U.S. patent application number 13/675954 was filed with the patent office on 2013-04-04 for stem cells from adipose tissue, and differentiated cells from said cells.
This patent application is currently assigned to Yves Saint Laurent Parfums. The applicant listed for this patent is Centre National De La Recherche Scientifique, Universite de Nice Sophia Antipolis, Yves Saint Laurent Parfums. Invention is credited to Gerard Ailhaud, Christian Dani, Anne-Marie Rodriguez.
Application Number | 20130084270 13/675954 |
Document ID | / |
Family ID | 31497207 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130084270 |
Kind Code |
A1 |
Rodriguez; Anne-Marie ; et
al. |
April 4, 2013 |
STEM CELLS FROM ADIPOSE TISSUE, AND DIFFERENTIATED CELLS FROM SAID
CELLS
Abstract
The invention concerns adult multipotent human stem cells,
characterized in that they have: i) significant telomerase
activity, ii) an HLA Class I negative phenotype, iii) a normal
karyotype, iv) a capacity to become quiescent, v) a capacity for
self-renewal preserved for at least 130 population doublings.
Inventors: |
Rodriguez; Anne-Marie;
(Nice, FR) ; Dani; Christian; (Nice, FR) ;
Ailhaud; Gerard; (Gonfaron, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yves Saint Laurent Parfums;
Centre National De La Recherche Scientifique;
Universite de Nice Sophia Antipolis; |
Neuilly Sur Seine
Paris Cedex
Nice Cedex |
|
FR
FR
FR |
|
|
Assignee: |
Yves Saint Laurent Parfums
Neuilly Sur Seine
FR
Universite de Nice Sophia Antipolis
Nice Cedex
FR
Centre National De La Recherche Scientifique
Paris Cedex
FR
|
Family ID: |
31497207 |
Appl. No.: |
13/675954 |
Filed: |
November 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10632581 |
Jul 31, 2003 |
8334135 |
|
|
13675954 |
|
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Current U.S.
Class: |
424/93.7 ;
435/29; 435/366 |
Current CPC
Class: |
C12N 2501/39 20130101;
C12N 2502/1305 20130101; A61K 35/12 20130101; C12N 2501/125
20130101; G01N 2333/918 20130101; C12N 2510/00 20130101; C12N
2500/42 20130101; C12N 2501/33 20130101; C12N 2500/38 20130101;
G01N 33/5017 20130101; C12N 2500/25 20130101; C12N 2501/135
20130101; G01N 33/5008 20130101; C12N 2501/70 20130101; C12N
2501/115 20130101; C12N 5/0667 20130101; C12N 2501/11 20130101;
C12N 2501/13 20130101; G01N 33/5023 20130101; G01N 33/5073
20130101; C12N 2503/02 20130101; C12N 2501/01 20130101 |
Class at
Publication: |
424/93.7 ;
435/366; 435/29 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2002 |
FR |
0209799 |
Feb 28, 2003 |
FR |
0302657 |
Claims
1. An adult multipotent human stem cell, characterized in that it
has: i) significant telomerase activity, ii) an HLA Class I
negative phenotype, iii) a normal karyotype, iv) a capacity to
become quiescent, v) a capacity for self-renewal preserved for at
least 130 population doublings. wherein the stem cell is
characterized in that it can express at least one transgene.
2.-12. (canceled)
13. A method for obtaining multipotent human stem cells comprising
the following steps: (a) culturing cells from a human adipose
tissue sample, (b) selecting two cell sub-populations termed a "CA"
population and a "CS" population, the "CA" population having an
adhesion rate of less than 12 hours, and the "CS" population having
an adhesion rate of more than 12 hours, (c) enriching the "CA"
population until a quiescent cell population is obtained, and (d)
inducing proliferation of stem cells of the "CA" population.
14. A method according to claim 13, comprising the following
additional steps prior to step (a): (i) enzymatically digesting a
sample of adipose tissue; and (ii) recovering a cell fraction that
is free of adipocytes, containing all of the cell types present in
the preparation obtained in (i) with the exception of adipocytes;
wherein the culturing in step (a) is carried out using the cell
fraction obtained in step (ii) for at least 12 hours.
15. A method according to claim 13, characterized in that the
adipose tissue sample derives from a healthy child under 10 years
of age.
16. A method according to claim 13, characterized in that the
adipose tissue sample is a sample of extramedullary tissue derived
from the umbilical region, from the pubic region, from the inguinal
region, from the perineal region, from the abdominal region, or
from the subcutaneous region.
17. A method according to claim 13, characterized in that
proliferation of stem cells in step (d) is an intensive
proliferation induced by adding a growth factor.
18. A method according to claim 14, characterized in that the
enzymatic digestion in step (i) is carried out by bringing the
adipose tissue sample into contact with a collagenase preparation
for a maximum period of 10 minutes.
19. A method according to claim 14, characterized in that the
recovery of the cell fraction that is free of adipocytes in step
(ii) is carried out by centrifugation.
20. A method according to claim 14, characterized in that the cell
fraction that is cultured does not undergo any filtration step
before culturing.
21. A method according to claim 14, characterized in that the
culturing step is carried out in a culture medium supplemented with
foetal calf serum without the addition of other growth factors.
22. A method according to claim 14, characterized in that during
the culturing step, cell transfer is carried out when the cells
reach 80% confluence, transfer being carried out at a seeding
density of about 1000 to 3500 cells/cm.sup.2.
23. A method according to claim 13, characterized in that the "CA"
population becomes quiescent after about 60 population
doublings.
24. A method according to claim 17, characterized in that the
growth factor employed is selected from the group consisting of
bFGF, PDGF, EGF, NGF and SCF.
25.-28. (canceled)
29. A therapeutic product for use in in vivo tissue regeneration
which comprises adult multipotent human stem cells having: i)
significant telomerase activity, ii) an HLA Class I negative
phenotype, iii) a normal karyotype, iv) a capacity to become
quiescent, v) a capacity for self-renewal preserved for at least
130 population doublings.
30.-32. (canceled)
33. A method for producing differentiated cells of the mesodermal
lineage, characterized in that adult multipotent human stem cells
are cultivated from confluence in the presence of a differentiation
medium, wherein the stem cells have: i) significant telomerase
activity, ii) an HLA Class I negative phenotype, iii) a normal
karyotype, iv) a capacity to become quiescent, v) a capacity for
self-renewal preserved for at least 130 population doublings.
34. A method according to claim 33, characterized in that the stem
cells are seeded at a density of about 10 000 to 25 000
cells/cm.sup.2.
35. A method according to claim 33, characterized in that the
differentiation medium is a medium allowing differentiation into
adipocytes.
36. A method according to claim 33, characterized in that the
differentiation medium is a medium allowing differentiation into
osteoblasts.
37. A method according to claim 33, characterized in that the
differentiation medium is a medium allowing differentiation into
myocytes, or an angiogenic medium.
38. A screening method to identify agents that can modulate the
differentiation of cells into cells of the mesodermal lineage,
characterized by: a) culturing adult multipotent human stem cells
under culture conditions that allow their differentiation into
cells of the mesodermal lineage, in the presence of a candidate
agent, wherein the stem cells have: i) significant telomerase
activity, ii) an HLA Class I negative phenotype, iii) a normal
karyotype, iv) a capacity to become quiescent, v) a capacity for
self-renewal preserved for at least 130 population doublings; b)
comparing the differentiation of cells in the presence of a
candidate agent with differentiation in the absence of the
candidate agent.
39.-42. (canceled)
43. A screening method for identifying agents that may have a
lipolytic activity, characterized by: a) culturing adult
multipotent human stem cells under conditions allowing their
differentiation into adipocytes, wherein the stem cells have: i)
significant telomerase activity, ii) an HLA Class I negative
phenotype, iii) a normal karyotype, iv) a capacity to become
quiescent, v) a capacity for self-renewal preserved for at least
130 population doublings; b) bringing the adipocytes thus obtained
into contact with a candidate agent, c) evaluating the lipolytic
activity of the candidate agent.
44. A screening method for identifying agents that may have an
anti-lipolytic activity, characterized by: a) culturing adult
multipotent human stem cells under conditions allowing their
differentiation into adipocytes, wherein the stem cells have: i)
significant telomerase activity, ii) an HLA Class I negative
phenotype, iii) a normal karyotype, iv) a capacity to become
quiescent, v) a capacity for self-renewal preserved for at least
130 population doublings; b) bringing the adipocytes thus obtained
into contact with a candidate agent, in the presence of a lipolytic
agent, c) evaluating the anti-lipolytic activity of the candidate
agent.
45. A screening method for identifying agents that may have an
insulin-sensitizing activity, characterized by: a) culturing adult
multipotent human stem cells under conditions allowing their
differentiation into adipocytes, wherein the stem cells have: i)
significant telomerase activity, ii) an HLA Class I negative
phenotype, iii) a normal karyotype, iv) a capacity to become
quiescent, v) a capacity for self-renewal preserved for at least
130 population doublings; b) bringing the adipocytes obtained into
contact with a candidate agent, c) evaluating the
insulins-sensitizing activity of the candidate agent.
46. (canceled)
47. A cosmetic composition comprising a plurality of adult
multipotent human stem cells having: i) significant telomerase
activity, ii) an HLA Class I negative phenotype, iii) a normal
karyotype, iv) a capacity to become quiescent, v) a capacity for
self-renewal preserved for at least 130 population doublings; in
association with an excipient, vehicle, solvent, colorant,
fragrance, antibiotic or other additives acceptable in cosmetic
products.
48.-54. (canceled)
55. A pharmaceutical composition comprising a cell population and a
physiologically acceptable excipient, wherein the cell population
comprises a plurality of adult multipotent human stem cells having:
i) significant telomerase activity, ii) an HLA Class I negative
phenotype, iii) a normal karyotype, iv) a capacity to become
quiescent, v) a capacity for self-renewal preserved for at least
130 population doublings; characterized in that it is free of
adipocytes, fibroblasts, preadipocytes, endothelial cells,
pericytes, mastocytes, and smooth muscle cells.
Description
[0001] The present invention relates to mulitpotent human stem
cells that can be isolated from human adipose tissue and to the use
of said cells in therapy and cosmetology. The invention also
concerns a method for isolating said stern cells from adult human
adipose tissue and to a method for differentiating them into cells
of endodermal or ectodermal or mesodermal origin. Finally, the
invention relates to screening methods for identifying agents that
are capable of exerting an effect either on cell differentiation or
on the function of differentiated cells.
[0002] The presence of adult multipotent "stem" cells has been
demonstrated in a large number of tissues, for example the bone
marrow, blood, liver, muscle, the nervous system, and in adipose
tissue. Adult "stem" cells, which in theory are capable of infinite
self-renewal, have great cell plasticity, i.e. the ability to
differentiate into tissues other than those for which it was
believed they were destined. The properties of said cells, which
are similar to those of embryonic stem cells (ES), open up
considerable therapeutic perspectives especially as their use does
not pose the problems of compatibility and ethics, encountered with
ES cells.
[0003] Unfortunately, their medical application (transplantation)
is currently extremely limited for two main reasons: [0004]
firstly, it is very difficult to isolate said cells. Indeed Stem
cells are very rare in an organism and very little is currently
known about them, in particular at a molecular level, rendering
direct purification impossible. There is a method for enriching
multipotent cells (a population known as "SP" for "side
population") based on the capacity to exclude a vital stain
(Goodell M A et al. (1996), J Exp Med, vol 83, 1797-1806; Zhou S et
al. (2001) Nature Medecine, vol 7, 1028-1034). Other methods
involve positive or negative selection, based on the presence or
absence of cell markers. As an example, International patent
application WO 01/11011 describes the depletion of bone marrow
cells of CD45+ glycophorin A+ cells followed by culturing
CD45-/GlyA-cells in the presence of growth factors. A similar
method has been described by Reyes et al (Blood, November 2001,
vol. 98, n.sup.o 9, 2615-2625). [0005] secondly, prior
amplification of said cells in the undifferentiated state in vitro
poses a major problem.
[0006] While many investigators have successfully demonstrated the
presence of multipotent human cells in a large number of tissues,
no-one has been able to maintain these cells in vitro in the
undifferentiated state beyond 50 to 80 population doublings.
Self-renewal capacity is a doubly important indicator: first,
because of the very limited number of stem cells naturally present
in adult tissue, a quantity of stem cells sufficient for
therapeutic use can only be obtained if they can be multiplied in
vitro while preserving their original characteristics. Second, the
self-renewal capacity is closely related to the very definition of
a true stem cell (which is immortal) and thus can be indirectly
correlated with extended cell plasticity. Thus, it is highly
desirable to be able to isolate multipotent cells with a
self-renewal capacity that is conserved beyond 100 population
doublings.
[0007] International patent application WO 01/11011 (Furcht,
Verfaillie and Reyes) describes human multipotent cells isolated
from bone marrow. Said cells have a normal caryotype, a negative
HLA class I phenotype and can be maintained in culture in vitro up
to 40 population doublings. However, some rare cells can reach 70
population doublings. The authors indicate that these cells can
differentiate into cells of the mesodermal lineage, for example
into osteoblasts, chondroblasts, adipocytes and myocytes, and also
into cells of the ectodermal lineage and of the endodermal lineage.
Said cells, however, are capable of a limited number of divisions,
and so the authors suggest that, to allow the production of a large
quantity of cells, a heterologous gene coding for telomerase should
be introduced. The heterologous gene must be excised before using
in transplantation. This reversible immortalization technique
remains highly controversial, however. There is in fact a risk of
malignant transformation of cells when an exogenous telomerase
activity is introduced at a level of expression that is
non-physiological and not controlled by the cell (Wong Jing et al.,
Nature, 405, June 2000, 755-756). Similar studies have also been
described by Reyes et al (Blood, November 2001, vol. 98, n.sup.o 9,
2615-2625).
[0008] Jiang et al (Nature, Advance Online Publication 20 Jun.
2002, doi:10.1098/nature00870) describe the production of
multipotent mouse and rat cells which can be maintained in culture
in vitro beyond 100 population doublings. The authors also make
reference to a human multipotent population which could be
maintained in vitro beyond 80 population doublings. However, no
supplemental information is given on the subject of these human
cells.
[0009] Qu-Petersen et al (J. Cell Biol. 157, 5, 2002, 851-864)
report the production of multipotent murine cells from muscle
tissue. Said cells, termed "MDSC" cells ("Muscle Derived Stem
Cells"), have a normal caryotype and have a self-renewal capacity
while retaining their multipotentiality for about 30 population
doublings. They can differentiate into cells from different
lineages. However, the multipotent character disappears beyond 40
population doublings, at which stage the cells become senescent and
die. This work strongly suggests that said multipotent cells
display immunoprivileged behavior. Indeed, injection of said cells
into the muscles of dystrophic mice which are immunologically
different from those from which the MDSC cells are derived, results
in substantial muscle regeneration even in the absence of
immunosuppressive substances of the cyclosporine type.
Surprisingly, the authors observed that the transplantation did not
cause infiltration of tissue by CD4.sup.+ and CD8.sup.+ lymphocytes
of the grafted mouse. This tolerance is at least in part explained
by the negative MHC class I phenotype of the MDSC cells. This work
thus shows that MDSC cells are not recognized by the T lymphocytes
of the (immunologically incompatible) receiver, and suggests that
said cells could be used in allo-transplantation. This study was
not extended to human cells.
[0010] Two hypotheses can be proposed to explain the limited
self-renewal capacity shown by the different multipotent cells
isolated up to now: [0011] firstly, it can be assumed that the
various studies were not carried out on true "stem" cells but
rather on intermediate precursors. This hypothesis is all the more
supported by on the fact that stem cells can readily be confused
with precursors in terms of plasticity. Further, contamination of
the culture by precursors is facilitated by their abundance
compared with "stem" cells, but their lifetime is limited; [0012]
secondly, it is also possible to envisage that the "stem" cells
could not be maintained in vitro in the undifferentiated state as
the culture conditions were unsuitable.
[0013] It should also be noted that to date, many of the methods
carried out to obtain multipotent cells use bone marrow as the cell
source. However, removing cells from the bone marrow is a difficult
operation involving risks for the patient and representing a meager
source of stem cells. Thus, it is a technique that is poorly suited
to the large scale production of stem cells.
[0014] Several teams of investigators have therefore attempted to
develop methods that allow isolation of multipotent cells from
other, more abundant tissues, which methods do not run major risks
for the patients. From this standpoint, adipose tissue a priori
constitutes a promising source. However, to date, while the
presence of multipotent cells has been demonstrated in human
adipose tissue, the results have been fairly disappointing. The
cell populations obtained are often heterogeneous and cannot be
maintained in culture in vitro beyond two or three population
doublings. Further, to date, no investigators have reported the
production from human adipose tissue of multipotent cells with a
negative HLA class I phenotype. This characteristic, which is not
required for self-grafting use, becomes indispensable if cells are
intended for broader therapeutic use, in particular for
allo-transplantation.
[0015] For example, International patent applications WO 01/62901
(Artecel Sciences Inc) and EP 1 077 254 (Zen Bio Inc) describe the
production, from adipose tissue, of populations of stromal cells
with a multipotent character. Said populations are heterogeneous
and contain, inter alia, pericytes, endothelial cells and smooth
muscle cells (see Erickson et al., Biochem. and Biophys. Res. Com.
290, 763-769, (2002)). Their self-renewal capacity is extremely
limited and an analysis of the expression of surface markers
confirms that they are positive HLA class I. The characteristics of
said cell populations are thus not compatible with their use in
therapy.
[0016] American patent US 2002/0076400 (Katz et al.) and WO
00/53795 (University of Pittsburgh and the Regents of the
University of California) also describe the production of
multipotent cell populations from human adipose tissue. Said cell
populations can be differentiated into adipocytes, osteoblasts,
chondrocytes and myocytes. According to the authors, they can be
maintained in culture in vitro for at least 15 cell transfers
without losing their multipotent character. No information is given
regarding the corresponding population doubling. Before subjecting
the cells to successive transfers, a telomerase activity was
detected in said population, which was heterogeneous. This activity
was not measured after successive transfers. No surface marker
analyses were carried out. The expression of HLA class I antigens
was thus not determined.
[0017] The present invention overcomes the disadvantages of the
techniques described above.
[0018] The present inventors have developed a method that can
reproducibly isolate multipotent "stem" cells from the adipose
tissue of young children and multiply them in the undifferentiated
state in large quantities in vitro for more than 200 population
doublings. Their therapeutic use thus becomes possible. In its
major aspect, the invention concerns a method for producing stem
cells from adipose tissue and also the use of the stem cells
obtained.
[0019] In the context of the present invention the following terms
signify: [0020] Self-renewal: the capacity of division without
altering the initial characteristics of the cell. [0021] stem cell:
multipotent cell with a high self renewal capacity, with a
telomerase activity and capable of becoming quiescent. [0022] adult
stem cell: stem cell other than an embryonic stem cell deriving,
for example, from a newborn, a child or an adult. [0023] multi
potent or multipotential: capable of differentiating into at least
two cell types. [0024] quiescent: the capacity of a cell to remain
in a non-proliferating and non-senescent state.
[0025] More particularly, the invention concerns a method for
producing human multipotent stem cells from adult tissue in
particular from adult adipose tissue. In a first step, the method
comprises culturing cells deriving from a tissue sample, preferably
adult adipose tissue. Other types of tissue which can be used
include the muscle, bone marrow, liver, and nervous system. After
12 hours of culture, the cells are separated into two
sub-populations depending on their adhesion rate, a first cell
population "CA" adhering in less than 12 h, and a second cell
population "CS" adhering more slowly and occurring, after 12 hours
of culture, in suspension in the culture medium. The "CA"
population is then enriched until a population of cells that is
capable of becoming quiescent is obtained. From this stage,
intensive proliferation of stem cells of the "CA" population can
then be induced.
[0026] In a preferred variant of the invention, the method for
producing multipotent human stem cells comprises the following
steps: [0027] a) enzymatic digestion of a sample of adipose tissue;
[0028] b) recovering a cell fraction that is free of adipocytes,
containing all of the cell types present in the preparation
obtained in (a) with the exception of adipocytes; [0029] c) in
vitro culture for at least 12 hours of the cell fraction obtained
in step (b), [0030] d) selection of two cell sub-populations termed
population "CA" and population "CS", population "CA" having an
adhesion rate of less than 12 hours, and population CS having an
adhesion rate of more than 12 hours; [0031] e) enriching population
"CA" until a population of cells is obtained that are capable of
becoming quiescent; [0032] f) optionally, inducing enhanced
proliferation of stem cells of the "CA" population, for example by
adding a growth factor.
[0033] FIG. 19 shows a diagram of a preferred variant of the method
of the invention.
Step (a): Enzymatic Digestion of a Sample of Adipose Tissue:
[0034] The enzymatic digestion step is preferably carried out by
bringing the adipose tissue sample into contact with an enzymatic
preparation such as collagenase for a short period, i.e. a maximum
of 10 minutes, and more preferably 5 to 10 minutes, or 5 to 8
minutes. This allows complete dissociation of the tissue while
avoiding damage to certain cell types and thus results in better
viability of all of the cell types.
[0035] Regarding the nature of the adipose tissue, it preferably
derives from a healthy individual, preferably a healthy young
child, preferably below 10 years of age, for example a newborn or a
child of 2 to 3 months to 8 years old. The child may be male or
female.
[0036] The age of the donor appears to be an important point.
Indeed, a certain amount of data obtained from hematopoietic "stem"
cells strongly suggests that the "stem" cells not only reduce in
number with the age of the individual but also undergo an ageing
process resulting in a loss of functionality (Geiger H and Van Zant
(2002), Nature, vol 3, n.sup.o 4, 329-333).
[0037] If this data is extrapolated, the adipose tissue of young
children appears to constitute a more abundant and more functional
source of "stem" cells than the adipose tissue of adult
individuals.
[0038] The adipose tissue sample can derive from any anatomical
site, but is preferably a sample of tissue of extramedullary
origin, more particularly from the umbilical region or from the
pubic region or from the inguinal region or from the perineal
region or from the abdominal region or from the subcutaneous
region. The pubic, pre-pubic, inguinal and umbilical regions are
more particularly preferred.
Step (b): Recovery of a Cell Fraction Free of Adipocytes:
[0039] The adipose tissue that has undergone enzymatic digestion is
then treated to remove the adipocytes. An adipocyte free cell
fraction is then recovered, containing all of the cell types
present in the adipose tissue (for example pre adipocytes, stem
cells, endothelial cells, pericytes, mastocytes . . . ) with the
exception of adipocytes.
[0040] The adipocytes can be eliminated by any appropriate means.
Centrifuging is particularly effective since all of the cells of
interest can be found in the centrifugation pellet while adipocytes
float in the supernatant.
[0041] It is important to note that this step in the procedure is
carried out without filtering, enabling all cell types other than
the adipocytes to be preserved in culture. In conventional
techniques for preparing cells from adipose tissues, filtration
steps, successive or otherwise, are generally carried out;
depending on the authors, before or after centrifuging, to
eliminate waste. This step, however, risks the loss of certain cell
types.
Step (c): In Vitro Culture:
[0042] The cell fraction obtained during step (b) is then cultured
for at least 12 hours, preferably for 12 to 80 hours, for example
12 to 72 hours.
[0043] For this step of the procedure, the cells are seeded at a
density in the range 1000 to 5000 cells/cm.sup.2, for example 1000
to 3500 cells/cm.sup.2. Multipotent cells are not actually present
in great numbers compared with other cell types, and high density
seeding thus ensures that each dish contains this cell type.
[0044] The culture medium used for this step of the method is
normally a DMEM type culture medium supplemented with foetal calf
serum without the addition of other growth factors. As an example,
a particularly suitable medium is: DMEM+10% decomplemented foetal
calf serum+antibiotics (100 U/ml penicillin, 100 .mu.g/ml
streptomycin).
[0045] The cell yield for this step varies depending on the sample:
1000 to 5000 cells per mg of tissue.
Step (d): Selecting Two Cell Sub-Populations:
[0046] The step of enrichment of multipotent stem cells starts by
the separation, at the start of the culture step (c), of two cell
sub-populations as a function of their adhesion rate: [0047] a cell
population CA adhering in less than 12 h [0048] a cell population
CS adhering more slowly (48 to 72 h).
[0049] After 12 hours of culture, population CS occurs in
suspension in the culture medium while population CA adheres to the
dishes.
[0050] "Stem" cells are found only in sub-population CA while the
second sub population contains multipotent precursors which die
after about 60 population doublings. While population CS therefore
cannot be used for the production of stem cells, it can
nevertheless be used for other applications. The CS population has
the following characteristics: [0051] i) it is multipotent, [0052]
ii) it has a negative HLA Class I phenotype, [0053] iii) it has a
normal caryotype, [0054] iv) its self-renewal capacity is preserved
for about 40 to 60 population doublings, [0055] v) its
proliferation rate is not affected by Leukemia Inhibitory Factor
(LIF).
[0056] This cell population thus lends itself to therapeutic and
cosmetic uses comparable to those normally known for multipotent
cells of the prior art.
[0057] The step of selection of the rapidly adhering population
(population CA) is important as it allows, from the start of
culture, a lesser dilution of the "stem" cells with respect to the
bulk of the different precursor cells by carrying out an initial
selection.
Step (e): Enrichment for Stem Cells:
[0058] The CA and CS populations are then cultured in identical
conditions. For population CA, this culture produces a substantial
enrichment in stem cells. This enrichment step is based on the fact
that the precursors have a lifetime that is limited compared with
the stem cells (which are immortal, in theory). During the initial
population doublings, the precursors will multiply much more
rapidly than the stem cells, then they will start to die, by 50 to
80 population doublings. At this stage, the population is highly
enriched in stem cells.
[0059] During this step, each cell transfer is carried out when the
cells reach 80% confluence and high density seeding is carried out,
i.e. at a density in the range 1000 to 5000 cells/cm.sup.2,
preferably in the range 1000 to 3500 cells/cm.sup.2, and more
particularly in the range 2000 to 2500 cells/cm.sup.2.
[0060] At each cell transfer, the cells are diluted by a maximum of
2 or 3 for about 50-80 population doublings (stage at which the CA
population is highly enriched in "stem" cells and the CS population
dies, which corresponds to the death of the precursors). This step
is indispensable based on the hypothesis that the true "stem" cell,
which is quiescent in its normal state, divides more slowly than
the precursor. Greater dilution of the cells during the
trypsination steps could run the risk of losing these multipotent
cells in certain culture dishes.
[0061] After about 50 to 80 population doublings (for example 60
population doublings), the CS population has the characteristics of
a senescent population (loss of proliferative potential and loss of
multipotentiality) and dies. In contrast, the CA population at the
same stage proliferates more slowly compared with the first
population doublings (population doubling time about 72 hours
compared with an initial mean doubling time of about 36 hours) and
is capable of becoming quiescent.
[0062] The CA population can be considered to have reached
quiescence when it exhibits the following characteristics: [0063]
spontaneous stoppage of proliferation at about 70% confluence;
[0064] confluence may be achieved at this stage in the presence of
bFGF or other growth factors. A reduction in the population
doubling time from 72 hours to about 36 hours may be observed;
[0065] the effect of bFGF or other growth factors on the doubling
time is reversible.
[0066] Further, a measurement of the endogenous X-gal activity
determined at a pH of 6 in the CA population is negative (less than
0.05%), confirming that this population is in the quiescent state
rather than in the senescent state.
[0067] The culture medium employed for this enrichment step is
typically a medium without added growth factors, for example the
culture medium DMEM+10% decomplemented foetal calf
serum+antibiotics (100 U/ml of penicillin, 100 .mu.g/ml of
streptomycin).
Step (f): Induction of Stem Cell Proliferation in the
Undifferentiated State:
[0068] After reaching quiescence, proliferation of cells in the CA
population is induced by trypsination and dilution of the cells in
new dishes. Preferably, the cells are subjected to a trypsin
treatment at 80% confluence and are diluted 2 to 10 times,
preferably 5 to 10 times in identical new culture dishes.
[0069] The addition of a growth factor at this stage, for example
basic fibroblast growth factor (bFGF), PDGF, EGF, NGF or SCF allows
intensive proliferation of "stem" cells of the CA population. The
addition of human bFGF at this stage is particularly preferred.
[0070] The addition of growth factors such as bFGF incorporated,
for example, at a concentration of about 3 to 20 ng/ml of medium,
in particular 5 to 10 ng/ml, not only halves the population
doubling time (for example the population doubling time without
bFGF is about 72 hours, while it is about 36 hours with bFGF), but
also allows the population to reach confluence. Without growth
factors, proliferation of a quiescent population of stem cells of
the invention can be provoked by trypsination and dilution, but
proliferation will spontaneously stop at about 70% confluence.
Since confluence is indispensable in vitro for initiating the
differentiation of many cell types, the use of growth factors such
as bFGF must be envisaged for in vitro production of this type of
differentiated cell.
[0071] This step of the method is clearly distinguished from prior
art methods. In the method of the invention, growth factors such as
bFGF are only used after obtaining a population that can become
quiescent. In contrast, bFGF is generally used from the start of
culture of freshly isolated cells ((Tsutsumi S et al., Biochemical
and Biophsysical Research Communications 288,413-419 (2001)). This
very early use of hFGF has a peverse effect as it stimulates not
only proliferation of true "stem" cells but also of all precursors.
This has the result of further increasing the precursor/stem cell
ratio, resulting in a loss of this rare cell type by dilution.
According to the invention, an innovating point is the use of bFGF
when the mass of precursors has disappeared and the population,
highly enriched in "stem" cells, becomes quiescent.
[0072] Inducing proliferation of stem cells can produce large
quantities of these multipotent cells. The cells produced can be
recovered from the culture media using conventional methods.
[0073] In summary, the method of the invention comprises a number
of innovating elements that can optimize the production of stem
cells: [0074] rapid digestion with an enzymatic preparation such as
collagenase (step a)) which allows complete dissociation of the
tissue while preventing damage to certain cell types; [0075] the
absence of filtering steps in step b), to avoid loss of certain
cell types on the filters; [0076] high density cell seeding during
culture steps d) and e). Indeed, multipotent cells are in small
supply compared with other cell types; [0077] isolation of 2 sub
populations "CA" and "CS", as a function of their adhesion rate;
[0078] late use of a growth factor such as bFGF after the cells
have become quiescent (step f).
[0079] In carrying out the method of the invention, the present
inventors have established a plurality of human multipotent lines
from the adipose tissue of young children. The technique of the
invention has been validated for a several adipose tissue samples,
for example those shown in Table 1 (see the Examples below).
[0080] The cells of the invention have many characteristics of stem
cells, for example: the capacity to become quiescent; quiescence
stops if induced to do so with bFGF or other growth factors,
resulting in an intense resumption of proliferation (reversible
effect of said growth factors secreted in vivo into the organism
during damage to the body); maintenance of multipotentiality over a
large number of population doublings; significant telomerase
activity; normal karyotype.
[0081] More particularly, the cells of the invention are
multipotent adult human stem cells, characterized in that they
have: [0082] i) a capacity for self-renewal that is retained over
at least 80 population doublings, and preferably over at least 100
population doublings; [0083] ii) significant telomerase activity
[0084] iii) a negative HLA class I phenotype; [0085] iv) a normal
karyotype; [0086] v) a capacity to become quiescent.
[0087] The self-renewal capacity of the cells of the invention is
preserved over at least 80 population doublings, preferably at
least 100 or 130 population doublings, and more particularly over
at least 200 population doublings. This means that the cells of the
invention are capable of undergoing at least 80 or 130 or 200
population doublings without losing their original characteristics.
In other words, the multipotentiality, telomerase activity,
negative HLA class I phenotype, normal caryotype and the capacity
to become quiescent are retained throughout all of these population
doublings.
[0088] The telomerase activity of the cells of the invention, which
can be measured by conventional techniques, is particularly
important. In accordance with the definition of a "stem" cell by
Watt and Hogan (Science, vol 287, February 2000), telomerase
activity normally means that the cells obtained should be capable
of infinite self-renewal. Telomerase activity is only present in
embryonic cells and, in the adult, in tumour cells and "stem"
cells. This activity thus confirms that the cells are stem
cells.
[0089] The level of the endogenous telomerase activity of the cells
of the invention preferably corresponds to at least 20%, for
example 20% to 50% of the telomerase activity of a reference cell
line, more particularly 22% to 50%. The reference line is typically
a transformed line having endogenous telomerase activity, such as
the transformed human line HEK293T (Human Embryonic Kidney 293
immortalised with T antigen). This activity can be measured at any
stage. It is preferably measured after 30 or 40 population
doublings, for example at the quiescent stage after about 60
population doublings.
[0090] Regarding the immunological characteristics of the cells of
the invention, they differ from a conventional somatic cell. These
cells do not express molecules of the HLA class I system on their
surface (confirmed by flow cytometry), nor do they express HLA
class II on the surface. The molecules of the HLA class I system
present self and non self peptides to CD8+cytotoxic T lymphocytes,
hence their critical role in graft rejection reactions. The absence
of surface HLA class I molecules suggests the "universal" nature of
the stem cells of the invention in transplantation. Said cells
could be used for allo-transplantation with no risks of rejection
by the host, independently of its genotype.
[0091] The negative HLA class I and/or class II phenotype can be
analysed by any conventional technique. It is preferably measured
after 30 or 40 population doublings, for example at the quiescent
stage at about 60 population doublings, or at a later stage, i.e.,
after quiescence, for example at 100 or 120 population doublings.
During the first population doublings, surface expression of HLA
class I molecules is low but significant, then disappears at later
stages (for example after quiescence between 50 and 80 population
doublings) corresponding to the stage at which early precursors
disappear.
[0092] In the context of the invention, the expression "HLA
negative" means that the stem cells of the invention have a level
of surface expression of HLA class I molecules that cannot be
detected by flow cytometry with single labeling (using a
fluorochrome). Preferably, the "HLA negative" cells of the
invention also have a level of surface expression of HLA class II
molecules that cannot be detected by flow cytometry with single
labeling.
[0093] Further, thanks to a phenomenon of immunoprivileged
behavior, the cells of the invention in the differentiated state
most probably do not induce a rejection reaction in a host,
independently of its genotype.
[0094] The cells of the invention have a normal karyotype,
confirming that they are not transformed.
[0095] In the normal state, the cells of the invention are
quiescent (Blau H M et al, Cell, 105, 829-841, June 29, (2001)) and
start proliferating again in the presence of bFGF. Quiescence is a
particular characteristic of stem cells. Beyond a certain number of
population doublings, "non-stem" cells become senescent. The
quiescent state can, in theory, be maintained indefinitely. For the
cells of the invention, the inventors have maintained this state
for periods of up to one year. Proliferation can then be induced by
trypsination and dilution, optionally accompanied by adding a
growth factor. At quiescence, the cells of the invention stop
proliferating spontaneously before reaching confluence, for example
between 50% and 90% of confluence, more particularly between 60%
and 70% of confluence.
[0096] One important characteristic of the cells of the invention
is their multipotentiality. They are capable of differentiating
into at least two cell types. More particularly, they are capable
of differentiating into cells of endodermal origin (for example the
liver) or ectodermal origin (nerve cells: astrocytes,
oligodendrocytes and neurones) or of mesodermal origin. Examples of
cells of the mesodermal lineage that can be cited are adipocytes,
osteoblasts, myocytes, endothelial cells and chondrocytes.
[0097] It has been demonstrated that the cells of the invention,
even at late stages, are capable of differentiating into functional
adipocytes (demonstrated by lipolysis, GPDH activity, adipocyte
markers), into functional osteoblasts (demonstrated by the presence
of osteoblast markers and calcification of the extracellular
matrix), into functional myocytes and into endothelial cells. This
differentiation can take place in vitro or in vivo.
[0098] It has been demonstrated that CA cells of the invention
have, at the single cell level, the capacity of differentiating
into adipocytes, osteoblasts, myocytes and into endothelial cells,
i.e. each CA cell of the invention is capable of differentiating
into these four cell types. In contrast to adipocytes, osteoblasts
and myocyte cells which belong to the "Limb bud mesoderm",
endothelial cells derive from the visceral mesoderm. The cells of
the invention are thus not limited to differentiation into cells of
the mesenchymal lineage but can more generally differentiate into
cells of the mesodermal lineage.
[0099] The inventors have investigated the presence of markers on
the stem cells of the invention. They have established that they
express the transcription factors Oct-4 and Rex-1, and the surface
antigen ABCG2 (ABC transporter responsible for the SP phenotype,
Zhou S et al., Nature Medicine, 7, n.sup.o 9, September 2001,
1028-1034). The Oct-4 and Rex-1 transcription factors are expressed
specifically in embryonic stem cells of mice and humans. Oct-4 is
indispensable to maintaining the pluripotentiality of mouse
embryonic stem cells. It has also been shown that Oct-4 is
expressed by human embryonic stem cells.
[0100] It has also been observed that the stem cells of the
invention do not react to Leukemia Inhibitory Factor (LIF), at
concentrations of about 10 ng/ml. LIF produces no changes in
morphology or proliferation of the cells. In accordance with
results obtained by other investigators with human stem cells, in
particular embryonic stem cells, it can therefore be concluded that
the cells of the invention most probably do not express the
receptor for LIF (LIF-R) and are thus LIF-R negative.
[0101] Preferably, the cells of the invention, after having reached
quiescence, stably exhibit the following phenotype in vitro: [0102]
HLA class I negative, [0103] HLA class II negative, [0104] CD3
negative, [0105] CD13 positive, [0106] Oct-4 positive, [0107] Rex-1
positive, [0108] ABCG2 positive.
[0109] These phenotype characteristics are associated with a normal
caryotype and significant telomerase activity. Preferably, the
cells are also LIF-R negative. This phenotype is stably conserved
in vitro, i.e. in the absence or presence of FGF-2 and at
concentrations of foetal calf serum that may exceed 10%. The
phenotype is also preserved at high seeding densities, and beyond
140 population doublings.
[0110] The doubling time for the cell populations of the invention
varies as a function of the proportion of stem cells present. As an
example, before reaching quiescence, the doubling time is about 36
to 40 hours, reflecting the presence of precursors in the CA
population. As the stem cell proportion increases, the doubling
time also increases, and reaches about 70 to 80 hours at
quiescence. Stem cells divide much more slowly than precursors.
After quiescence, adding growth factors such as bFGF can
significantly reduce the doubling time, for example to about 36
hours, allowing intensive and rapid production of stem cells.
[0111] The cells of the invention can be genetically modified then
selected to introduce or cause expression of a novel
characteristic, for example by ablation or modification of an
endogenous gene or for expression of a transgene such as a reporter
gene or a gene the expression product of which has therapeutic
properties, under the control of a suitable promoter, for example
ubiquitous or tissue-specific promoter.
[0112] The expression of a transgene or DNA or RNA can be either
constitutive or reversibly or irreversibly inducible. The
heterologous DNA or RNA of interest can be carried by any
expression vector, for example a viral vector (including retroviral
vectors), inert vectors, plasmid vectors or an episomal vector.
[0113] The vectors can be introduced into the cells by
transfection, for example using chemical agents such as calcium
phosphate, by lipofection or by using a physical agent such as
electroporation, micro-injection, etc. . . . Said vector can be
maintained in the cell either in the epsiomal form or integrated
into the genome, randomly or in a targeted manner.
[0114] Said genetically modified cells can be used in gene therapy
to supply an expression product of a heterologous gene to an
individual. Thanks to the multipotent nature and HLA class I
negative nature, the stem cells of the invention are particularly
suitable for this type of application.
[0115] When the cells of the invention are transduced or
transfected by a reporter gene, they can be used to carry out a
number of studies. As an example, the plasticity of multipotent
adipose tissue stem cells can be investigated using stem cells
transfected with the lacZ gene coding for .beta.-galactosidase
(blue after staining with Xgal). These labeled cells can be used
for in vitro and in vivo experiments.
[0116] For example, in vitro, the plasticity of said cells can be
studied by co-culture experiments. In particular, the capacity of
said cells (of mesodermal origin) to differentiate into cells of
endodermal origin (for example the liver) and into cells of
ectodermal origin (nerve cells: astrocytes, oligodendrocytes and
neurons) can be studied.
[0117] In vivo, the labeled cells can also be transplanted into the
athymic (nude) mouse which has a deficient immune system and which
thus cannot reject these cells. The transplanted cells do not
produce a tumor. The regenerating power of said cells is only
visible if suitable lesions are initially produced in the mouse to
be transplanted.
[0118] The stem cells of the invention do not express surface HLA
class I molecules, in contrast to the majority of somatic cells.
The absence of said proteins the immune function of which is
crucial, suggests that said stem cells can be transplanted
universally without any rejection reaction.
[0119] Indeed, the inventors have demonstrated that the cells of
the invention can be transplanted into immunocompetent mice without
a rejection reaction 6 months after transplantation.
[0120] The CA cells of the invention are thus characterized in that
in vivo they have an immunoprivileged behavior, i.e. they do not
give rise to a rejection reaction when they are transplanted into
an immunocompetent mammal (such as a mouse), even after more than
10 days following transplantation, preferably after 80 days, and
more preferably after 6 months. Transplantation can be allogenic or
xenogenic. The absence of a rejection reaction can be determined
using techniques that can demonstrate the absence of lymphocyte
infiltration, for example using anti-CD3 antibodies or using a
hematoxylin stain.
[0121] Surprisingly, it has also been shown that in vivo, the cells
of the invention have the capacity to migrate in the
undifferentiated state. 50 days after transplantation of CA cells
of the invention into the Anterior Tibialis of an immunocompetent
mouse, the presence of said cells was observed in the damaged
tissue adjacent to the injection site. This behavior suggests that
the cells of the invention can contribute, by recruitment, to
restoration of a normal phenotype at anatomical sites other than
the injection site.
[0122] The invention also concerns enriched populations of
multipotent cells, characterized in that they comprise the stem
cells of the invention, and in that they are free of adipocytes,
Fibroblasts, pre-adipocytes, endothelial cells, pericytes,
mastocytes and smooth muscle cells.
[0123] The populations of the invention are preferably entirely
homogeneous, i.e. they contain only stem cells. More particularly,
the populations are clonal populations.
[0124] The invention also concerns the production of differentiated
cells from the stem cells of the invention.
[0125] For example, the invention concerns the production of
differentiated cells of the mesodermal lineage, characterized in
that stem cells of the invention are cultivated from confluence, in
the presence of a suitable differentiation medium.
[0126] The following medium can be cited as a medium that allows
adipocyte differentiation: [0127] DMEM medium/Ham's F12 (vol/vol,
1:1), supplemented with antibiotics, for example 100 U/ml of
penicillin, 100 .mu.g/ml of streptomycin, [0128] 5 .mu.g/ml human
insulin (Sigma), [0129] 10 .mu.g/ml of human transferrin (Sigma),
[0130] PPAR.gamma. activator, for example 1 .mu.M of BRL49653, or 2
.mu.m of Ciglitazone (Biomol), [0131] 100 to 250 .mu.M of
isobutyl-methylxanthine (IBMX) [0132] 1 .mu.M of dexamethasone
[0133] 0.2 nM of triiodothyronin (T3 Sigma). [0134] 48 to 72 hours
later, this medium is replaced by the same medium containing no
IBMX or dexamethasone.
[0135] The following medium can be cited as a medium that allows
osteoblast differentiation: [0136] DMEM supplemented with
antibiotics, for example 100 U/ml of penicillin, 100 .mu.g/ml of
streptomycin, [0137] 10% of decomplemented foetal calf serum,
[0138] 0.1 .mu.M of dexamethasone (SIGMA), [0139] 10 mM of
.beta.-glycerophosphate (SIGMA) [0140] 50 .mu.g/ml of ascorbic acid
(SIGMA). [0141] The medium is replaced every 2-3 days over a period
of between 15 and 20 days.
[0142] The following medium can be cited as a medium that allows
myocyte differentiation: [0143] The medium sold under the trade
name PromoCell, or [0144] DMEM medium [0145] 2% of decomplemented
foetal calf serum [0146] antibiotics (for example 100 U/ml of
penicillin, 100 .mu.g/ml of streptomycin) [0147] The medium is
replaced every 2-3 days over 4 to 6 weeks. [0148] The following
medium can be cited as a medium that allows differentiation into
endothelial cells: [0149] DMEM medium supplemented with
antibiotics, [0150] 10 ng/ml of human VEGF.sub.121 (SIGMA).
[0151] For adipocyte, osteoblast and myocyte differentiation, the
stem cells are normally seeded at a density of about 10 000 to 25
000 cells/cm.sup.2.
[0152] Prior to the differentiation step, the cells are normally
seeded at a density of 25000 cells/cm.sup.2 in a proliferation
medium (DMEM supplemented with 10% FCS and 2.5 ng/ml of FGF-2). Two
days later, the culture medium is changed in the absence of FGF-2
for 48 hours. The cells are then maintained in a differentiation
medium for 10 days.
[0153] The stem cells of the invention are particularly suitable
for use in therapy or in cosmetology.
[0154] The therapeutic use of the stem cells of the invention
include, inter alia, use in transplantation and in gene
therapy.
[0155] For example, for use in transplantation, the cells of the
invention are multiplied in the undifferentiated state in vitro
followed by introducing the cells into an individual. The cells can
either be injected into the circulation or implanted into an
anatomical site. The cells then differentiate in vivo as a function
of the damaged anatomical site. As an example, intramuscular
transplantation of the stem cells of the invention into an
individual with muscle lesions will give rise to muscle
differentiation and regeneration. Similarly, the regeneration of
adipose tissue can be envisaged by in vivo differentiation of cells
to adipocytes.
[0156] Transplantation of the cells of the invention can thus be
used to regenerate tissue in vivo, for example bone tissue, adipose
tissue or muscle tissue.
[0157] If necessary, transplantation can be accompanied by
implantation of a matrix that can improve tissue regeneration, for
example by supplying a physical support for the proliferation of
cells or by supplying substances such as growth factors, etc. The
matrix may be biodegradable.
[0158] In accordance with the invention, transplantation may be
autologous or allogenic. The cells of the invention are
particularly suited to allo-transplantations because of their HLA
class I negative nature. The cells can thus be used in any
individual independently of genotype without risking rejection.
[0159] In a further variant, the stem cells of the invention can be
used in the differentiated state, for example as adipocytes,
chondrocytes, osteoblasts, myocytes etc. In this variant, the stem
cells are subjected to differentiation in vitro followed by
introduction of the differentiated cells into the individual.
[0160] For the therapeutic applications of the invention, the stem
cells may or may not be genetically modified. When the cells are
genetically modified, they can be used in gene therapy to supply an
expression product to a patient, for example a heterologous
protein. The modified cells can be cultivated in vitro in the
undifferentiated state then introduced into the recipient.
Alternatively, the cells can be multiplied in vitro in the
differentiated state and then introduced to the recipient.
[0161] The invention also concerns the implementation of surgical
and therapeutic methods using the stem cells of the invention. It
also concerns pharmaceutical compositions comprising the stem cells
of the invention in association with a physiologically acceptable
excipient.
[0162] The stem cells of the invention can also be used for in
vitro production of proteins, which may or may not be recombinant,
particularly therapeutic proteins. In fact, the cells of the
invention can be cultivated in vitro for at least 100, for example
at least 200 population doublings, and thus constitute an almost
inexhaustible source of expression products. The proteins in
question can be expression products of genes endogenous to the stem
cells, or alternatively, can be expression products of heterologous
genes.
[0163] The cells of the invention can also be used in screening
systems for the identification of active agents, for example gene
products, seric extracts, conditioned media, products of animal or
plant origin, libraries of pharmacological agents, etc.
[0164] For example, the invention comprises a screening method for
identifying agents that can modulate the differentiation of cells
into cells of a mesodermal line, characterized by: [0165] a)
culturing stern cells in accordance with the invention under
conditions allowing their differentiation into cells of the
mesodermal lineage (for example adipocytes, osteoblasts or
myocytes) in the presence of a candidate agent, [0166] b) comparing
the differentiation of cells in the presence of the candidate agent
with differentiation in the absence of the candidate agent.
[0167] The test agent may be an agent that can enhance
differentiation or an agent that can prevent or slow or reduce
differentiation (anti-differentiation substance) or a substance
that can modify the differentiation route.
[0168] The invention also comprises a screening method that can
identify agents that may have a lipolytic activity, characterized
by: [0169] a) culturing stem cells of the invention under
conditions allowing them to differentiate into adipocytes; [0170]
b) bringing the adipocytes thus obtained into contact with a
candidate agent and determining the lipolytic activity of the
candidate agent.
[0171] The invention also comprises a screening method that can
identify agents that may have an anti-lipolytic activity,
characterized by: [0172] a) culturing stem cells of the invention
under conditions allowing them to differentiate into adipocytes;
[0173] b) bringing the adipocytes thus obtained into contact with a
candidate agent in the presence of a lipolytic agent; [0174] c)
determining the anti-lipolytic activity of the candidate agent.
[0175] The invention also comprises a screening method that can
identify agents that may have an insulin-sensitising activity,
characterized by: [0176] a) culturing stem cells of the invention
under conditions allowing them to differentiate into adipocytes;
[0177] b) bringing the adipocyte thus obtained into contact with a
candidate agent; [0178] c) determining the insulin-sensitising
activity of the candidate agent compared with untreated
adipocytes.
[0179] The invention also pertains to the use of stem cells in
cosmetology.
[0180] In so far as the stem cells of the invention can
differentiate into adipocytes, they can be used in esthetic surgery
or repair surgery, for example to reduce the wrinkled appearance of
the skin, to reduce scars or various skin blemishes, or to carry
out tissue repair. The invention therefore concerns the
implementation of said surgical methods using the cells of the
invention.
[0181] The cells can also be included in cosmetic compositions
comprising excipients, vehicles, solvents, colorants, fragrances,
antibiotics or other products and additives that are normally used
in cosmetic products. The inclusion of the cells in creams,
pomades, ointments, gels, various fluids, etc, allows them to be
applied directly to the skin or other tissues or phanera. Thus, the
invention also pertains to cosmetic compositions containing stem
cells of the invention in an undifferentiated state, or containing
differentiated cells derived from the stem cells.
KEY TO FIGURES
[0182] Differents aspects of the invention are shown in the
Figures:
[0183] FIG. 1: Endogenous .beta.-galactosidase activity of CA and
CS cells detected at pH 6.
[0184] Xgal staining, which reveals endogenous .beta.-galactosidase
activity (signifying cell senescence) carried out at the 60
population doubling stage, corresponding to cell transfer 20
("T20"), reveals that the CS population is senescent (degree of
senescence 0.415.+-.0.025%), while the CA population is simply
quiescent (degree of senescence 0.045.+-.0.01%).
[0185] FIG. 2: Effect of bFGF (basic Fibroblast Growth Factor) at a
concentration of 5 ng/ml of medium) on CA and CS populations at
late stages, i.e. after 50 population doublings.
[0186] FIG. 2 shows the proliferation of CA.+-.bFGF and CS.+-.bFGF
(Abscissa: days after plating at 12 000 cells/35 mm diameter dish;
Ordinate: number of cells (.times.1000)/dish). Only cells of the CA
population effectively respond to bFGF. After 50 population
doublings, bFGF has no significant effect on the CS population.
These observations confirm the quiescent state of the CA population
and the state of senescence in the CS population.
[0187] FIG. 3: Morphology of CA cells at late stages (after 50
population doublings) in the absence of bFGF and in the presence of
bFGF.
[0188] "W/O FGF"=without bFGF
[0189] "+FGF"=with bFGF (5 ng/ml)
[0190] "FCS"=foetal calf serum
[0191] bFGF causes a change in cell morphology. When quiescent, the
cells are flat and enlarged. In the presence of bFGF, and thus in
the proliferative phase, they take on a fibroblast form.
[0192] The effect of bFGF is reversible.
[0193] FIG. 4: Karyotype of Primo 2CA cells.
[0194] Primo 2 cells were karyotyped for the 2 sub populations CA
and CS, with or without bFGF and at different passages. For Primo
2CA cells, the caryotypes were produced at the following stages:
T21=80 population doublings; T23=90 population doublings; T34=130
population doublings. In all cases, the karyotypes were normal.
FIG. 4 shows an example of a karyotype for Primo 2CA cells.
[0195] FIG. 5: In vitro differentiation of cells of Primo 2CA and
of Primo 2CS into adipocytes at early stages:
[0196] CST1: population CS at passage 1 (corresponding to 3
population doublings);
[0197] CST7: population CS at passage 7 (corresponding to 21
population doublings);
[0198] CAT5: population CA at passage 5 (corresponding to 15
population doublings). Oil Red O stain.
[0199] FIG. 6: Transcriptional expression of adipogenic markers
during Primo 2 cell differentiation. Northern Blot analysis.
[0200] J0, J7, J12=0, 7 and 12 days, respectively, after induction
of differentiation,
[0201] CA T14: Primo 2 CA population at passage 14 (corresponding
to 42 population doublings).
[0202] CS T16: Prima 2 CS population at passage 16 (corresponding
to 48 population doublings);
[0203] FIG. 7: In vitro differentiation into osteoblasts of CA and
CS cells at early passage:
[0204] CST1: CS population at passage 1 (corresponding to 3
population doublings);
[0205] CST7: CS population at passage 7 (corresponding to 21
population doublings);
[0206] CAT5: CA population at passage 5 (corresponding to 15
population doublings). Alizarin Red stain.
[0207] FIG. 8: Differentiation capacity of CA cells at late
passage.
[0208] A: adipocyte differentiation of Primo 2CA cells. Induction
of Adipocyte differentiation was carried out when the cells were at
the T30 stage (about 130 population doublings). Oil Red O
stain.
[0209] B: osteoblast differentiation of Primo 2CA cells. Induction
of Osteoblast differentiation was carried out when the cells were
at the T30 stage (about 130 population doublings). Alizarin Red
stain.
[0210] FIG. 9: Functionality of adipocytes and osteoblasts:
Transcriptional expression of specific markers either for adipocyte
differentiation or for osteoblast differentiation.
[0211] Transcriptional expression of the markers hPPAR.gamma.-2,
haP2 and hOC were determined using RT-PCR. The cells used were i)
adipocytes (left hand side of Figure), from Primo2CA, adipocyte
induction having taken place at stage T32 (about 140 population
doublings), and ii) osteoblasts (right hand side of Figure), from
Primo2CA, osteoblast induction having taken place at stage T32
(about 140 population doublings).
[0212] hPPAR.gamma.-2: human peroxisome proliferator activated
receptor .gamma.: adipocyte marker.
[0213] haP2: human fatty acid binding protein: adipocyte
marker,
[0214] hOC human osteocalcin (osteoblast marker)
[0215] FIG. 10: Functionality of adipocytes: lipolysis capacity of
Primo 2CA cells.
[0216] Lipolyses were carried out on adipocytes obtained from Primo
2CA T32 cells (about 140 population doublings), with agonists
specific for different .beta.-adrenergic receptors. FIG. 10 shows
the lipolysis rates obtained and confirms the absence of .beta.3
adrenergic receptors.
[0217] FIG. 11: Functionality of osteoblasts: Detection of calcium
associated with the extracellular matrix.
[0218] The extracellular matrix present in culture dishes after
lysis of the cell mat was dried then incubated with the solution
from a "SIGMA calcium detection kit". The quantity of calcium
secreted by the osteoblasts was quantified by reading the solution
using a spectrophotometer (OD 575). Osteoblast functionality was
confirmed.
[0219] The quantity of calcium secreted by the osteoblasts varied
as a function of the serum batch (211707, 210407, 210811, 210812,
3903, conventional). These batches may contain non characterized
cytokines, hormones or growth factors present in varying
proportions.
[0220] The adipocytes did not secrete significant quantities of
calcium.
[0221] FIG. 12: Morphology of Primo2CA cells as a function of the
number of population doublings
[0222] A: 40 population doublings
[0223] B: 100 population doublings: quiescent
[0224] C: 150 population doublings: quiescent
[0225] D: 150 population doublings+bFGF: proliferative phase.
[0226] bFGF causes a change in the morphology of the cells. When
quiescent, they are flattened and enlarged. In the presence of
bFGF, and thus in the proliferative phase, they take on the form of
fibroblasts (see also FIG. 3).
[0227] FIG. 13: Adipocyte differentiation capacity of Primo 1CA,
Primo 3CA and Primo 6CA: Adipocytes after 8 days of
differentiation.
[0228] A: Primo 1: differentiation induced at 50 population
doublings
[0229] B: Primo 3: differentiation induced at 40 population
doublings
[0230] C: Primo 6: differentiation induced at 40 population
doublings
[0231] FIG. 14: Adipocyte differentiation capacity of Primo 1CA,
Primo 3CA and Primo 6CA: Oil red O stain
[0232] A: Primo 1: differentiation induced at 40 population
doublings
[0233] B: Primo 3: differentiation induced at 25 population
doublings
[0234] C: Primo 6: differentiation induced at 25 population
doublings
[0235] FIG. 15: Cell marking and flow cytometry analysis.
[0236] Demonstration of HLA Class I negative nature of Primo 2CA
stem cells.
[0237] Single label: FITC
[0238] 1. HELA: Human tumour cells. HLA Class I positive.
[0239] 2. SVF: Adult adipose tissue, no population doublings. HLA
Class I positive
[0240] 3. Primo 2CA: 120 population doublings
[0241] 4. Primo 2CS: 45 population doublings
[0242] Black line: Mouse IgG: negative antibody control
[0243] Gray line: Anti-HLA Class I W6/32 antibodies
[0244] FIG. 16: Obtaining multipotent clones from CA cells.
[0245] Clones CA1 and CA3 were placed in a culture medium
permitting differentiation into adipocytes and osteoblasts. The
adipocytes were revealed by staining with red oil and the
osteoblasts were revealed with Alizarin red.
[0246] FIG. 17: Expression of a transgene in CA stem cells.
[0247] CA stem cells were transduced by a retrovirus expressing a
gene for resistance to an antibiotic, puromycin, and the reporter
gene LacZ. They were then selected in the presence of puromycin.
The selected cells all expressed the LacZ gene, revealed in situ by
.beta.-galactosidase activity.
[0248] FIG. 18: Expression of Oct-4, Rex-1 and ABCG2 in CA stem
cells:
[0249] Left hand side photos: Expression of Oct-4 and ABCG2 RNA in
CA cells and clone CA 1.
[0250] RNA are extracted from CA and CA1 cells and the expression
of Oct-4 and ABCG2 is:
[0251] amplified by RT-PCR
[0252] then detected by hybridization.
[0253] Right hand side photos: Expression of Rex-1 transcription
factor by Primo 2CA cells after 80 and 160 population doublings.
The transcription factor Rex-1 is involved in maintaining the
undifferentiated state of embryonic stem cells. The numbers "1",
"2" and "3" mean "-RT (negative control)", "CA cells", and "CA1
cells", respectively.
[0254] The PCR conditions are:
[0255] for Oct-4: 94.degree. C., 1 min; 57.degree. C., 1 min;
72.degree. C., 1 min for 45 cycles.
[0256] Primers: 5'-GACAACAATGAAAATCTTCAGGAGA-3' and
5'-TTCTGGCGCCGGTTACAGAACCA-3',
[0257] internal primer 5'-CACTCGGTTCTCGATACTGG-3' for a 220-bp
fragment
[0258] for ABCG2: 94.degree. C., 1 min; 60.degree. C., 1 min;
72.degree. C., 1 min for 31 cycles,
[0259] Primers: 5'-GGCCTCAGGAAGACTTATGT-3' and
5'-AAGGAGGTGGTGTAGCTGAT-3'
[0260] for Rex-1: 94.degree. C., 1 min, 60.degree. C. 1 min,
72.degree. C. 1 min, Number of cycles 31; 72.degree. 5 min, 1
cycle
[0261] Primer: 5'-CTCTCCAGTATGAACCAGG-3' and
5'-GAAAGGATCAGAACAACAGC-3',
[0262] internal primer, 5'-GGCATTGACCTATCAGATCC-3' for a 400-bp
fragment.
[0263] FIG. 19: Schematic representation of a preferred variant of
the method for producing adult stem cells from human adipose
tissue.
[0264] FIG. 20: Characteristics of Primo2 CA cells in terms of
surface markers.
[0265] Primo2 CA cells at the 80, 120 and 160 population doubling
stages were labelled with anti-HLA I, anti-HLA-DR anti-CD3,
anti-CD13 antibodies previously coupled with FITC or phycoerythrin.
Control=IgG; the following antibodies were used: HLA class I
conjugated with fluorescein (FITC); HLA-DR (HLA class II)
conjugated with phycoerythrin (PE); CD3 (marker for T lymphocytes)
conjugated with PE; CD13 (markers for stromal cells of bone marrow,
endothelial cells, early progenitors of granulocytes/monocytes and
their descendance) conjugated with FITC.
[0266] Thin line: IgG control
[0267] Thick line: antibody of interest.
[0268] FIG. 21: In vitro myocyte differentiation after 4 days.
[0269] Detection by immuno-histochemistry of myogenin, an early
factor in myocyte differentiation. FIG. 21 illustrates micrographs
of Primo2 CA cells at 150 population doublings after 4 days in the
presence of myocyte differentiation medium. The cells are fixed
with 4% paraformaldehyde for 10 minutes at ambient temperature and
permeabilized in the presence of PBS/0.1% Triton X100 for 10
minutes; the endogenous peroxidase activity is then blocked by
incubating the cells with 3% H.sub.2O.sub.2 for 5 minutes. The
cells are then incubated with the primary antibody: anti-myogenin
antibody (mouse anti-human IgG) (1:100) between 30 minutes and one
hour at ambient temperature then with secondary antibody
(anti-mouse IgG coupled with peroxydase).
[0270] FIG. 22: In vitro myocyte differentiation after 21 days.
[0271] After 21 days in the presence of myocyte differentiation
medium, the fast twitch myosin or FT myosin, a late marker for
myocyte differentiation (intracellular marker) was detected.
Detection by FACS: after detaching the Primo2CA cells (150
population doublings), they are fixed in the presence of PBS/1%
formaldehyde for 15 minutes at ambient temperature then
permeabilized with a digitonin solution (10 .mu.g/ml of PBS) for 7
to 8 minutes at ambient temperature. Antibody labeling is then
carried out using the protocol described for expression of surface
markers (FIG. 20). The antibody used is a mouse antibody directly
conjugated to phycoerythrin and recognizing human FT myosin.
[0272] Thin line: IgG control
[0273] Thick line: FT-myosin
[0274] FIG. 23: In vitro differentiation of endothelial cells
[0275] Detection of von Willebrandt factor (vWF), a specific marker
for endothelial cells, by immuno-histochemistry. FIG. 23
illustrates the expression of vWF by Primo2 CA cells at 150
population doublings after 21 days in the presence of angiogenic
medium. The cells are fixed with 4% paraformaldehyde for 10 minutes
at ambient temperature and permeabilized in the presence of
PBS/0.1% Triton X100 for 10 minutes; the endogenous peroxidase
activity is then blocked by incubating the cells with 3%
H.sub.2O.sub.2 for 5 minutes. The cells are then incubated with the
primary antibody: anti-vWF (goat IgG recognizing both human, rat
and mouse vWF) between 30 minutes and one hour at ambient
temperature then with secondary antibody, anti-goat IgG coupled
with peroxydase (1: 100). After maintaining for 21 days in a medium
composed of DMEM and hVEGF121 (10 ng/ml), the Primo2CA cells
express vWF.
[0276] FIGS. 24 to 27: In vivo muscle regenerating power of Primo
2CA cells after 10 days, 50 days, 80 days and 6 months
transplantation in the mdx mouse with no immunosuppressor: FIGS.
24, 25, 26 and 27 illustrate co-localization of human nuclei with
the muscle fibers re-expressing dystrophin 10 days (FIG. 24), 50
days (FIG. 25), 80 days (FIG. 26) and 6 months (FIG. 27) after
transplantation of Primo 2CA cells into the Tibialis Anterior. This
co-localization was carried out by double labelling dystrophin by
immunofluorescence and human nuclei by FISH. The transplanted cells
are Primo 2CA cells at 160 population doublings, 150 000 in
number.
[0277] The dystrophin immunofluorescence detection step was carried
out before labelling the human nuclei by FISH:
[0278] Detection of dystrophin by immunofluorescence: the muscle
sections are incubated for one hour with an antibody recognizing
human dystrophin, previously coupled with fluorescein. The
antibodies used are as follows:
[0279] either an antibody specific for human and mouse dystrophin
(mouse anti-human IgG1: NCL-DYS2 from Novocastra, directed against
the C-terminal end of human and mouse dystrophin) or,
[0280] an antibody specific for human dystrophin (mouse anti-human
IgG2a: NCL-DYS3 from Novocastra, directed against the N-terminal
end of human dystrophin)
[0281] Detection of human nuclei by FISH: The probe used to detect
the human nuclei is a specific probe for human centromers
(.alpha.-Satellite) coupled with digoxigenin (CP5095-DG.5,
Appligene Oncor). The detection step consists of applying an
anti-digoxigenin antibody coupled with rhodamine to slides. Before
analysing the sections, the nuclei are completely stained using a
DAPI solution (blue stain). The slides are then observed under a
fluorescence microscope (Axiophot Zeiss) with a 100 watt bulb and a
system of filters (Perceptive Scientific International).
[0282] Green: dystrophin;
[0283] Red: human centromers
[0284] Blue: nuclei (human and murine)
[0285] TA: Tibialis Anterior
[0286] G: Gastrocnemius
[0287] FIG. 24 (10 days): anti-dystrophin antibody: NCL-DYS2
[0288] Left Hand Side photos: untreated Tibialis Anterior
(control);
[0289] Center photos: Tibialis Anterior of a mdx mouse treated with
cyclosporin, 10 days after transplantation;
[0290] Right Hand Side photos: Tibialis Anterior of an
immunocompetent mouse (no cyclosporin), 10 days after
transplantation;
[0291] FIG. 25 (50 days): anti-dystrophin antibodies: NCL-DYS2
[0292] Left Hand Side photos: untreated Tibialis Anterior
(control);
[0293] Center photos: Tibialis Anterior of a mdx mouse treated with
cyclosporin, 50 days after transplantation;
[0294] Right Hand Side photos: Gastrocnemius of an immunocompetent
mdx mouse (no cyclosporin), 50 days after transplantation, in
adjacent TA;
[0295] FIG. 26 (80 days): anti-dystrophin antibodies: NCL-DYS2
[0296] Left Hand Side photos: Tibialis Anterior of an
immunocompetent mdx mouse (no cyclosporin) 80 days after
transplantation;
[0297] Right Hand Side photos: Gastrocnemius of an immunocompetent
mouse (no cyclosporin), 80 days after transplantation, in adjacent
TA;
[0298] FIG. 27 (6 months): anti-dystrophin antibodies: NCL-DYS2
[0299] Left Hand Side photos: Tibialis Anterior of an
immunocompetent mdx mouse (no cyclosporin), 6 months after
transplantation;
[0300] Right Hand Side photos: Tibialis Anterior of an untreated
mouse (reference).
[0301] FIG. 28: Demonstration by comparative immunodetection of the
human origin of the dystrophin expressed in the myofibers of
transplanted muscle: An analysis of the presence of myofibers
expressing dystrophin and the subcellular localization in the
tibialis anterior 10 days after transplantation was carried out
using the following antibodies:
[0302] (a): an antibody directed against the C-terminal end of
human and mouse dystrophin (mouse anti-human IgG1: NCL-DYS2 from
Novocastra,),
[0303] (b) and (c): an antibody directed against the N-terminal end
of human dystrophin (mouse anti-human IgG2a: NCL-DYS3 from
Novocastra,),
[0304] (d) and (e): an antibody specific for mouse collagen
III.
[0305] The scale bar corresponds to 15 .mu.m in FIGS. 28(a) and
28(b), and to 1 .mu.m in FIGS. 28(c) to (e). A star * indicates a
section from the same myofiber.
[0306] The similarity between FIGS. 28(a) and (b) indicates the
human origin of the expressed dystrophin. The human dystrophin is
located beneath the sarcolemma. In contrast, the mouse collagen III
is present in the extracellular space between the myofibers (FIGS.
28(c) to (e)).
[0307] FIG. 29: Absence of cellular and humoral immune reactions 10
days after transplantation of the stem cells of the invention: The
existence of any lymphocyte infiltration following transplantation
of Primo 2CA cells into an immunocompetent mdx mouse was studied
using hematoxylin (FIGS. 29(a), (b) and (c)), or mouse anti-CD3
antibody (FIGS. 29(a'), (b') and (c')).
[0308] FIGS. 29(a) and (a'): Tibialis anterior of untreated mdx
immunocompetent mouse (control);
[0309] FIGS. 29 (b) and (b'): Tibialis anterior of mouse, 10 days
after transplantation of Primo 2CA cells, 150,000 in number;
[0310] FIGS. 29 (c) and (c'): Tibialis anterior of mouse, 10 days
after transplantation of unpurified human stromal-vascular cells
isolated from adipose tissue;
[0311] The scale bar corresponds to 50 .mu.m in FIGS. 29(a) to (c),
and to 20 .mu.m in FIGS. 29(a') to (c').
[0312] 10 days after transplantation of the Primo 2CA cells, no
lymphocyte infiltration (CD3') is observed (see FIG. 29(b) and
(b'), compared with FIGS. 29 (a) and (a')). In contrast,
transplantation of unpurified stromal-vascular cells isolated from
human adipose tissue induced a cytotoxic and humoral reaction (FIG.
29(c) and (c')).
[0313] FIG. 30: Absence of cellular and humoral immune reactions 6
months after transplantation of Primo 2CA cells: The existence of a
possible immune reaction 6 months after transplantation of Primo
2CA cells into a mdx immunocompetent mouse was studied by
application of the same techniques as those described for FIG. 29.
Left Hand Side photos: Tibialis Anterior of mdx immunocompetent
mouse, 6 months after transplantation of Primo 2CA cells, labelling
with hematoxylin;
[0314] Right Hand Side photos: Tibialis anterior of mdx
immunocompetent mouse, untreated (control), labelling with
hematoxylin.
[0315] The absence of infiltration by CD3+T lymphocytes was
determined in the muscle transplanted with the Primo 2CA cells,
signifying the absence of a rejection reaction 6 months after
transplantation.
[0316] FIG. 31: In vivo muscle regeneration power of Primo1CA cells
and Primo 3 CA cells 10 days after transplantation without
immunosuppressor into the mdx mouse: The in vivo muscle
regeneration capacity was evaluated by co-localisation of human
nuclei will muscle fibers re-expressing dystrophin. The
co-localisation technique is identical to that described for the
Primo 2CA cells (see legends to FIGS. 24 to 27), but the
transplanted cells are Primo 1CA or Primo 3CA cells.
[0317] photo PRIMO1: Tibialis Anterior of an mdx immunocompetent
mouse (no cyclosporin), 10 days after transplantation of Primo 1CA
cells at 50 and at 120 population doublings, 150 000 in number. The
antibody used to detect the dystrophin is an antibody specific for
human dystrophin (mouse anti-human IgG2a: NCL-DYS3 from Novocastra,
directed against the N-terminal end of human dystrophin)
[0318] PRIMO3 photos: Tibialis Anterior of an mdx immunocompetent
mouse (no cyclosporin), 10 days after transplantation of Primo 3CA
cells at 45, 80 and 110 population doublings, 150 000 in number.
The antibody used to detect the dystrophin is also NCL-DYS3 from
Novocastra.
[0319] After 10 days of transplantation, a potential for muscle
regeneration was visible, both for the Primo 1 CA cells and for the
Primo 3 CA cells.
[0320] FIG. 32: Absence of cellular and humoral immune reactions 10
days after transplantation of Primo 3 CA cells:
[0321] The existence of a possible immune reaction 10 days after
transplantation of Primo 3CA cells in an immunocompetent mdx mouse
was studied by applying the same techniques as those described for
Primo 2CA cells (see legends to FIGS. 29 and 30).
[0322] Primo 3 photo: Tibialis anterior of mdx immunocompetent
mouse, 10 days after transplantation of Primo 3CA cells (110
population doublings, 150 000 in number), labeling with
hematoxylin.
[0323] An absence of lymphocyte infiltration is observed with the
Primo 3CA cells, suggesting behavior identical to that of the Primo
2CA cells.
EXAMPLES
1--Method for Obtaining and for In Vitro Expansion of Multipotent
Stem Cells from Adipose Tissue
1.1. Description of Adipose Tissue Samples Obtained
[0324] Six samples of adipose tissue were obtained from young
children aged 1 month to 7 years; the sexes and anatomical
locations were different.
[0325] Table 1 shows the origin of each sample, the weight thereof
and the number of cells obtained using the technique described
below.
TABLE-US-00001 TABLE I Human adipose tissue samples used for the
production of multipotent stem cells Anatomical Sample Number of
Sample name Sex Age location weight cells obtained Primo 1 F 2
years Umbilical 300 mg 400 000 7 months region Primo 2 M 5 years
Pubic region 400 mg 500 000 Primo 3 M 4 months Prepubic 210 mg 400
000 region Primo 4 F 7 years Inguinal region 2.1 g 2 000 000 Primo
5 M 1 month unknown 200 mg 1 000 000 Primo 6 M 18 months unknown
200 mg 350 000
[0326] The Examples below describe obtaining multipotent stem cells
from samples Primo 1 to 6. Said stem cells were obtained by
carrying out the following steps [0327] isolating multipotent cells
from the sample [0328] in vitro enriching of the cell culture in
multipotent cells [0329] obtaining a population of quiescent stem
cells [0330] inducing intensive proliferation of stem cells
[0331] The stem cells obtained were characterized by [0332]
measurement of telomerase activity [0333] producing Karyotypes at
different passage [0334] studying cell plasticity (differentiation
into different cell types) [0335] determining presence or absence
of cell markers [0336] cloning stem cells
[0337] The methodology and results are described in detail
below.
1.2 Method for Isolating Multipotent Cells from the Adipose Tissue
of Young Children
[0338] After surgery, the sample is stored in DMEM medium
(Dulbecco's Modified Eagle's Medium). +10% foetal calf serum, at
ambient temperature. The tissue is rinsed in PBS (Phosphate Buffer
Saline) at 37.degree. C. then drained and weighed. The sample is
then very finely chopped to optimize the enzymatic digestion
step.
[0339] The digestion medium is composed of DMEM medium (Dulbecco's
Modified Eagle's Medium) containing antibiotics (100 U/ml of
penicillin and 100 .mu.g/ml of streptomycin), 2 mg/ml of
collagenase (Boerhinger reference 103586) and 20 mg/ml of bovine
serum albumin fraction 5 (Sigma A reference 2153). The digestion
volume is a function of the tissue weight; generally, 1 ml of
digestion medium for 100 to 200 mg of tissue. Digestion is carried
out at 37.degree. C. under gentle agitation. In contrast to
conventional techniques for the preparation of human preadipocytes,
the digestion period is very rapid, 5 to 10 minutes, which
corresponds to complete dissociation of the tissue by the
collagenase. The collagenase activity is then inhibited by adding
foetal calf serum (200 .mu.l/ml of digestion medium).
[0340] The cell preparation is then centrifuged for 5 min at 1000
rpm, a step which enables the separation of the adipocytes (which
float) from other cell types contained in the adipose tissue
(pre-adipocytes, stem cells, endothelial cells, pericytes,
mastocytes . . . ). It is important to note that this step in the
procedure is carried out without filtration, which means that all
of the cell typos contained in the adipose tissue are preserved
(with the exception of the adipocytes).
[0341] The cell pellet obtained after centrifuging is re-suspended
in the culture medium: DMEM+decomplemented foetal calf
serum+antibiotics (100 U/ml of penicillin, 100 .mu.g/ml of
streptomycin). The number of cells obtained is counted. The cell
yield varies depending on the samples: 1000 to 5000 cells per mg of
tissue. The cells are seeded at high density, 1000 to 3000 cells
per cm.sup.2 into plastic dishes (crystalline polystyrene,
Greiner).
[0342] When put into culture, two cell sub-populations are isolated
according to their adhesion rates. The first sub-population,
designated "CA", is constituted by cells which adhere very rapidly
(less than 12 h). The second sub-population, CS, is constituted by
cells that adhere much more slowly (48 to 72 h).
[0343] In practice, 12 h after starting the culture, certain cells
have adhered to the plastic. Those cells constitute the CA
population. The cells constituting the CS population are at the
same moment in suspension in the culture medium. That culture
medium is removed and deposited in a new culture dish. After 72 h,
the CS cells have adhered.
1.3 Enriching the Culture in Multipotent Stem Cell
1.3.1 Obtaining a Population of Quiescent Stem Cells:
[0344] The two sub-populations, CA and CS, are maintained in
culture in the same way. Said cells, in the early stages
(corresponding to about 50-60 population doublings) have similar
characteristics in terms of plasticity, proliferation and
morphology.
[0345] The cells are maintained in the culture medium DMEM+10%
decomplemented foetal calf serum+antibiotics (100 U/ml of
penicillin, 100 .mu.g/ml of streptomycin).
[0346] When the cells reach 80% confluence, they are treated with
trypsin (Trypsine-EDTA, Invitrogen) and taken up into culture in
three new dishes of identical diameter. The seeding density
corresponds to 1000 to 3500 cells/cm.sup.2. The cells are
deliberately not diluted further to preserve the multipotent cells,
which theoretically divided more slowly than the precursors, in all
of the dishes.
[0347] The cells were maintained under these conditions until they
stop proliferating. For Primo 2, the CA and CS cells stop
proliferating after cell transfer 20 (T20) corresponding to 60
population doublings.
[0348] Xgal staining (revealing the endogenous .beta.-galactosidase
activity detected at pH 6 and demonstrating cell senescence)
revealed that the CS population was senescent while the CA
population was simply quiescent (see FIG. 1).
[0349] For this enrichment step, different culture media were
tested. It was observed that the stem cells of the invention did
not react to "Leukemia Inhibitory Factor" (LIF) (10 ng/ml). LIF
produces no change in the morphology or proliferation of the cells.
This tends to confirm that the cells do not express the LIF
receptor (LIF-R).
1.3.2 Induction of Intensive Stem Cell Proliferation
[0350] After establishing a population of quiescent CA cells, human
bFGF (basic Fibroblast Growth Factor) is added to the culture
medium described above in a concentration of 5 ng/ml of medium.
[0351] As indicated in FIG. 2, only cells of the CA population
effectively respond to bFGF. In contrast, bFGF had practically no
effect on the CS population at late stages (i.e. after 50
population doublings).
[0352] These observations confirm once again the quiescent state of
the CA population and the senescent state of the CS population.
[0353] The CA cells, treated with bFGF, are subjected to trypsin
treatment at 80% confluence, this time diluted 5 to 10 times in new
identical culture dishes.
[0354] Two supplemental points can be made regarding bFGF: [0355]
bFGF causes a change in the morphology of the cells. When
quiescent, they are flattened and enlarged. In the presence of
bFGF, and thus in the proliferative phase, they take on a
fibroblast form (FIG. 3, FIG. 12) [0356] further, the effect of
bFGF is reversible (FIG. 3). 1.4. Freezing Cells from the Two
Sub-Populations CA and CS
[0357] Cells from the two sub-populations CA and CS are frozen
regularly to constitute a stock of each cell population and to
allow its development during cell transfers to be followed.
Cryoconservation does not change the properties of said cells.
[0358] In practice, the cells were trypsinated, centrifuged and
re-suspended in a freezing medium constituted by foetal calf serum
supplemented with 10% DMSO. These cells were then placed at
-20.degree. C. for 1 h then at -80.degree. C. overnight and finally
stored in liquid nitrogen at -180.degree. C.
2. Measurement of Telomerase Activity of the Stem Cells
2.1 Methodology for Determining Telomerase Activity:
[0359] The telomerase activity quantified using a TeloTAGGG
Telomerase PCR Elisa.sup.PLUS kit (Roche).
[0360] The telomerase activity is quantified in two steps: [0361]
i) The first step is an amplification/elongation step (or TRAP
assay) wherein the telomerase adds telomeric motifs (TTAGGG) to the
3' end of a biotinylated primer. [0362] ii) The second step is
detection and quantification by Elisa.
[0363] The PCR products obtained in step 1 are hybridized with a
specific primer for telomeric ends labeled with digoxigenin.
Further, Elisa microplates were treated with streptavidin to
immoblise the products via the biotin. The immobilized amplicons
were detected with an anti-digoxigenin antibody conjugated with an
anti-DIG-HRP and the peroxidase substrate TMB.
[0364] The intensity of the photometric reaction was estimated
using an Elisa microplate reader (absorbance at 450 nm with a
reference wavelength of 690 nm).
[0365] The relative telomerase activity of the sample is then
calculated with respect to the telomerase activity of a positive
control (cells from the HEK293 line, Human Embryonic Kidney
293).
2.2 Results of Telomerase Activity Determination:
[0366] Using the "TeloTAGGG telomerase PCR Elisa Plus" kit sold by
Roche, the telomerase activity present in the 2 sub-populations CA
and CS of Primo2 was quantified.
[0367] A significant telomerase activity was detected in the stem
cells of the invention. For Primo 2CA (T25: cell transfer 25,
corresponding to about 100 population doublings), the telomerase
activity was about 20% compared with the telomerase activity of the
transformed human line HEK293T (Human Embryonic Kidney 293
immortalized by T antigen). The HEK293T line is used in this kit as
a reference.
[0368] In contrast, no significant telomerase activity was detected
in the CS cells, for example the cells from Primo 2CS (T20) had an
activity of about 5% compared with that of HEK293T.
3. Karyotype of Stem Cells
[0369] The karyotype allows observation and classification of the
chromosomes present during metaphase.
3.1 Methodology for Determining Karyotype:
[0370] Metaphases are obtained using conventional cytogenic
techniques. After accumulating the cells in metaphase by blocking
the fusorial apparatus (incubation in the presence of colchicine
for 3 h), chromosome dispersion is carried out in the cytoplasm by
the action of a hypotonic solution (75 mM KCl for 40 min at
37.degree. C.) followed by fixing with methanol/acetic acid (3/1).
The chromosomes are then identified using RHG-banding
techniques.
3.2 Results of Karyotype Determination:
[0371] The cell karyotype was determined. These karyotypes were
carried out on the 2 sub-populations CA and CS in the presence or
absence of bFGF and at different passages (T21, T23 and T34 for
Primo 2CA).
[0372] In all cases, the karyotypes were completely normal. Thus,
the cells had not undergone any chromosomal rearrangement. FIG. 4
shows the karyotype of Primo 2CA cells.
4. Cell Plasticity of the Stem Cells
[0373] Stem cell plasticity is evaluated using the following
techniques:
4.1 Methodology for Evaluating Cell Plasticity:
[0374] 4.1.1 Conditions for Differentiation into Different Cell
Types:
[0375] The cells are trypsinated then seeded at 20000
cells/cm.sup.2. The cells reach confluence 24 to 48 hours later. As
confluence is a critical step for differentiation, the cells are
maintained at confluence for an additional 24 h before proceeding
to differentiation (adipocytes, osteoblasts, myocytes).
[0376] i) Conditions for Differentiation into Adipocytes [0377] The
confluent cells are incubated in a DMEM/Ham's F12 (vol/vol, 1:1)
medium supplemented with 100 U/ml of penicillin, 100 .mu.g/ml of
streptomycin, 5 .mu.g/ml of human insulin (Sigma), 10 .mu.g/ml of
human transferrin (Sigma), 1 .mu.M of PPAR activator (for example
BRL49653), 100 to 250 .mu.M of isobutyl-methylxanthine (IBMX) and 1
.mu.M of dexamethasone. 48 to 72 hours later, this medium is
replaced by the medium described above but containing no IBMX and
dexamethasone. This differentiation medium is replaced every 2-3
days for a period of 15 to 20 days corresponding to an optimum
adipocyte differentiation.
[0378] ii) Conditions for Differentiation into Osteoblasts [0379]
Cells that have been confluent for 24 h are incubated with an
osteoblast differentiation medium composed of DMEM, 100 U/ml of
penicillin, 100 .mu.g/ml of streptomycin, 10% of decomplemented
foetal calf serum, 0.1 .mu.M of dexamethasone (SIGMA), 10 mM of
.beta.-glycerophosphate (SIGMA) and 50 .mu.M of ascorbic acid
(SIGMA). [0380] The medium is replaced every 2-3 days over a period
of 15 to 20 days.
[0381] iii) Conditions for Differentiation into Myocytes [0382]
Cells that have been confluent for 24 h are incubated either in
DMEM medium or in PromoCell medium in the presence of 2% of
decomplmented foetal calf serum and antibiotics (100 U/ml of
penicillin, 100 .mu.g/ml of streptomycin). The medium is replaced
every 2-3 days over 3 to 6 weeks particularly every 3 days over 21
days.
[0383] iv) Conditions for Differentiation into Endothelial Cells
[0384] The cells are seeded at 20 000/cm.sup.2 in a DMEM medium
containing 10 ng/ml of human VEGF.sub.121 (SIGMA). The
differentiation medium is replaced every 2-3 days for 21 days.
4.1.2 Stains
[0385] i) Oil Red O Stain (Adipocytes: Staining of Intracellular
Lipids) [0386] After fixation in a PBS/0.25% glutaraldehyde
solution, the cells are incubated for 5 minutes at ambient
temperature in a solution of Oil Red O 2% (weight/volume). The
cells are then washed and stored in 70% glycerol.
[0387] ii) Alizarin Red Stain (Osteoblasts: Calcification of
Extracellular Matrix) [0388] After fixation in a PBS/0.25%
glutaraldehyde solution, the cells are incubated for 5 minutes at
ambient temperature in a solution of Alizarin Red O 1%
(weight/volume). The cells are then washed with water and stored
dry.
4.1.3. Transcriptional Analysis
[0389] i) Extraction d'RNA [0390] Cellular RNAs are extracted using
Tri Reagent (Euromedex, Ref TR-118)
[0391] ii) Northern Blot [0392] 20 .mu.g of RNA/well are deposited
on an agarose gel (1.2%)/MOPS (1.times.)/formaldehyde (1.1M). After
electrophoresis in a MOPS (1.times.) migration buffer, the RNA is
transferred to a membrane of nylon Hybond N+ (Amersham Pharmacia).
[0393] The membrane is then hybridized in the presence of a
specific probe labelled with .sup.32P [dCTP] using the
Rediprime.TM. II Random Prime Labelling system (Amersham
Pharmacia).
[0394] iii) RT-PCR [0395] Reverse transcription PCR reaction was
carried out using a OneStep RT-PCR kit from Qiagen.
4.1.4. Analysis of the Expression of Intracellulars Markers:
[0395] [0396] This technique was used to quantify the number of
Primo2CA cells capable of in vitro differentiation into myocytes.
The marker that was analysed was fast twitch myosin ou FT myosin, a
late marker for myogenesis. [0397] After detaching the cells, they
are fixed in the presence of PBS/1% formaldehyde for 15 min at
ambient temperature then permeabilized with a solution of digitonin
(10 .mu.g/ml of PBS) for 7 to 8 min at ambient temperature. [0398]
Antibody labeling is then carried out, using the protocol described
for detecting surface markers (see Examples 7 and 11 below). The
antibody used is a mouse antibody directly conjugated with
phycoerythrin and recognizing human FT myosin.
4.1.5. Immunohistochemistry
[0398] [0399] The cells are fixed with 4% paraformaldehyde for 10
min at ambient temperature. When the desired protein is nuclear
(such as myogenin, for example), the cells are permeabilized in the
presence of PBS/0.1% Triton X100 for 10 min. The activity of the
endogenous peroxidase is then blocked by incubating the cells with
3% H.sub.2O.sub.2 for 5 min. [0400] The cells are then incubated
with the primary antibody for between 30 min and 1 h at ambient
temperature, then with the secondary antibody (anti mouse IgG
coupled with peroxidase (Vector Laboratories) or anti-goat IgG
coupled with peroxidase (Santa Cruz Biotechnology). [0401] The
primary antibody used in our experiments, von Willebrand factor
(vWF) (goat IgG, recognizing both human, rat and mouse vWF) (Santa
Cruz Biotechnology) and Myogenin (mouse anti human IgG) (Santa Cruz
Biotechnology), were used in a proportion of 1:100.
4.2 Results of Cell Plasticity Analysis
4.2.1. Plasticity of the Two Cell Sub-Populations CA and CS at
Early Passages
[0402] At early stages (for example T1, T5, T7 corresponding to 3,
15 and 21 population doublings, respectively), the CA and CS
populations have the same characteristics in terms of plasticity,
morphology and proliferation.
[0403] i) Differentiation into Adipocytes [0404] The results for
the experiments involving Oil Red O staining are shown in FIG. 5
(for Primo 2CA and Primo 2CS) and FIG. 14 for Primo 1CA (40
population doublings), Primo 3CA (25 population doublings) and
Primo 6CA (25 population doublings). Further, the adipocytes from
Primo 1CA, Primo 3CA and Primo 6CA after 50, 40 and 40 population
doublings, respectively, are shown in FIG. 13. A comparison of
FIGS. 13 and 14 clearly shows that the higher the number of
population doublings, the more homogeneous the differentiation.
[0405] A "Northern Blot" analysis (FIG. 6) demonstrates the
transcriptional expression of adipogenic markers (aP2 and
PPAR.gamma.2) during differentiation: CA T14 (42 population
doublings) and CS T16 (48 population doublings).
[0406] ii) Differentiation into Osteoblasts [0407] FIG. 7 shows the
differentiation of CS and CA cells into osteoblasts. Alizarin Red
stain.
4.2.2. Evolution of Cell Plasticity at Late Passages
[0408] Cells of Primo 2 CS, after transfer 20 (corresponding to
about 60 population doublings) become senescent. They
simultaneously lose their proliferative potential and their
differentiation capacity.
[0409] In contrast, the cells of the CA population at the same
stage become quiescent. They proliferate in the presence of bFGF
and retain their plasticity. Said plasticity remains unchanged at
transfer 40 (corresponding to about 200 population doublings). FIG.
8 shows adipocyte and osteoblast differentiation for Primo2 CA T30
(about 130 population doublings).
[0410] At late passages, the CA population also retains
transcriptional expression of the different specific markers either
for adipocyte differentiation, or for osteoblast differentiation.
(FIG. 9) Primo2 CA T32 (140 population doublings).
4.2.3--Capacity of Primo2 CA Cells to Differentiate into Myocytes
and Endothelial Cells In Vitro
[0411] Under appropriate culture conditions, Primo 2CA cells are
capable of differentiating in vitro after about 3 weeks into
myocytes and endothelial cells.
[0412] i) Myocyte Differentiation [0413] After 4 days in the
presence of myocyte differentiation medium, Primo2CA cells express
early markers for myocyte differentiation such as myogenin
(immunohistochemical labeling, cf FIG. 21). After 7 days, myogenin
expression is not longer detectable. [0414] After 21 days, 95% of
the Primo2 cells cultivated in this medium express a late marker
for myocyte differentiation, namely Fast Twitch myosin
(intracellular labeling and FACS analysis, cf FIG. 22).
[0415] ii) Differentiation into Endothelial Cells [0416] In
contrast to adipocytes, osteoblast and myocyte cells which belong
to the "Limb bud mesoderm", endothelial cells derive from the
visceral mesoderm. [0417] After being maintained for 21 days in a
medium composed of DMEM and hVEGF121 (10 ng/ml), the Primo2 CA
cells express von Willebrand factor, a specific marker for
endothelial cells (immunohistochemistry, cf FIG. 23).
5. Characterization of Adipocyte Functionality by Enzymatic
Assay
5.1 Methodology for Characterizing Adipocyte Functionality
5.1.1 Measuring Glycerophosphate Dehydrogenase Activity (GPDH)
[0418] Firstly, the cells which are to have the GPDH activity
measure, are lysed. The principle of the assay can be summarized in
the following scheme:
##STR00001##
[0419] The initial rate of disappearance of NADH is determined at
340 nm (in the presence of NADH, DHAP and cell lysate), which
allows the quantity of degraded substrate and hence the specific
enzymatic activity (after assaying the proteins) to be
calculated.
[0420] The reading is made with a spectrophotometer, allowing
kinetic measurements to be carried out (KONTRON Uvicon 860
thermostatted at 37.degree. C.).
5.1.2. Lipolysis Test
[0421] This test consists of measuring the radiolabelled glycerol
liberated by adipocytes in the presence of adrenergic receptor
agonists. The method used is that described by Bradley D C and
Kaslow H R (Anal Biochem, 1989, 180, 11-16). The glycerol liberated
is phosphorylated in the presence of glycerokinase and ATP and ATP
labeled with .sup.32P in the .gamma. position. The residual ATP is
then hydrolyzed in an acid medium at 90.degree. C. and precipitated
with ammonium molybdate and triethylamine. The radioactivity
incorporated in the form of glycerophosphate labelled with .sup.32P
is estimated by counting in a .beta. counter and the values are
expressed in pmol using a calibration curve.
5.2 Results of Characterization of Adipocyte Functionality
5.2.1. Glycerophosphate Dehydrogenase Activity (GPDH)
[0422] Primo 2CA Cells (T24 in the Presence of Human bFGF): [0423]
After 11 days of differentiation (2 experiments) [0424] Control: 77
nmol/min/mg of protein [0425] In the presence of an agonist of
PPAR.gamma. (BRL49653): 290 nmol/min/mg of protein [0426] After 16
days of differentiation (3 experiments) [0427] Control: 20
nmol/min/mg of protein [0428] In the presence of an agonist for
PPAR.gamma. (BRL49653): 390 nmol/min/mg of protein
[0429] Primo 2CS Cells (T22 in the Presence of Human bFGF) [0430]
After 13 days of differentiation (3 experiments) [0431] Control: 22
nmol/min/mg of protein [0432] In the presence of an agonist for
PPAR.gamma. (BRL49653): 30 nmol/min/mg of protein
5.2.2. Lipolysis Capacity of Primo 2CA Cells
[0433] Lipolyses were carried out on Primo 2CA T32 cells with
specific agonists for the different .beta. adrenergic receptors,
namely: [0434] Isoproterenol: .beta.1, .beta.2 adrenergic [0435]
Dobutamine: .beta.1 adrenergic [0436] Terbutaline: .beta.2
adrenergic [0437] CL316243 .beta.3 adrenergic:
[0438] The following lipolysis rates were obtained (using a
glycerol calibration curve): [0439] Control: 5.76 nmol/h/mg of
protein [0440] Dobutamine: 60.1 nmol/h/mg of protein [0441]
Terbutaline: 93.78 nmol/h/mg: 60.1 nmol/h/mg of protein [0442]
CL316243: 17.1 nmol/h/mg of protein
[0443] The results are shown in FIG. 10.
[0444] The lipolysis experiments show the presence of .beta.1 and
.beta.2 adrenergic receptors and the absence of .beta.3 adrenergic
receptors; these results are in accordance with in vivo
observations (Galitzky et al; (1997) British J. Pharmacol 122:
1244-1250).
6. Characterization of Osteoblast Functionality by Detecting
Calcium Associated with the Extracellular Matrix
6.1 Methodology for Characterizing Osteoblast Functionality:
[0445] To detect the calcium secreted by osteoblasts, the cells
were cultivated in the osteoblast differentiation medium described
above.
[0446] After optimum differentiation, the cell mat is lysed with a
0.1 N NaOH solution for 45 min. A neutralization step is then
carried out by adding 1N HCl (0.2 vol/1 vol of NaOH). The dishes in
which the extracellular matrix remains are dried then incubated
with the solution from a "SIGMA calcium detection kit". The
quantity of calcium secreted by the osteoblasts is quantified by
measuring said solution using a spectrophotometer (DO575)
6.2. Results: Osteoblast Functionality
[0447] The functionality of the osteoblasts was demonstrated by the
technique for detecting calcium associated with the extracellular
matrix (FIG. 11).
[0448] We should also emphasize the importance of the batch of
foetal calf serum in osteoblast differentiation. This reflects the
crucial role in osteoblast differentiation of certain cytokines,
hormones or growth factors that are not characterized and which are
present in varying proportions depending on the serum batch.
7. Cell Labeling and Flow Cytometry Analysis
[0449] The HLA Class I negative nature of the stem cells of the
invention was demonstrated by flow cytometry using a conventional
single label system:
7.1 Single Labeling
[0450] The cells are detached then washed in PBS. After
centrifuging, the cells are re-suspended and incubated with the
primary antibody at a concentration of 10 .mu.g/ml for 30 min at
4.degree. C. The antibodies used are either monoclonal mouse
antibodies directed against class I HLA molecules (W6/32,
Novocastra), or a mouse IgG antibody (Santa Cruz) used as a
negative control. The number of cells used for each condition is
5.times.10.sup.5 to 10.sup.6. The following cells were used for
this analysis: [0451] HeLa: Human tumor cells (positive HLA Class
I: positive control). [0452] SVF: adult adipose tissue, no
population doubling (positive HLA Class I) [0453] Primo 2CA: 120
population doublings [0454] Primo 2CS: 45 population doublings
[0455] The cells are then washed and incubated for 20 min at
4.degree. C. with an antibody (secondary) which is a mouse anti IgG
antibody coupled with FITC (0.2 .mu.g/10.sup.6 cells) (Caltag).
[0456] The cells are then washed and their fluorescence analyzed by
flow cytometry (Scan FACS Becton Dickinson).
[0457] The results are shown in FIG. 15 and show that the stem
cells of the invention (for example Primo 2CA) have a level of HLA
class I molecule expression that cannot be detected by single label
flow cytometry. The stem cells of the invention are thus "HLA class
I negative".
[0458] This flow cytometry experiment was also carried out with
stern cells from Primo1CA and Primo3CA and with cells from Primo2CS
(FIG. 15). In all cases, surface expression of HLA class I was
negative.
8. Obtaining multipotent clones from CA cells
[0459] Primo 2CA cells were seeded in a limiting dilution
condition, namely 1/3 of cells per well of 24 well plates, then
maintained in the presence of 10% FCS containing 5 ng/ml bFGF.
[0460] Ten days later, the clones were isolated and amplified.
These clones preserved their undifferentiated phenotype until made
to differentiate. Their capacity for differentiation into
adipocytes and osteoblasts was then demonstrated.
[0461] FIG. 16 shows two clones, CA1 and CA3, which were placed in
a culture medium allowing differentiation into adipocytes and
osteoblasts. The adipocytes were revealed by Red Oil stain and the
osteoblasts were revealed by Alizarin red stain. The clones
analyzed by flow cytometry were also revealed to be HLA class I and
II negative.
9. Expression of a Transgene in Stem Cells
[0462] The stem cells of the invention, in particular cells from
clone CA1 (obtained as described in Example 7), were transduced at
the 21 population doublings stage by a retrovirus expressing a gene
for antibiotic resistance, puromycin, and the reporter gene LacZ
under the control of an LTR promoter.
[0463] The infectious virions were produced from 293 cells stably
transfected with a PVPack-GP vector (containing gag and pol
sequences) which was co-transfected with the plasmid pFB-Neo-lacZ
and the vector PVPack-VSVG expressing vector, containing the G
protein of the virus for vesicular stomatitis.
[0464] FIG. 17 shows that the transduced cells subsequently
selected in the presence of puromycin all express the LacZ gene, as
revealed in situ by .beta.-galactosidase activity.
10. Expression of Oct-4, ABCG2 and Rex-1 in CA Stem Cells
[0465] Oct-4 is a transcription factor that is specifically
expressed in mouse embryonic stem cells and is indispensable to
maintaining their pluripotentiality. It has also been shown that
Oct-4 is expressed by human embryonic stem cells.
[0466] The transcriptional expression of Oct-4 was demonstrated in
the stem cells of the invention. RNA was extracted from CA cells
(homogeneous populations and clonal populations) and Oct-4
expression was amplified by RT-PCR then detected by hybridization.
The PCR conditions were: 94.degree. C., 1 min; 57.degree. C., 1
min; 72.degree. C., 1 min for 45 cycles. -RT: negative control.
[0467] Similarly, transcriptional expression of ABCG2 was
demonstrated in the stem cells of the invention. RNA was extracted
from CA cells (homogeneous populations and clonal populations) and
ABCG2 expression was amplified by RT-PCR then detected by
hybridization. The PCR conditions for ABCG2 were: 94.degree. C., 1
min; 60.degree. C., 1 min; 72.degree. C., 1 min for 31 cycles.
[0468] FIG. 18 shows the results obtained with Primo 2CA cells at
transfer 16 (48 population doublings) and for cells of the Primo
2CA1 clone at transfer 5 (15 population doublings). They express
Oct-4 and ABCG2.
[0469] Primo 2CA cells also express the transcription factor Rex-1
(FIG. 18, right hand side). The transcription factor Rex-1 is a
specific marker for mouse and human embryonic stem cells. The PCR
conditions for Rex-1 are as follows:
[0470] 94.degree. C. 1 min., 60.degree. C. 1 min., 72.degree. C. 1
min., number of cycles 31, 72.degree. 5 min 1 cycle.
11. Characterization of CA Cells of the Invention in Terms of
Surface Markers
[0471] Characterization of the CA cells of the invention,
particular of Primo 2CA cells, was further investigated in terms of
surface markers using flow cytometry.
[0472] 11.1. Methodology for Analyzing the Expression of Surface
Markers by Flow Cytometry: [0473] The labelling protocol was
described above (see Example 7). The antibodies used were as
follows: [0474] HLA class I conjugated with fluorescein (FITC);
[0475] HLA-DR (HLA class II) conjugated with phycoerythrin (PE);
[0476] CD-3 (marker for T lymphocytes) conjugated with PE; [0477]
CD13 (marker for stromal cells of bone marrow, endothelial cells,
early progenitors of granulocytes/monocytes and of their
descendance) conjugated with FITC
[0478] 11.2--Results of Characterization of CA Cells of the
Invention in Terms of Surface Markers: [0479] The Primo 2CA cells
are surface negative for the expression of CD3, HLA class I and
HLA-DR. In contrast, they are CD13 positive (marker expressed,
inter alia, in bone marrow stromal cells (cf FIG. 20). [0480] The
absence of surface HLA class I and H molecules strongly suggests
the non immunogenicity of Primo 2CA. [0481] It is interesting to
note that Primo 2CA cells differ from the human bone marrow cells
described by Reyes et al (Blood, November 2001). [0482] Those
cells, termed MPC, for "Mesodermal Progenitor Cells" are IRA class
I and class II negative and CD13 positive only when cultivated in
low densities in a culture medium containing a low concentration of
foetal calf serum (2%) with the obligatory presence of EGF and
PDGF. [0483] In contrast, cultivated in the presence of 10% foetal
calf serum (concentration used to amplify the cells of the
invention, in particular Primo 2CA cells), hMPCs display the
inverse phenotype, namely HLA class I and HLA-DR positive and CD13
negative. The hMPCs, cultivated in the presence of 10% foetal calf
serum or bFGF (FGF-2), lose their proliferation potential very
rapidly and die.
12. In Vivo Differentiation of CA Stem Cells of the Invention into
Endothelial Cells and into Myocytes
[0484] Example 4 demonstrated that cells of the invention are
capable of differentiating in vitro after three weeks into
endothelial and cells and into myocytes.
[0485] The examples presented below demonstrate that the human CA
cells of the invention (particular the cells of Primo 2CA, Primo
1CA and Primo 3CA) injected into the muscle of mdx mice, an animal
model for Duchenne's disease in man, are capable of regenerating
normal fibers after only 10 days. Surprisingly, said cells are not
rejected when transplanted into the non immunosuppressed mouse.
Said cells, by dint of their in vivo regeneration capacity and
their absence of immunogenicity, offer a number of therapeutic
perspectives in an allogenic context.
12.1 Transplantation Protocol and Analysis of In Vivo Regeneration
by Immunofluorescence and FISH (Fluorescence In Situ
Hybridization)
12.1.1. Transplantation Protocol
[0486] To analyse the in vivo regeneration potential of the CA
cells of the invention (more particularly cells of Primo2 CA, Primo
1CA and Primo 3CA), the mdx mouse animal model (C57BL/10ScSn
DMD.sup.mdx/J), was used.
[0487] These mdx mice (X-chromosome-linked muscular dystrophy)
constitute a good model for studying Duchenne myopathies in man as
they have a point mutation of the gene for dystrophin (located on
the X chromosome) causing non-translation of dystrophin. In man,
the absence of dystrophin, a protein with a little known role,
causes a cascade of events that are currently little understood,
leading to the progressive disappearance of muscle fibers and
death.
[0488] The following cells were used for the in vivo muscle
regeneration experiments: [0489] Primo 2CA cells obtained between
80 and 160 population doublings (more precisely 80, 120 and 160
population doublings); [0490] Primo 1CA cells obtained at 50 and at
120 population doublings; [0491] Primo 3CA cells at 45, 80 and 110
population doublings.
[0492] In all of the experiments 3 to 4 month old mdx mice of both
genders were used.
[0493] The left Anterior Tibialis muscle was transplanted with 150
000 Primo 2CA, Primo 1CA or Primo 3CA cells taken up in a volume of
50 .mu.l of HBSS (Hank's Buffered Saline solution). The same volume
of HBSS was injected into the right muscle to serve as a negative
control.
[0494] To analyse the muscle regeneration potential of the cells of
the invention, in a first step we treated transplanted mice with an
immunosuppressor, cyclosporin, to avoid the risk of immune
rejection. The cyclosporine was administered intraperitoneally once
a day in a concentration of 10 mg/kg of animal weight/day as of
transplantation.
[0495] The transplanted mdx mice simultaneously treated with
cyclosporine were sacrificed after 10 days.
[0496] In parallel, to test the non immunogenicity of Primo2 CA,
Primo 1CA or Primo 3CA cells, the inventors used the same
transplantation protocol but on mdx mice with a normal immune
system, i.e. not treated with cyclosporine.
[0497] The transplanted muscles and the control muscles (injected
only with 50 .mu.l HBSS) were recovered and frozen in isopentane
then liquid nitrogen.
12.1.2 Detection of Dystrophin by Immunofluorescence
[0498] 12 .mu.m seriated sections were prepared from the frozen and
dehydrated muscles by successive passages of 10 minutes in 50, 75
and 100% ethanol.
[0499] To carry out dystrophin labeling, the frozen sections were
fixed in methanol/glacial acetic acid (70/30, v/v) for 15 min at
ambient temperature. After washing with PBS (phosphate buffered
saline) and incubating for 30 min in a blocking solution (PBS+3%
BSA (bovine serum albumin)), the sections were incubated for one
hour with a specific antibody for human dystrophin (mouse
anti-human IgG2a, Novocastra NCL-DYS3), or an antibody that
recognized both human, rat and mouse dystrophin (mouse IgG1
NCL-DYS2, Novacastra). To reduce background noise, the antibody was
first coupled with fluorescein (Alexa Fluor 488), using "Zenon
Alexa Fluor 488 Mouse IgG2a or IgG1 Labeling Kit", depending on the
antibodies (Molecular Probes).
[0500] The sections were then washed with PBS and then with water
and analyzed using a fluorescence microscope (Olympus BH2).
12.1.3. Detection of Human Transplanted Nuclei by FISH
(Fluorescence In Situ Hybridization)
[0501] The slides were dehydrated by successive passages of 2 min
in baths of ethanol in increasing concentrations (70, 80 and 100%),
then denatured at 73.degree. C. in a 70% formamide/2.times.SSC
solution (citrated saline solution) for 2 min 30 s. The slides were
then rehydrated (ethanol baths in decreasing concentrations of 100,
80 and 70%) before proceeding to hybridization.
[0502] The probe used to detect human nuclei was a specific probe
for all human centromers (.alpha.-Satellite) coupled with
digoxigenin (CP5095-DG.5, Appligene Oncor).
[0503] The probe, initially diluted in the hybridization buffer
Hybrisol VI (Appligene Oncor) (1 .mu.l of probe for 10 .mu.l of
buffer), was denatured for 5 min at 72.degree. C. then deposited on
the slides. The hybridization step was carried out at 37.degree. C.
in a moist chamber for 12 h.
[0504] The slides were then washed: 1 wash in 50%
formamide/2.times.SSC at 43.degree. C. for 15 min followed by
washing in 2.times.SSC at 37.degree. C. for 8 minutes.
[0505] The final step was a detection step consisting of applying
to the slides an anti-digoxigenin antibody coupled with rhodamine
(Appligene Oncor) (5 min in the dark).
[0506] Before analyzing the sections, the nuclei were stained
completely using a DAPI solution (blue stain).
[0507] The slides were then observed under a fluorescence
microscope (Axiophot Zeiss) with a 100 watt lamp and a filter
system (Perceptive Scientific International).
12.1.4. Double Immunofluorescence Labeling/FISH
[0508] In order to co-localize human nuclei with muscle fibers
re-expressing dystrophin, the inventors carried out double labeling
(dystrophin/human nuclei). To this end, the dystrophin detection
step was carried out before labeling the human nuclei by FISH.
12.2--Results: Muscle Regeneration Capacity and Non Immunogenicity
of CA Cells of the Invention In Vivo:
12.2.1 Muscle Regeneration Capacity In Vivo
[0509] Many studies regarding the multipotentiality of adult stem
cells in vivo have been questioned, in particular after
demonstrating the fusion power of said cells (Terada et al, Nature
2002; Wurmser et al, Nature, 2002 and Ying et al, Nature, 2002).
Further, certain studies suggest that the capacity of said cells to
express specific markers for the tissue into which they have been
transplanted is an extremely rare event (Wagers et al, Science,
2002; Morshead et al, Nature, 2002).
[0510] To avoid the problems mentioned above, mdx mice, an animal
model for Duchenne's disease, were used. These mice are deficient
in dystrophin (a point mutation in the gene). (In reality, there
exists a small number of reverting fibers that re-express
dystrophin but the percentage of those fibers does not exceed 1%
(Hoffman et al., J Neurol Sci, 1990; Gillis, J Muscle Research and
Cell Motility, 1999)).
[0511] Firstly, to avoid any rejection problems, the mdx mice were
treated with an immunosuppressor, cyclosporin.
[0512] Injection of a small number of Primo 2CA cells (100000 to
150000 cells) into the Tibialis Anterior muscle resulted in
restoration of dystrophin in about 50% of the fibers in only 10
days. These positive fibers were collected in clusters
(corresponding to the injection point).
[0513] In agreement with the above results, using FISH, human
nuclei with muscle fibers positive for dystrophin (cf FIGS. 24 and
25) could be located.
[0514] Similar results were obtained for Primo 2CA cells between 60
and 160 population doublings, treated or not treated with bFGF
(FGF-2). Similar results were obtained with the CA1 clone derived
from the Primo 2CA population.
12.2.2 Non Immunogenicity of CA Cells In Vivo:
[0515] In contrast to the majority of somatic cells, the CA cells
of the invention are free of HLA class 1 markers (see Example 7)
and surface HLA class II markers (Example 11). This extremely rare
phenotype strongly suggests the non-immunogenicity of the CA cells
of the invention.
[0516] To validate the non-immunogenicity of these cells in vivo,
an experimental approach similar to that described in section
12.2.1 was used, except that non immunosuppressed mdx mice were
used.
[0517] After 10 days of transplantation, the muscle of the
transplanted and non-immunosuppressed mice re-expresses dystrophin
levels comparable with that of mice treated with cyclosporin. A
muscle regeneration potential was observed, both for Primo 2CA
cells and for Primo 1CA cells and Primo 3CA cells in the absence of
an immunosuppressor (see FIG. 24 for Primo 2CA cells and FIG. 31
for Primo 1CA cells and Primo 3CA cells).
[0518] After 50 days of transplantation, in the absence of
immunosuppressor, the number of muscle fibers positive for
dystrophin continued to increase in the injected Tibialis Anterior
but fibers were also found in other muscles such as the
gastrocnemius (cf FIG. 25). These results strongly suggest that
Primo 2CA cells are capable not only of regenerating muscle at the
injection point but also of migrating to repair the surrounding
muscles. An increase in the percentage of peripheral nuclei of
human origin within the fibers and a reduction in the percentage of
central nuclei was observed 10 to 50 days after transplantation
(73% a 85%, and 27% a 15% respectively), indicating that the
injected cells participate in terminal differentiation of
myocytes.
[0519] Using FISH, the human nuclei were still present and
co-localised with the dystrophin-positive fibers. Further, the
localization of a certain number of human nuclei at the outer
periphery of the positive fibers suggests the presence of human
satellite cells and/or of endothelial cells of human origin.
[0520] By comparison, transplantation of a large quantity of human
myoblasts (4 million) into non-immunosuppressed mice resulted in
complete rejection after one month (Huard et al, Muscle and Nerve,
1994) and thus no muscle regeneration.
[0521] After 80 days transplantation, the number of muscle fibers
positive for dystrophin continued to increase in the injected
muscle, and fibers were still found in the gastrocnemius cf FIG.
26).
[0522] After 6 months transplantation, more than 80% of the fibers
expressed human dystrophin as opposed to 50% at earlier times (FIG.
27). Further, within the fiber, we determined much more regular
expression of dystrophin compared with earlier transplantation
times in which dystrophin expression was irregular in the same
fiber. Further, the transplanted muscle exhibited a substantial
improvement in fiber morphology (more regular and an absence of
necrosis, an important process in mdx mice of this age).
[0523] These observations indicate that the transplanted cells do
not give rise to any rejection reaction in the immunocompetent
mouse. The non-immunogenic nature of the transplanted cells has
been demonstrated using hematoxylin stain. No rejection reaction
(absence of infiltration by CD3+T lymphocytes) was observed after
10 days (FIG. 29), 50 days, 80 days (results not shown) or after 6
months transplantation of Primo 2CA cells (see FIG. 30). Similarly,
the absence of lymphocyte infiltration could also be observed 10
days after transplantation of Primo 1CA and Primo 3CA cells,
confirming the absence of a rejection reaction. The same results
were obtained with Primo 1CA cells. The immunoprivileged behavior
of Primo 1CA cells and Primo 3CA cells was thus identical to that
observed with Primo 2CA. These results were also confirmed using an
anti-CD3 antibody, demonstrating the absence of lymphocyte
infiltration.
[0524] In contrast, transplantation of unpurified human
stromal-vascular cells isolated from human adipose tissue induced a
cytotoxic and humoral immune reaction (FIG. 29(c) and (c')).
[0525] In conclusion, after six months transplantation, the cells
caused a substantial improvement in the transplanted muscle with a
high percentage of fibers expressing human dystrophin and an
absence of necrosis which is observed in untreated mdx mice of the
same age; this was in the absence of an immunosuppressor.
[0526] The human origin of the dystrophin expressed in the
myofibers of the transplanted muscle has been demonstrated by
comparative immunodetection, using an antibody specific for human
dystrophin (directed against the N-terminal end of human
dystrophin: mouse anti-human IgG2a: NCL-DYS3 from Novocastra,), and
an antibody capable of recognizing both human dystrophin and murine
dystrophin (directed against the C-terminal end of human and murine
dystrophin: mouse anti-human IgG1: NCL-DYS2 from Novocastra,).
[0527] The results of this comparative immunodetection are shown in
FIG. 28. The presence of myofibers expressing dystrophin and the
subcellular location in the Tibialis Anterior 10 days after
transplantation is visible. The similarity between FIGS. 28(a) and
(b) indicates the human origin of the expressed dystrophin. The
human dystrophin is located beneath the sarcolemma. In contrast,
mouse collagen III is present in the extracellular space between
the myofibers (FIGS. 28(c) to (e)).
[0528] The mechanisms involved in the tolerance of the CA cells of
the invention in a xenogenic context, i.e. in an organism that is
immunologically very different (mdx mouse) still have to be
elucidated.
[0529] However, it may be supposed that a certain number of cells
located at the outer periphery of the muscle fibers play a key role
in this tolerance. These cells may play a local immunosuppression
role by synthesizing immunosuppressive factors, for example
anti-inflammatory type Th2 cytokines such as IL10 and/or by
expressing surface proteins leading to the absence of recognition
by alloreactive lymphocytes of the host (Jorgensen et al,
Engineering mesenchymal stem cells for immunotherapy, Gene Therapy
10, 928-931 (2003)).
[0530] These cells can also induce generalized tolerance by
re-educating the host's immune system. The presence of human CA
cells in the thymus and spleen of the host reinforces this
hypothesis (Fandrich F et al, "Preimplantation-stage stem cells
induce long-term allogeneic graft acceptance without supplementary
host conditioning", Nat. Med 8, 171-178 (2002).
[0531] These results demonstrate the immunoprivilege of the human
CA cells of the invention which are capable of regenerating muscle
without being rejected. These cells thus offer many prospects for
cell therapies in allotransplantation. In particular, for genetic
diseases such as myopathies where autotransplantations are
impossible, the use of cells similar to Primo 2CA would constitute
a good therapeutic alternative.
Sequence CWU 1
1
8125DNAartificial sequenceprimer 1gacaacaatg aaaatcttca ggaga
25223DNAartificial sequenceprimer 2ttctggcgcc ggttacagaa cca
23320DNAartificial sequenceprimer 3cactcggttc tcgatactgg
20420DNAartificial sequenceprimer 4ggcctcagga agacttatgt
20520DNAartificial sequenceprimer 5aaggaggtgg tgtagctgat
20619DNAartificial sequenceprimer 6ctctccagta tgaaccagg
19720DNAartificial sequenceprimer 7gaaaggatca gaacaacagc
20820DNAartificial sequenceprimer 8ggcattgacc tatcagatcc 20
* * * * *