U.S. patent application number 17/523325 was filed with the patent office on 2022-03-03 for methods for assessing the purity of a mesenchymal stem cells preparation.
The applicant listed for this patent is CLINIQUES UNIVERSITAIRES SAINT-LUC, NOVADIP BIOSCIENCES SA, UNIVERSITE CATHOLIQUE DE LOUVAIN. Invention is credited to Denis DUFRANE.
Application Number | 20220064729 17/523325 |
Document ID | / |
Family ID | 1000005962824 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064729 |
Kind Code |
A1 |
DUFRANE; Denis |
March 3, 2022 |
METHODS FOR ASSESSING THE PURITY OF A MESENCHYMAL STEM CELLS
PREPARATION
Abstract
The present invention relates to a method for assessing,
evaluating and/monitoring the purity of a mesenchymal stem cells
preparation, in particular of an adipose stem cells preparation,
comprising measuring the expression level of at least one growth
factor.
Inventors: |
DUFRANE; Denis; (Wavre,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE CATHOLIQUE DE LOUVAIN
CLINIQUES UNIVERSITAIRES SAINT-LUC
NOVADIP BIOSCIENCES SA |
Louvain la Neuve
Bruxelles
Mont-Saint-Guibert |
|
BE
BE
BE |
|
|
Family ID: |
1000005962824 |
Appl. No.: |
17/523325 |
Filed: |
November 10, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15571461 |
Nov 2, 2017 |
11203785 |
|
|
PCT/EP2016/060351 |
May 9, 2016 |
|
|
|
17523325 |
|
|
|
|
62158875 |
May 8, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12N 5/0662 20130101; C12Q 1/6881 20130101; G01N 33/53 20130101;
G01N 2333/475 20130101; G01N 33/5005 20130101; C12N 5/0667
20130101; G01N 33/74 20130101 |
International
Class: |
C12Q 1/6881 20060101
C12Q001/6881; C12N 5/0775 20060101 C12N005/0775; G01N 33/74
20060101 G01N033/74; G01N 33/53 20060101 G01N033/53; G01N 33/50
20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2015 |
EP |
15179417.9 |
Claims
1-14. (canceled)
15. An in vitro method for assessing, evaluating and/or monitoring
the purity of a cell preparation comprising mesenchymal stem cells
(MSC), wherein said method comprises measuring the expression level
of at least one growth factor expressed by said cell preparation,
wherein said at least one growth factor is SDF-1.alpha. and/or
VEGF.
16. The in vitro method according to claim 15, wherein said method
further comprises comparing the measured expression level with a
reference expression level.
17. The in vitro method according to claim 15, wherein said
mesenchymal stem cells are isolated from tissues selected from the
group comprising adipose tissue, bone marrow, umbilical cord blood,
amniotic fluid, Wharton's jelly, placenta, peripheral blood,
fallopian tube, corneal stroma, lung, muscle and fetal liver.
18. The in vitro method according to claim 15, wherein said
mesenchymal stem cells are adipose stem cells (ASC).
19. The in vitro method according to claim 15, wherein said
expression level is assessed at the protein level, preferably by
the detection and/or quantification of said at least one growth
factor secreted in the cell culture supernatant.
20. The in vitro method according to claim 15, wherein said
expression level is assessed at the RNA level, preferably by
RT-PCR, RT-qPCR, Northern Blot and/or hybridization techniques.
21. The in vitro method according to claim 15, wherein said cell
preparation is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml.
22. The in vitro method according to claim 15, wherein said cell
preparation is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml; and/or the VEGF expression level is of at least
200 pg/ml in the cell culture medium, preferably of at least 250,
260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290
pg/ml; wherein said cell preparation is cultured in hypoxic
conditions, preferably at about 0.1% O.sub.2, and at high
concentration of glucose, preferably at about 4.5 g/l of glucose,
before measuring the expression level.
23. The in vitro method according to claim 15, wherein said cell
preparation is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml; and/or the VEGF expression level is of at least 90
pg/ml in the cell culture medium, preferably of at least 95, 100,
105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml,
wherein said cell preparation is cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and at high concentration
of glucose, preferably at about 4.5 g/l of glucose, before
measuring the expression level.
24. The in vitro method according to claim 15, wherein said method
is for assessing the quality or purity of a cell preparation
comprising mesenchymal stem cells (MSC), wherein said cell
preparation comprising MSC is to be used as MSC-based cell therapy
product in regenerative medicine.
25. A cell population identified by an in vitro method for
assessing, evaluating and/or monitoring the purity of a cell
preparation comprising mesenchymal stem cells (MSC), wherein said
method comprises measuring the expression level of at least one
growth factor expressed by said cell preparation, wherein said at
least one growth factor is SDF-1.alpha. and/or VEGF.
26. A kit for implementing an in vitro method for assessing,
evaluating and/or monitoring the purity of a cell preparation
comprising mesenchymal stem cells (MSC), wherein said method
comprises measuring the expression level of at least one growth
factor expressed by said cell preparation, wherein said at least
one growth factor is SDF-1.alpha. and/or VEGF, wherein said kit
comprises means for determining or measuring the expression level
of at least one growth factor, optionally further comprising the
reference for comparing the expression level of said at least one
growth factor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/571,461, filed Nov. 2, 2017, which is a 35
U.S.C. .sctn. 371 filing of International Patent Application No.
PCT/EP2016/060351, filed May 9, 2016, which claims priority to U.S.
Provisional Patent Application Ser. No. 62/158,875, filed May 8,
2015, and European Patent Application No. 15179417.9, filed Jul.
31, 2015. Each of the aforementioned applications is incorporated
herein by reference in its entirety.
FIELD OF INVENTION
[0002] The present invention relates to methods for assessing the
purity of a cell preparation, in particular of a mesenchymal stem
cells preparation. In particular, the present invention relates to
growth factors as biomarker of the purity of a mesenchymal stem
cells preparation.
BACKGROUND OF INVENTION
[0003] The use of stem cell-based therapies for the repair and
regeneration of various tissues and organs offers alternative
therapeutic solutions for a number of diseases. Mesenchymal stem
cells (MSC) are plastic-adherent stromal cells characterized by
their ability to differentiate into mesenchymal tissues such as
bone, cartilage and fat. Because of these properties, MSC seem to
be an ideal population of stem cells for practical regenerative
medicine. One of the richest sources of MSC is adipose tissue and
adipose-tissue derived stem cells (ASC) are widely investigated for
development of new therapies in the field of regenerative medicine
(such as, for example, for wound healing, bone/cartilage
regeneration, Crohn's disease, . . . ).
[0004] Cell therapy products must be manufactured following the
"Good Manufacturing Practice" recommendations which required a
purity analysis of the final cellular products. However,
fibroblasts may be common cell contaminants that affect the purity
of mesenchymal stem cell preparations. Currently, cell therapy
products remain non-characterized in term of cellular purity for
fibroblastic cells. Indeed, current defined criteria for
mesenchymal stem cells, including ASC, characterization are (i)
plastic adherence for cell spreading and proliferation; (ii)
surface markers profile (CD44+, CD45-, CD73+, CD90+, CD105+); and
(iii) differentiation capacity toward
adipose/osteogenic/chondrogenic lineages. However, mesenchymal stem
cells and fibroblasts are considered nowadays as a subfamily
without any capacities to differentiate according to these criteria
(Hematti, Cytotherapy, 2012; 14:516-521). Fibroblasts are
ubiquitous cells, present in variable tissues (dermis, adipose
tissue, muscle . . . ). They are also plastic-adherent, express the
similar surface marker phenotype as MSC and have the capacity to
differentiate towards the mesenchymal lineages when cultivated in
specific media.
[0005] Few studies relate to the identification of fibroblasts from
other cells. Pilling et al. discloses the identification of markers
that discriminate between human peripheral blood monocytes, tissue
macrophages, fibrocytes and fibroblasts (PLoS One. 2009,
4(10):e7475). However mesenchymal stem cells are not included in
this study. Moreover Goodpaster et al. describes that fibroblasts
may be positively identified by the TE-7 antibody which
specifically recognizes growing and quiescent fibroblasts in
formalin-fixed, paraffin-embedded tissue samples (Journal of
Histochemistry & Cytochemistry. 2008, 56(4):347-358).
Nevertheless the TE-7 antibody was not tested on mesenchymal stem
cells.
[0006] Accordingly, there is currently no quantitative and
objective method to distinguish MSC from fibroblasts. Fibroblasts,
however, are associated with cancer cells at all stages of cancer
progression. A cell therapy product comprising fibroblasts is thus
potentially carcinogenic. It therefore exists a need of tools to
assess the purity of an MSC preparation with respect to
fibroblastic contamination, in particular in context of cell
therapy products preparation.
[0007] The inventors herein surprisingly demonstrate that
mesenchymal stem cells and fibroblasts can be differentiated
according to their capacity to secrete factors such as SDF-1.alpha.
and VEGF.
[0008] Therefore, the present invention relates to a method for
assessing the presence of fibroblasts in a preparation of
mesenchymal stem cells, e.g. a composition comprising cells, and
for quantifying the purity of such a preparation.
SUMMARY
[0009] This invention relates to an in vitro method for assessing,
evaluating and/or monitoring the purity of a cell preparation
comprising mesenchymal stem cells (MSC), wherein said method
comprises measuring the expression level of at least one growth
factor expressed by said cell preparation, wherein said at least
one growth factor is SDF-1.alpha. and/or VEGF.
[0010] According to one embodiment, the method of the invention
further comprises comparing the measured expression level with a
reference expression level.
[0011] In one embodiment, the mesenchymal stem cells of the cell
preparation of the invention are isolated from tissues selected
from the group comprising adipose tissue, bone marrow, umbilical
cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral
blood, fallopian tube, corneal stroma, lung, muscle and fetal
liver. In a particular embodiment, the mesenchymal stem cells are
adipose stem cells (ASC).
[0012] According to one embodiment, the expression level of at
least one growth factor expressed by said cell preparation of the
invention is assessed at the protein level, preferably by the
detection and/or quantification of said at least one growth factor
secreted in the cell culture supernatant. In a particular
embodiment, the expression level is assessed at the RNA level,
preferably by RT-PCR, RT-qPCR, Northern Blot and/or hybridization
techniques.
[0013] According to one embodiment, the cell preparation of the
invention is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml.
[0014] According to another embodiment, the cell preparation of the
invention is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml; and/or the VEGF expression level is of at least
200 pg/ml in the cell culture medium, preferably of at least 250,
260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290
pg/ml; wherein said cell preparation is cultured in hypoxic
conditions, preferably at about 0.1% O.sub.2, and at high
concentration of glucose, preferably at about 4.5 g/l of glucose,
before measuring the expression level.
[0015] According to another embodiment, the cell preparation of the
invention is substantially pure when the SDF-1.alpha. expression
level is of at most 100 pg/ml, preferably of at most 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml; and/or the VEGF expression level is of at least 90
pg/ml in the cell culture medium, preferably of at least 95, 100,
105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml,
wherein said cell preparation is cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and at high concentration
of glucose, preferably at about 4.5 g/l of glucose, before
measuring the expression level.
[0016] In one embodiment, the method of the invention is for
assessing the quality or purity of a cell preparation comprising
mesenchymal stem cells (MSC), wherein said cell preparation
comprising MSC is to be used as MSC-based cell therapy product in
regenerative medicine.
[0017] This invention also relates to the use of a growth factor,
preferably SDF-1.alpha. and/or VEGF, as a biomarker of the quality
of a cell preparation comprising mesenchymal stem cells (MSC), in
particular of a cell preparation comprising MSC to be used as
MSC-based cell therapy product in regenerative medicine.
[0018] Another object of the invention is a cell population
identified by the in vitro method as described hereinabove.
[0019] This invention also relates to a substantially pure
mesenchymal stem cells population, preferably an adipose stem cells
population.
[0020] This invention also encompasses to a kit for implementing
the in vitro method as described hereinabove, wherein said kit
comprises means for determining or measuring the expression level
of at least one growth factor, optionally further comprising the
reference for comparing the expression level of said at least one
growth factor.
Definitions
[0021] In the present invention, the following terms have the
following meanings: [0022] "Purity of a cell preparation" refers to
the enrichment of cells of interest from a heterogeneous population
(also called mixed population). In one embodiment, cells of the
invention are mesenchymal stem cells, preferably adipose tissue
mesenchymal stem cells. In one embodiment, the purity according to
the invention is expressed in percentage of mesenchymal stem cells,
preferably adipose tissue mesenchymal stem cells, from a mixed
population comprising other cell types, preferably fibroblasts.
[0023] "Mesenchymal stem cells" or MSCs, are multipotent stem cells
that can differentiate into a variety of cell types including:
osteogenic, chondrogenic, adipogenic, myelosupportive stroma,
myogenic, or neurogenic lineages. Mesenchymal stem cells can be
isolated from tissues including, without limitation, adipose
tissue, bone-marrow, umbilical cord tissue, amniotic fluid,
Wharton's jelly, placenta, peripheral blood, fallopian tube,
corneal stroma, lung, muscle, skin, bone, dental tissue,
pre-menstrual fluid, foreskin and fetal liver, and the like. [0024]
"Adipose tissue" refers to any fat tissue. The adipose tissue may
be brown, yellow or white adipose tissue. Preferably, the adipose
tissue is subcutaneous white adipose tissue. Adipose tissue
includes adipocytes and stroma. Adipose tissue may be found
throughout the body of an animal. For example, in mammals, adipose
tissue may be present in the omentum, bone marrow, subcutaneous
space, fat pads (e.g., scapular or infrapatellar fat pads), and
surrounding most organs. Cells obtained from adipose tissue may
comprise a primary cell culture or a progenitor cell line. The
adipose tissue may be from any organism having fat tissue. [0025]
"Adipose tissue-derived cell" refers to a cell that originates from
adipose tissue. In particular, "adipose tissue mesenchymal stem
cells" (ASC) refer to stromal cells that originate from adipose
tissue which can serve as precursors to a variety of different cell
types such as, but not limited to, adipocytes, osteocytes,
chondrocytes. [0026] "Growth factor" refers to any substance
participating in the regulation of the cellular proliferation or
differentiation. [0027] "Tissular oxygen levels" refers to oxygen
levels from about 3% to about 6%, preferably at an oxygen level of
about 5%. [0028] "Hypoxic environment" refers to oxygen levels from
about 0% to about 1%, preferably at an oxygen level of about 0.1%.
[0029] "Normoglycemic conditions" refers to a concentration of
glucose of from about 0.5 g/l to about 1.5 g/l, preferably at a
concentration of glucose of about 1 g/1. [0030] "Hyperglycaemic
conditions" refers to a concentration of glucose from about 2 g/l
to about 10 g/l, preferably from about 3 g/l to about 6 g/l, more
preferably at a concentration of glucose of about 4.5 g/l. [0031]
"About" preceding a value means plus or less 10% of said value.
[0032] "Passaging", also known as subculture or splitting cells,
refers to transferring a small number of cells into a new vessel
when cells are at confluence or almost, to prolong the life and/or
expand the number of cells in the culture. In one embodiment, the
passage 0 (P0) is the point at which cells were initially placed in
culture. [0033] "Late passaged mesenchymal stem cell" refers to a
cell exhibiting a less immunogenic characteristic when compared to
an earlier passaged cell. The immunogenicity of a mesenchymal stem
cell corresponds to the number of passages. Preferably, the cell
has been passaged up to at least the fourth passage, more
preferably, the cell has been passaged up to at least the sixth
passage, and most preferably, the cell has been passaged up to at
least the eight passage.
DETAILED DESCRIPTION
[0034] This invention relates to a method, preferably an in vitro
method, for assessing, evaluating and/or monitoring the purity of a
cell preparation comprising mesenchymal stem cells (MSC), e.g. a
composition of cells comprising MSC, wherein said method comprises
determining or measuring the expression level of at least one
growth factor expressed by said cell preparation. In one
embodiment, MSC are adipose tissue-derived mesenchymal stem cells
(ASC).
[0035] In one embodiment, the method of the invention is for
assessing the presence of fibroblasts in a cell preparation
comprising mesenchymal stem cells. According to this embodiment,
the method of the invention may thus correspond to a quality
control method, aiming at checking, for example, the purity of a
cell preparation comprising MSC with respect to fibroblastic
contamination. Those MSC may be, for example, used for MSC-based
cell therapy product.
[0036] In one embodiment, the method of the invention is for
quantifying the purity of a cell preparation comprising mesenchymal
stem cells. In one embodiment, the cell preparation is a
heterogeneous preparation comprising mesenchymal stem cells and
other cell types. In a particular embodiment, the cell preparation
comprises mesenchymal stem cells and fibroblasts.
[0037] In one embodiment, the method of the invention is for
quantifying the percentage of mesenchymal stem cells from a
heterogeneous population comprising mesenchymal stem cells and
other cell types, preferably fibroblasts.
[0038] The Applicant demonstrated that with progressive
contaminations of MSC by fibroblasts, the expression level of
growth factors tends to increase or decrease. Consequently, this
invention demonstrates the capacity to discriminate MSC from
fibroblasts, based on their specific growth factors expression. In
particular, the discrimination may be based on their growth factors
expression in specific conditions of oxygenation and/or
glycemia.
[0039] Therefore, in one embodiment, the method of the invention is
for assessing, evaluating and/or monitoring the purity of a cell
preparation comprising MSC with respect to fibroblastic
contamination, based on their specific growth factors
expression.
[0040] Examples of growth factors include, but are not limited to,
adipocytokines, angiopoietins, angiopoietin-like proteins and
receptors thereof, chemokines and receptors thereof, common beta
chain receptors, common gamma chain receptors, EGF, FGF, hedgehog
proteins, IGF, interferons, interleukins and receptors, PDGF, TGF,
TNF, VEGF, SDF-1 and Wnt.
[0041] In one embodiment, the method of the invention comprises
measuring the expression level of SDF-1 (stromal cell-derived
factor 1, also known as C-X-C motif chemokine 12 or CXCL12, Pre-B
Cell Growth-stimulating Factor (PBSF), SCYB12 or TLSF) and/or VEGF
(vascular endothelial growth factor). Preferably, SDF-1 is in the
form of SDF-1.alpha..
[0042] In one embodiment, the method according to the invention
comprises measuring the expression level of SDF-1.alpha.. In
another embodiment, the method according to the invention comprises
measuring the expression level of VEGF. In another embodiment, the
method according to the invention comprises measuring the
expression level of SDF-1.alpha. and VEGF.
[0043] According to one embodiment, MSC are isolated from tissues
selected from the group comprising adipose tissue, bone marrow,
umbilical cord blood, Wharton's jelly (such as, for example,
Wharton's jelly found within the umbilical cord), placenta,
peripheral blood, fallopian tube, corneal stroma, lung, muscle
skin, bone, dental tissue and fetal liver, or the like. In a
particular embodiment, MSC are isolated from adipose tissue. In a
preferred embodiment, MSC are adipose stem cells (ASC).
[0044] In one embodiment, MSC are isolated from any warm-blooded
animal tissues, preferably from human tissues. In a particular
embodiment, MSC are human ASC.
[0045] In one embodiment, the cells are cells in culture,
preferably are cell lines and/or are derived from primary cells,
i.e. cells isolated straight from the tissue. In one embodiment,
the cells are recovered from a sample from an individual, obtained
for example by biopsy. Preferably, the step of recovering the
sample from an individual is not part of the method of the present
invention.
[0046] Isolation of mesenchymal stem cells may be accomplished by
any acceptable method known to one of ordinary skill in the art.
Examples of methods for isolating MSC include, but are not limited
to, digestion by collagenase, trypsinization, or explant
culture.
[0047] In a particular embodiment, mesenchymal stem cells are
isolated from adipose tissue by digestion of the tissue, for
example by collagenase.
[0048] According to one embodiment of the invention, after
isolation, the cell preparation comprising MSC is cultured in any
culture medium designed to support the growth of the cells known to
one of ordinary skill in the art. As used herein, such culture
medium is called "proliferation medium" or "growth medium".
Examples of growth medium include, without limitation, MEM, DMEM,
IMDM, RPMI 1640, FGM or FGM-2, 199/109 medium, HamF10/HamF12 or
McCoy's 5A, preferably DMEM or RPMI.
[0049] In one embodiment, the growth medium may further comprise
any supplementary factors known by the person skilled in the art
that may be used in cell culture. Examples of supplementary factors
include, but are not limited to, FBS; glycine; amino acids, such as
glutamine, asparagine, glutamic acid, aspartic acid, serine,
proline or alanine, preferably the L-configuration of amino acids;
and antibiotics, such as streptomycin or penicillin.
[0050] In a particular embodiment, the cell preparation comprising
MSC is cultured in DMEM supplemented with fetal bovine serum,
glutamine, preferably L-glutamine, and antibiotics such as
penicillin, streptomycin and amphotericin B.
[0051] In one embodiment, the cell preparation comprising MSC may
be contaminated by other types of cells, such as, for example, by
fibroblasts. In a particular embodiment, the cell preparation
comprising MSC is contaminated by fibroblasts.
[0052] In one embodiment, the cell preparation comprising MSC is
cultured in growth medium up to at least 2 passages, preferably at
least 3 passages, more preferably at least 4 passages. As used
herein, the term "cultured up to at least 4 passages" means that
the cell preparation is detached and transferred into a new vessel
up to at least 4 times. In one embodiment, the mesenchymal stem
cells of the cell preparation are late passaged mesenchymal stem
cells.
[0053] For passaging cells, cells may be detached by one of several
methods known to one of ordinary skill in the art, including
trypsin treatment to break down the proteins responsible for
surface adherence, chelating sodium ions with EDTA which disrupts
some protein adherence mechanisms, or mechanical methods like
repeated washing or use of a cell scraper. The detached cells are
then resuspended in fresh medium.
[0054] In one embodiment, the cell preparation comprising MSC is
cultured for at least 24 hours, preferably for at least 36, 48, 60
or 72 hours. In another embodiment, the cell preparation comprising
MSC is cultured for at least 1 day, preferably for at least 2, 3,
4, 5, 6 or 7 days. In another embodiment, the cell preparation
comprising MSC is cultured for at least 10, 15, 20, 25, 30, 35 or
40 days.
[0055] According to one embodiment, the cell preparation comprising
MSC is cultured in standard culture conditions. As used herein,
"standard culture conditions" means at a temperature of about
37.degree. C., and at a tension of about 21% O.sub.2 and of about
5% CO.sub.2.
[0056] In one embodiment, the step of culturing the cell
preparation comprising MSC is not part of the method of the present
invention.
[0057] In one embodiment, culture conditions of the cell
preparation comprising MSC are changed before measuring the
expression level of at least one growth factor.
[0058] As used herein, the term "before measuring the expression
level of at least one growth factor" means at least 6 hours,
preferably at least 9, 12, 15, 18 or 24 hours between the last
passage and the measurement of the expression level of at least one
growth factor.
[0059] According to one embodiment, the cell preparation comprising
MSC is cultured at an oxygen tension of no more than 21%,
preferably at most of 15%, more preferably at most of 10% before
measuring the expression level of at least one growth factor. In a
particular embodiment, the cell preparation comprising MSC is
cultured at an oxygen level from about 3% to about 6%, preferably
at about 5% O.sub.2 corresponding to tissular oxygen tension before
measuring the expression level of at least one growth factor. In
another embodiment, the cell preparation comprising MSC is cultured
at an oxygen level from about 0% to about 1%, at about 0.1% O.sub.2
corresponding to hypoxic environment before measuring the
expression level of at least one growth factor.
[0060] According to another embodiment, the cell preparation
comprising MSC is cultured in a medium comprising from about 0.1 to
about 10 g/l of glucose, preferably from about 0.5 to about 6 g/l
of glucose, more preferably from about 1 to about 4.5 g/l of
glucose before measuring the expression level of at least one
growth factor.
[0061] In one embodiment, the cell preparation comprising MSC is
cultured in a medium comprising a low concentration of glucose,
corresponding to normal blood sugar levels in vivo, i.e. in a
medium comprising from about 0.5 to about 1.5 g/l of glucose,
preferably about 1 g/l of glucose before measuring the expression
level of at least one growth factor. In another embodiment, the
cell preparation comprising MSC is cultured in a medium comprising
a high concentration of glucose, corresponding to hyperglycaemic
conditions, i.e. in a medium comprising from about 2 to about 10
g/l of glucose, preferably from about 3 to about 6 g/l of glucose,
more preferably about 4.5 g/l of glucose before measuring the
expression level of at least one growth factor.
[0062] In one embodiment, the cell preparation comprising MSC is
cultured at about 21% O.sub.2 and about 1 g/l glucose before
measuring the expression level of at least one growth factor. In
another embodiment, the cell preparation comprising MSC is cultured
at about 21% O.sub.2 and about 4.5 g/l glucose before measuring the
expression level of at least one growth factor. In another
embodiment, the cell preparation comprising MSC is cultured at
about 5% O.sub.2 and about 1 g/l glucose before measuring the
expression level of at least one growth factor. In another
embodiment, the cell preparation comprising MSC is cultured at
about 5% O.sub.2 and about 4.5 g/l glucose before measuring the
expression level of at least one growth factor. In another
embodiment, the cell preparation comprising MSC is cultured at
about 0.1% O.sub.2 and about 1 g/l glucose before measuring the
expression level of at least one growth factor. In another
embodiment, the cell preparation comprising MSC is cultured at
about 0.1% O.sub.2 and about 4.5 g/l glucose before measuring the
expression level of at least one growth factor.
[0063] According to another embodiment, culture conditions of the
cell preparation comprising MSC are always the same during all
steps of the method of the invention.
[0064] In one embodiment of the invention, the method comprises the
following steps: [0065] a) culturing a cell preparation comprising
mesenchymal stem cells, [0066] b) optionally changing the culture
conditions, and [0067] c) quantifying expression level of at least
one growth factor, preferably SDF-1.alpha. and/or VEGF.
[0068] In a preferred embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0069] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0070] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0071] c) quantifying SDF-1.alpha. and/or VEGF
expression level.
[0072] In a particular embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0073] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0074] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0075] c) quantifying SDF-1.alpha. expression
level.
[0076] In a particular embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0077] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0078] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0079] c) quantifying SDF-1.alpha. and VEGF expression
level.
[0080] As used herein, the term "expression" may refer
alternatively to the transcription of a growth factor (i.e.
expression of the RNA) or to the translation (i.e. expression of
the protein) of a growth factor, or to the presence of the growth
factor in the supernatant of the cells in culture (i.e. to the
secretion of the growth factor).
[0081] Methods for determining the expression level are well-known
from the skilled artisan, and include, without limitation,
determining the transcriptome (in an embodiment wherein expression
relates to transcription of a growth factor) or proteome (in an
embodiment wherein expression relates to translation or secretion
of a growth factor) of a cell.
[0082] In one embodiment of the invention, the expression of the
growth factor is assessed at the RNA level. Methods for assessing
the transcription level of a growth factor are well known in the
prior art. Examples of such methods include, but are not limited
to, RT-PCR, RT-qPCR, Northern Blot, hybridization techniques such
as, for example, use of microarrays, and combination thereof
including but not limited to, hybridization of amplicons obtained
by RT-PCR, sequencing such as, for example, next-generation DNA
sequencing (NGS) or RNA-seq (also known as "Whole Transcriptome
Shotgun Sequencing") and the like.
[0083] In another embodiment of the invention, the expression of
the growth factor is assessed at the protein level. Methods for
determining a protein level in a sample are well-known in the art.
Examples of such methods include, but are not limited to,
immunohistochemistry, Multiplex methods (Luminex), western blot,
enzyme-linked immunosorbent assay (ELISA), sandwich ELISA,
fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay
(EIA), radioimmunoassay (RIA), flow cytometry (FACS) and the like.
Preferably determining the protein level of the growth factor is
assessed by enzyme-linked immunosorbent assay (ELISA).
[0084] In one embodiment of the invention, determining the
expression level of the growth factor specifically corresponds to
the detection and quantification of said growth factor secreted in
the cell culture supernatant. In a particular embodiment, the
method of the invention comprises measuring expression level of
SDF-1.alpha. and/or VEGF in the cell culture supernatant of the
cell preparation comprising MSC.
[0085] In one embodiment, the step of measuring the expression
level of at least one growth factor is performed when the MSC
comprised in the cell preparation are at a density leading to about
80% confluence, preferably to about 85, 90, 95, 99 or 100%
confluence.
[0086] In the meaning of the invention, the cell preparation
comprising MSC is substantially pure when said cell preparation
comprises less than 25% of fibroblasts, preferably less than 24,
23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11% of
fibroblasts. In one embodiment, the cell preparation comprising MSC
is substantially pure when said cell preparation comprises less
than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of fibroblasts.
[0087] According to one embodiment, the cell preparation comprising
MSC is substantially pure when the SDF-1.alpha. expression level of
the cell preparation comprising MSC is at most of 100 pg/ml,
preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0088] In one embodiment, the cell preparation comprising MSC is
substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at about 21% 02, is at
most of 50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0089] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, is at most of 50 pg/ml,
preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0090] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured in hypoxic condition,
preferably at about 0.1% O.sub.2, is at most of 50 pg/ml,
preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0091] In one embodiment, the cell preparation comprising MSC is
substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at low concentration of
glucose, preferably at about 1 g/l of glucose, is at most of 100
pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0092] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at high concentration of
glucose, preferably at about 4.5 g/l of glucose, is at most of 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0093] In one embodiment, the cell preparation comprising MSC is
substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at about 21% O.sub.2 and
at low concentration of glucose, preferably at about 1 g/l of
glucose, is at most of 100 pg/ml, preferably at most of 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 pg/ml.
[0094] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at about 21% O.sub.2 and
at high concentration of glucose, preferably at about 4.5 g/l of
glucose, is at most of 50 pg/ml, preferably at most of 40, 30, 25,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 pg/ml.
[0095] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and at low concentration
of glucose, preferably at about 1 g/l of glucose, is at most of 100
pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0096] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and at high concentration
of glucose, preferably at about 4.5 g/l of glucose, is at most of
50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0097] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured in hypoxic condition,
preferably at about 0.1% O.sub.2, and at low concentration of
glucose, preferably at about 1 g/l of glucose, is at most of 100
pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0098] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC cultured in hypoxic condition,
preferably at about 0.1% O.sub.2, and at high concentration of
glucose, preferably at about 4.5 g/l of glucose, is at most of 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0099] According to one embodiment, the cell preparation comprising
MSC is substantially pure when the VEGF expression level of the
cell preparation comprising MSC cultured in hypoxic conditions,
preferably at about 0.1% O.sub.2, and at high concentration of
glucose, preferably at about 4.5 g/l of glucose, is at least of 200
pg/ml, preferably at least of 250, 260, 270, 280, 281, 282, 283,
284, 285, 286, 287, 288, 289 or 290 pg/ml.
[0100] According to another embodiment, the cell preparation
comprising MSC is substantially pure when the VEGF expression level
of the cell preparation comprising MSC cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and at high concentration
of glucose, preferably at about 4.5 g/l of glucose, is at least of
90 pg/ml, preferably at least of 95, 100, 105, 110, 111, 112, 113,
114, 115, 116, 117, 188, 119 or 120 pg/ml.
[0101] According to another embodiment, the cell preparation
comprising MSC is substantially pure when the VEGF expression level
of the cell preparation comprising MSC cultured tissular oxygen
tension, preferably at about 5% O.sub.2, and at low concentration
of glucose, preferably at about 1 g/l of glucose, is at least of
160 pg/ml, preferably at least of 161, 162, 163, 164, 165, 166,
167, 168 or 169 pg/ml, more preferably at least of 170 pg/ml.
[0102] In one embodiment, the cell preparation comprising MSC is
substantially pure when the cell preparation comprising MSC
cultured in hypoxic condition, preferably at about 0.1% 02, and at
high concentration of glucose, preferably at about 4.5 g/l of
glucose, presents a SDF-1.alpha. expression level of at most 50
pg/ml, preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF
expression level of at least 200 pg/ml, preferably of at least 250,
260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290
pg/ml.
[0103] In another embodiment, the cell preparation comprising MSC
is substantially pure when the cell preparation comprising MSC
cultured at tissular oxygen tension, preferably at about 5%
O.sub.2, and at high concentration of glucose, preferably at about
4.5 g/l of glucose, presents a SDF-1.alpha. expression level of at
most 50 pg/ml, preferably of at most 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and
a VEGF expression level of at least 90 pg/ml, preferably of at
least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 188,
119 or 120 pg/ml.
[0104] In another embodiment, the cell preparation comprising MSC
is substantially pure when the cell preparation comprising MSC
cultured at tissular oxygen tension, preferably at about 5%
O.sub.2, and at low concentration of glucose, preferably at about 1
g/l of glucose, presents a SDF-1.alpha. expression level of at most
100 pg/ml, preferably of at most 50, 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and
a VEGF expression level of at least 160 pg/ml, preferably at least
of 161, 162, 163, 164, 165, 166, 167, 168 or 169 pg/ml, more
preferably at least of 170 pg/ml.
[0105] In one embodiment of the invention, the method comprises the
following steps: [0106] a) culturing a cell preparation comprising
mesenchymal stem cells, [0107] b) optionally changing the culture
conditions, and [0108] c) quantifying secretion of at least one
growth factor in the cell culture supernatant, preferably
SDF-1.alpha. and/or VEGF.
[0109] In a preferred embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0110] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0111] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0112] c) quantifying SDF-1.alpha. and/or VEGF
secretion in the cell culture supernatant.
[0113] In a particular embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0114] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0115] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0116] c) quantifying SDF-1.alpha. secretion in the
cell culture supernatant.
[0117] In a particular embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0118] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0119] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, and [0120] c) quantifying SDF-1.alpha. and VEGF secretion
in the cell culture supernatant.
[0121] In one embodiment, the method of the invention further
comprises a step of comparing the measured expression level with a
reference level.
[0122] As used herein, the term "reference" broadly encompasses any
suitable reference expression level which may be used as a basis
for comparison with respect to the measured expression level.
[0123] In one embodiment, the reference is a pure fibroblasts
preparation. As used herein, "a pure fibroblasts preparation", e.g.
a composition comprising fibroblasts, means a preparation known to
be free from any other types of cells than fibroblasts. According
to one embodiment, the pure fibroblasts preparation is cultured in
the same conditions as the cell preparation comprising MSC.
[0124] In one embodiment, the cell preparation comprising MSC is
substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC is significatively lower than the
SDF-1.alpha. expression level of the pure fibroblasts preparation.
As used herein, the term "significatively lower" means at least 1.5
fold lower, preferably at least 2, 3 or 4 fold lower, more
preferably at least 5, 6, 7 or 8 fold lower.
[0125] In another embodiment, the cell preparation comprising MSC
is substantially pure when the VEGF expression level of the cell
preparation comprising MSC is significatively higher than the VEGF
expression level of the pure fibroblasts preparation. As used
herein, the term "significatively higher" means at least 1.5 fold
higher, preferably at least 2 fold higher, more preferably at least
2.1, 2.2, 2.3, 2.4 or 2.5 fold higher.
[0126] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC is significatively lower than the
SDF-1.alpha. expression level of the pure fibroblasts preparation
and when the VEGF expression level of the cell preparation
comprising MSC is significatively higher than the VEGF expression
level of the pure fibroblasts preparation.
[0127] In another embodiment, the reference sample is a pure
mesenchymal stem cells preparation. As used herein, "a pure
mesenchymal stem cells preparation", e.g. a composition comprising
mesenchymal stem cells, means a preparation known to be free from
fibroblasts. According to one embodiment, the pure mesenchymal stem
cells preparation is cultured in the same conditions as the cell
preparation comprising MSC to be tested.
[0128] In one embodiment, the cell preparation comprising MSC is
substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC is at least 80%, preferably at
least 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or
100% of the SDF-1.alpha. expression level of the pure MSC
preparation.
[0129] In a particular embodiment, the cell preparation comprising
MSC is totally pure, i.e. without any fibroblastic contamination,
when the SDF-1.alpha. expression level of the cell preparation
comprising MSC and of the pure MSC preparation are the same.
[0130] In another embodiment, the cell preparation comprising MSC
is substantially pure when the VEGF expression level of the cell
preparation comprising MSC is at least 80%, preferably at least
85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or 100%
of the VEGF expression level of the pure cell preparation
comprising MSC.
[0131] In a particular embodiment, the cell preparation comprising
MSC is totally pure, i.e. without any fibroblastic contamination,
when the VEGF expression level of the cell preparation comprising
MSC and of the pure MSC preparation are the same.
[0132] In another embodiment, the cell preparation comprising MSC
is substantially pure when the SDF-1.alpha. expression level of the
cell preparation comprising MSC is at least 80%, preferably at
least 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or
100% of the SDF-1.alpha. expression level of the pure MSC
preparation and when the VEGF expression level of the cell
preparation comprising MSC is at least 80%, preferably at least
85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or 100%
of the VEGF expression level of the pure MSC preparation.
[0133] In one embodiment of the invention, the method comprises the
following steps: [0134] a) culturing a cell preparation comprising
mesenchymal stem cells, [0135] b) optionally changing the culture
conditions, [0136] c) quantifying expression level of at least one
growth factor, preferably SDF-1.alpha. and/or VEGF, and [0137] d)
comparing the expression level measured in step (c) with a
reference expression level.
[0138] In a preferred embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0139] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0140] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, [0141] c) quantifying SDF-1.alpha. and/or VEGF expression
level, and [0142] d) comparing the expression level measured in
step (c) with SDF-1.alpha. and/or VEGF expression levels of a
reference population of cells cultured in the same conditions as
MSC.
[0143] In a particular embodiment, the method of the invention for
assessing the purity of a cell preparation comprising mesenchymal
stem cells, preferably adipose stem cells, comprises the following
steps: [0144] a) culturing a cell preparation comprising
mesenchymal stem cells, preferably adipose stem cells, [0145] b)
optionally changing the culture conditions, preferably the O.sub.2
tension, [0146] c) quantifying SDF-1.alpha. and VEGF expression
level, and [0147] d) comparing the expression level measured in
step (c) with SDF-1.alpha. and VEGF expression levels of a
reference population of cells cultured in the same conditions as
MSC.
[0148] The invention also relates to a cell population identified
by the method of the invention as described hereinabove.
[0149] In one embodiment, the cell population thus identified is
assessed for its purity with respect to fibroblastic
contamination.
[0150] In one embodiment, the cell population thus identified is
substantially pure, i.e. said cell population comprises less than
25% of fibroblasts, preferably less than 24, 23, 22, 21, 20, 19,
18, 17, 16, 15, 14, 13, 12 or 11% of fibroblasts. In one
embodiment, the cell preparation thus identified of the invention
comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of
fibroblasts.
[0151] According to one embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 100
pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.
[0152] According to one embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at about 21% O.sub.2.
[0153] According to another embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at tissular oxygen tension,
preferably at about 5% O.sub.2.
[0154] According to another embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured in hypoxic conditions,
preferably at about 0.1% O.sub.2.
[0155] According to one embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 100
pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the
cell preparation comprising MSC is cultured at low concentration of
glucose, preferably at about 1 g/l of glucose.
[0156] According to another embodiment, the cell population thus
identified presents a SDF-1.alpha. expression level of at most 50
pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 10 pg/ml when the
cell preparation comprising MSC is cultured at high concentration
of glucose, preferably at about 4.5 g/l of glucose.
[0157] In one embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 100 pg/ml,
preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at about 21% O.sub.2 and at
low concentration of glucose, preferably at about 1 g/l of
glucose.
[0158] In another embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 50 pg/ml,
preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at about 21% O.sub.2 and at
high concentration of glucose, preferably at about 4.5 g/l of
glucose.
[0159] In one embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 100 pg/ml,
preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at tissular oxygen tension,
preferably at about 5% O.sub.2, and at low concentration of
glucose, preferably at about 1 g/l of glucose.
[0160] In another embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 50 pg/ml,
preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured at tissular oxygen tension,
preferably at about 5% O.sub.2, and at high concentration of
glucose, preferably at about 4.5 g/l of glucose.
[0161] In one embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 100 pg/ml,
preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured in hypoxic conditions,
preferably at about 0.1% O.sub.2, and at low concentration of
glucose, preferably at about 1 g/l of glucose.
[0162] In another embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 50 pg/ml,
preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell
preparation comprising MSC is cultured in hypoxic conditions,
preferably at about 0.1% O.sub.2, and at high concentration of
glucose, preferably at about 4.5 g/l of glucose.
[0163] According to one embodiment, the cell population thus
identified presents a VEGF expression level of at least of 200
pg/ml, preferably at least of 250, 260, 270, 280, 281, 282, 283,
284, 285, 286, 287, 288, 289 or 290 pg/ml when the cell preparation
comprising MSC is cultured in hypoxic conditions, preferably at
about 0.1% O.sub.2, and high concentration of glucose, preferably
at about 4.5 g/l of glucose.
[0164] According to another embodiment, the cell population thus
identified presents a VEGF expression level of at least of 90
pg/ml, preferably at least of 95, 100, 105, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119 or 120 pg/ml when the cell preparation
comprising MSC is cultured at tissular oxygen tension, preferably
at about 5% O.sub.2, and high concentration of glucose, preferably
at about 4.5 g/l of glucose.
[0165] According to another embodiment, the cell population thus
identified presents a VEGF expression level of at least of 160
pg/ml, preferably at least of 161, 162, 163, 164, 165, 166, 167,
168 or 169 pg/ml, more preferably at least of 170 pg/ml when the
cell preparation comprising MSC is cultured at tissular oxygen
tension, preferably at about 5% O.sub.2, and low concentration of
glucose, preferably at about 1 g/l of glucose.
[0166] In one embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 50 pg/ml,
preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF
expression level of at least 200 pg/ml, preferably of at least 250,
206, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290
pg/ml, when the cell preparation comprising MSC is cultured in
hypoxic conditions, preferably at about 0.1% O.sub.2, and at high
concentration of glucose, preferably at about 4.5 g/l of
glucose.
[0167] In another embodiment, the cell population thus identified
presents a SDF-1.alpha. expression level of at most 50 pg/ml,
preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF
expression level of at least 90 pg/ml, preferably of at least 95,
100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120
pg/ml, when the cell preparation comprising MSC is cultured at 5%
O.sub.2 and high concentration of glucose, preferably at about 4.5
g/l of glucose.
[0168] In another embodiment, the cell preparation thus identified
presents a SDF-1.alpha. expression level of at most 100 pg/ml,
preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF
expression level of at least 160 pg/ml, preferably at least of 161,
162, 163, 164, 165, 166, 167, 168 or 169 pg/ml, more preferably at
least of 170 pg/ml, when the cell preparation comprising MSC is
cultured at 5% O.sub.2 and low concentration of glucose, preferably
at about 1 g/l of glucose.
[0169] Another object of the invention is a substantially pure
mesenchymal stem cells population, preferably an adipose stem cells
population.
[0170] In one embodiment, the substantially pure cell population of
the invention comprises less than 25% of fibroblasts, preferably
less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11%
of fibroblasts. In one embodiment, the cell preparation of the
invention comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of
fibroblasts.
[0171] According to one embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 100 pg/ml, preferably at most of 50, 40, 30, 25,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 pg/ml.
[0172] According to one embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured at about 21% O.sub.2.
[0173] According to another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured at tissular oxygen tension, preferably at about
5% O.sub.2.
[0174] According to another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured in hypoxic conditions, preferably at about 0.1%
O.sub.2.
[0175] According to one embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 100 pg/ml, preferably at most of 50, 40, 30, 25,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 pg/ml when cultured at low concentration of glucose,
preferably at about 1 g/l of glucose.
[0176] According to another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured at high concentration of glucose, preferably at
about 4.5 g/l of glucose.
[0177] In one embodiment, the substantially pure cell population of
the invention presents a SDF-1.alpha. expression level of at most
100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when
cultured at about 21% O.sub.2 and at low concentration of glucose,
preferably at about 1 g/l of glucose.
[0178] In another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured at about 21% O.sub.2 and at high concentration
of glucose, preferably at about 4.5 g/l of glucose.
[0179] In one embodiment, the substantially pure cell population of
the invention presents a SDF-1.alpha. expression level of at most
100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when
cultured at tissular oxygen tension, preferably at about 5%
O.sub.2, and at low concentration of glucose, preferably at about 1
g/l of glucose.
[0180] In another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured at tissular oxygen tension, preferably at about
5% O.sub.2, and at high concentration of glucose, preferably at
about 4.5 g/l of glucose.
[0181] In one embodiment, the substantially pure cell population of
the invention presents a SDF-1.alpha. expression level of at most
100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when
cultured in hypoxic conditions, preferably at about 0.1% O.sub.2,
and at low concentration of glucose, preferably at about 1 g/l of
glucose.
[0182] In another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably at most of 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml when cultured in hypoxic conditions, preferably at about 0.1%
O.sub.2, and at high concentration of glucose, preferably at about
4.5 g/l of glucose.
[0183] According to one embodiment, the substantially pure cell
population of the invention presents a VEGF expression level of at
least of 200 pg/ml, preferably at least of 250, 260, 270, 280, 281,
282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml when cultured
in hypoxic conditions, preferably at about 0.1% O.sub.2, and high
concentration of glucose, preferably at about 4.5 g/l of
glucose.
[0184] According to another embodiment, the substantially pure cell
population of the invention presents a VEGF expression level of at
least of 90 pg/ml, preferably at least of 95, 100, 105, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml when cultured
at tissular oxygen tension, preferably at about 5% O.sub.2, and
high concentration of glucose, preferably at about 4.5 g/l of
glucose.
[0185] According to another embodiment, the substantially pure cell
population of the invention presents a VEGF expression level of at
least of 160 pg/ml, preferably at least of 161, 162, 163, 164, 165,
166, 167, 168 or 169 pg/ml, more preferably at least of 170 pg/ml
when cultured at tissular oxygen tension, preferably at about 5%
O.sub.2, and low concentration of glucose, preferably at about 1
g/l of glucose.
[0186] In one embodiment, the substantially pure cell population of
the invention presents a SDF-1.alpha. expression level of at most
50 pg/ml, preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF
expression level of at least 200 pg/ml, preferably of at least 250,
206, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290
pg/ml, when cultured in hypoxic conditions, preferably at about
0.1% O.sub.2, and high concentration of glucose, preferably at
about 4.5 g/l of glucose.
[0187] In another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 50 pg/ml, preferably of at most 40, 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
pg/ml; and a VEGF expression level of at least 90 pg/ml, preferably
of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119 or 120 pg/ml, when cultured at tissular oxygen tension,
preferably at about 5% O.sub.2, and high concentration of glucose,
preferably at about 4.5 g/l of glucose.
[0188] In another embodiment, the substantially pure cell
population of the invention presents a SDF-1.alpha. expression
level of at most 100 pg/ml, preferably of at most 50, 40, 30, 25,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 pg/ml; and a VEGF expression level of at least 160 pg/ml,
preferably at least of 161, 162, 163, 164, 165, 166, 167, 168 or
169 pg/ml, more preferably at least of 170 pg/ml, when cultured at
tissular oxygen tension, preferably at about 5% O.sub.2, and low
concentration of glucose, preferably at about 1 g/l of glucose.
[0189] Another object of the invention is a kit for implementing
the method of the invention, wherein said kit comprises means for
determining or measuring the expression level of at least one
growth factor of a mesenchymal stem cells (MSC) preparation,
preferably SDF-1.alpha. and/or VEGF.
[0190] In one embodiment, the expression level of at least one
growth factor is assessed at the protein level, and the kit of the
invention may comprise means for detecting the at least one growth
factor, preferably SDF-1.alpha. and/or VEGF. In one embodiment,
said means for detecting the at least one growth factor is an
antibody specific of said at least one growth factor, preferably
SDF-1.alpha. and/or VEGF. In one embodiment, the kit of the
invention may also comprise means for detecting the expression
level of at least one normalization protein.
[0191] In another embodiment, the expression level of at least one
growth factor is assessed at the RNA level, and the kit of the
invention may comprise means for total RNA extraction, means for
reverse transcription of total RNA, and means for quantifying the
expression of RNA of at least one growth factor, preferably VEGF
and/or SDF-1.alpha.. In one embodiment, the means for quantifying
the expression of RNA of at least one growth factor, preferably
SDF-1.alpha. and/or VEGF are PCR or qPCR primers specific for said
growth factor, preferably SDF-1.alpha. and/or VEGF. In one
embodiment, the kit also comprises reagents for carrying out a
quantitative PCR (such as, for example, buffers, enzyme, and the
like). In one embodiment, the kit of the invention may also
comprise means for detecting the expression level of at least one
normalization gene at the RNA level.
[0192] According to one embodiment, the kit of the invention
further comprises the reference for comparing the measured
expression level of the at least one growth factor.
[0193] In one embodiment, the kit of the invention further
comprises a pure fibroblasts preparation. In another embodiment,
the kit of the invention further comprises a pure MSC
preparation.
[0194] In one embodiment, the kit of the invention comprises the
supernatant of a pure fibroblasts preparation. In another
embodiment, the kit of the invention comprises the supernatant of a
pure MSC preparation. In another embodiment, the kit of the
invention comprises a range of dilution of pure MSC preparation
supernatant and pure fibroblasts preparation supernatant. An
example of dilution range is, without limitation, 100/0, 75/25,
50/50, 25/75, 0/100.
[0195] The present invention also relates to a growth factor,
preferably SDF-1.alpha. and/or VEGF, as a biomarker of the quality
or purity of a cell preparation comprising MSC, in particular of a
cell preparation comprising MSC to be used as MSC-based cell
therapy product in regenerative medicine.
[0196] In a particular embodiment, the invention concerns a growth
factor, preferably SDF-1.alpha. and/or VEGF, as a biomarker of the
quality or purity of an ASC preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0197] FIG. 1 is a photograph showing ASC and DF in proliferation
medium (A) and in osteogenic differentiation medium (B).
[0198] FIG. 2 is a graph showing the cell proliferation of ASC and
DF according to the number of passages.
[0199] FIG. 3 is a histogram showing the cell survival of ASC and
DF in proliferation medium without FBS, at 0.1 or 5% O.sub.2.
[0200] FIGS. 4A-4D are a set of histograms showing KGF secretion
(FIG. 4A), b-FGF secretion (FIG. 4B), IGF-1 secretion (FIG. 4C),
and HGF secretion (FIG. 4D) of 5 different ASC/DF dilutions in
proliferation medium with 4.5 g/l glucose, at 0.1 or 5%
O.sub.2.
[0201] FIGS. 5A-5B are a set of histograms showing VEGF secretion
(FIG. 5A) and SDF-1.alpha. secretion (FIG. 5B) of 5 different
ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at
0.1% O.sub.2.
[0202] FIGS. 6A-6B are a set of histograms showing VEGF secretion
(FIG. 6A) and SDF-1.alpha. secretion (FIG. 6B) of 5 different
ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at
5% O.sub.2.
[0203] FIG. 7 is a histogram showing SDF-1.alpha. secretion of 5
different ASC/DF dilutions in proliferation medium with 4.5 g/l
glucose, at 21% O.sub.2.
[0204] FIGS. 8A-8B are a set of histograms showing VEGF secretion
(FIG. 8A) and SDF-1.alpha. secretion (FIG. 8B) of 5 different
ASC/DF dilutions in proliferation medium with 1 g/l glucose, at
0.1% O.sub.2.
[0205] FIGS. 9A-9B are a set of histograms showing VEGF secretion
(FIG. 9A) and SDF-1.alpha. secretion (FIG. 9B) of 5 different
ASC/DF dilutions in proliferation medium with 1 g/l glucose, at 5%
O.sub.2.
[0206] FIG. 10 is a histogram showing SDF-1.alpha. secretion of 5
different ASC/DF dilutions in proliferation medium with 1 g/l
glucose, at 21% O.sub.2.
EXAMPLES
[0207] The present invention is further illustrated by the
following examples.
Example 1
Materials and Methods
[0208] This study was performed according to the guidelines of the
Belgian Ministry of Health. All procedures were approved by the
Ethical Committee of the Medical Faculty (Universite Catholique de
Louvain) for tissue procurement and clinical study (B40320108280).
All materials were obtained from Lonza (Verviers, Switzerland),
Sigma-Aldrich (St. Louis, Mo., USA), or Invitrogen (Carlsbad,
Calif., USA) unless otherwise noted.
ASC and DF Isolation and Culture
[0209] A combined harvesting of human adipose (mean: 7.4 g) and
dermal (mean: 1.5 cm.sup.2) tissues were performed in 8 patients
(Table 1) undergoing elective plastic surgery after informed
consent and serologic screening, by lipoaspiration using the
Coleman technique, and skin biopsy, respectively. Adipose tissue
and skin samples were kept in sterile conditions for a maximum of
60 minutes at 4.degree. C. before adipose-derived stem cells (ASC)
and dermal fibroblasts (DF) isolation.
TABLE-US-00001 TABLE 1 Coupled ASC/DF donors characteristics Donor
1 2 3 4 5 6 7 8 Age 19 44 40 62 56 46 45 41 (years) Sex F F F F F F
F F Clinical mamma- abdomino- mamma- Lat. abdomino- mamma- mamma-
mamma- indication plasty plasty plasty Dorsi plasty plasty plasty
plasty flap
[0210] The adipose tissue was digested with collagenase (1/2 w/v)
in a water bath at 37.degree. C. for 60 minutes. Collagenase was
inactivated in Dulbecco's modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum. Collected tissue was centrifuged for
10 minutes at 1500 rpm at room temperature. The pellet was
suspended in a proliferation medium made up of DMEM supplemented
with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics
(100 U/ml penicillin, 100 .mu.g/ml streptomycin, and 1 .mu.l/ml
amphotericin B) and filtered through a 500-.mu.m mesh screen. The
collected suspension was then seeded in 25 cm.sup.2 culture flasks
with proliferation medium.
[0211] DF were isolated by extraction from de-epidermized dermal
biopsies, minced in 2 mm.times.2 mm fragments and placed in plastic
well. Small volume of the proliferation medium was added to avoid
detachment from the plastic surface.
[0212] After 24 hours of incubation at 37.degree. C. and 5%
CO.sub.2, the proliferation media were replaced. This initial
passage of the primary cells is referred to as passage 0. Dermal
pieces were removed from the culture dish when adherent cells were
visible on the plastic surface surrounding tissue fragments. Cells
were maintained in proliferation medium (changed 2 times/week) up
to passage 4, after sequential trypsinizations. Cells from 3 donors
were cultivated until passage 15 to study the proliferation profile
in standard culture conditions (37.degree. C., 21% O.sub.2, 5%
CO.sub.2, 4.5 g/l glucose).
Membrane Marker Profile Characterization
[0213] At passage 4, ASC and DF were characterized for standard
cell surface markers (CD44, CD45, CD73, CD90, CD105, stro-1, CD106,
CD146, CD166, CD19, CD31, CD11b, CD79.alpha., CD13, HLA-DR, CD14,
CD34) [Dominici et al., Cytotherapy. 2006; 8(4):315-317; Bourin et
al., Cytotherapy. 2013; 15:641-648] by fluorescence-activated cell
sorting (FACScan; BD Biosciences, San Jose, Calif.).
[0214] Briefly, ASC were stained with saturating amounts of
monoclonal antibodies: anti-Stro-1, anti-CD90, anti-CD106,
anti-CD105, anti-CD146, anti-CD166, anti-CD44, anti-CD19, anti-CD45
(Human Mesenchymal Stem Cell marker antibody panel, R&D System,
Minneapolis, Minn., USA), anti-CD44 (PE mouse anti-human CD44, BD
Bioscience, Franklin Lakes, N.J., USA), anti-CD73 (FITC mouse
anti-human CD73, BD Bioscience), anti-CD31 (FITC, mouse anti-human,
Abcam, Cambridge, UK), anti-CD11b (FITC, mouse anti-human, Abcam,
Cambridge, UK), anti-CD79a (PE, mouse anti-human, Abcam, Cambridge,
UK), anti-CD13 (FITC, mouse anti-human, Abcam, Cambridge, UK),
anti-HLA-DR (FITC, mouse anti-human, Abcam, Cambridge, UK),
anti-CD14 (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD34
(PE, mouse anti-human, Abcam, Cambridge, UK). At least 10,000 gated
events were analyzed by flow cytometry with CellquestPro software.
Results are expressed in mean fluorescence intensity (MFI), and
expressed as percentage of positive cells (threshold: 95% of
isotype).
Differentiation Capacity
[0215] ASC and DF were tested at passage 4 in specific media to
assess the capacity of differentiation toward osteogenic lineage.
The differentiation was evaluated by Alizarin red staining after
culturing the cell during 3 weeks in specific differentiation
medium (proliferation medium supplemented with dexamethasone (1
.mu.M), sodium ascorbate (50 .mu.g/ml), and sodium dihydrophosphate
(36 mg/ml) [Qu et al., In Vitro Cell Dev Biol Anim. 2007;
43:95-100]. Osteogenic differentiation was confirmed by staining
for calcium phosphate with Alizarin red after formalin fixation. In
addition, immunohistochemistry for osteocalcin was performed to
confirm the bone phenotype.
Impact of Oxygen Tension and Fetal Bovine Serum (FBS) on Cell
Proliferation: EdU Assay
[0216] Cell proliferation capacity was tested by direct DNA
synthesis measurement by 5-ethynyl-2'-deoxyuridine incorporation
using Click-iT.RTM. EdU Alexa Fluor.RTM. 488 Flow Cytometry Assay
Kit (Life Technology, Waltham, Mass., USA). ASC (n=3) and DF (n=3)
were seeded in 21.5 cm.sup.2 culture dishes at a density of 5000
cells/cm.sup.2, and cultured for 24 hours in 10% FBS, 21% O.sub.2.
Cells proliferation was then stopped by replacing the proliferation
medium by the same, without FBS, for 24 hours. The cells were
finally placed for 48 hours in the specific conditions: 0.1%
O.sub.2, 5% O.sub.2 and 21% O.sub.2 in proliferation medium
supplemented with 1% FBS or 5% FBS and EdU
(5-ethynyl-2'-deoxyuridine, a nucleoside analog of thymidine and
incorporated into DNA during active DNA synthesis) was added. After
revelation with Alexa Fluor.RTM. 488, positive cells were counted
by flow cytometry (FACS can; BD Biosciences, San Jose, Calif.).
Growth Factor Secretion Profile
[0217] After trypsinization, cells (after passage 3) were counted
and 5 progressive dilutions were obtained: 100% ASC+0% DF; 75%
ASC+25% DF; 50% ASC+50% DF; 25% ASC+75% DF; and 0% ASC+100% DF, and
seeded in 12-well culture plates with cells at a density leading to
about 80% to 95% confluence in triplicate for incubation in hypoxic
chambers (Modular Incubator Chamber MIC-101; Billups-Rothenberg,
Del Mar, Calif., USA) at 0.1% O.sub.2 and 5% O.sub.2, corresponding
to highly hypoxic environment and tissular oxygen tension,
respectively. The cells were exposed (for each dilution and oxygen
tension) to normoglycaemic (1 g/L) or hyperglycaemic (4.5 g/L)
proliferation media. After incubation for 24 hours in these
controlled conditions; cell culture supernatants were harvested
individually and stored at -20.degree. C. for further growth factor
quantification by enzyme-linked immunosorbent assay (VEGF, HGF,
IGF-1, SDF-1.alpha. and basic FGF by Quantikine ELISA kit; R&D
System, Minneapolis, Minn., USA). Cellular viability was assessed
immediately after the hypoxic stress by
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium solution (MTS; Promega, Leiden, the Netherlands)
assay. Hypoxic/glycaemic stress tests and growth factor
quantifications were performed in triplicate and duplicate,
respectively. Results are expressed in picograms per
millimeter.
Statistical Analysis
[0218] The one-sample Kolmogorov test and Q-Q plots were used to
assess the normal distribution of values. Statistically significant
differences between groups (with normal distribution) were tested
by paired t-test and one-way analysis of variance with the
Bonferroni post hoc test. Statistical tests were performed with
PASW 18 (SPSS; IBM, New York, N.Y., USA); p<0.05 was considered
significant.
Results
[0219] Surface marker profiles do not allow the distinction between
the two cell populations (Table 2).
TABLE-US-00002 TABLE 2 Surface marker characterization of human ASC
and DF ASC DF % of positive % of positive cells cells Mesenchymal
(stromal) cells markers CD13 99.06 99.86 CD44 95.53 99.97 CD73
93.78 99.86 CD90 98.63 100.00 CD105 96.86 99.78 CD166 60.74 96.51
Bone marrow-derived MSC markers CD106 5.41 2.83 Stro-1 4.03 5.73
CD146 7.16 33.91 Endothelial cells markers CD31 5.59 5.41
Hematopoietic lineage markers CD14 6.75 28.27 CD45 5.15 0.62 CD11b
5.80 8.65 CD34 5.53 0.54 Human leukocyte antigens HLA-DR 6.52 1.65
CD19 4.51 2.05 CD79.alpha. 5.10 0.37
[0220] ASC and DF were positive (>90% of positive cells) for
mesenchymal cell markers (CD13, CD44, CD73, CD90, CD105, CD166),
negative for endothelial (CD31), bone-marrow-derived stromal cells
(CD106, Stro-1, CD146) and hematopoietic markers (CD14, CD45,
CD11b, CD34), and for HLA-DR, CD79a and CD19. After culture in
specific differentiation media (FIG. 1), osteogenic differentiation
capacity was demonstrated for both ASC and DF by Alizarin red
staining and osteocalcin immunohistochemistry.
[0221] ASC and DF had similar proliferation profile until passage
15 (FIG. 2, NS).
[0222] ASC and DF viability was not significantly impacted after 24
hours of culture at 0.1% O.sub.2 and 5% O.sub.2 without FBS (FIG.
3). At 5% O.sub.2, DF viability was reduced when compared to ASC
(87.04% of ASC survival, p<0.05).
[0223] The study of HGF, IGF-1, bFGF and KGF secretion (at 0.1% and
5% 02, 4.5 g/l glucose) from the sequential dilutions of ASC and DF
did not demonstrate any significant curve (FIGS. 4A-4D).
[0224] However, for VEGF and SDF-1.alpha., linear regressions
following ASC "contamination" by DF were observed. Indeed,
SDF-1.alpha. secretion level decreases with increasing ASC
proportion. This result is found in different conditions of oxygen
tension (21%, 5% or 0.1%) or of glucose concentrations (1 g/l or
4.5 g/l) (FIGS. 5 to 10). Moreover, in high glucose culture
conditions and at 0.1% O.sub.2 and 5% O.sub.2 VEGF secretion level
increases with increasing ASC proportion (FIGS. 5A and 6A). The
same measurements in low glucose conditions demonstrated
significant linear regressions for VEGF secretion at 5% O.sub.2
(FIG. 8A).
[0225] The relations were inversed since DF release higher levels
of SDF-1.alpha. and VEGF was produced in higher rates by ASC,
allowing the measurement of the cell proportion (ASC purity).
* * * * *