U.S. patent application number 15/440170 was filed with the patent office on 2017-06-08 for progenitor cells from wharton's jelly of human umbilical cord.
This patent application is currently assigned to Tissue Regeneration Therapeutics Inc.. The applicant listed for this patent is Tissue Regeneration Therapeutics Inc.. Invention is credited to Dolores BAKSH, John E. DAVIES, Morris HOSSEINI, Antony D.S. LICKORISH, Rahul SARUGASER.
Application Number | 20170157180 15/440170 |
Document ID | / |
Family ID | 32869477 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170157180 |
Kind Code |
A1 |
DAVIES; John E. ; et
al. |
June 8, 2017 |
PROGENITOR CELLS FROM WHARTON'S JELLY OF HUMAN UMBILICAL CORD
Abstract
Human progenitor cells are extracted from perivascular tissue of
human umbilical cord. The progenitor cell population proliferates
rapidly, and harbours osteogenic progenitor cells and MHC-/-
progenitor cells, and is useful to grow and repair human tissues
including bone.
Inventors: |
DAVIES; John E.; (Toronto,
CA) ; BAKSH; Dolores; (Mississauga, CA) ;
SARUGASER; Rahul; (Toronto, CA) ; HOSSEINI;
Morris; (Braunschweig, DE) ; LICKORISH; Antony
D.S.; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tissue Regeneration Therapeutics Inc. |
Toronto |
|
CA |
|
|
Assignee: |
Tissue Regeneration Therapeutics
Inc.
Toronto
ON
Tissue Regeneration Therapeutics Inc.
Toronto
ON
|
Family ID: |
32869477 |
Appl. No.: |
15/440170 |
Filed: |
February 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14884339 |
Oct 15, 2015 |
9611456 |
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15440170 |
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13903575 |
May 28, 2013 |
9567564 |
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14884339 |
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12482963 |
Jun 11, 2009 |
8481311 |
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13903575 |
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10961919 |
Oct 8, 2004 |
7547546 |
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12482963 |
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PCT/CA04/00182 |
Feb 10, 2004 |
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10961919 |
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60446275 |
Feb 11, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0654 20130101;
C12N 5/0668 20130101; C12N 5/0653 20130101; C12N 2509/00 20130101;
A61K 35/51 20130101; C12N 2523/00 20130101; C12N 5/0605
20130101 |
International
Class: |
A61K 35/51 20060101
A61K035/51; C12N 5/073 20060101 C12N005/073 |
Claims
1. A Wharton's jelly extract, wherein the extract comprises human
progenitor cells and is obtained by enzymatic digestion of the
perivascular tissue proximal to the vasculature of human umbilical
cord.
2. A Wharton's jelly extract according to claim 1, wherein the
extract is essentially free from cells of umbilical cord blood.
3. A Wharton's jelly extract according to claim 2, wherein the
extract is obtained by subjecting umbilical cord vasculature
bearing proximal Wharton's jelly to enzymatic digestion in a
suitable cell extraction medium.
4. A Wharton's jelly extract according to claim 3, wherein the step
of enzymatic digestion results in the release of cells from the
collagen matrix of the Wharton's jelly.
5. A Wharton's jelly extract according to claim 4, in which the
extraction is performed at 37.degree. C. in phosphate buffered
saline and 0.5-10.0 mg/mL collagenase for a period of 1 to 24
hours.
6. A method for obtaining a human progenitor cell, comprising the
step of isolating said cell from the Wharton's extract according to
claim 1.
7. A method for producing a cell population comprising human
progenitor cells, the method comprising the step of culturing an
isolated human progenitor cell obtained by the method according to
claim 6.
8. A cell population comprising human progenitor cells, whenever
obtained by the method according to claim 7.
9. A cell population useful as a source of human progenitor cells,
wherein the cell population is extractable from perivascular tissue
of human umbilical cord, and has the characteristics of (1) rapid
proliferation, (2) the presence of osteoprogenitor cells, and (3)
the presence of immuno-incompetent cells.
10. A cell population enriched for osteoprogenitor cells obtained
by the method according to claim 7.
11. A cell population enriched for immuno-incompetent human
progenitor cells obtained by the method according to claim 7.
12. A method for producing bone tissue, comprising the step of
subjecting a cell population according to claim 9 to culturing in
the absence of osteogenic supplements.
13. A method for producing a population of MHC double negative
human progenitor cells, comprising the step of culturing a
previously frozen cell population defined in claim 9.
14. An isolated population of human progenitor cells, in the form
of human umbilical cord perivascular cells having a 3G5+, CD45-
phenotype, the population comprising cells characterized by rapid
proliferation, the presence of human osteoprogenitor cells, and the
presence of human progenitor cells that are negative for markers of
MHC class I and MHC class H.
15. An isolated population of human progenitor cells, the
population comprising cells that upon culturing give rise
spontaneously to cells of a type selected from bone cells and fat
cells, the population comprising the non-adherent fraction of the
cell population according to claim 14.
16. An isolated population of human progenitor cells, wherein at
least 50% of the cells in said population have an MHC double
negative phenotype.
17. An isolated population of human progenitor cells according to
claim 16, wherein at least 80% of the cells in said population have
an MHC double negative phenotype.
18. An isolated population of human progenitor cells according to
claim 17, wherein at least 90% of the cells in said population have
an MHC double negative phenotype.
19. A composition comprising an effective amount of a cell
population according to claim 14, and a carrier suitable for
delivering said population to a tissue site.
20. A method of tissue engineering, comprising the step of
delivering to a tissue site at which engineering is desired, a
composition according to claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and is a continuation
of, U.S. patent application Ser. No. 12/482,963, filed Jun. 11,
2009, which claims priority to U.S. patent application Ser. No.
10/961,919, filed Oct. 8, 2004, which is a continuation-in-part of
International Application No. PCT/CA2004/000182, filed Feb. 10,
2004, which claims benefit to U.S. Provisional Application No.
60/446,275, filed Feb. 11, 2003. Each of these applications are
hereby incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] This invention focuses on the harvesting of a population of
rapidly proliferating human cells from the connective tissue of the
umbilical cord (UC); the culture of such cells in osteogenic,
chondrogenic, adipogenic and myogenic conditions; the demonstration
of a high percentage of cells within these populations that are
immunologically incompetent, as shown by their lack of cell surface
histocompatibility antigens; and the ability of these cells to be
used as a source of multipotent progenitor cells for various
cell-based therapies.
BACKGROUND OF THE INVENTION
[0003] The UC is one of the first structures to form following
gastrulation (formation of the three embryonic germ layers). As
folding is initiated, the embryonic disc becomes connected, by the
primitive midgut (embryonic origin) to the primitive yolk sac
(extra-embryonic origin) via the vitelline and allantoic vessels
which in turn develop to form the umbilical vessels (Haynesworth et
al., 1998; Pereda and Motta, 2002; Tuchmann-Duplessis et al.,
1972). These vessels are supported in, and surrounded by, what is
generally considered a primitive mesenchymal tissue of primarily
extra-embryonic derivation called Wharton's Jelly (WJ) (Weiss,
1983). From this early stage, the UC grows, during gestation, to
become the 30-50 cm cord seen at birth. It can be expected
therefore, that WJ contains not only the fibroblast-like, or
myo-fibroblast-like cells which have been described in the
literature (see below), but also populations of progenitor cells
which can give rise to the cells of the expanding volume of WJ
necessary to support the growth of the cord during embryonic and
fetal development.
[0004] WJ was first described by Thomas Wharton, who published his
treatise Adenographia in (1656) (Wharton T W. Adenographia.
Translated by Freer S. (1996). Oxford, U.K.: Oxford University
Press, 1656; 242-248). It has subsequently been defined as a
gelatinous, loose mucous connective tissue composed of cells
dispersed in an amorphous ground substance composed of
proteoglycans, including hyaluronic acid (Schoenberg et al., 1960),
and different types of collagens (Nanaev et al., 1997). The cells
dispersed in the matrix have been described as "fibroblast-like"
that are stellate in shape in collapsed cord and elongate in
distended cord (Parry, 1970). Smooth muscle cells were initially
observed within the matrix (Chacko and Reynolds, 1954), although
this was disputed by Parry (1970) who described them as somewhat
"unusual fibroblasts" which superficially resemble smooth muscle
cells. Thereafter, little work had been done on characterizing
these cells until 1993 when Takechi et al. (1993) performed
immunohistochemical investigations on these cells. They described
the cells as "fibroblast-like" that were "fusiform or stellate in
shape with long cytoplasmic processes and a wavy network of
collagen fibres in an amorphous ground substance" (Takechi et al.,
1993). For the immunohistochemical staining, they used primary
antibodies against actin and myosin (cytoplasmic contractile
proteins), vimentin (characteristic of fibroblasts of embryonic
mesenchyme origin) and desmin (specific to cells of myogenic
origin) in order to determine which types of myosin are associated
with the WJ fibroblasts. They observed high levels of chemically
extractable actomyosin; and although fibroblasts contain
cytoplasmic actomyosin, they do not stain for actin or myosin,
whereas the WJ fibroblasts stained positively for both.
Additionally, positive stains for both vimentin and desmin were
observed leading to the conclusion that these modified fibroblasts
in WJ were derived from primitive mesenchymal tissue (Takechi et
al., 1993). A subsequent, more recent study by Nanaev et al. (1997)
demonstrated five steps of differentiation of proliferating
mesenchymal progenitor cells in pre-term cords. Their findings
supported the suggestion that myofibroblasts exist within the WJ
matrix. The immunohistochemical characterization of the cells of
WJ, shows remarkable similarities to that of pericytes which are
known to be a major source of osteogenic cells in bone
morphogenesis and can also form bone nodules referred to as colony
forming unit-osteoblasts (CFU-O) (Aubin, 1998) in culture (Canfield
et al., 2000).
[0005] Recent publications have reported methods to harvest cells
from UC, rather than UC blood. Mitchell at al. (Mitchell at al.,
2003) describe a method in which they first remove and discard the
umbilical vessels to harvest the remaining tissue. The latter,
which will include both the remaining WJ (some of which will have
been discarded with the vessels, since the umbilical vessels are
entirely enveloped in WJ) and the amniotic epithelium, is then
diced to produce small tissue fragments that are transferred to
tissue culture plates. These tissue fragments are then used as
primary explants from which cells migrate onto the culture
substratum.
[0006] In another publication, Romanov et al. (2003) indicate they
were successful in isolating mesenchymal stem cell-like cells from
cord vasculature, although they also indicate their cultures do not
contain cells from WJ. Specifically, they employ a single, 15 min,
collagenase digestion from within the umbilical vein, which yields
a mixed population of vascular endothelial and sub-endothelial
cells. Romanov et al. show that sparse numbers of fibroblast-like
cells appear from this cell harvest after 7 days.
[0007] Also, U.S. Pat. No. 5,919,702 describes a method of
isolating "pre-chondrocytes" from the WJ of human UC, and their use
to produce cartilage. Particularly, the method comprises slicing
open a one inch section of cord longitudinally, dissecting away the
blood vessels and `casing`, which are then discarded, and
collecting the WJ into a sterile container where it was cut into
2-3 mm.sup.3 sections for culturing. In a preferred method, cells
are isolated by placing a 2-3 mm.sup.3 section of the WJ on a glass
slide on the bottom of a Petri dish, covering it with another
slide, and culturing it for 10-12 days in order to allow the
`pre-chondrocytes` to migrate out to the culture dish surface.
[0008] It is an object of the present invention to provide a cell
population comprising human progenitor cells.
[0009] It is another object of the present invention to provide a
source from which human progenitor cells can be extracted.
[0010] It is a further object of the present invention to provide a
method for the isolation of human progenitor cells.
[0011] It is a further object of the present invention to provide
human osteoprogenitor cells useful for the production of bone
tissue.
[0012] It is a further object of the present invention to provide
human mesenchymal progenitor cells useful for the production of
cartilage tissue, adipose tissue and muscle tissue.
[0013] It is a further object of the present invention to provide
human immuno-incompetent progenitor cells useful
therapeutically.
SUMMARY OF THE INVENTION
[0014] There has now been devised a procedure for extracting cells
from Wharton's jelly of human umbilical cord, which yields a unique
cell population characterized by rapid proliferation, the presence
of osteoprogenitor and other human progenitor cells, including
immuno-incompetent cells which display neither of the major
histocompatibility markers (human leukocyte antigen (HLA) double
negative). The cell population is a useful source of progenitor
cells from which to grow bone and other connective tissues
including cartilage, fat and muscle, and for autogenic and
allogeneic transfer of progenitor cells to patients, for
therapeutic purposes.
[0015] More particularly, and according to one aspect of the
present invention, there is provided a Wharton's jelly extract,
wherein the extract comprises human progenitor cells and is
obtained by enzymatic digestion of the Wharton's jelly proximal to
the vasculature of human umbilical cord, in a region usefully
termed the perivascular zone of Wharton's jelly. The tissue within
this perivascular zone, and from which the present progenitor cells
are extracted, can also be referred to as perivascular tissue. The
extraction procedure suitably results in an extract that is
essentially free from cells of umbilical cord blood, epithelial
cells or endothelial cells of the UC and cells derived from the
vascular structure of the cord, where vascular structure is defined
as the tunicae intima, media and adventia of arteriolar or venous
vessels. The resultant extract is also distinct from other
Wharton's jelly extracts isolated from the bulk Wharton's jelly
tissue that has been separated from the vascular structures.
[0016] In accordance with another of its aspects, the present
invention provides a method for obtaining a human progenitor cell,
comprising the step of isolating the cell from the Wharton's jelly
extract obtained in accordance with the invention.
[0017] In a related aspect, the present invention provides a cell
population obtained by culturing of the cells present in the
Wharton's jelly extract. In embodiments, there is provided a
population of osteoprogenitor cells. In other embodiments, there is
provided a population of immuno-incompetent progenitor cells.
[0018] In one embodiment, the extracted progenitor cell population
is characterized as an adherent cell population obtained following
culturing of the extracted cells under adherent conditions. In
another embodiment, the extracted progenitor cell population is
characterized as a non-adherent (or "post-adherent") (PA) cell
population present within the supernatant fraction of extracted
cells grown under adherent conditions. This PA fraction is derived
by transferring the supernatant of the initially plated HUCPV cells
into a new T-75 flask to allow the as yet non-adhered cells to
attach to the culture surface. This process is repeated with this
new T-75 flask, transferring its media into another new T-75 flask
in order to harvest any remaining PA cells. This PA cell population
comprises, according to another aspect of the invention, a
subpopulation of progenitor cells that, when cultured under
adherent conditions, proliferates rapidly and forms bone nodules
and fat cells spontaneously. This embodiment provides a means to
increase the yield of adherent cells isolated from the enzymatic
digest cell population.
[0019] Also provided by the present invention is a population of
committed osteoprogenitor cells characterized by the property of
differentiating into bone cells when cultured in the absence of
supplements otherwise required for such differentiation.
[0020] In another of its aspects, the present invention provides a
method for producing connective tissue, including bone tissue,
cartilage tissue, adipose tissue and muscle tissue, which comprises
the step of subjecting cells obtained from the Wharton's jelly
extract to conditions conducive to differentiation of those cells
into the desired connective tissue phenotype. In this respect, the
invention further provides for the use of such cells in cell-based
therapies including cell transplantation-mediated treatment of
medical conditions, diseases and disorders.
[0021] More particularly and according to another aspect of the
invention, there is provided a composition and the use thereof in
tissue engineering, comprising progenitor cells in accordance with
the invention or their differentiated progeny, and a carrier
suitable for delivering such cells to the chosen tissue site.
[0022] These and other aspects of the invention will now be
described in greater detail with reference being had to the
accompanying drawings, in which:
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a light micrograph representing the three distinct
zones of tissue represented in the human UC;
[0024] FIG. 2 is a representative illustration of the looped vessel
in the collagenase solution;
[0025] FIG. 3 is a light micrograph of the cells isolated from the
WJ that have attached to the polystyrene tissue culture
surface;
[0026] FIG. 4 is a light micrograph illustrating the initial
formation of a CFU-O;
[0027] FIG. 5 is a light micrograph illustrating a mature
CFU-O;
[0028] FIG. 6 demonstrates tetracycline-labeled CFU-O's under UV
fluorescence on a 35 mm polystyrene tissue culture dish;
[0029] FIG. 7 illustrates side by side a phase-contrast light
micrograph and a fluorescence micrograph of the same
tetracycline-labeled CFU-O;
[0030] FIG. 8 is a scanning electron micrograph of a mature CFU-O
on the tissue culture polystyrene surface;
[0031] FIG. 9 is a scanning electron micrograph of a cross-section
of a CFU-O exposing the underlying matrix;
[0032] FIG. 10 is a scanning electron micrograph of the lightly
mineralized collagen fibres located on the advancing edge of the
CFU-O;
[0033] FIG. 11 is a scanning electron micrograph of the
non-collagenous matrix (seen as globules) laid down on the
polystyrene interface by differentiating osteogenic cells;
[0034] FIG. 12 is a scanning electron micrograph of heavily
mineralized collagen that comprises the centre of a mature
CFU-O;
[0035] FIG. 13 illustrates the flow cytometry data demonstrating
that WJ-derived cells are 77.4% MHC I and MHC II negative;
[0036] FIG. 14 is a black and white reproduction of a Masson's
trichome-stained transverse section of bone nodule showing the
distribution of collagen within which cells have become entrapped
(osteocytes), and multilayering of peripheral cells some of which
are becoming surrounded by the elaborated extracellular matrix;
[0037] FIG. 15 shows the potential expansion of the adherent
perivascular WJ population in relation to the expansion of the
committed osteoprogenitor subpopulation and total osteoprogenitor
subpopulation;
[0038] FIG. 16 shows proliferation of the perivascular WJ cells
from 0-144 hours illustrating a normal growth curve with a lag
phase from 0-24 hrs, log phase from 24-72 hours, and plateau phase
from 72-120 hours. The doubling time during the entire culture
period is 24 hours, while during log phase it is 16 hours;
[0039] FIG. 17 shows major histocompatibility complex (MHC)
expression of the WJ cells shown over 5 passages, the change in
their expression due to free-thawing, and subsequent expression due
to reculture;
[0040] FIG. 18 shows the CFU-F frequency of HUCPV cells;
[0041] FIG. 19 shows the doubling time of HUCPV cells from P0
through P9. HUCPV cells demonstrate a relatively stable and rapid
doubling time of 20 hours from P2 to P8; and
[0042] FIG. 20 shows the proliferation of HUCPV cells demonstrating
that >10.sup.14 cells can be derived within 30 days of culture.
With this rapid expansion, 1,000 therapeutic doses (TDs) can be
generated within 24 days of culture; and
[0043] FIG. 21 shows the effects of collagenase concentration and
digestion time on cell harvest.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention provides an extract of Wharton's jelly
(WJ), as a source of a rapidly proliferating cell population
comprising human progenitor cells including osteoprogenitor cells,
as well as immuno-incompetent cells.
[0045] For purposes of this description, the extracted cell
population can be referred to as human umbilical cord perivascular
(HUCPV) cells. The HUCPV cell population constitutes a rich source
of multipotent progenitor cells that are unique in their phenotype,
particularly as revealed by the variety of cell subpopulations
contained therein. Also for purposes of this description, the
perivascular zone of the Wharton's jelly from which the present
cells are extracted can be referred to as perivascular tissue.
[0046] As used herein, the term "progenitor cells" refers to cells
that will differentiate under controlled and/or defined conditions
into cells of a given phenotype. Thus, an osteoprogenitor cell is a
progenitor cell that will commit to the osteoblast lineage, and
ultimately form bone tissue when cultured under conditions
established for such commitment and differentiation. A progenitor
cell that is "immuno-incompetent" or "non-immunogenic" is a cell
having a phenotype that is negative for surface antigens associated
with class I and class II major histocompatibility complexes (MHC).
Such a progenitor cell is also referred to herein as an HLA double
negative.
[0047] The HUCPV cell population extracted from WJ is also
characterized by "rapid proliferation", which refers to the rate at
which the extracted cells will grow relative to other known
progenitor cell populations, under conditions that are standard for
progenitor cell expansion. As will be appreciated from the
experimental results presented herein, and as shown in FIG. 16, the
present progenitor cell population can double within at least about
25 hours and as quickly as 7-15 hours, and thus expands far more
rapidly than other known osteoprogenitor cell populations and other
progenitor cell populations extracted from WJ.
[0048] The cells and cell populations of the present invention can
be obtained by extraction from WJ of human umbilical cord. Unlike
the prior art, and in accordance with the present invention, such
cells are extracted from the WJ that is associated with, i.e.,
proximal to, the exterior wall of the umbilical vasculature. The
Wharton's jelly that is associated with or very near to the
external surface of the cord vasculature lies within a region
termed the perivascular zone, and typically remains associated with
the vasculature when the vessels are excised from the cord, as is
done for instance either to extract Wharton's jelly from the cord,
or to extract the vessels from the cord and associated Wharton's
jelly. It has remarkably been found that the Wharton's jelly within
this perivascular zone, and which has typically been discarded in
prior art practice, is a rich source of progenitor cells having the
characteristics herein described. Accordingly, the present
invention exploits the tissue from this perivascular zone of the
Wharton's jelly as a source for useful human progenitor cells,
termed HUCPV cells.
[0049] In embodiments, the HUCPV cell population is characterized
by the presence of progenitor cells having many markers indicative
of a functional mesenchymal (non-hematopoietic) phenotype, i.e.,
CD45-, CD34-, SH2+, SH3+, Thy-1+ and CD44+. Of particular
significance, the population is characterized generally as
harbouring cells that are positive for 3G5 antibody, which is a
marker indicative of pericytes. The extracted cell population
generally is a morphologically homogeneous fibroblastic cell
population, which expresses alpha-actin, desmin, and vimentin, and
provides a very useful source from which desired cell
subpopulations can be obtained through manipulation of culturing
conditions and selection based for instance on cell sorting
principles and techniques.
[0050] To extract such perivascular cells from human umbilical
cord, in a preferred embodiment, care is taken during the
extraction process to avoid extracting cells of the umbilical cord
blood, epithelial cells or endothelial cells of the UC, and cells
derived from the vascular structure of the cord, where vascular
structure is defined as the tunicae intima, media and adventia of
arterial or venous vessels. Obtaining an extract that is
essentially free of these unwanted cells can be achieved by careful
flushing and washing of the umbilical cord prior to dissection,
followed by careful dissection of the vessels from within the cord.
The vessels can also be carefully pulled away from the surrounding
cord tissue in which case the perivascular tissue is excised with
the vessels. It will be appreciated that, with care being taken to
avoid extracting these unwanted cells, they may still be present to
a small extent in the resulting extract. This is acceptable
provided they occur at a frequency too low to interfere with the
observed results presented herein, i.e., observation of cell
colonies derived from mesenchymal and specifically mesodermal
origin, frequency and rapidity of formation of CFU-F, CFU-O and
CFU-A, and characterization of HLA phenotypes observed in the
cultured population.
[0051] The tissue that lies within the perivascular zone is the
Wharton's jelly proximal to the external wall of the umbilical
vasculature, and lies typically within a zone extending to about 3
mm from the external wall of the vessels. Suitably, the target
extraction zone can lie within about 2 mm, e.g., about 1 mm from
the external wall of any one of the three vessels. The extraction
of WJ from this region can be readily achieved using the technique
described in the examples. In this technique the vessels are used
as a carrier for the WJ, and the vessels per se are used as the
substrate from which the progenitor cells are extracted. Thus, in
embodiments of the invention, cord vessels bearing a thin coating
of perivascular tissue are excised either surgically or manually
from fresh umbilical cord that has been washed thoroughly to remove
essentially all cord blood contaminants. The vessels bearing the
proximal perivascular tissue, or sections thereof, are then
incubated at about 37.degree. C. in an extraction medium such as
phosphate buffered saline (PBS) containing an enzyme suitable for
digesting the collagen matrix of the perivascular tissue in which
the desired cells reside. For this purpose, digestion with a
collagenase is suitable, at a concentration within the range from
about 0.1 mg/mL to 10.0 mg/mL or more, e.g., 0.5 mg/mL. The enzyme
type, concentration and incubation time can vary, and alternative
extraction conditions can be determined readily simply by
monitoring yield of cell phenotype and population under the chosen
conditions. For instance, a higher collagenase concentration of 4
mg/mL (e.g., 1-4 mg/mL) is also suitable over a shorter digestion
period of about 3 hours (e.g., 1-5 hours). During the extraction,
the ends of the vessels are tied, or clipped, off and can be
suspended above the extraction medium to avoid contamination by
agents contained within the vessel. It will thus be appreciated
that the present Wharton's jelly extract is essentially free from
cord blood cells, umbilical cord epithelial cells, vessel
endothelial cells and vessel smooth muscle cells.
[0052] Other digestive enzymes that can be used in the isolation
procedure are 0.1 to 10 mg/ml hyaluronidase, 0.05 to 10 mg/ml
trypsin as well as EDTA. The optimum collagenase concentration is 4
mg/ml for a digestion period of 3 hours, although a less expensive
alternative is to use 0.5 mg/ml for 18-24 hours. Still other
alternatives to collagenase concentrations are illustrated in FIG.
21. Desirably, digestion is halted at or before the vessels begin
to degrade which, as shown in FIG. 21, occurs at different time
points depending on the collagenase concentration.
[0053] After about 24 hours in the 0.5 mg/mL collagenase extraction
medium, e.g., 12-36 hours, such as 18-24 hours, or after about 3
hours in the 4.0 mg/mL collagenase extraction medium, the vessels
are removed, leaving a perivascular tissue extract that contains
human progenitor cells. These cells are expanded under conditions
standard for expansion of progenitor cells. The cells can, for
instance, be selected on polystyrene to select for adherent cells,
such as in polystyrene dishes or flasks and then maintained in a
suitable culturing medium. In an embodiment of the invention, the
extracted cells are cultured for expansion, with or without prior
selection for adherent cells, under conditions of stirred
suspension, as described for instance by Baksh et al in
WO02/086104, the disclosure of which is incorporated herein by
reference.
[0054] In a particular embodiment of the present invention, the
extracted population of HUCPV cells is cultured under adherent
conditions, and non-adherent cells resident in the supernatant are
recovered for further culturing. These "post-adherent" cells are
characterized as a subpopulation by a propensity to form bone
nodules and fat cells spontaneously, and constitute a valuable
embodiment of the present invention. Thus, in this respect, the
present invention further provides an isolated population of
progenitor cells extracted from perivascular tissue, the cells
having the propensity to form at least one of several
differentiated cell types including bone cells, cartilage cells,
fat cells and muscle cells, wherein such progenitor cells
constitute the non-adherent fraction of the HUCPV cells cultured
under adherent conditions. Such cells are obtained by culturing the
perivascular tissue-extracted HUCPV cells under adherent
conditions, selecting the non-adherent cell population, and then
culturing the non-adherent cell population under conditions useful
to (1) expand said population or (2) to cause differentiation
thereof into a desired cell phenotype. Culturing conditions useful
therein are those already established for such expansion and
differentiation, as exemplified herein.
[0055] It will also be appreciated that the present invention
includes HUCPV subpopulations that are cultured and expanded under
standard adherent culturing conditions. As is revealed herein, such
adherent cell populations are known to comprise the
immunoincompetent or non-immunogenic, progenitors, and mesenchymal
progenitors, which constitute valuable embodiments of the present
invention.
[0056] The cells present in the extract can, either directly or
after their expansion, be sorted using established techniques to
provide expandable subpopulations enriched for cells of a given
phenotype. Thus, the present invention further provides
perivascular tissue extracted cell populations that are enriched
for multipotent mesenchymal progenitor cells, osteoprogenitor
cells, cell populations that are enriched for immuno-incompetent
progenitor cells, and cell populations that are enriched for
multipotent and osteoprogenitor cells that are immuno-incompetent.
Further, the cells can be enriched to select for only those that
are positive for the pericyte marker 3G5, using antibody thereto,
and to select only for those that are negative for either one or
both of the MHC class I and class II markers. The cell population
can also be enriched by selection against other surface markers,
such as by depletion of those bearing CD45 to remove hematopoietic
cells, for instance. Such enriched cell populations are valuable
embodiments of the present invention.
[0057] As is revealed in FIG. 17, it has been found that the
distribution of MHC markers within the progenitor cell population
is altered by freeze-thawing. Upon passaging of fresh cells, the
frequency of MHC double negative cells is relatively
constant/marginally increased. However, it has been found, as noted
in the examples herein, that the frequency of MHC double negative
cells in the progenitor population is increased significantly in
cells plated following freezing. Thus, in the present progenitor
cell population, cells of the MHC double negative phenotype are
further characterized by the propensity to increase in frequency
following freezing. Such freezing is performed in the usual manner,
by first preparing a cell aliquot, and then storing the cell
preparation for the desired period. It will be appreciated that
such cells can be stored for many years if desired.
[0058] In an embodiment, the present invention thus further
provides a method for producing MHC double negative progenitor
cells, by obtaining a perivascular tissue extract as herein
described, or an MHC double negative-enriched fraction thereof;
subjecting the extract or fraction thereof to freezing, and then
culturing the frozen cells. The resulting cells as noted are
potentially useful to induce tissue formation or repair in human
subjects.
[0059] The cell populations obtained from the extract or from a
suitably enriched fraction thereof, are useful either directly or
following their expansion to provide differentiated cell
populations. All of the procedures suitable for their fractionation
and enrichment, and for their expansion are established in the
prior art, and are exemplified herein. Expansion can proceed, for
instance, in the presence of factors such as IL-3 and Stem Cell
Factor, and similar agents known in the art. In one embodiment, the
present cell population, and particularly the osteoprogenitor cells
therein, are subjected to differentiation using conditions
established for the growth of bone tissue therefrom. Remarkably, a
subpopulation of osteoprogenitor cells that arise from the
culturing of the present progenitor cell population, referred to as
committed osteoprogenitors, have shown the ability to differentiate
in the absence of osteogenic supplements. Alternatively, the
osteoprogenitor cells are cultured in a medium supplemented with
one or more agents that stimulate osteogenesis, such as
dexamethasone. In addition, the progenitor cells can also be
cultured with supplements suitable for stimulating differentiation
into other mesenchymally-derived connective tissues (Caplan, 1991),
including cartilage, muscle, tendon, adipose etc., all in
accordance with standard practice in the art.
[0060] As a practical alternative to in vitro culturing of cells in
the present cell population, it will be appreciated that the cells
can be transplanted in vivo to induce the formation of a desired
tissue directly within a patient. By this route, the in situ
formation of bone is provided by implanting osteoprogenitor, for
the benefit of patients suffering from various bone conditions,
diseases and disorders, particularly including bone fracture and
osteoporosis. Such therapies can also be applied to attenuating
maladies of other connective tissues such as, but not limited to,
cartilage, fat and muscle. The immuno-incompetent progenitor cells
present in the cell population are particularly valuable in this
respect, given the substantially reduced rejection response that
can be expected following their implantation.
[0061] For use in transplantation, the present cells can be
provided as a composition, further comprising a carrier useful for
their delivery to the tissue site selected for engineering. The
cells are presented in a dose effective for the intended effect. It
is expected that an effective cell dose will lie in the range from
10.sup.3 to 10.sup.7 cells, e.g., 10.sup.4-10.sup.6 such as
2.times.10.sup.5 cells, per dose. The carrier selected for delivery
of those cells can vary in composition, in accordance with
procedures established for delivery of viable cells. In
embodiments, the cells are exploited for purposes of bone tissue
engineering. In one embodiment, the cells are presented with a
carrier in the form of a scaffold material that serves to localize
the cells as an implant at a bone site that is defective or
fractured, or is surgically prepared to receive the implant. A
variety of materials are suitable as carriers for this purpose. In
a particular embodiment, the carrier is formed of resorbable
material such as calcium phosphate, PLGA or mixtures thereof.
Equivalent materials can be used, provided they allow for the cells
to remain viable during formation and delivery of the composition,
and are otherwise physiologically compatible at the implantation
site.
[0062] Still other carriers suitable for delivery of the progenitor
cells will include vehicles such as PBS and gels including
hyaluronic acid, gelatin and the like with equivalents being useful
provided they possess the pH and other properties required for cell
viability.
[0063] It will also be appreciated that the present cells are
useful as hosts for delivering gene expression products to the
desired tissue site. That is, the present cells can in accordance
with embodiments of the present invention, be engineered
genetically to receive and express genes that upon expression yield
products useful in the tissue repair process, such as the various
growth factors which, in the case of bone tissue, can usefully
include PTH, the BMPs, calcitonin, and the like. The cells can also
be developed as transgenics for other purposes, such as by
introduction of genes that alter the cell phenotype, to make it
more robust, or more suitable to a given end-use.
[0064] Embodiments of the invention are described in the following
examples.
[0065] Harvest of Progenitor Cells from Human Wharton's Jelly
[0066] The UCs were collected from full-term caesarian section
infants immediately upon delivery at Sunnybrook & Women's
College Hospital, Toronto, Canada. The UC was transferred by the
surgeon into a sterile vessel containing medium (80% .alpha.-MEM,
20% antibiotics), and immediately transported to our laboratories
at the Institute of Biomaterials & Biomedical Engineering,
University of Toronto.
[0067] All procedures from this point on were performed aseptically
in a biological safety cabinet. The UC was washed in Phosphate
Buffered Saline (PBS) (--Mg.sup.2+, --Ca.sup.2+) three times to
remove as much of the UC blood as possible, and transferred back
into a container with medium. A length of approximately 6 cm of
cord was cut with sterile scissors and placed onto a sterile cork
dissection board. The remaining cord (30-45 cm) was returned to the
medium-filled container and placed into an incubator at 37.degree.
C. The 6 cm section of cord was `twisted` against its helix, and
pinned at both ends to reveal a smooth and straight surface of the
UC epithelium. Using fine scissors, the UC was cut approximately
1-2 mm deep along its length to reveal the WJ. Starting with each
`flap` of cut epithelium, the WJ was teased from its inner surface
using the blunt edge of a scalpel, and the teased away epithelium
(approximately 0.5 mm thick) was pinned down. This procedure
resulted in the WJ being exposed, and with its three vessels
embedded in it running straight from end to end rather than
helically along its longitudinal axis. Care was taken to constantly
bathe the section with 37.degree. C. PBS. Isolating one of the ends
of a vessel with forceps, it was teased away from the WJ along its
length until it was free of the bulk of the WJ matrix.
Alternatively, the middle of the vessel could be dissected from the
matrix, held with tweezers, and teased from the matrix in each
direction toward its ends. Once freed by either method, the vessel
was surrounded with approximately 1-2 mm of the cell-bearing WJ
matrix. The dissected vessel was then clipped at both ends with
either a surgical clamp, mosquito clip or sutured to create a
`loop,` blocking the passage of fluid either into or out of the
vessel. The `loop` was immediately placed along with the scissors
into a 50 ml tube containing a 0.5 mg/ml collagenase solution with
PBS (--Mg.sup.2+, --Ca.sup.2+), and placed into an incubator at
37.degree. C. The remaining two vessels were dissected in a similar
fashion, looped, and also placed in the collagenase solution in the
incubator. Subsequent to the removal of the vessels, strips of WJ,
constituting perivascular tissue, could easily be dissected off the
epithelium and placed into 50 ml tubes with the collagenase
solution. The remaining epithelial layer was then disposed of in a
biohazard waste container. The same protocol was used with the
remaining 30-45 cm of UC, producing 15 to 25 tubes with either
`loops` or perivascular tissue strips.
[0068] Initiation of Wharton's Jelly Progenitor Cell Cultures
[0069] After 18-24 hours, the `loops` were removed with the aid of
their attached suspension clamp or suture and a pipette, and the
remaining suspensions were then diluted 2-5 times with PBS and
centrifuged at 1150 rpm for 5 minutes to obtain the cell fraction
as a pellet at the bottom of the tube/s. After removal of the
supernatant, the cells were resuspended in eight times volume of 4%
NH.sub.4Cl for 5 minutes at room temperature in order to lyse any
contaminating red blood cells. The suspensions were then
centrifuged again at 1150 rpm for 5 minutes to isolate the cell
fraction as a pellet, and the supernatant was removed. After
counting the cells with the use of hemocytometer, they were plated
directly onto T-75 cm.sup.2 tissue culture polystyrene dishes, and
allowed to incubate at 37.degree. C. for 24-72 hours in order to
allow the cells to attach to the polystyrene surface. The medium
was then changed every two days.
[0070] The results detailed below have been reproduced using the
procedure described above, but in which collagenase-based digestion
proceeded either at 4 mg/mL for 3 hours, 2 mg/ml for six hours and
at 1 mg/mL for 12 hours.
[0071] The attached cells were passaged using 0.1% trypsin solution
after 7 days, at which point they exhibited 80-90% confluency, as
observed by light microscopy, and there was evidence of
`mineralized` aggregate formation, as revealed under phase
microscopy and indicated by expected changes in optical properties.
Upon passage, cells were plated either in 35 mm tissue culture
polystyrene dishes or 6 well plates at 4.times.10.sup.3
cells/cm.sup.2 in supplemented media (SM) (75% .alpha.-MEM or
D-MEM, 15% FBS, 10% antibiotics) and treated with 10.sup.-8M Dex, 5
mM .beta.-GP and 50 .mu.g/ml ascorbic acid to test the osteogenic
capacity of these cells. These plates were observed on days 2, 3, 4
and 5 of culture for CFU-O otherwise referred to as `bone nodule`
formation.
[0072] In order to test the chondrogenic capacity of these cells,
2.times.10.sup.5 cells were centrifuged at 1150 rpm for 5 minutes
in order to obtain the cells as a pellet. Once the supernatant was
removed, the cells were maintained in SM supplemented with 10 ng/ml
transforming growth factor-beta (TGF-.beta.) (and optionally with
10.sup.-7M dexamethasone). The supplemented medium was replaced
every two days, maintaining the cultures for 3-5 weeks, at which
point they were harvested for histology (by fixation with 10%
neutral formalin buffer (NFB)), embedded in paraffin, cut into 6
.mu.m section, and stained for the presence of collagen II
(antibody staining) and the presence of glycosaminoglycans (alcian
blue staining). To assess the adipogenic differentiation capacity
of the cells, they were initially cultured in 6-well plates in SM
(with D-MEM), which was replaced every 2 days, until they reached
60% confluence. At that point the medium was replaced with the
adipogenic induction medium (AIM) (88% D-MEM, 3% FBS, 33 .mu.M
Biotin, 17 .mu.M Pantothenate, 5 .mu.M PPAR-gamma, 100 nM Bovine
insulin, 1 .mu.M Dexamethasone, 200 .mu.M Isobutyl methylxanthine
and 10% antibiotics). The AIM was replaced every 2 days for 10 days
at which point the cells were fixed in 10% NFB and stained with Oil
Red O which stains the lipid vacuoles of adipocytes red. Finally,
in order to assess the myogenic capacity of the cells, they were
initially cultured in T-75 cm.sup.2 tissue culture flasks in SM
(with D-MEM) until they reached 80-90% confluence, at which point
the medium was replaced with myogenic medium (MM) (75% D-MEM, 10%
FBS, 10% Horse serum, 50 .mu.M hydrocortisone and 10% antibiotics).
The MM was replaced every 2 days. After 3-5 weeks in culture, the
cells were removed from the culture surface (see subculture
protocol), lysed in order to obtain their mRNA, and assessed by
rtPCR for the presence of several myogenic genes, including: MyoG,
MyoD1, Myf5, Myosin heavy chain, myogenin and desmin.
[0073] Another useful approach to obtaining the perivascular
tissue-derived progenitor cell cultures has been adopted, using the
following protocol: [0074] 1. Obtain sterile umbilical cord (UC)
from caesarian-section patient and transport to biological safety
cabinet in media (80% .alpha.-MEM, 20% antibiotics) [0075] 2. Wash
the UC 3.times. in sterile 37.degree. C. phosphate buffered saline
(PBS) [0076] 3. Cut the UC into approximately 1-2 inch sections
with a sharp pair of scissors [0077] 4. Wash each section of UC
2.times. in sterile 37.degree. C. PBS to remove as much residual
umbilical cord blood (UCB) as possible. [0078] 5. Isolate one of
the UC sections on a dry sterile dish [0079] 6. Using two sets of
forceps, grasp the epithelium approximately 2 mm apart, and pull
away from each other, tearing the epithelium. [0080] 7. Grasping
the epithelium along the length of the UC section, continue to tear
the epithelium away, exposing the WJ underneath [0081] 8. Similarly
to step 6, continue tearing the epithelium away in `strips` until
approximately half of the epithelium has been torn away. [0082] 9.
The umbilical vessels should be clearly visible through the WJ, and
the ends loose on the cut edges of the UC section. [0083] 10. By
grasping a remaining part of the epithelium with one set of
forceps, and the end of a vessel with the other, the vessel can be
`pulled` from the bulk WJ with its surrounding perivascular tissue
(PVT). [0084] 11. This process is repeated with each vessel, until
all three are free of the underlying WJ matrix. [0085] 12. Once
released, each vessel is placed into 37.degree. C. PBS. [0086] 13.
Steps 5-12 are repeated with each section of UC until all the
vessels have been isolated in a sterile 37.degree. C. PBS-filled
beaker. [0087] 14. Then, by placing each vessel individually on a
clean, sterile surface, the ends can be ligated together with a
suture using a double knot into a `loop` [0088] 15. Once all of the
vessels have been ligated into loops, the loops are placed into a
0.5 mg/ml collagenase solution in a sterile 50 ml tube [0089] 16.
The 50 ml tube is placed into a rotator in a 37.degree. C.,
5%/CO.sub.2 incubator overnight. [0090] 17. The following day, the
collagenase is inactivated with 1 ml fetal bovine serum (FBS), and
the loops removed from the suspension. [0091] 18. The remaining
suspension is diluted with PBS, centrifuged at 1150 rpm for 5
minutes, and the supernatant removed. [0092] 19. The pellet is then
resuspended in 8 times volume of 4% NH.sub.4Cl for 5 minutes at
room temperature to lyse all contaminating red blood cells, then
centrifuged at 1150 rpm for 5 minutes, and the supernatant removed.
[0093] 20. The cells remaining in the pellet are resuspended in
supplemented media (SM) (75% .alpha.-MEM, 15% FBS, 20%
antibiotics), and aliquot is counted on a hemocytometer. [0094] 21.
The cell suspension is then plated onto a T-75 tissue-culture
polystyrene flask, and allowed to proliferate. [0095] 22. After 2
days, the supernatant from the flask is transferred to a new T-75
flask in order to harvest the post adherent"" (PA) cells. [0096]
23. After 2 days, the supernatant from the first PA flask is
transferred to a new T-75 flask in order to harvest any remaining
PA cells. [0097] 24. The SM is replaced in all three T-75 flasks
every 2 days until the cells reach sub-confluence (1-2 weeks), at
which point they are sub-cultured (passaged).
[0098] Progenitor Assays
[0099] Cell Proliferation Assay
[0100] During the weekly passage procedure (occurring every 6
days), aliquots of 3.times.10.sup.4 cells were plated into each
well of 24 6-well tissue culture polystyrene plates. On days 1, 2,
3, 4, 5 and 6 days of culture, four of the 6-well plates were
passaged and the cells were counted. The exponential expansion of
these cells was plotted, and the mean doubling time for the cells
in these cultures was calculated. Results are shown in FIG. 16. It
will be noted that the doubling time for the PVWJ cell culture is
about 24 hours across the entire culturing period. During the log
phase, the doubling time is a remarkable 16 hours. This compares
with literature reported doubling times of about 33-36 hours for
bone marrow mesenchymal cells (Conget and Minguell, 1999), and
about 3.2 days for mesenchymal stem cells derived from adipose
tissue (Sen et al., 2001). For observation of proliferation with
successive passaging, 3.times.10.sup.5 cells were plated into 4
T-75 flasks (n=4) and fed with SM which was replaced every 2 days.
After 6 days of culture the cells were subcultured (see subculture
protocol above), and counted with the use of a hemocytometer.
Aliquot of 3.times.10.sup.5 cells were seeded into 4 new T-75
flasks, cultured for 6 days, and the process of counting was
repeated. This process was repeated from P0 through P9 for 4 cord
samples.
[0101] FIG. 18 illustrates the CFU-F frequency of HUCPV cells. The
frequency of 1:300 is significantly higher than that observed for
other mesenchymal progenitor sources including neonatal BM
(1:10.sup.4) (Caplan, 1991), and umbilical cord blood-derived
"unrestricted somatic stem cells" (USSCs) (Kogler et al., 2004)
which occur at a frequency of 1:2.times.10.sup.8. FIG. 19
illustrates the proliferation rate of HUCPV cells with successive
passaging. The initial doubling time of 60 hours at P0 drops to 38
hours at P1, which drops and maintains itself at 20 hours from
P2-PS. The cells begin to enter senescence thereafter and their
proliferation rate begins to drop rapidly. Interestingly, when
observed during the first 30 days of culture (FIG. 20), HUCPV cells
derive 2.times.10.sup.10 cells within 30 days. As one therapeutic
dose (TD) is defined as 2.times.10.sup.5 cells (Horwitz et al,
1999) (Horwitz E M, Prockop D J, Fitzpatrick L A, Koo W W, Gordon P
L, Neel M et al. Transplantability and therapeutic effects of bone
marrow-derived mesenchymal cells in children with osteogenesis
imperfecta. Nat Med 1999; 5:309-313.), HUCPV cells can derive 1 TD
within 10 days of culture, and 1,000 TDs within 24 days of
culture.
[0102] As shown in FIG. 15, the perivascular tissue-derived
progenitors comprise different sub-populations of progenitor cells.
Within this population, there are the so-called "committed"
osteoprogenitor cells characterized by an ability to form bone
nodules, as shown herein, in the absence of the culturing
supplements normally required to induce such differentiation, such
as culturing in the presence of dexamethasone. These committed
osteoprogenitors thus differentiate spontaneously to form bone
nodules. Also within the progenitor population are osteogenic cells
that can be induced to form bone nodules, when cultured in the
presence of the required factors, such as dexamethasone,
JP-glycerophosphate and ascorbic acid. Thus, the "total osteogenic"
sub population graphed in FIG. 15 includes a "committed osteogenic
progenitor" population, as well as an "uncommitted osteogenic
progenitor" population, and reveals the total number of cells that
can be induced to differentiate along the osteogenic
differentiation pathway. The actual ratio between the "committed"
and "uncommitted" population is approximately 1:1, and so the ratio
between the "total osteogenic progenitor" population and the
"committed osteogenic progenitor" population is about 2:1. Analysis
of the bone forming properties of the progenitors was performed as
noted below.
[0103] Chondrogenic, adipogenic and myogenic differentiation of the
cells has also been observed. Although osteogenic and occasional
adipogenic differentiation has been observed spontaneously, the
other phenotypes were only observed when cultured under specific
induction conditions for the respective phenotype.
[0104] Serial Dilution and CFU-F Assays
[0105] Dilutions of 1.times.10.sup.5, 5.times.10.sup.4,
2.5.times.10.sup.4, 1.times.10.sup.4, 5.times.10.sup.3,
1.times.10.sup.3, HUCPV cells were seeded onto 6-well tissue
culture plates (Falcon#353046) and fed every two days with SM. The
number of colonies, comprising >16 cells, were counted in each
well on day 10 of culture, and confirmed on day 14. CFU-F
frequency, the average number of cells required to produce one
colony, was consequently determined to be 1 CFU-F/300 HUCPV cells
plated. Based on this frequency, the unit volume required to
provide 300 HUCPV cells (done in triplicate from each of 3 cords)
was calculated, and 8 incremental unit volumes of HUCPV cells were
seeded into individual wells on 6-well plates. Again, colonies
comprising >16 cells (CFU-Fs) were counted on day 10 of culture
to assay CFU-F frequency with incremental seeding.
[0106] CFU-O Assay
[0107] During the weekly passage procedure, aliquots of test cell
populations were directly plated on tissue-culture polystyrene in
bone forming medium containing 75% .alpha.-MEM, 15% FBS (StemCell
Batch #: S13E40), 10% antibiotic stock solution containing
penicillin G (167 units/ml), gentamicin (50 .mu.g/ml) and
amphotericin B (0.3 .mu.g/ml), and Dex (10.sup.-8M),
.beta.-glycerophosphate (5 mM) and L-ascorbic acid (50 ug/ml), at a
cell seeding density of 1.times.10.sup.4 cells/cm.sup.2. Cultures
were re-fed every two days for a period of 12 days. The cultures
were maintained until mineralized nodular areas, detected as bone
nodules, were observed (usually 3 days) at which point the cultures
were re-fed with tetracycline containing medium at the last culture
re-feed, then fixed in Karnovsky's fixative and prepared for
analysis. A Leitz Aristoplan microscope (Esselte Leitz GmbH &
Co KG, Stuttgart, Germany) was used to visualize the tetracycline
labelled cultures under phase contrast as well as UV fluorescence
and a Hitachi S-2000 scanning electron microscope at an
accelerating voltage of 15 kV was used to generate images to
demonstrate the presence of morphologically identifiable bone
matrix.
[0108] CFU-C Assay
[0109] In order to test the chondrogenic capacity of these cells,
2.times.10.sup.5 cells were centrifuged at 1150 rpm for 5 minutes
in order to obtain the cells as a pellet. Once the supernatant was
removed, the cells were maintained in SM supplemented with 10 ng/ml
transforming growth factor-beta (TGF-.beta.) (and optionally with
10.sup.-7M dexamethasone). The supplemented medium was replaced
every two days, maintaining the cultures for 3-5 weeks, at which
point they were harvested for histology (by fixation with 10%
neutral formalin buffer (NFB)), embedded in paraffin, cut into 6
.mu.m section, and stained for the presence of collagen II
(antibody staining) and the presence of glycosaminoglycans (alcian
blue staining). Staining confirmed the formation of chondrocytes
under induction conditions.
[0110] CFU-A Assay
[0111] To assess the adipogenic differentiation capacity of the
cells, they were initially cultured in 6-well plates in SM (with
D-MEM), which was replaced every 2 days, until they reached 60%
confluence. At that point the medium was replaced with the
adipogenic induction medium (AIM) (88% D-MEM, 3% FBS, 33 .mu.M
Biotin, 17 .mu.M Pantothenate, 5 .mu.M PPAR-gamma, 100 nM Bovine
insulin, 1 .mu.M Dexamethasone, 200 .mu.M Isobutyl methylxanthine
and 10% antibiotics). The AIM was replaced every 2 days for 10 days
at which point the cells were fixed in 10% NFB and stained with Oil
Red O which stains the lipid vacuoles of adipocytes red. Staining
confirmed the formation of adipocytes, not only under induction
conditions, but also in their absence, i.e., spontaneously
[0112] CFU-M Assay
[0113] In order to assess the myogenic capacity of the cells, they
were initially cultured in T-75 cm.sup.2 tissue culture flasks in
SM (with D-MEM) until they reached 80-90% confluence, at which
point the medium was replaced with myogenic medium (MM) (75% D-MEM,
10% FBS, 10% Horse serum, 50 .mu.M hydrocortisone and 10%
antibiotics). The MM was replaced every 2 days. After 3-5 weeks in
culture, the cells were removed from the culture surface (see
subculture protocol), lysed in order to obtain their mRNA, and
assessed by rtPCR for the presence of several myogenic genes,
including: MyoG, MyoD1, Myf5, Myosin heavy chain, myogenin and
desmin. Results confirmed the presence of myocytes under these
induction conditions.
[0114] Data Analysis
[0115] Tetracycline Stain
[0116] Tetracycline (9 .mu.g/ml) was added to the cultures prior to
termination. At termination, the cells were fixed in Karnovsky's
fixative overnight and then viewed by UV-excited fluorescence
imaging for tetracycline labeling of the mineral component of the
nodular areas.
[0117] Scanning Electron Microscopy (SEM)
[0118] Representative samples of CFU-O cultures were prepared for
SEM by first placing them in 70%, 80%, 90% and 95% ethanol for 1
hour, followed by immersion in 100% ethanol for 3 hours. They were
then critical point dried. A layer of gold approximately 3 nm layer
was sputter coated with a Polaron SC515 SEM Coating System onto the
specimens, which were then examined at various magnifications in a
Hitachi S-2000 scanning electron microscope at an accelerating
voltage of 15 kV. The images generated are used to demonstrate the
presence of morphologically identifiable bone matrix.
[0119] Flow Cytometry for HLA-Typing
[0120] Test cell populations of >1.times.10.sup.5 cells were
washed in PBS containing 2% FBS (StemCell Batch #: S13E40) and
re-suspended in PBS+2% FBS with saturating concentrations (1:100
dilution) of the following conjugated mouse IgG1 HLA-A,B,C-PE and
HLA-DR,DP,DQ-FITC for 30 minutes at 4.degree. C. The cell
suspension was washed twice with PBS+2% FBS, stained with 1 g/ml
7-AAD (BD Biosciences) and re-suspended in PBS+2% FBS for analysis
on a flow cytometer (XL, Beckman-Coulter, Miami, Fla.) using the
ExpoADCXL4 software (Beckman-Coulter). Positive staining was
defined as the emission of a fluorescence signal that exceeded
levels obtained by >99% of cells from the control population
stained with matched isotype antibodies (FITC- and PE-conjugated
mouse IgG1,.kappa. monoclonal isotype standards, BD Biosciences).
For each sample, at least 10,000 list mode events were collected.
All plots were generated in EXPO 32 ADC Analysis software.
[0121] In addition to HLA typing, the HUCPV cell population was
also assessed for other markers, with the following results:
TABLE-US-00001 Marker Expression CD105 (SH2) + + CD73 (SH3) + +
CD90 (Thy1) + + CD44 + + CD117 (c-kit) 15% + MHC I 75% + MHC II -
CD106 (VCAM1) - STRO1 - CD123 (IL-3) - SSEA-4 - Oct-4 - HLA-G -
CD34 - CD235a (Glycophorin A) - CD45 -
[0122] Results
[0123] Light Micrographs of Bone Nodule Colonies
[0124] FIGS. 3, 4 and 5 illustrate CFU-O's that were present in the
cultures on day 3 and day 5. They demonstrated the confluent layer
of "fibroblast-like" cells surrounding a nodular area represented
by an `aggregation` of polygonal cells that were producing the
bone-matrix. These CFU-O's were observed in both the Dex (+) and
Dex (-) cultures, and displayed similar morphology over successive
passages.
[0125] Tetracycline Labeling of CFU-O Cultures
[0126] Tetracycline labeling of cultures was used for labeling
newly formed calcium phosphate associated with the biological
mineral phase of bone. The tetracycline labeling of the cultures
coincide with the mineralized nodular areas, which is visualized by
exposing the cultures to UV light. FIGS. 6 and 7 depict
tetracycline labeled CFU-O cultures of Day 3 and Day 5 cultures of
progenitor cells. These images were generated by UV-excited
fluorescence imaging, and photographed.
[0127] Scanning Electron Microscopy
[0128] The CFU-O's were observed under SEM for formation of
mineralized collagen matrix which demonstrates the formation of the
CFU-O's from the initial stages of collagen formation through to
the densely mineralized matrix in the mature CFU-O. FIGS. 8, 9, 10,
11, 12 and 14 represent scanning electron micrographs of the
CFU-Os.
[0129] Flow Cytometry & HLA-Typing
[0130] The flow cytometry, identifying cell-surface antigens
representing both Major Histocompatibility Complexes (MHCs)
demonstrated 77.4% of the population of isolated cells as
MHC.sup.-/-. FIG. 13 illustrates the flow cytometry results in
relation to the negative control. FIG. 17 shows the impact of
freeze-thawing on the frequency of MHC-/- cells in the progenitor
population. The effect of freeze-thawing was studied as
follows:
[0131] Test cell populations of >1.times.10.sup.5 cells were
washed in PBS containing 2% FBS and re-suspended in PBS+2% FBS with
saturating concentrations (1:100 dilution) of the following
conjugated mouse IgG1 HLA-A,B,C-PE (BD Biosciences #555553, Lot
M076246) (MHC I), HLA-DR,DP,DQ-FITC (BD Biosciences #555558, Lot
M074842) (MHC II) and CD45-Cy-Cychrome (BD Biosciences #555484, Lot
0000035746) for 30 minutes at 4.degree. C. The cell suspension was
washed twice with PBS+2% FBS and re-suspended in PBS+2% FBS for
analysis on a flow cytometer (XL, Beckman-Coulter, Miami, Fla.)
using the ExpoADCXL4 software (Beckman-Coulter). Positive staining
was defined as the emission of a fluorescence signal that exceeded
levels obtained by >99% of cells from the control population
stained with matched isotype antibodies (FITC-, PE-, and
Cy-cychrome-conjugated mouse IgG1,.kappa. monoclonal isotype
standards, BD Biosciences), which was confirmed by positive
fluorescence of human BM samples. For each sample, at least 10,000
list mode events were collected. All plots were generated in EXPO
32 ADC Analysis software.
[0132] Sub-Culture & Cell Seeding
[0133] The attached cells were sub-cultured (passaged) using 0.1%
trypsin solution after 7 days, at which point they exhibited 80-90%
confluency as observed by light microscopy. Upon passage, the cells
were observed by flow cytometry for expression of MHC-A,B,C,
MHC-DR,DP,DQ, and CD45. They were then plated in T-75 tissue
culture polystyrene flasks at 4.times.10.sup.3 cells/cm.sup.2 in
SM, and treated with 10.sup.-8M Dex, 5 mM .beta.-GP and 50 .mu.g/ml
ascorbic acid to test the osteogenic capacity of these cells. These
flasks were observed on days 2, 3, 4, 5 and 6 of culture for CFU-O
or bone nodule, formation. Any residual cells from the passaging
procedure also were cryopreserved for future use.
[0134] Cryopreservation of Cells
[0135] Aliquots of 1.times.10.sup.6 PVT cells were prepared in 1 ml
total volume consisting of 90% FBS, 10% dimethyl sulphoxide (DMSO)
(Sigma D-2650, Lot#11K2320), and pipetted into 1 ml polypropylene
cryo-vials. The vials were placed into a -70.degree. C. freezer
overnight, and transferred the following day to a -150.degree. C.
freezer for long-term storage. After one week of cryo-preservation,
the PVT cells were thawed and observed by flow cytometry for
expression of MHC-A,B,C, MHC-DR,DP,DQ, and CD45. A second protocol
was used in which the PVT cells were thawed after one week of
cryopreservation, recultured for one week, sub-cultured then
reanalyzed by flow cytometry for expression of MHC-A,B,C,
MHC-DR,DP,DQ, and CD45.
[0136] The results are presented in FIG. 17. It will be noted that
the frequency of MHC-/- within the fresh cell population is
maintained through several passages. When fresh cells are frozen
after passaging, at -150.degree. C. for one week and then
immediately analyzed for MHC phenotype, this analyzed population
displays a remarkably enhanced frequency of cells of the MHC-/-
phenotype. Thus, and according to an embodiment of the present
invention, cells of the MHC-/- phenotype can usefully be enriched
from a population of PVT cells by freezing. Still further
enrichment is realized upon passaging the cultures of the
previously frozen cells. In particular, and as seen in FIG. 17,
first passage of cryopreserved cells increases the relative
population of MHC-/- cells to greater than 50% and subsequent
freezing and passaging of those cells yields an MHC-/- population
of greater than 80%, 85%, 90% and 95%. The frozen PVT cells per se
are potentially very useful in human therapy, given their non
immunogenic nature.
[0137] Harvest of Post Adherent HUCPV Cell Fraction
[0138] The yield of progenitors recovered from the perivascular
tissue can be enhanced in the following manner. In order to harvest
the "post adherent" (PA) fraction of HUCPV cells, the supernatant
of the initially seeded HUCPV harvest was replated onto a new T-75
flask, and incubated at 37.degree. C., 5% CO.sub.2 for 2 days. The
initially seeded HUCPV flask was then fed with fresh SM. After 2
days this supernatant was again transferred to a new T-75 flask,
and the attached cells fed with fresh SM. Finally, the supernatant
of the third seeded flask was aspirated, and this flask fed with
fresh SM. (Consequently, for each cord, 3 flasks are generated: the
initially seeded flask, the first PA fraction and the second PA
fraction.) Similar to identical characteristics of these cells are
seen compared to the initially seeded cells, confirming that higher
cell yields are obtained by isolating these PA fractions. Similar
to the initially seeded HUCPV cells, these PA cells have a rapid
proliferation rate, spontaneously produce bone nodules in culture,
and can be induced to differentiate into the other three lineages:
cartilage, fat and muscle.
[0139] Tissue Engineering Compositions Comprising the Progenitor
Cells
[0140] In this example, the cells are combined with a carrier in
the form of CAP/PLGA used commonly in the bone engineering field.
In order to seed the CAP/PLGA scaffolds, they were cut into 5 mm by
5 mm cylinders. Then, a 200 .mu.l suspension of 2.times.10.sup.5
HUCPV cells was placed into a sterile 1.5 ml eppendorf tube, and
the scaffold placed into the suspension. Using a modified pipette
tip (with a suction diameter of 5 mm), the suspension of cells was
suctioned and washed through the scaffold several times by
pipetting up and down. The scaffolds were then incubated in this
suspension for 4 hours at 37.degree. C., 5% CO.sub.2, to allow for
attachment of the cells to the scaffold. After the 4 hours, the
scaffolds were removed from the suspensions and placed into
individual wells of a non-tissue culture treated 24-well plate, fed
with 1 ml of SM and incubated at 37.degree. C., 5% CO.sub.2 for 14
days, the SM being replaced every 2 days. The scaffolds were then
fixed in Kamovsky's fixative and prepared for SEM analysis (see
above). After 14 days of culture, the HUCPV cells completely
covered the scaffold.
[0141] As noted, the PVT progenitor cell population may also be
exploited to give rise to mesenchymal cells and tissues other than
bone, by culturing under conditions appropriate for such
differentiation. To generate adipocytes, for instance, the
progenitors are prepared at a concentration of 10.sup.4
cells/cm.sup.2 and plated in 35 mm tissue culture dishes. The cells
are maintained in Preadipocyte Medium (PM) (DMEM/Ham's F-10 (1:1,
vol/vol), 10% fetal calf scrum, 15 mM HBPES, 100 U/ml penicillin,
100 .mu.g/ml streptomycin, 0.25 .mu.g/ml amphotericin B) for 3
days. After 3 days, the PM is removed, and the cells are fed with
Adipogenic medium (DMEM/Ham's F-10 nutrient broth, 1:1, v/v; HEPES
buffer (15 mM); Fetal Bovine Serum (3%); Biotin (33 .mu.M),
Pantothenate (17 .mu.M), human insulin (100 nM), dexamethasone (0.5
.mu.M), PPARY agonist (1 .mu.M) and antibiotics), and cultured for
3 days. After the 3 day induction, the Adipogenic medium is
removed, and the cultures are maintained in Adipocyte Medium (AM)
(DMEM/Ham's F-10 (1:1, vol/vol), 3% fetal calf serum, 1 .mu.M
dexamethasone, 100 nM human insulin, 33 .mu.M D-biotin, 17 .mu.M
Na-pantothenate, 15 mM HEPES, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 0.25 .mu.g/ml amphotericin B), with regular feeding
every 3 days, ensuring to only remove half the medium, replenishing
with an equal volume of AM since adipocytes will float if all the
media is removed. After four feedings (12 days), cells appear
rounded with lipid droplets. Positive identification of
differentiated mesenchymal cells into adipocytes can be confirmed
by staining with Oil Red O and Nile Red.
[0142] Similarly, chondrocytes may be generated using cell
suspensions prepared at a concentration of 10.sup.4 cells/cm.sup.2
and plated in 35 mm tissue culture dishes. To promote chondrogenic
cells are cultured without serum and with transforming growth
factor-.beta.3. The cell pellets develop a multilayered matrix-rich
morphology and histologically show an increased proteoglycan-rich
extracellular matrix during culture.
[0143] To generate myoblasts, cell suspensions are prepared at a
concentration of 10.sup.4 cells/cm.sup.2 and plated in 35 mm tissue
culture dishes. The cells are maintained in MCDB 120 medium
completed with 15% fetal bovine serum (FBS) for 1 week (myoblast
proliferation medium, MPM). At 1 week, the serum level in the basal
medium (MPM) is dropped to 2% (myoblast differentiation medium,
MDM) and the cultures are terminated after 7 days. The cultures are
re-fed 3-times a week with appropriate culture medium.
[0144] It will thus be appreciated that the present invention
provides human progenitor cells having properties useful in the
production of various connective tissues including bone, and
further provides progenitor cells that are immune incompetent and
ideal for transplantation into human patients to treat connective
tissue conditions including bone diseases and disorders. The human
progenitor cells are generated from extracts of a particular zone
of human umbilical cord Wharton's jelly, termed the perivascular
zone, extending proximally from the external wall of the cord
vessels. The cell population extracted from this zone displays
remarkable properties, including rapid proliferation, changes in
cell morphology, as witnessed by the formation of cell colonies
occurring before day 7 in all subcultured flasks (approximately
7-10 doublings) and the appearance of bone nodule formation without
the addition of osteogenic supplements to the culture medium, as
well as relatively high frequency of MHC double negative cells, the
frequency of which is increased upon culturing of cells that have
been frozen.
[0145] The following references are incorporated herein by
reference:
REFERENCES CITED
[0146] Aubin J E, 1998, Bone stem cells: J Cell Biochem Suppl, v.
30-31, p. 73-82. [0147] Canfield, A E, M J Doherty, B A Ashton,
2000, Osteogenic potential of vascular pericytes, in J E Davies
(ed), Bone Engineering: Toronto, E M Squared, Inc., p. 143-151.
[0148] Caplan, A I, 1991, Mesenchymal stem cells: J Orthop. Res, v.
9, p. 641-650. [0149] Chacko, A W, S R M Reynolds, 1954,
Architecture of deistended and nondistended human umbilical cord
tissues, with special reference to the arteries and veins: Carnegie
Institution of Washington, Contributions to Embryology, v. 35, p.
135-150. [0150] Conget, P, J J Minguell, 1999, Phenotypical and
functional properties of human bone marrow mesenchymal progenitor
cells: J. Cell Physiol, v. 181, p. 67-73. [0151] Haynesworth, S E,
D Reuben, A I Caplan, 1998, Cell-based tissue engineering
therapies: the influence of whole body physiology: Adv Drug Deliv
Rev, v. 33, p. 3-14. [0152] Kogler, G, S Sensken, J A Airey, T
Trapp, M Muschen, N Fedhahn, S Liedtke, R V Sorg, J Fischer, C
Rosenbaum, S Greschat, A Knipper, J Bender, O Degistirici, J Gao, A
I Caplan, E J Colletti, G Almeida-Porada, H W Muller, E Zanjani, P
Wernet, 2004, A new human somatic stem cell from placental cord
blood with intrinsic pluripotent differentiation potential: J Exp.
Med., v. 200, p. 123-135. [0153] Mitchell, K E, M L Weiss, B M
Mitchell, P Martin, D Davis, L Morales, B Helwig, M Beerenstrauch,
K Abou-Easa, T Hildreth, D Troyer, 2003, Matrix cells from
Wharton's jelly form neurons and glia: Stem Cells, v. 21, p. 50-60.
[0154] Parry, B E W, 1970, Some electron microscope observations on
the mesenchymal structures of full-term umbilical cord: Journal of
Anatomy, v. 107, p. 505-518. [0155] Pereda, J, P M Motta, 2002, New
advances in human embryology: morphofunctional relationship between
the embryo and the yolk sac: Medical Electron Microscopy, v. 32, p.
67-78. [0156] Romanov, Y A, V A Svintsitskaya, V N Smimov, 2003,
Searching for alternative sources of postnatal human mesenchymal
stem cells: Candidate MSC-like cells from umbilical cord: Stem
Cells, v. 21, p. 105-110. [0157] Schoenberg, M D, A Hinman, R D
Moore, 1960, Studies on connective tissue V, Feber formation in
Wharton's Jelly: Laboratory Investigation, v. 9, p. 350-355. [0158]
Sen, A, Y R Lea-Currie, D Sujkowska, D M Franklin, W O Wilkison, Y
D Halvorsen, J M Gimble, 2001, Adipogenic potential of human
adipose derived stromal cells from multiple donors is
heterogeneous: J. Cell Biochem., v. 81, p. 312-319. [0159] Takechi,
K, Y Kuwabara, M Mizuno, 1993, Ultrastructural and
immunohistochemical studies of Wharton's jelly umbilical cord
cells: Placenta, v. 14, p. 235-245. [0160] Tuchmann-Duplessis, H, O
David, P Haegel, 1972, Illustrated Human Embryology, New York,
Springer-Verlag, p. 54-61. [0161] Weiss, L, 1983, Histology: cell
and tissue biology, New York, Elseiver Biomedical, p. 997-998.
[0162] Wharton, T W, 1656, Adenographia, Translated by Freer S.
(1996). Oxford, U.K., Oxford University Press, p. 242-248.
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