control and was
injected with PBS.
[0325] Surgical Procedures
[0326] 1-1.5 cm incision was made in the skin in the inguinal area.
The femoral artery was ligated twice with 6-0 silk and transected
distal to the ligature. The wound was closed with 3-0 silk and the
mice were allowed recovering. Five hours post-operative excision of
one femoral artery, mice received an IM injection of
1.times.10.sup.6 placental derived adherent cells (PLX-C) in a
total volume of 50 .mu.l at 2 administration Sites. Control group
animals were identically injected with PBS (Gibco), see Table 6
below.
TABLE-US-00006 TABLE 6 Pilot study of PLX-C in Mouse Hind limb
Ischemia Model No. of Mice Time of Sacrifice Test Group Treatment
per group Cell Dose Lot Post-dosing 21 days 1 PLX-C i.m n = 10 1
.times. 10.sup.6 C.G.13.0 n = 10 2 PBS i.m n = 10 0 N/A n = 10
[0327] Follow Up
[0328] Blood flow on legs from both sides was measured 3
consecutive times with a non contact laser Doppler just after the
operation, and on days 6, 9, 14 and 21 post operation, and is
expressed as the ratio of the flow in the ischemic limb to that in
the normal limb [Tokai. J. et al].
[0329] Macroscopic Evaluation of Ischemic Severity
[0330] The ischemic limb was macroscopically evaluated at days 1,
6, 9, 14, 21, till study termination by using graded morphological
scales for necrotic area; grade 0: absence of necrosis, grade I:
necrosis limiting to toes (toes loss), grade II: necrosis extending
to a dorsum pedis (foot loss), grade III: necrosis extending to a
crus (knee loss), grade IV: necrosis extending to a thigh (total
hind-limb loss) [Tokai. J. et al].
[0331] In Vivo Assessment of Limb Function and Ischemic Damage
[0332] Semiquantitative assessment of impaired use of the ischemic
limb was be performed serially as follows: 3=dragging of foot, 2=no
dragging but no plantar flexion, 1=plantar flexion, and 0=flexing
the toes to resist gentle traction of the tail (Rutherford et al.,
1997).
[0333] Molecular and Biochemical Analysis
[0334] In addition to clinical evaluation, molecular and
biochemical samples were obtained at day 21 and are currently being
analyzed in order to better understand the molecular mechanisms
underlying improved healing in the placental derived adherent cells
(PLX-C) injected group.
[0335] Experimental Results
[0336] Implantation of Placental Derived Adherent Cells
Significantly Induces Blood Flow in the Hip and Foot of the
Ischemic Hind Limb Model
[0337] To test the efficacy of the adherent cells in vivo, mice
were subjected artery ligation followed by intramuscular injections
of placental derived adherent cells and the blood flow was measured
in the hips and foot (both body sides) using a non contact laser
Doppler at pre-determined time period following treatment. As is
shown in FIG. 17, injection of PLX-C markedly improved blood flow
(BF) to the damaged limb, as determined by blood flow assessments,
increase in limb function, increase in capillary density, decrease
in oxidative stress and endothelial damage. In terms of blood flow,
the effect was demonstrated 9 days following injection and was
observed throughout the entire study. In the PLX-C treated group,
BF increased from 24.+-.2.3 to 80.+-.4.7%, while in the control,
vehicle-treated group BF was in the range of 35.+-.2 to
54.+-.4.5%--in the hip/implantation area (day 0 vs. day 21,
respectively). Similarly to the hip area, but to a lesser extent,
an increase in BF was also demonstrated in the paw area of PLX-C
treated mice. Thus, in the vehicle treated group BF increased from
12.+-.0.6 to 46.+-.4.9%, while in the PLX-C group BF increased from
10.+-.0.7 to 52.+-.5.5% (day 0 vs. day 21 respectively), as shown
in FIG. 17.
[0338] The Adherent Cells are Capable of Improving Limb Function In
Vivo
[0339] To further evaluate the in vivo effects of the placental
derived adherent cells, the limb functions in the treated mice were
assessed using the scoring system described under Materials and
Experimental Methods, hereinabove. As is shown in FIG. 18, mice
treated with the adherent cells exhibited a significant improvement
in the limb function (2.5.+-.0.2 vs. 2.1.+-.0.2 control vs. PLX-C
group, respectively, note the significant effect at day 21 post
treatment). However, the degree of improvement in limb function
during the 21 days observation was comparable, suggesting that
PLX-C. under the conditions of the present study did not exhibit a
major change of function recovery.
[0340] Macroscopic assessment of ischemic severity revealed that in
the control, vehicle treated group, necrosis limited to the toes
was observed in two animals on day 6. In the PLX-C treated group,
necrosis limited to the toes, was demonstrated only in one animal
and only after 14 days. Post mortem immunohistochemical analyses of
the limbs treated with PLX-C indicated a significant increase in
the number of new capillaries (vessels) supplying the limb and
suggesting PLX-C possess the ability to promote angiogenesis (FIG.
19).
[0341] Finally, a decreased oxidative stress and a reduction in
endothelial inflammation (which was a surrogate parameter for
improved endothelial function) in the treated animals were observed
in the PLX-C treated mice (FIGS. 20A-B). This was likely due to
increased oxygen supply in mice treated with PLX-C cells, but not
in control mice treated with PBS.
[0342] In conclusion, when compared to control, PBS-injected mice,
none of the PLX-C injected mice exhibited any adverse clinical
signs or symptoms in response to intra mascular (i.m.) cell
administration. Thus, PLX-C induces an increase in blood flow,
likely resulting from angiogenesis as supported by histological
evaluation of the damaged limb. In addition, the delay in
development of necrosis and the difference in number of affected
animals are suggestive of a clinical response.
[0343] Implantation of Placental Derived Adherent Cells
[0344] Another efficacy study was carried out in Balb/C mice
comprising safety endpoints (i.e., gross necmrpsis and
histopathological analysis of selected organs) as was described in
the materials and methods section above.
[0345] In this study, seven groups of mice, each consisting of 10
male Balb/c mice (Ischemic hind limb) were used as detailed in
Table 7, hereinbelow. A single group of mice did not have ischemia
induced (in order to test the overall safety and tolerability of
PLX-C cells in normal, healthy animals). Following induction of
ischemia, control buffer or PLX-C cells were administered i.m. to
the affected limb, and mice were observed for up to 1 month post
dosing. A single group of mice received two separate injections in
the affected limb, separated by 1 week (Days 1 and 8). Blood flow
was monitored by Laser Doppler analysis, and ischemic severity was
assessed macroscopically and behaviorally out to 30 days post-dose,
at which time, mice were sacrificed, and tissues retained for
histological analysis.
TABLE-US-00007 TABLE 7 Efficacy study of PLX-C in Mouse Hind limb
Ischemia Model Cell Amount Number of Time of Sacrifice Test Group
no. Treatments (Dose) Treatments Lot Post-dosing 30 days 1 PLX-C 1
.times. 10.sup.6 1 C.G.13.0 10 2 PLX-C 1 .times. 10.sup.6 1
C.G.25.0 10 3 PLX-C 1 .times. 10.sup.6 2 C.G.25.0 10 4 PLX-C 0.5
.times. 10.sup.6 1 C.G.13.0 10 5 PLX-C 0.1 .times. 10.sup.6 1
C.G.13.0 10 6 Control N/A 1 N/A 10 Freezing Medium 7* PLX-C 1
.times. 10.sup.6 1 C.G.13.0 10 C.G.25.0
[0346] In this study, different PLX-C batches at three
concentrations were administered. The results showed that
0.1.times.10.sup.6 and 0.5.times.10.sup.6 PLX-C had a minor
therapeutic benefit. A noticeable improvement in blood flow was
observed by day 29 (end of experiment) in animals treated with
1.times.10.sup.6. This improvement in blood flow was significant
(p<0.05) in group 2M (batch G.C25) in comparison to control,
vehicle injected mice. Additionally, a second injection of the same
batch of cells significantly improved BF on day 15 compared to the
single injection (55.+-.24 compared to 31.+-.12.9 and 27.+-.12.5%,
respectively). Macroscopic assessment of ischemic severity revealed
that there was a trend for improvement in the groups receiving
1.times.10.sup.6 (1 M & 2M) in comparison to the control
vehicle treated group (6M).
[0347] Altogether, these results demonstrate the efficacy of the
adherent cells in inducing vascularization (e.g., blood flow) and
improving limb function in hind limb ischemic mice model and
suggest the use of these cells (e.g., placental derived adherent
cells) for treating ischemic limb diseases.
Example 7
PLX-C for the Treatment of Stroke
[0348] The aim of this study was to evaluate the therapeutic
efficacy of systemic (intravenous) human PLX-C--placenta derived
adherent cells transplantation in the treatment of stroke.
[0349] Materials and Experimental Methods
[0350] Subjects, Surgery and Transplantation
[0351] Male spontaneously hypertensive rats, suffering from
hypertension, hypercholesterolemia, diabetes and microangiopathies
were used. The animals were kept under constant conditions
concerning temperature, air humidity and light/dark cycle. Subjects
were assigned experimental groups randomly (see Table 8,
below).
TABLE-US-00008 TABLE 8 Rat treatment groups in the treatment of
stroke Group no. Treatment No. of animals 1 PLX-C, batch 1 single
administration N = 8 2 PLX-C, batch 1 double administration N = 7 3
PLX-C, batch 2 single administration N = 8 4 PLX-C, batch 2 double
administration N = 7 5 Control- Vehicle solution N = 12
[0352] Animals received a single or double dose of 1.times.10.sup.6
PLX-C of different batches. All transplantation procedures were
conducted intravenously. The double injected group was transplanted
10 and 24 hours following brain ischemia, while single
transplantations were performed 24 hours upon stroke. All
transplanted cells were pre-labeled with the fluorescence dye
PKH26.
[0353] Experimental brain ischemia was conducted via permanent
occlusion of the right cerebral artery. Of note, one animal died
following anesthesia.
[0354] Magnetic Resonance Investigation (MRI)
[0355] MRI of lesion development was carried out on days 1, 8, 29
and 60 using a 1.5T scanner (Philips). Infarct volumetry and brain
atrophy was measured and calculated as means of values obtained by
three blinded investigators using coronal T2- sequences.
[0356] Behavioral Tests
[0357] Functional changes were measured by using two dependent
behavioral test arrays. The Beam Walk test is a common test used to
quantify sensory-motor deficits. The rats were conditioned to run
across a horizontal mounted bar with the rat's home cage at the
end. The time of transit was measured for five times and documented
as diurnal mean value. Hanging at the beam was assessed with 20
seconds and falling down with 30 seconds. Measurements took place
daily within the first week and every seventh day until the end of
the observation period.
[0358] The second test, the modified neurological severity score
(mNSS) contained additional sensory, motor and reflex items.
Outcome of the mNSS was expressed as a score between 1 and 18,
whereas points between 1 and 6 implied a mild, 7 to 12a moderate
and 13 to 18a sever injury. Evaluation of mNSS score was performed
on days 1, 4, 7, 14, 21, 28, 35, 42, 49 and 56 following brain
ischemia.
[0359] Histology
[0360] Subsequent to the end of the experimental period, all rats
were sacrificed and transcardially perfused with 4% formalin
solution. Extracted brains were cryopreserved and cut into 30 .mu.m
thick sections. For the appraisal of glia reaction, an
immunohistochemical investigation was performed with primary
antibody against GFAP. A 750 .mu.m broad area was examined
(semi-quantitatively) close to the infarct border for density of
GFAP+ cells. For inspection of astroglial reactivity, 15 regions
where included with average interspaces of 0.6 mm.
[0361] Statistics
[0362] All data concerning weight. MRI analysis and histological
examinations were investigated for Gaussian distribution and
analyzed for statistically significant differences using the ANOVA
and accordingly the ANOVA on ranks.
[0363] Data collected in the Beam Walk and mNSS test were subjected
to detailed statistical analysis making allowance for repeated
measurements of subjects as well as for temporal development of
subjects individually (stratified analysis). For balancing of
inter-individual differences concerning the degree of brain damage
a random intercept model was used. The data collected within the
Beam Walk test therefore had to be transformed to a categorical
system. Here, time values of less than 5 seconds were considered as
category (0), 5 to 10 seconds as category (1), 10 to 15 seconds as
category (2), 15 to 20 seconds as category (3), hanging as category
(4) and falling as category (5).
[0364] Experimental Results
[0365] Weight
[0366] Periodic weighing allowed a good estimation of the subject's
general state of health. An initial reduction of weight was
observed in all groups due to anesthesia and the surgical
intervention (data not shown). Subsequently, a quick normalization
of weight and a stable course until the end of the experiment at
day 60 was observed (data not shown). The experimental groups
showed a homologous progression of body weight.
[0367] Beam Walk test
[0368] All experimental groups showed a significant reduction of
Beam Walk categories during the course of the experiment (data not
shown). A significant lower decrease of Beam Walk categories were
observed in the experimental group 1 (PLX-C batch 1 single
administration) compared to the control group (-0.01247 vs.
-0.02931, respectively). There was no evidence for statistically
significant differences between the experimental group 3 (PLX-C
batch 2 single administration) and the control group (data not
shown).
[0369] Modified Neurological Severity Score (mNss)
[0370] All experimental groups displayed a significant reduction of
neurological score points (data not shown). Comparing the mNSS
results of subjects treated with PLX-2 (PLX-C double
administration) revealed a statistically significant superiority
compared to the control group. The duplicate transplantation of
batch2 (group 3) showed a significant improvement in the mNSS test
compared to the simple injection of the same batch (data not
shown).
[0371] Infarct Volumetry
[0372] Magnetic resonance imaging is a highly sophisticated method
to estimate the degree of brain damage and tissue lost in vivo.
Taking inter-individual fluctuations into consideration, the
development of infarct volume was denoted as percentage of the
infarct volume at day 1, individually. The infarct volume on day 1
did not differ significantly between the experimental groups. The
general development of infarct volume displayed an approximate
decrease of 0.50% between day 1 and day 8. This was mainly due to a
retrogression of the initial brain edema. Examination of lesion
development in vivo using MRI revealed that group 4 (PLX-C batch 2
double administration) subjects showed a significant reduced
infarct quotient at Day 60 (0.48 t 0.02 vs. 0.60.+-.0.03,
respectively, results not shown).
[0373] Taken together. these results indicate that intravascular
administration of PLX-C resulted in a significant improvement of
functional recovery in both behavioral tests in the treatment of
stroke. Moreover, a considerable and as well statistically
significant superiority of PLX-C double transplantations was
observed compared to the analogous single injection.
[0374] A corroboration of measured behavioral improvements by MRI
was evident in subjects treated twice with PLX-C. In addition, a
significant reduction of the infarct volume and the brain atrophy
was observed at the end of the experiment. Moreover, in both
functional tests a stable improvement of functional recovery
following double-dosed transplantation of PLX-C was observed
compared to controls and unverifiable effects upon single
injections.
Example 8
Treatment of Pathologies Requiring Connective Tissue Regeneration
and/or Repair
[0375] Treating Pathologies Requiring Bone Regeneration and/or
Repair Using the Adherent Cells of the Invention
[0376] Animal models (e.g., Mature New Zealand white rabbits) are
used to examine the effect of the adherent cells of the invention
(which are derived from placenta or adipose tissue and are obtained
from a 3D culture, e.g. PLX-C cells) on the healing of
critical-sized segmental defects in the femora. The animals are
randomly assigned to one of three groups. Animals of group A, are
injected with 1-10.times.10.sup.6 of the adherent cells (PLX-C
cells) into the defect site. Animals of group B are injected with
PBS. In animals of group C, the defect was left untreated.
Radiographs are made immediately after the operation and at
one-week intervals. At 12 weeks, the animals are sacrificed, the
involved femora are removed, and undecalcified histological
sections from the defects and adjacent bone are prepared.
Mechanical. histological and histomorphometric studies are carried
out to examine the healing of the defects and the formation of bone
in and around the defected site. In addition a reverse
transcription-polymerase chain reaction (RT-PCR) is performed to
detect mRNA of type-I and type-II collagen.
[0377] Treating Pathologies Requiring Tendon Regeneration and/or
Repair Using the Adherent Cells of the Invention
[0378] Animal models (e.g., skeletally mature New Zealand white
rabbits) are used to examine the effect of the adherent cells of
the invention (e.g., PLX-C cells) on the healing of tendons. The
hallucis longus tendons are translated into 2.5-mm diameter
calcaneal bone tunnels. The bone tunnels are treated with or
without PLX-C. The animals are randomly assigned to one of three
groups. Animals of group A, are injected with 1-10.times.10.sup.6
PLX-C cells into the defect site or IV. Animals of group B are
injected with PBS. In, animals of group C the defect is left
untreated. Three specimens from each group are harvested at 2, 4,
and 6 weeks postoperatively and evaluation for morphologic
characteristics of the healing tendon to bone interface is
performed by the use of conventional histology and
immunohistochemical localization of collagen Types I, II, and
III.
[0379] Treating Pathologies Requiring Cartilage Regeneration and/or
Repair Using the Adherent Cells of the Invention
[0380] Animal models (e.g., skeletally mature New Zealand white
rabbits) are used to examine the effect of the adherent cells of
the invention (e.g., PLX-C cells) on the healing of cartilage. A
full-thickness defect of the articular cartilage of the patellar
groove of the left distal femur is performed. A flap of about 6 mm
is removed from the fascia overlying the quadriceps muscle and
sutured to the peripheral rim of the artificial defect with 6-0
catgut. The animals are randomly assigned to one of three groups.
Animals of group A are injected with 1-10.times.10.sup.6 PLX-C
cells into the defect site or IV. Animals of group B are injected
with PBS. In animals of group C, the defect is left untreated. The
animals are sacrificed. Fourteen weeks after the implantation of
the PLX-C cells onto the osteochondral defect, the distal femora
are reacted and histological evaluation are performed and the
specimens are graded semiquantitatively based on the predominant
nature of the repair tissue, matrix staining, regularity of the
surface, structural integrity, thickness of the repair, apposition
between the repaired cartilage and surrounding normal cartilage,
freedom from degenerative signs in repair tissue, and freedom from
degenerative changes of the surrounding normal cartilage.
[0381] Treating Pathologies Requiring Ligament Regeneration and/or
Repair Using the Adherent Cells of the Invention
[0382] Animal models (e.g., skeletally mature New Zealand white
rabbits) are used to examine the effect of the adherent cells of
the invention (e.g., PLX-C cells) on the healing of ligament.
Unicortical circular defects of 8 mm in diameter will be performed.
The animals are randomly assigned to one of three groups. Animals
of group A are injected with 1-10.times.10.sup.6 PLX-C cells into
the defect site or IV. Animals of group B are injected with PBS. In
animals of group C, the defect is left untreated. The animals are
sacrificed fourteen weeks after the implantation of the PLX-C cells
onto the oligamental defect. Histological evaluation are performed
and the specimens are graded semiquantitatively based on the
predominant nature of the repaired tissue.
[0383] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0384] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications and GenBank Accession
numbers mentioned in this specification are herein incorporated in
their entirety by reference into the specification, to the same
extent as if each individual publication, patent or patent
application or GenBank Accession number was specifically and
individually indicated to be incorporated herein by reference. In
addition, citation or identification of any reference in this
application shall not be construed as an admission that such
reference is available as prior art to the invention.
REFERENCES
Additional References are Cited in Text
[0385] Bauer. Thomas W. Muschler, George F., Bone Graft Materials:
An Overview of the Basic Science. Clinical Orthopaedics &
Related Research. 371:10-27, February 2000. [0386] Carstanjen B,
Desbois C. Hekmati M, and Behr L. Successful engraftment of
cultured autologous mesenchymal stem cells in a surgically repaired
soft palate defect in an adult horse. Can J Vet Res. 2006 April:
70(2): 143-147. [0387] Bruder S P, et al. 1998 The effect of
implants loaded with autologous mesenchymal stem cells on the
healing of canine segmental bone defects. J Bone Joint Surg Am.
80(7):985-96 [0388] Chao Wan, Qiling He, Gang Li, 2006. Allogenic
peripheral blood derived mesenchymal stem cells (MSCs) enhance bone
regeneration in rabbit ulna critical-sized bone defect model.
Journal of Orthopaedic Research 24 (4) 610-618. [0389] Herthel D.
J. 2001, Enhanced Suspensory Ligament Healing in 100 Horses by Stem
Cells and Other Bone Marrow Components. AAEP PROCEEDINGS/Vol. 47.
[0390] Gordon et al. Tendon Regeneration Using Mesenchymal Stem
Cells, p 313-320 in Tendon Injuries. Springer London. 2005. [0391]
Horwitz et al. 1999. Transplantability and therapeutic effects of
bone marrow derived mesenchymal cells in children with osteogenesis
imperfecta. Nat. Med. 5:309-313. [0392] Horwitz et al., 2002.
Isolated allogeneic bone marrow-derived mesenchymal cells engraft
and stimulate growth in children with osteogenesis imperfecta:
Implications for cell therapy of bone. PNAS 99(13)8932-8937. [0393]
Livingston, T. L. 2003 Mesenchymal stem cells combined with
biphasic calcium phosphate ceramics promote bone regeneration.
Journal of Materials Science: Volume 14 (3):211-218. [0394] Young
et al. 1998. Use of mesenchymal stem cells in a collagen matrix for
Achilles tendon repair. J Orthop Res. 16(4):406-13.
* * * * *
References