U.S. patent application number 15/120432 was filed with the patent office on 2017-03-23 for materials and methods for expansion of stem cells.
The applicant listed for this patent is FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. Invention is credited to TENG MA.
Application Number | 20170081638 15/120432 |
Document ID | / |
Family ID | 54009684 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170081638 |
Kind Code |
A1 |
MA; TENG |
March 23, 2017 |
MATERIALS AND METHODS FOR EXPANSION OF STEM CELLS
Abstract
The subject invention concerns novel and translatable materials
and methods for expansion of stem cells, such as mesenchymal stem
cells (MSC), that significantly improve translational success of
the cells in the treatment of various conditions, such as stroke.
The subject invention utilizes cell self-aggregation as a
non-genetic means to enhance their therapeutic potency in a
microcarrier bioreactor. The subject invention integrates a cell
aggregation process in a scalable bioreactor system. In one
embodiment of the method, thermally responsive microcarriers (TRMs)
are utilized in conjunction with a bioreactor system. Cells are
cultured in a container or vessel in the presence of the TRMs
wherein cells adhere to the surface of the TRMs. Once cells are
adhered to the TRMs they can be cultured at a suitable temperature
for cell growth and expansion, e.g., at about 37.degree. C. After a
period of time sufficient for cell growth and expansion on the
TRMs, the cell culture temperature is reduced so that the cells
detach from the TRMs. The detached cells are allowed to form cell
clusters that are then cultured under conditions such that the
clusters aggregate to form 3D aggregates. The 3D aggregates can be
collected and treated to dissociate the cells (e.g., using
enzymatic treatment, such as trypsinization). Dissociated cells can
then be used for transplantation in methods of treatment or for in
vitro characterization and study.
Inventors: |
MA; TENG; (TALLAHASSEE,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION, INC. |
TALLAHASSEE |
FL |
US |
|
|
Family ID: |
54009684 |
Appl. No.: |
15/120432 |
Filed: |
February 27, 2015 |
PCT Filed: |
February 27, 2015 |
PCT NO: |
PCT/US2015/018166 |
371 Date: |
August 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61946415 |
Feb 28, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2527/00 20130101;
C12N 2531/00 20130101; C12N 2533/30 20130101; C12N 2500/02
20130101; C12N 2501/39 20130101; C12N 2533/70 20130101; C12M 25/16
20130101; C12M 27/10 20130101; C12N 2501/999 20130101; C12N 2533/54
20130101; C12N 2500/42 20130101; C12N 2513/00 20130101; C12N 5/0075
20130101; A61K 35/28 20130101; C12N 5/0062 20130101; C12N 2539/10
20130101; C12N 2501/727 20130101; C12N 2501/998 20130101; C12M
33/00 20130101; C12N 5/0663 20130101; C12N 2501/33 20130101; C12N
2509/00 20130101; C12M 27/16 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A61K 35/28 20060101 A61K035/28; C12M 3/04 20060101
C12M003/04; C12N 5/00 20060101 C12N005/00; C12M 3/06 20060101
C12M003/06 |
Claims
1. A method for expanding a stem cell, wherein said method
comprises culturing stem cells in a bioreactor system in the
presence of a thermally responsive microcarrier (TRM), wherein stem
cells adhere to the surface of said TRM; growing the adhered stem
cells for a sufficient period of time for the stem cells to
increase in numbers; detaching the stem cells from the TRM by
reducing the culture temperature to a critical solution temperature
that results in said adhered cells detaching from the surface of
said TRM; providing said detached cells sufficient time to
aggregate and form three-dimensional (3D) stem cell aggregates,
wherein said 3D stem cell aggregates exhibit improved therapeutic
potency.
2. The method according to claim 1, wherein said bioreactor system
comprises a spinner flask bioreactor or a rocking platform
bioreactor, such as the WAVE Bioreactor.
3. The method according to claim 1, wherein said culture conditions
provide for rocking and/or agitation of said cells.
4. The method according to claim 1, wherein said TRM is a
microcarrier bead coated with or comprising a thermally responsive
material selected from one or more of polyN-isopropylacrylamide
(PNIPAAm), poly(allylamine
hydrochloride)-co-poly(N-isopropylacrylamide), or poly(styrene
sulfonate)-co-poly(N-isopropylacrylamide).
5. The method according to claim 1, wherein said TRM comprises one
or more of glass, polystyrene, poly(carprolactone), nylon,
poly(ethylene terephthalate) (PET), gelatin, or dextran.
6. The method according to claim 4, wherein said TRM optionally
comprises a terminal coating of a layer of positively charged
allylamine hydrochloride (PAH), or negatively charged styrene
sulfonic acid (PSS), or serum, such as fetal bovine serum
(FBS).
7. The method according to claim 1, wherein said TRM has a diameter
of between about 50 .mu.m to about 500 .mu.m; or from about 100
.mu.m to about 200 .mu.m.
8. The method according to claim 1, wherein said stem cells are
cultured in said bioreactor under hypoxic or low oxygen
conditions.
9. The method according to claim 8, wherein said hypoxic or low
oxygen conditions comprise O.sub.2 tension at between about 1% and
about 10%; or between about 1% and about 3%.
10. (canceled)
11. The method according to claim 1, wherein said method further
comprises collecting said 3D aggregates and treating said 3D
aggregates to dissociate said aggregates into individual cells.
12. The method according to claim 11, wherein said treating step
comprises using an enzymatic agent.
13. The method according to claim 12, wherein said enzymatic agent
is trypsin.
14. The method according to claim 1, wherein said stem cells are
cultured in a container or vessel that comprises a surface or
coating, wherein said surface or coating inhibits or prevents
attachment of said stem cells to said container or vessel.
15. The method according to claim 1, wherein the cells from said 3D
cell aggregates exhibit one or more of the following: upregulated
CXCR-4 expression; migration towards SDF-1; increased resistance to
ischemic conditions; and/or enhanced expression of one or more
anti-inflammatory cytokines and/or growth factors, such as IL-10,
HGF, stanniocalcin 1 (STC-1), PGE-2, and/or IL-6.
16. The method according to claim 11, wherein said cells are
transplanted into a person or animal in need of treatment.
17. (canceled)
18. The method according to claim 1, wherein said stem cells are
MSC from bone marrow.
19. The method according to claim 1, wherein said stem cells are
mammalian or human stem cells, or wherein said stem cells are
mesenchymal stein cells (MSC).
20. (canceled)
21. A stem cell prepared according to a method that comprises
culturing stem cells in a bioreactor system in the presence of a
thermally responsive microcarrier (TRM), wherein stem cells adhere
to the surface of said TRM; growing the adhered stem cells for a
sufficient period of time for the stem cells to increase in
numbers; detaching the stem cells from the TRM by reducing the
culture temperature to a critical solution temperature that results
in said adhered cells detaching from the surface of said TRM;
providing said detached cells sufficient time to aggregate and form
three-dimensional (3D) stem cell aggregates, wherein said 3D stem
cell aggregates exhibit improved therapeutic potency; or a kit or
article of manufacture comprising one or more containers and
comprising one or more of said stem cells.
22. The stem cell according to claim 21, wherein said stem cell is
a human or mammalian cell.
23. The stem cell according to claim 21, wherein said stem cell is
a mesenchymal stem cell (MSC).
24. The stem cell according to claim 21, wherein said stem cell is
MSC from bone marrow.
25. The stem cell according to claim 21, wherein said stem cell is
a human mesenchymal stem cell (hMSC).
26. The stem cell according to claim 21, wherein said stem cell is
provided in a cell culture or cell storage medium, or wherein said
stem cell is provided in a vessel or container.
27. (canceled)
28. A thermally responsive microcarrier (TRM), wherein said TRM is
coated with or comprises a thermally responsive material that
allows for cell adhesion at a first temperature but that provides
for cell detachment at a second temperature, wherein said second
temperature is less than said first temperature; or a bioreactor
system comprising a cell culture vessel or container that contains
said TRM, and optionally a cell culture fluid or medium.
29-41. (canceled)
42. A method for treating a disease or condition amenable to
treatment with a stem cell, wherein the method comprises
administering to a person or animal in need of treatment an
effective amount of stem cells prepared according to a method that
comprises culturing stem cells in a bioreactor system in the
presence of a thermally responsive microcarrier (TRM), wherein stem
cells adhere to the surface of said TRM; growing the adhered stem
cells for a sufficient period of time for the stem cells to
increase in numbers: detaching the stem cells from the TRM by
reducing the culture temperature to a critical solution temperature
that results in said adhered cells detaching from the surface a
said TRM; providing said detached cells sufficient time to
aggregate and form three-dimensional (3D) stem cell aggregates,
wherein said 3D stem cell aggregates exhibit improved therapeutic
potency.
43. The method according to claim 42, wherein said stem cell is a
human or mammalian stem cell, or wherein said stem cell is a
mesenchymal stem cell.
44-49. (canceled)
50. A method for increasing or enhancing aggregation of stem cells
during in vitro cell culture, the method comprising culturing said
stem cells under conditions or in the presence of one or more
compounds that increases or enhances actin-mediated contractility
or polymerization in said stem cells.
51. The method according to claim 50, wherein said stem cell is a
human or mammalian cell.
52. The method according to claim 50, wherein said stem cell is a
mesenchymal stem cell.
53. The method according to claim 50, wherein said stem cell is a
human mesenchymal stem cell.
54. The method according to claim 50, wherein said compound is
selected from insulin, ecdysterone, ATP, fesselin, surfactant
proteins A and D, cortactin, sphingosine-1-phosphate (S1P), or j
asplakinolide (JASP).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 61/946,415, filed Feb. 28, 2014,
which is hereby incorporated by reference herein in its entirety,
including any figures, tables, nucleic acid sequences, amino acid
sequences, or drawings.
BACKGROUND OF THE INVENTION
[0002] Human mesenchymal stem or stromal cells (hMSCs) have been
tested in over 400 clinical trials in a wide range of diseases
(clinicaltrials.gov). As hMSCs translate into clinics, there is an
increasing demand on technology for scalable production of
therapeutically competent hMSCs in scalable bioreactor system.
However, rapid hMSCs expansion has been found to induce cellular
senescence and reduce their therapeutic potency. As a result, hMSC
expansion technology must not only meet the demand in quantity but
also preserve hMSC therapeutic potency during expansion.
[0003] Recent studies have shown that hMSC have unique properties
to self-assemble into three-dimensional (3D) aggregates. The
formation of 3D aggregates is beneficial for hMSC properties in
several ways. First, hMSC 3D aggregation is found to activate the
secretion of anti-inflammatory cytokines and other growth factors
that enhance their therapeutic outcome. Second, hMSC 3D aggregation
also reduces hMSC size, thereby reducing the risk for obstructing
vascular system post-transplantation. Third, hMSC aggregation is a
self-selection process that enhances hMSC's resistance to ischemic
stress, prolonging their life-span in vivo. Finally, hMSC
aggregation is a cell-mediated functional activation process that
does not involve genetic materials, which facilitates their
translation to clinical application. Together, these properties
make hMSC aggregation an attractive method to promote hMSC
viability and functions.
[0004] In the translation of hMSC-based therapy into clinics,
advanced cell expansion technology is a crucial barrier that must
be addressed. hMSC are rare in the bone marrow (.about.1 in
10.sup.5 mononuclear cells) and that the biomolecules released from
MSC are in the range of .about.10 ng based on a clinical scale mass
of MSC (.about.10.sup.8 cells), which is orders of magnitude lower
than that of other biologics (Parekkadan et al. (2010)). Currently,
large doses of MSC, ranging from 0.5.times.10.sup.6/kg to
10.times.10.sup.6/kg are required for clinical application.
Transplantation of culture-expanded hMSC has very low engraftment
and homing efficiency, with less than 0.001% of total injected
cells survived and homed to the ischemic cortex in rats. Thus, ex
vivo expansion needs not only expand cell population but also
preserve their innate properties. However, it has been
well-documented that expansion of hMSC in adherent culture is
associated with gradual loss of chemokine receptors such as CXCR4,
decreases in MSC's responsiveness to stimuli and migratory
capacity, reduced cytokine secretion, increased senescence, and an
increase in cell size with reduced mobility (Rombouts et al.
(2003)) (Li et al. (2008)) (Copland et al. (2011); Chavakis et al.
(2011)). Therefore, advanced cell expansion technology that
maintains hMSC properties in a regulatory-compliant bioreactor
system is crucial for hMSC's translational success.
[0005] To mitigate the culture-induced changes in hMSC properties,
strategies from genetic engineering to pharmacological and hypoxic
preconditioning have been actively pursued to maintain or augment
hMSC therapeutic potency. For example, pre-treatment of MSC with
growth factors or pharmacological drugs have shown to increase cell
secretory properties, in vivo persistence, and functional recovery
in ischemic stroke or cardiac injuries (Numasawa et al. (2011);
Shinmura et al. (2011)). Additionally, hypoxic pre-conditioning of
MSC by short-term exposure to sub-lethal hypoxia (0.5% O.sub.2) has
been shown to improve therapeutic outcomes of stroke animals by
initiating a wide spectrum of actions, including increased
expression of trophic factors such as BDNF and GDNF,
down-regulation of inflammatory genes, and increased cell viability
under ischemic stress (Wei et al. (2012)). Recent discoveries
suggest that 3D hMSC aggregates, which are tightly packed cell
clusters with 500-10,000 cells in each aggregate, have activated
anti-inflammatory protein expression, increased resistance to
ischemic stress, and increased expression of migratory cytokines
such as CXCR4 (Bartosh et al. (2010); Potapova et al. (2008); Lee
et al. (2012); Ylostalo et al. (2012)). Studies further
demonstrated that both aggregates and aggregate-derived hMSC were
more effective than hMSC from adherent cultures in modulating
inflammatory reactions and improved recovery of stroke and
myocardial infarction in mice (Bartosh et al. (2010); Lee et al.
(2009); Guo et al. (2014)). Additionally, hMSC dissociated from the
3D aggregates are smaller in size due to compaction with about
one-half the size of the adherent cells, thereby facilitating in
vivo migration. In the translation of hMSC therapy to clinical
treatment, preserving and enhancing hMSC's innate properties in the
culturing stage and its implementation in a scalable process can
significantly improve its potential in translational success
because of its minimal safety and regulatory concerns.
BRIEF SUMMARY OF THE INVENTION
[0006] The subject invention concerns novel and translatable
materials and methods for expansion of stem cells, such as MSC,
that significantly improve the cells' translational success in the
treatment of various conditions, such as ischemic cardiovascular
and cerebral injuries. The subject invention utilizes cell
self-aggregation in a microcarrier bioreactor as a non-genetic
means to enhance stem cell therapeutic potency. The subject
invention integrates a cell aggregation process in a scalable
bioreactor system.
[0007] One aspect of the present invention concerns methods for
expanding and growing stem cells, such as MSC, in a manner that
provides for improved or enhanced therapeutic potency of the cells.
In one embodiment of the method, thermally responsive microcarriers
(TRMs) are utilized in conjunction with a scalable bioreactor
system, such as the spinner flask bioreactor or a rocking
bioreactor (e.g., WAVE BIOREACTOR.TM. (General Electric Healthcare
Life Sciences)). Cells are cultured in a container or vessel in the
presence of the TRMs wherein cells adhere to the surface of the
TRMs. Once cells are adhered to the TRMs they can be cultured at a
suitable temperature for cell growth and expansion, e.g., at about
37.degree. C. After a period of time sufficient for cell growth and
expansion on the TRMs, the cell culture temperature is reduced so
that the cells detach from the TRMs. The detached cells can form
cell clusters that are then cultured under conditions such that the
clusters aggregate to form 3D aggregates. Following 3D aggregation
of the cells, the 3D aggregates can be collected and treated to
dissociate the cells (e.g., using enzymatic treatment, such as
trypsinization). Dissociated cells can then be used for
transplantation in methods of treatment or for in vitro
characterization and study.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1. Outline of the overall experimental design. The
spinner flask or clinical WAVE BIOREACTOR.TM. (GE Healthcare
Bio-Sciences Corp., Piscataway, N.J.) can be used for integrated
hMSC expansion and activation using thermal responsive
microcarriers (TRMs). The TRMs can be fabricated by coating
commercial polystyrene microcarrier beads with thermal-responsive
PNIPAAm for hMSC adhesion and expansion at about 37.degree. C.
under hypoxia in a single-use CELLBAG.TM. in the WAVE
BIOREACTOR.TM.. After cell expansion, bioreactor temperature is
reduced to about 20.degree. C. to detach hMSCs from the TRMs. The
detached hMSC clusters coalesce and form 3D aggregates under
controlled agitation or rocking and mixing condition, which
promotes cell aggregation and activates hMSC secretory and
migration properties. hMSC can then be dissociated for in vitro
characterization and transplantation.
[0009] FIGS. 2A and 2B. (FIG. 2A) Enhanced FGF-2 secretion and
binding with ECM under hypoxia. (FIG. 2B) hMSC exhibited increased
proliferation on decellularized ECM with the highest proliferation
on the hypoxic ECM.
[0010] FIGS. 3A-3D. (FIG. 3A) Enzymatically detached hMSCs formed
aggregates (EDA) on non-adherent surface. (FIG. 3B) hMSC also
formed aggregates (TLA) after detachment from thermal responsive
surface at 20.degree. C. (FIG. 3C) TLAs have reduced Caspase-3/7
compared to the tissue culture plastics (TCP) and the EDAs;
exposure to in vitro ischemic condition increased Caspase-3/7.
(FIG. 3D) hMSC dissociated from TLAs had lower Caspase-3/7 than TCP
and EDA under standard and ischemic conditions (Kim et al.
(2013a)).
[0011] FIGS. 4A-4D. (FIGS. 4A and 4B) Oxygen profiles in the
aggregates containing 5,000 (FIG. 4A) and 500 (FIG. 4B) cells (x, y
axis are aggregate radius in meter). (FIG. 4C) Generation of a
hypoxic zone in the aggregates requires a hypoxic culture
environment (2% O.sub.2). (FIG. 4D) Aggregates of different cell
number and corresponding radius (left to right): 500 cells, 94
.mu.m; 2000 cell, 152 .mu.m; 5,000 cells, 224 .mu.m. (FIG. 4E)
Confocal images showed BrdU (green) incorporating cells in the TLAs
of different sizes (Blue: DAPI) (Kim et al. 2013a).
[0012] FIGS. 5A-5F. CytoD interrupts hMSC aggregation. (FIG. 5A).
CytoD treatment prevented hMSC aggregation at high concentration.
CytoD treatment reduced compaction of hMSC aggregates at low
concentration and there is no significant difference in compaction
between (FIG. 5B) 2D-pretreated and (FIG. 5C) 3D-treated
aggregates. (FIG. 5D) Dose-dependent decline of aggregate diameters
following 3D-treatment. However, 0.6 .mu.M cytoD 3D-treatment
significantly reduced cell viability (FIG. 5E) and packing density
(FIG. 5F) compared to control aggregates at day 5. *: p<0.05;
**: p<0.01; ***: p<0.005; NS: No statistical difference.
[0013] FIGS. 6A-6C. Aggregate treatment by nocodazole. (FIGS. 6A
and 6B). Nocodazole-treated hMSC aggregates underwent faster
compaction the control aggregates. (FIG. 6C). F-actin structure was
stained with phalloidin rhodamine (red) and nuclei counterstained
with DAPI (blue). Nocodazole treatment significantly altered hMSC
morphology on planar surface.
[0014] FIGS. 7A-7E. LPA treatment has limited effects on hMSC
aggregate survival and compaction. Both (FIG. 7A) 2D pretreatment
and (FIG. 7B) 3D treatment by LPA have minimal impact on aggregate
compaction. (FIG. 7C) Projected areas of the LPA-treated aggregates
declined at the same rate as control. Following 5 days of LPA
3D-treatment, the LPA-treated aggregates have comparable cell
survival (FIG. 7D) as well as (FIG. 7E) cell packing density as the
control.
[0015] FIGS. 8A-8D. Aggregate treatment by Rock inhibitor Y-27632.
Treatment by Y-27632 prevented aggregate compaction (FIG. 8A) with
reduced decline in diameter of aggregates (FIG. 8B). (FIG. 8C).
After 5 days of treatment, cell survival in the 10 .mu.M Y-27632
treated aggregates is lower than non-treated control. (FIG. 8D). By
day 5, cell packing density of Y-27632-treated aggregates is
significantly lower compared to control.
[0016] FIGS. 9A-9D. Caspase expression and inhibition. (FIG. 9A).
Formation of 3D aggregates significantly increased caspase 3/7
expression compared to 2D adherent culture for all treatment
groups. Treatment by pan caspase inhibitor Q-VD-OPh significantly
reduced cell compaction (FIG. 9B), improved cell survival (FIG.
9C), and increased cell packing density (FIG. 9D).
[0017] FIGS. 10A-1, 10A-2, 10B, and 10C. Morphology of 3D hMSC
aggregates. (FIGS. 10A-1, 10A-2). Treatment by CytoD and Y-27632
significantly altered cell morphology in the aggregates as shown by
SEM images. Cells in the control aggregates spread on aggregate
surface with intimate cell-cell contacts. However, both cytoD- and
Y-27632-treated aggregates have large interstitial space and
exhibit round morphology. (FIG. 10B). H&E staining of control
aggregates showed intact cortical actomyosin outer boundary,
whereas disintegrated outer boundaries were observed in the
Y-27632- and CytoD-treated aggregates. (FIG. 10C). F-actin staining
of the histological sections indicated discontinuous outer boundary
for the Y-27632- and CytoD-treated aggregates compared to control
aggregate.
[0018] FIGS. 11A and 11B. Aggregate spreading on glass coverslip
(FIG. 11A) and fusion on ULA surface (FIG. 11B). Aggregates treated
by nocodazole, LPA, and Y-27632 for 5 days readily re-adhered and
spread on glass coverslips and fused when adjacently placed on ULA
surface. Aggregates treated by cytoD for 5 days or
pre-differentiated into osteoblasts did not spread on glass
coverslips nor fused when placed in close contact. OD: hMSC
aggregates treated with osteogenic induction medium for 7 days.
[0019] FIGS. 12A-12H. Cell size, morphology, migration, and hypoxia
resistance of aggregate-derived hMSCs. hMSCs dissociated from
aggregates are significantly smaller measured by flow cytometry
(FIG. 12A) and light microscope (FIG. 12B) compared to 2D adherent
control. When replated and cultured for 24 hours,
aggregate-dissociated hMSCs are smaller compared to 2D adherent
control (FIG. 12C). hMSCs dissociated from aggregates have
significantly higher CXCR-4 expression measured by mean fluorescent
intensity by flow cytometery (FIG. 12D) and higher migration toward
SDF-1 in transwell assay (FIG. 12E). (FIG. 12F) hMSCs dissociated
from aggregates have higher resistance to in vitro ischemia
compared to 2D adherent control. hMSC aggregates have significantly
higher levels of IL-6 (FIG. 12G) and PGE-2 (FIG. 12H) expression
compared to 2D adherent control, but the elevated expressions were
attenuated by Q-VD-OPh, a pan caspase inhibitor to basal levels.
CytoD and Y-27632 treatment have limited effects on IL-6 and PGE-2
secretion. ND: Not detectable.
[0020] FIGS. 13A and 13B. Intracellular ATP and mitochondrial
membrane potential. (FIG. 13A). Aggregates of 500, 2,000, and 5,000
cells/aggregate have comparable levels of intracellular ATP/cell
but all are significantly lower than that of 2D adherent cells.
(FIG. 13B). Formation of aggregates significantly increased TMRM
mean fluorescent intensity, indicating reduced mitochondrial
membrane potential.
[0021] FIG. 14. Fusion of hMSC and osteogenic aggregates on ULA
surface. Contacting hMSC aggregates fused within 24 hours, but OD
aggregates did not fuse when placed in close contact on ULA surface
after 108 hours. When placed in close contact, MSC aggregates
enveloped the OD aggregates but no change in OD aggregates was
observed after 48 hours. hMSC: hMSC aggregates treated with
.alpha.-MEM for 7 days. OD: hMSC aggregates treated with osteogenic
induction medium for 7 days.
[0022] FIG. 15. Effects of culture time on fusion of hMSC
aggregates on ULA surface. Extended culture time had little effects
on hMSC fusion on ULA surface with only a slight delay after 21
days of culture.
[0023] FIGS. 16A-16F. FIG. 16F: hBMSC readily form 3D aggregates on
ultra-low adhesion (ULA) plates and re-adhere and fuse with
adjacent aggregates; actin inhibition by cytochalasin D abrogates
these properties. FIGS. 16A-16E. 3D aggregation increased hBMSC
CXCR-4 expression, migration toward SDF-1 after dissociation, and
resistance to in vitro ischemia (FIGS. 16A-16C). Levels of IL-10,
HGF, STC-1, PGE-2, and IL-6 are significantly higher in hASC
aggregates compared to 2D control. (FIG. 16D). Hypoxia (2% O.sub.2)
preconditioning (HPC) increases hASC viability after 3 days of
culture (FIG. 16E). **: p<0.01.
[0024] FIG. 17. Preparation of thermal responsive coating using
PSS-co-PNIPAM and PAH-co-PNIPAM. Reducing temperature from
37.degree. C. to 20.degree. C. triggers pNIPAAm conformation change
from collapsed to expanded state, triggering cell release.
[0025] FIG. 18. After seeding, hBMSCs were expanded in the TRM
bioreactor and expanded for 1 week at 37.degree. C. At day 7,
temperature is reduced to 20.degree. C. to trigger release of hBMSC
from microcarriers. The detached MSC clusters spontaneously
coalesce and form 3D aggregates in suspension overnight.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The subject invention concerns novel and translatable
materials and methods for expansion of stem cells, such as MSC,
that significantly improve the cells' translational success in the
treatment of various conditions. 3D aggregation was found to
activate the secretion of anti-inflammatory cytokines and other
growth factors by the cells. 3D aggregation also reduces cell size,
thereby reducing the risk for obstructing a vascular system.
Aggregation also enhances cell resistance to ischemic stress,
thereby prolonging cell life span in vivo. The subject invention
integrates a cell aggregation process in a scalable bioreactor
system. FIG. 1 outlines one embodiment of the present
invention.
[0027] One aspect of the present invention concerns methods for
expanding stem cells, such as MSC, in a manner that provides for
improved or enhanced therapeutic potency. Cells for use in the
invention can be isolated from a human or other animal, for
example, from bone marrow and/or adipose tissue. In a specific
embodiment, the cells are bone marrow MSC. In one embodiment of the
method, thermally responsive microcarriers (TRMs) are utilized in
conjunction with a bioreactor system, such as the spinner flask
bioreactor or a rocking bioreactor, such as the WAVE
BIOREACTOR.TM., and cells are cultured in a container or vessel in
the presence of the TRMs wherein cells adhere to the surface of the
TRMs. In one embodiment, the surface of the container or vessel is
one that inhibits or prevents cell attachment thereto. In one
embodiment, the surface is an ultra-low attachment surface, such as
a surface having a hydrogel layer that is hydrophilic and/or
neutrally charged. The cells and TRMs can be cultured under
conditions that provide for rocking and/or agitation of the cells
and TRMs in the culture system. In an exemplified embodiment,
agitation or a wave motion is imparted to the cell culture system.
Bioreactor systems of the invention can provide for controlled gas
content (air, O.sub.2, N.sub.2, and/or CO.sub.2), flow rates,
temperature, pH, agitation rate, and circulation rate. TRMs of the
invention are coated with or comprise a material that is responsive
to thermal conditions, such that the material supports cell
adhesion and growth at 37.degree. C., whereas lowering the
temperature to a lower critical solution temperature (LCST) causes
the material to become hydrophilic and swell to the point that
cells attached to the surface of the material are detached from the
surface. In a specific embodiment, TRMs are polystyrene
microcarrier beads comprising or coated with one or more layers of
PNIPAAm. Once cells are adhered to the TRMs they can be cultured at
a suitable temperature for cell growth and expansion, e.g., at
about 37.degree. C. In one embodiment, cells are cultured under
hypoxic or low oxygen (O.sub.2) conditions in a bioreactor system
with agitation. In a specific embodiment, oxygen tension during
cell culture is at about 2%. After a period of time sufficient for
cell growth and expansion on the TRMs, e.g., from several hours to
several days, the cell culture temperature is reduced, e.g., to
about 20.degree. C., so that the cells detach from the TRMs. In
another embodiment, cells are grown and cultured in a container or
vessel, wherein the surface of the container or vessel is one that
inhibits or prevents cell attachment to the surface and the cells
grow detached from the surface, forming clusters and aggregates.
The detached cells can form cell clusters. Optionally, the TRMs can
be removed from the cell culture container or vessel, or the
detached cells can be transferred to another container or vessel.
The detached cell clusters are then cultured at a temperature
(e.g., about 37.degree. C.) and for a period of time (e.g., several
hours to days) and under conditions, such as agitation, such that
the clusters aggregate to form 3D aggregates. Agitation and/or
rocking of the cell container or vessel can be adjusted to promote
cell aggregation. Following 3D aggregation of the cells, the 3D
aggregates can be collected and treated to dissociate the cells
(e.g., using enzymatic treatment, such as trypsinization).
Dissociated cells can then be used for transplantation in methods
of treatment or for in vitro characterization and study. In one
embodiment, the dissociated cells are smaller in diameter, have
higher levels of expression of CXCR-4, stronger migration to
stromal cell-derived factor-1 (SDF-1), and higher resistance to
ischemic stress than cells that were not from multicellular 3D
aggregates.
[0028] In one embodiment of the present methods, the stem cells are
cultured or grown in conditions wherein the O.sub.2 tension is
maintained at between about 1% and 10%. In a specific embodiment,
the O.sub.2 tension is maintained at between about 1% and 5%. In a
more specific embodiment, the O.sub.2 tension is maintained at
between about 1% and 3% (e.g., O.sub.2 tension could be about 1%,
2%, or 3%).
[0029] Microcarrier beads and containers or vessels utilized in the
present invention can be composed of any material suitable for
tissue culture, including, but not limited to, glass, polystyrene,
poly(carprolactone), nylon, poly(ethylene terephthalate) (PET),
gelatin, and dextran. Microcarrier beads of the invention can also
comprise a material that is magnetic or can become magnetic, such
as Fe.sub.3O.sub.4. Microcarrier beads of the invention can be of
any suitable size and/or shape for culturing cells. In one
embodiment, microcarrier beads can have a diameter of from about 50
.mu.m to about 500 .mu.m. In a further embodiment, microcarrier
beads can have a diameter of between about 100 .mu.m and about 200
.mu.m.
[0030] Any container or vessel suitable for cell culture is
contemplated within the scope of the invention. The container or
vessel can be of any suitable size, e.g., from a few hundred
milliliters in volume to 100 or more liters. In one embodiment, the
container or vessel is a bag compatible with the bioreactor system,
such as the CELLBAG.TM. (GE Healthcare Bio-Sciences Corp.). In one
embodiment, the surface of the container or vessel is one that
inhibits or prevents cell attachment thereto. In one embodiment,
the surface is an ultra-low attachment surface, such as a surface
having a hydrogel layer that is hydrophilic and/or neutrally
charged. The cell culture media utilized with the present invention
can be any suitable media for growth of stem cells, and can
optionally comprise a low concentration of an animal serum, or can
be serum-free.
[0031] In one embodiment of the present methods, a thermoresponsive
material of the TRM is one that provides for cell detachment from a
surface by modulating the temperature. Examples of thermoresponsive
materials include, but are not limited to,
polyN-isopropylacrylamide (PNIPAAm), poly(allylamine
hydrochloride)-co-poly(N-isopropylacrylamide), and poly(styrene
sulfonate)-co-poly(N-isopropylacrylamide), such as described in
Liao et al. (2010). In one embodiment, the surface of a
microcarrier comprises multiple layers of one or more
thermoresponsive films. The thermoresponsive materials can
optionally comprise a terminal coating of a layer of positively
charged allylamine hydrochloride (PAH), or negatively charged
styrene sulfonic acid (PSS), or serum, such as fetal bovine serum
(FBS).
[0032] The subject invention also concerns methods for treating
diseases and conditions in a person or animal by administering to
the person or animal an effective amount of stem cells, such as
MSC, that have been prepared using the methods of the subject
invention. Cells can be administered to the person or animal using
any suitable means, e.g., by injection, infusion, etc. Cells can be
provided in a physiologically acceptable fluid, carrier, or buffer.
Diseases and conditions contemplated for treatment using the
subject invention include, but are not limited to, stroke,
cardiovascular diseases, liver diseases, multiple sclerosis, graft
versus host disease, diabetes, Crohn's disease, and
neurodegenerative diseases. In one embodiment, the disease or
condition to be treated is one associated with ischemic stress,
such as ischemic cerebral (e.g., vascular dementia caused by
ischemia) and cardiovascular (e.g., myocardial ischemia) diseases,
or other ischemia of the bowel (large or small). In one embodiment,
a human is being treated and the cells are human cells. In another
embodiment, a canine animal is treated and the cells are canine
cells. The cells can be autologous cells, or donor matched cells,
or syngeneic or xenogeneic cells. Methods of the invention can
optionally include obtaining stem cells and expanding them using
the subject invention prior to administration of stem cells to the
person or animal.
[0033] The methods and compositions of the present invention can be
used in the treatment of humans and other animals. The other
animals contemplated within the scope of the invention include
domesticated, agricultural, or zoo- or circus-maintained animals.
Domesticated animals include, for example, dogs, cats, rabbits,
ferrets, guinea pigs, hamsters, pigs, monkeys or other primates,
and gerbils. Agricultural animals include, for example, horses,
mules, donkeys, burros, cattle, cows, pigs, sheep, and alligators.
Zoo- or circus-maintained animals include, for example, lions,
tigers, bears, camels, giraffes, hippopotamuses, and
rhinoceroses.
[0034] The subject invention also concerns stem cells, such as MSC,
that have been expanded and grown using a method of the present
invention. In one embodiment, the cells are mammalian cells. In a
specific embodiment, the cells are mammalian MSC. In a specific
embodiment, the cells are human cells. In an exemplified
embodiment, the cells are human MSC. The cells of the invention can
be provided in a container or vessel. In one embodiment, the
surface of the container or vessel is one that inhibits or prevents
cell attachment thereto. In one embodiment, the surface is an
ultra-low attachment surface, such as a surface having a hydrogel
layer that is hydrophilic and/or neutrally charged. In one
embodiment, the cells are provided in a suitable cell culture or
cell storage medium. In another embodiment, cells are provided in a
physiologically acceptable fluid, carrier, or buffer.
[0035] The subject invention also concerns microcarrier beads that
are coated with or comprise a material that is responsive to
thermal conditions. In one embodiment, a thermally responsive
material utilized in the invention supports cell adhesion and
growth at one temperature (e.g., 30-40.degree. C., and typically
about 37.degree. C.) but at a lower temperature (e.g.,
15-25.degree. C., and typically about 20.degree. C.) the material
changes such that cells adhered to the material then become
detached from the material. In one embodiment, the thermally
responsive material becomes hydrophilic and swells at lower
temperatures, thereby resulting in detachment of any cells adhered
to the surface of the material. Examples of thermally responsive
materials contemplated by the present invention include, but are
not limited to polyN-isopropylacrylamide (PNIPAAm), poly(allylamine
hydrochloride)-co-poly(N-isopropylacrylamide), and poly(styrene
sulfonate)-co-poly(N-isopropylacrylamide). In one embodiment, the
surface of a microcarrier comprises multiple layers of one or more
thermoresponsive films. The thermoresponsive materials can
optionally comprise a terminal coating of a layer of positively
charged allylamine hydrochloride (PAH), or negatively charged
styrene sulfonic acid (PSS), or serum, such as fetal bovine serum
(FBS). Microcarrier beads of the invention can be composed of any
tissue culture-compatible material, such as glass, polystyrene,
poly(carprolactone), nylon, poly(ethylene terephthalate) (PET),
gelatin, and dextran. Microcarrier beads can also comprise a
material that is magnetic or can become magnetic, such as
Fe.sub.3O.sub.4. In one embodiment, the microcarrier is a
polystyrene bead. Microcarrier beads of the present invention can
be of any suitable size or shape for culturing cells. In one
embodiment, the microcarrier beads can have diameters of from about
50 .mu.m to about 500 .mu.m. In a further embodiment, a
microcarrier bead of the invention can have a diameter of between
about 100 .mu.m and about 200 .mu.m. In one embodiment, a
microcarrier bead of the invention comprises a cell, such as a stem
cell (e.g., hMSC), adhered to it.
[0036] The subject invention also concerns a bioreactor system
comprising a cell culture vessel or container that contains TRMs of
the invention. The TRMs can be present in a sterile tissue culture
fluid or media that is suitable for culturing cells, such as MSC,
that is contained within the culture vessel or container. In one
embodiment, the bioreactor system comprises an apparatus for
rocking and/or agitating the culture vessel or container. In a
specific embodiment, the apparatus provides for a wave motion
within the container or vessel. The container or vessel can be of
any suitable size for cell culture. In one embodiment, the
container can have a volume of from about 100 milliliters to 100
liters or more. In one embodiment, the surface of the container or
vessel is one that inhibits or prevents cell attachment thereto. In
one embodiment, the surface is an ultra-low attachment surface,
such as a surface having a hydrogel layer that is hydrophilic
and/or neutrally charged. The bioreactor system can also provide
for controlled temperature and gas levels (e.g., O.sub.2) in the
container or vessel.
[0037] The subject invention also concerns articles of manufacture
and kits comprising one or more containers and one or more stem
cells, such as MSC, that have been prepared using the methods of
the present invention. Articles of manufacture and kits can
optionally comprise instructions or labeling that describes how to
maintain, store, and/or use the stem cells of the invention.
Articles of manufacture and kits can also optionally comprise media
for storage, maintenance, and/or use of the stem cells of the
invention. In one embodiment, articles of manufacture and a kit of
the invention comprises a syringe suitable for injection or
administration of the cells into a human or other animal. In one
embodiment, the cells are MSC. In a specific embodiment, the cells
are mammalian MSC. In an exemplified embodiment, the cells are
human MSC.
[0038] The subject invention also concerns methods for increasing
or enhancing aggregation of stem cells, such as MSC, during in
vitro cell culture. In one embodiment, a method of the invention
includes means for increasing actin-mediated contractility or
polymerization within cells. Any means or compound for increasing
or enhancing actin-mediated contractility or polymerization is
contemplated within the scope of the invention. For example,
compounds including, but not limited to, insulin, thapsigargin,
ecdysterone, ATP, fesselin, surfactant proteins A and D, cortactin,
sphingosine-1-phosphate (S1P), and jasplakinolide (JASP) are
contemplated for use with the present invention. Cells can be
cultured under conditions and/or in the presence of one or more
compounds that increase actin-mediated contractility or
polymerization in the cells. The methods for increasing or
enhancing aggregation of stem cells can be used in conjunction with
methods of the subject invention for expanding stem cells.
[0039] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0040] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Formation of a Promitotic Microenvironment Under Hypoxia and
Derivation of 3D hMSC Aggregates from Thermal Responsive
Surface
[0041] Hypoxic enhanced hMSC proliferation via HIF activation and
stimulated secretion of ECM and endogenous FGF-2, promoting the
formation of a pro-mitotic extracellular microenvironment (Grayson
et al. 2007; Kim et al. 2013a) (FIGS. 2A and 2B). The 3D hMSC
aggregates have been derived from thermal responsive culture plates
with higher resistance to ischemic stress (FIGS. 3A-3D). Contrary
to the common belief, modeling suggest a limited reduction of
oxygen tension in the 3D aggregates due to low hMSC specific oxygen
consumption and low cell packing density compared to tumor cells.
Consequently, oxygen tension in the bioreactor can be reduced in
order to create hypoxic regions in the aggregates (FIGS.
4A-4D).
[0042] Research Design and Methods:
[0043] hMSC proliferation and 3D aggregation can be achieved in a
spinner flask or WAVE BIOREACTOR.TM. system using TRMs and combined
3D aggregation and reduced oxygen tension. The methods of the
invention can restore CXCR4 functional expression and enhance hMSC
secretory and migratory properties in vitro
[0044] A novel bioreactor expansion strategy using TRMs in a
spinner flask or a WAVE BIOREACTOR.TM. system is used to achieve
hMSC expansion and spontaneous 3D aggregation under reduced oxygen
tension in the same bioreactor system. Microcarriers have been
traditionally used for scalable cell expansion such as in the WAVE
BIOREACTOR.TM., because these beads, with diameters of 100-200
.mu.m, provide large cell adhesion surfaces when suspended (Sharma
et al. (2011)). To date, however, conventional microcarrier culture
requires enzymatic cell detachment and is not conducive for cell
aggregation. We have developed novel TRMs using polystyrene beads
to achieve non-enzymatic cell detachment at reduced temperature
(i.e., 20.degree. C.) to induce spontaneous 3D aggregation. The
outcome is a translatable protocol for hMSC expansion and
functional activation in a clinical bioreactor system.
[0045] Reduced oxygen tension in the WAVE BIOREACTOR.TM. can
stimulate hMSC proliferation and enhance their secretory
properties. Our laboratory and others have shown that low oxygen
tension at 2% O.sub.2 (termed "hypoxia" in reference to 21% O.sub.2
in a standard incubator) extended hMSC proliferation and
significantly improved their multipotency and secretory functions.
We have also shown that hypoxia promoted the formation of a
pro-mitotic microenvironment by enhancing endogenous FGF-2
secretion and autocrine action on hMSC proliferation (Kim et al.
(2013a)). Importantly, hypoxia up-regulates CXCR4 expression in MSC
via HIF-1 activation (Liu et al. (2010)), and is an important
mediating factor that regulates stem cell recruitment and
retention. hMSCs can self-assemble and spontaneously form 3-D
aggregates in vitro in the absence of adherent surface, under
mechanical forces, or within confined spaces. Importantly, MSC
aggregation is a self-activation mechanism that enhances their
multi-lineage differentiation potential and secretion of
anti-inflammatory, pro-angiogenic, and trophic factors compared to
monolayer cultures (Ylostalo et al. (2012); Bhang et al. (2011)).
Among these factors, culturing hMSC as 3D aggregates significantly
enhanced CXCR4 expression, migration, and adhesion to endothelial
cells (Potapova et al. (2008)). To date, however, hMSC aggregates
have been obtained via laboratory techniques such as hanging drop
or centrifugation methods and a scalable aggregation process has
yet to be demonstrated. Thus, integration of hMSC aggregation in a
scalable bioreactor system, such as the clinical WAVE
BIOREACTOR.TM., is a non-genetic strategy that effectively enhances
hMSC therapeutic potential with minimal safety and regulatory
concerns.
Experiments: Development of TRMs with Optimal Thickness for hMSC
Expansion and Non-Enzymatic Detachment in a WAVE BIOREACTOR.TM.
System.
[0046] A layer-by-layer sequential coating strategy previously
developed in our laboratory (Liao et al. (2010)) using poly
N-isopropylacrylamide (NIPAM)-based thermoresponsive
polyelectrolyte multilayer (PEU) films can be used to coat
commercial polystyrene microcarriers (SoloHill, Ann Arbor, Mich.).
The number of PEU layers can be adjusted to control the thickness
of the coating, and can be characterized following established
methods (Moussallem et al. (2009); Olenych et al. (2005)). After
surface characterization, hMSC adhesion and detachment can be
assessed using the methods previous reported in static culture
(Liao et al. (2010)). After optimizing the TRM's surface
properties, the TRMs can be introduced to 0.5 L CELLBAG.TM.
(WAVE.TM., GE Life Healthcare) for hMSC seeding, expansion, and
aggregate formation.
[0047] P1 hMSCs can be obtained from Institute for Regenerative
Medicine, Texas A&M Health Science Center and expanded
following the method outlined in our prior publications (Grayson et
al. (2004); Zhao et al. (2005)). hMSCs at passage 3 or 4 can be
cultured in growth media on the TRMs. hMSC attachment efficiency on
the TRMs is comparable with commercial microcarriers whereas the
detachment efficiency after 15 minutes incubation at 20.degree. C.
may be above 95%. The impact of rocking pattern on hMSC expansion
kinetics can be measured by DNA assay to quantify cell number for
comparison.
[0048] Control of Oxygen Tension in the WAVE BIOREACTOR.TM..
[0049] Oxygen tension in the CELLBAG.TM. can be controlled by
passing the humidified gases with premixed O.sub.2, CO.sub.2 and
N.sub.2 through the O.sub.2/CO.sub.2/Air mix controller. Dissolved
oxygen concentration in the media can be continuously monitored
using optical oxygen probes (Oxford Optronix Ltd, Oxford, UK) and
controlled at, for example, 2% O.sub.2, 5% CO.sub.2, and balanced
nitrogen (i.e., hypoxia bioreactor) or at 21% O.sub.2, 5% CO.sub.2
and balanced nitrogen (i.e., control bioreactor), respectively. We
do not expect oxygen depletion in the aggregates because of limited
oxygen consumption based on our modeling results and short culture
time for aggregates.
[0050] Derivation of 3D hMSC Aggregates in the WAVE BIOREACTOR.TM.:
Influence of Temperature and Rocking Pattern.
[0051] After cell expansion, temperature in the bioreactor can be
reduced to 20.degree. C. to initiate cell detachment from the TRMs.
During this process, rocking pattern can be adjusted to improve
cell detachment efficiency. After cell detachment, the temperature
in the WAVE BIOREACTOR.TM. is increased to 37.degree. C. under
continued rocking to promote re-aggregation of the detached cells.
In our preliminary study (Kim et al. (2013a)), hMSC have strong
tendency to re-aggregate in suspension or low adherent surface. To
obtain aggregates with uniform size distribution, rocking pattern
and intensity can be adjusted to promote effective and uniform cell
aggregation in the bioreactor. From our preliminary study,
aggregates with diameters in the range from 100 to 300 .mu.m can be
obtained. After overnight culture, the hMSC aggregates can be
removed from the bioreactor and dissociated via trypsinization for
in vitro characterization and transplantation.
[0052] Quantification of Cytokines and Target Gene Expressions by
Real-Time PCR.
[0053] Target genes to be tested include Oct-4, CXCR-4, VEGF,
FGF-2, PDGF, Akt, ERK1/2, HIF-1,2. Total RNA can be isolated from
the hMSCs using the RNeasy Plus kit following the method reported
in our prior publication (Grayson et al. (2007)). The expression of
some of these target genes can be further validated by Western blot
or ELISA following the protocol established in our lab. To
characterize the activation state of ERK1/2 and Akt,
phospho-specific antibodies can be used. Secreted growth factors
including VEGF, BMP-2, FGF-2, and PDGF under different experimental
conditions can be assayed.
[0054] In Vitro Characterization of Dissociated hMSC: Cell
Viability, ROS Expression, and Flow Cytometry.
[0055] The aggregates can be dissociated in collagenase II solution
(Worthington Biochemical, NJ) at 37.degree. C. with intermittent
mixing. The aggregate morphology and size can be assayed by light
microscope, whereas total and spatial distribution of apoptotic
cells in the aggregates can be determined by measuring Caspase-3/7
activity and by TUNEL assay. Intracellular ROS can be measured with
DCFDA using a ROS Detection Kit as reported previously (Kim et al.
(2013a)). Cell surface markers including CXCR-4, CD90, CD146, CD271
and cell size can be measured using flow cytometry following the
method reported in our prior publication (Grayson et al.
(2004)).
Materials and Methods
[0056] Culture of hMSCs.
[0057] Frozen hMSCs at passage 1 in liquid nitrogen were obtained
from the Tulane Center for Stem Cell Research and Regenerative
Medicine. The hMSCs were isolated from the bone marrow of healthy
donors ranging in age from 19 to 49 years based on plastic
adherence, being negative for CD34, CD45, CD117 (all less than 2%)
and positive for CD29, CD44, CD49c, CD90, CD105 and CD147 markers
(all greater than 95%), and possessing tri-lineage differentiation
potential upon induction in vitro (Munoz et al. 2014). The hMSCs
were expanded with minimum essential medium-alpha (.alpha.-MEM)
(Life Technologies, Carlsbad, Calif.) supplemented with 1%
Penicillin/Streptomycin (Life Technologies) and 10% fetal bovine
serum (FBS) (Atlanta Biologicals, Lawrenceville, Ga.) on 150-mm
tissue culture petri dishes (Corning, Corning, N.Y.) at a density
of approximately 1,500 cells/cm.sup.2 in a standard 5% CO.sub.2
incubator. The culture media were changed every three days. hMSCs
from three different donors at passage 5 to 7 were used in the
experiments. All reagents were purchased from Sigma Aldrich (St.
Louis, Mo.) unless otherwise noted.
[0058] Aggregate Formation and Treatment with Cytoskeleton
Modulators.
[0059] hMSCs from monolayer culture were trypsinized and 100 .mu.L
media containing 5,000 cells were added in each well of an
ultra-low attachment (ULA) 96-well plate with round-bottom
(Corning, Corning, N.Y.) for overnight. To analyze size-dependent
ATP contents, aggregates containing 500, 2,000, and 5,000 cells
were used following the same method. Suspended single hMSCs
spontaneously self-assembled into one aggregate per well.
Aggregates were cultured individually up to 7 days with media
change every two days. The aggregates were tracked individually and
the morphologies were imaged with an Olympus IX70 microscope
(Center Valley, Pa.).
[0060] Cytochalasin D (cytoD), lysophosphatidic acid (LPA),
Y-27632, nocodazole were added into culture media to final
concentrations of 0.2 to 0.6 .mu.M, 2.0 to 10.0 .mu.M, 2.0 to 10
.mu.M, and 1.0 M, respectively. To test the temporal effects of
actin modulators on aggregate formation, cells were treated with
media containing cytoD and LPA during monolayer culture prior to
aggregate formation (termed "2D Pretreatment") or 12 hours after
aggregate formation in ULA plates (termed "3D Treatment). Only 3D
treatments were performed for Y-27632 and nocodazole.
[0061] Analysis of Aggregate Size, DNA Content, Fusion, and
Spreading on Adherent Surface.
[0062] Morphology of the aggregates was visualized using an Olympus
IX70 microscope and the images recorded. The images were processed
and the areas of the individual aggregates were calculated using
ImageJ software (rsb.info.nih.gov/ij/). The DNA content of the
individual aggregates was used to calculate cell number. Briefly,
individually collected hMSC aggregates were washed with phosphate
buffered saline (PBS) and digested with proteinase K overnight.
PicoGreen (Life Technologies) was added to triplicate samples and a
DNA standard in a 96-well plate. The cell numbers in aggregates
were determined by the PicoGreen fluorescence intensity using 9.3
pg/cell as determined in our prior study (Kim and Ma 2012). DNA
assay detects double-stranded DNA that may also be present in dead
cells with intact DNA. However, this population in the aggregates
is low, and the yield of live cells measured by DNA assay is close
to cell count using other methods such as flow cytometry (Ylostalo
et al. 2014). The packing densities were calculated using cell
number divided by aggregate volume assuming a spherical shape. At
least six aggregates were used in each condition.
[0063] To test re-adhesion and fusion, the aggregates from various
treatment conditions were plated on glass coverslip for re-adhesion
or placed adjacently on ULA surface for fusion. Images were
captured with Olympus IX70 microscope and analyzed by ImageJ
software.
[0064] Measurement of Caspase 3/7, Interleukin 6 (IL-6), and
Prostaglandin E2 (PGE-2).
[0065] Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay
systems (Promega, Madison, Wis.). Caspase-Glo 3/7 working buffer
was added to triplicate samples containing aggregates and culture
media in 96-well LUMITRAC 200 white immunology plate (Greiner
Bio-One, Monroe, N.C.). The results were read by a luminescence
plate reader and normalized to cell number (Biotek Instruments,
Winooski, Vt.). Secreted PGE-2 and IL-6 in conditioned media were
quantified using a PGE-2 Parameter Assay Kit and IL-6 DuoSet ELISA
kits, respectively (R&D Systems, Minneapolis, Minn.). Total
secreted PGE-2 and IL-6 were determined by subtracting cytokine
concentrations in culture media controls and normalized to the cell
number in the aggregates.
[0066] Osteogenic Induction, Scanning Electron Microscopy (SEM),
Immunohistochemistry, and Histology.
[0067] After aggregate formation in the ULA culture dishes, the
growth media were replaced with osteogenic media (high glucose
Dulbecco's modified eagle medium (DMEM) (Life Technologies)
supplemented with 10% FBS, 1% Penicillin/Streptomycin, 100 nM
dexamethasone, 10 nM sodium-b-glycerophosphate and 12.8-mg/l
ascorbic acid-2 phosphate) following the previously reported method
(Kim and Ma 2012). After 7 days of incubation, the OD-aggregates
were transferred to glass coverslip for adhesion or were placed
adjacently on ULA surface for fusion.
[0068] For SEM, the aggregate samples were fixed in 4%
paraformaldehyde (PFA), dehydrated through a graded series of
ethanol, incubated in hexamethyldisilazane, and vacuum dried
overnight. The samples were mounted onto carbon-coated chucks,
sputter-coated with gold in an argon atmosphere for 4 minutes at 2
kV, and analyzed on a SEM (JSM-7401F) (JEOL, Tokyo, Japan). For
immunostaining, the cells were fixed with 4% PFA, permeabilized
with 0.5% Triton X-100, blocked with 1.0% BSA, incubated with
primary antibody, and imaged with an Olympus IX70 (Center Valley,
Pa.) microscope. For histology, hematoxylin-eosin (H&E)
staining was performed following the prior reported method (Kim and
Ma 2012). hMSC aggregates were fixed in 10% formalin, dehydrated
and embedded in paraffin wax; 10 .mu.m sections were cut and
stained with Lerner-2 Hematoxylin (Lerner Laboratories, Pittsburgh,
Pa.) and Eosin-Y w/Phloxine (Richard-Allan Scientific, Kalamazoo,
Mich.) by standard procedures (Kim and Ma 2012). Images were
captured with Olympus IX70 microscope with MagnaFire SP 2.1B
software.
[0069] Flow Cytometry, Transwell Migration Assays, and In Vitro
Ischemia.
[0070] Aggregates were dissociated in trypsin, washed in PBS, and
fixed at 4% PFA at room temperature. Aliquots of 100 .mu.L, cell
suspension were incubated with fluorochrome-conjugated, anti-mouse
monoclonal antibody CXCR-4 (R&D Systems). For mitochondrial
membrane potential measurement, typsinized MSCs were washed by
centrifugation in warm HBSS. Cell suspension was incubated with
tetramethylrhodamine, methyl ester (TMRM) (Life Technologies),
washed with HBSS, and analyzed by flow cytometry (BD Biosciences,
San Jose, Calif.). Labeled samples were washed in PBS followed by
flow cytometry analysis with the isotype controls run in parallel
at the same concentration used for each antibody.
[0071] A transwell migration system (Neuro Probe, Md., USA) was
used to study the migration of aggregate-dissociated hMSCs and
monolayer cultured hMSCs in response to human recombinant SDF-1
(R&D systems). A cell migration assay kit using an 8-.mu.M pore
size was used. Resuspened cells in serum free medium were loaded in
the top chamber of the migration well. Serum-free medium containing
30 ng/mL of SDF-1 was added to the lower chamber. Cells were
incubated at 37.degree. C. in 5% CO.sub.2 for 4 hours. The
remaining cells on the top chamber were scratched, and the migrated
cells stained with Hoechst and counted.
[0072] To mimic ischemic conditions (IC), hMSCs dissociated from
the aggregates and adherent controls were incubated in serum-free
growth media at 1% O.sub.2 controlled in a C-Chamber (BioSpherix,
Lacona, N.Y.) for 6 h and then analyzed by Live/Dead staining (Life
Technologies). The combination of serum withdrawal and low oxygen
tension is known to induce hMSC apoptosis and has been used in
vitro to mimic ischemic condition (Kim and Ma 2013b).
[0073] Statistics.
[0074] Unless otherwise noted, all experiments were performed at
least in triplicate (n=3), and representative data were reported.
Experimental results were expressed as means.+-.standard deviation
(SD) of the samples. Statistical comparisons were performed by
one-way ANOVA and Tukey's post hoc test for multiple comparisons,
and significance was accepted at p<0.05.
[0075] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1
Actin-Mediated Contractility Influences the Assembly of
Multicellular hMSC Aggregates
[0076] While the initial steps of aggregation require close contact
and cell-cell adhesion via cadherin molecules, reorganization of
actin cortical network is crucial in the establishment of a mature
cell-cell contact (Amack and Manning 2012). To investigate the
temporal effects of actin-mediated contractility in hMSC aggregate
formation, hMSC cultures were treated with cytoD (1) in plastic
culture for 2 days prior to cell detachment (e.g., 2D pretreatment)
or (2) 12 hours after aggregate formation on ULA surfaces (i.e., 3D
treatment). In 2D pretreatment, hMSC displayed dose-dependent
response in which cytoD disrupted hMSC aggregation at concentration
above 0.6 .mu.M and prevented aggregate compaction at lower
concentrations (FIGS. 5A and 5B). In 3D treatment, hMSC aggregates
remained intact in cytoD concentration of 0.6 .mu.M and exhibited
dose-dependent reduction on compaction similar to those of 2D
pretreatment group (FIGS. 5B-5D). In contrast, cytoD treatment
prevented aggregate compaction but reduced cell viability, leading
to significantly lower cell survival and packing density compared
to control hMSC group at day 5 (FIGS. 5E and 5F).
[0077] To investigate the role of microtubule in hMSC aggregates,
the hMSC aggregates were treated by nocodazole, a chemical agent
that interferes with the polymerization of microtubules. Despite
the drastic changes of cell morphology on 2D surface,
nocodozole-treated hMSC aggregates have comparable or higher
compaction compared with the non-treated controls, suggesting
microtubule is not the major force during aggregate compaction
(FIGS. 6A-6C).
Example 2
Actin Mediates Aggregate Compaction but not Viability and Caspase
Expression
[0078] LPA is a naturally occurring bioactive phospholipid with
multiple biological functions that include its ability to initiate
cytoskeleton contraction by RhoA activation, promote cell survival
and proliferation, and to enhance survival of hypoxia-challenged
neonatal cardiomyocytes (Tigyi et al. 1994; Moolenaar 1995). LPA
has also been identified as a novel survival factor and protects
MSCs against hypoxia and serum deprivation-induced apoptosis (Chen
et al. 2008a). Treatment of hMSC aggregates by LPA, however, has
limited effects on aggregate compaction as well as viability at LPA
concentration up to 10 .mu.M (FIGS. 7A-7E).
[0079] The involvement of actin-myosin based contractility was
investigated by treatment of aggregates with Y-27632, which
inhibits the phosphorylation of Rho-associated kinase (ROCK) and
prevents cell compaction (Chen et al. 2010). As expected,
inhibition of ROCK kinase by 10 .mu.M Y-27632 reduced aggregate
compaction with a 6.7% increase in diameter by day 5 and a 38.7%
decrease in cell packing density compared to the control aggregates
(FIGS. 8A-8D). Despite significant reduction in aggregate
compaction, cell viabilities in the Y-27632-treated aggregates were
comparable or even less than those of control aggregates,
suggesting reduced compaction by Y-27632 treatment failed to rescue
hMSCs in the aggregates.
[0080] Prior studies have reported increased apoptosis in hMSC
aggregates (Bartosh et al. 2013; Kelm et al. 2012) but the origin
of the stress signals remains to be resolved. Measurement of
caspase 3/7 activity was used to quantify protein markers involved
in late-stage apoptosis owing to its capacity to discriminate
between apoptotic and necrotic cells (Chen et al. 2010). Compared
to their counterparts in adherent 2D culture, formation of
multi-cellular aggregates significantly increased caspase 3/7
activity for all treatment groups with the Y-27632-treated
aggregates having the highest caspase 3/7 activity. CytoD treatment
significantly increased caspase 3/7 activity in 2D adherent
culture, whereas cytoD-treated aggregates had comparable apoptotic
activity as hMSC control aggregates. Treatment by Y-27632 and LPA
also increased caspase 3/7 activity in both 2D culture and 3D
aggregates. These results suggest that prevention of aggregate
compaction by actin modulators did not significantly reduce
apoptosis, although the cytotoxic effects of the actin modulators
may be a contributing factor (FIG. 9A).
[0081] To determine the relation between apoptosis and aggregate
compaction, hMSC aggregates were treated with Q-VD-OPh, a pan
caspase inhibitor. After three days of culture, Q-VD-OPh-treated
aggregates have reduced compaction and significantly increased cell
viability compared to control, resulting in higher packing density
(FIGS. 9B-9D). These results suggest cellular apoptosis is upstream
of and partially contribute to aggregate compaction.
Example 3
Actin Mediates Aggregate Morphology, Interaction, and Spreading on
Adherent Surfaces
[0082] Disruption of actin significantly alters hMSC morphology in
the aggregates. As shown in FIGS. 10A-1, 10A-2, 10B and 10C, cells
in the hMSC aggregates were tightly packed and spread with limited
interstitial space at the boundary of the aggregate. In contrast,
cells in the cytoD- and Y-27632-treated aggregates were loosely
packed and exhibited spherical morphology with abundant
interstitial space as shown by SEM and histology (FIGS. 10A-1 and
10A-2). Histological sectioning also revealed contrasting
morphology of hMSC in the interior of control and cytoD and
Y-27632-treated aggregates. In the control aggregates, hMSCs are
morphologically heterogeneous with spindle-shaped cells at the
outer boundary and round and tightly packed cells in the interior,
indicating morphological polarization. In the cytoD and Y-27632
treated aggregates, cells are loosely packed with no spreading at
the boundary, indicating the absence of a mechanically polarized
outer boundary (FIGS. 10B and 10C).
[0083] The tendency of cellular aggregates to spontaneously form
spherical aggregates has been suggested to be analogous to the
behavior of liquid drops that spontaneously acquire a spherical
shape in suspension and fuse when placed in close contact to
minimize surface tension (Gonzalez-Rodriguez et al. 2012). When
placed in close contact, un-treated hMSC aggregates readily fused
and spread on glass coverslip, so did the ones treated by LPA and
nocodazole (FIGS. 11A and 11B). Y-27632-treated aggregates spread
on glass coverslips but have reduced tendency to fuse. However,
interrupting actin filaments by cytoD completely prevented
aggregate spreading on glass coverslip and fusion of adjacent
aggregates. Interestingly, after osteogenic induction, hMSC-OS
aggregates maintained spherical shape on rigid glass coverslip and
failed to fuse when in close contact. These results suggest that
the viscoelastic behavior of hMSC aggregates is primarily mediated
by actin.
Example 4
Functional Enhancement and Metabolic Alteration
[0084] To evaluate the effects of 3D aggregation on hMSC
properties, hMSC dissociated from the aggregates were
characterized. As shown in FIGS. 12A and 12B, aggregate-derived
hMSCs are significantly smaller than the 2D control with 36%
reduction in diameter in suspension. After replating the
aggregate-derived hMSCs on plastic culture dishes, they are also
significantly smaller than their 2D counterparts (FIG. 12C).
Aggregate-derived hMSCs also have significantly higher CXCR-4
expression and stronger migration ability toward SDF-1 indicated by
transwell migration assay (FIGS. 12D and 12E). To further
characterize hMSC's resistance to ischemic stress,
aggregate-derived hMSCs and control from 2D adherent cultures were
subject to in vitro ischemic (i.e., serum-free and 1% O.sub.2) that
mimics in vivo ischemic condition (FIG. 12F) (Kim and Ma 2013b).
The results showed that aggregate-derived hMSCs have significantly
higher survival under ischemic stress compared to their 2D
counterparts. Collectively, the enhanced expression of CXCR-4,
migration in response to SDF-1, resistance to ischemic stress,
along with reduced cell size suggest the beneficial impact of 3D
aggregation on hMSC properties in ischemic injuries.
[0085] Elevated secretion of anti-inflammatory cytokine such as
IL-6 and PGE-2 is thought to be due to hMSC stress response via
caspase-mediated mechanism (Bartosh et al. 2013; Ylostalo et al.
2012). To investigate whether caspase inhibition attenuates hMSC
secretory function, the aggregates were treated with Q-VD-OPh. As
shown in FIGS. 12G and 12H, formation of 3D aggregates
significantly upregulated the secretion of IL-6 and PGE-2 but this
enhancement is attenuated to basal levels in the presence of
Q-VD-OPh, suggesting the stress-induced functional activation in 3D
aggregates. In addition, secretion of IL-6 and PGE-2 is independent
from aggregate compaction because CytoD and Y-27632 treatment has
limited effects on the levels of IL-6 and PGE-2.
[0086] To identify the source of stress signal and determine
whether the impediment of oxygen and nutrients due to diffusion
alters bioenergetics and results in apoptosis, cellular ATP
contents/cell in aggregates of different sizes (e.g., 500, 2,000,
and 5,000 cells/aggregates) were measured and compared to the
adherent cells. All three aggregates have comparable ATP/cell
contents but all are less than 1/3 of the adherent cells (FIG.
13A). The aggregate-size independent ATP/cell contents suggest
diffusion limitation is not a primary factor for the reduced
metabolic activity but aggregation altered hMSC energy metabolism.
Indeed, accompanying the reduced ATP content, the aggregates have
significantly reduced mitochondrial membrane potential (MMP) as
measured by TMRM staining (FIG. 13B), suggesting increased
mitochondrial depolarization as the major contributing factor to
cellular apoptosis in 3D aggregates.
Example 5
Actin-Mediated Contractility Influences hMSC Aggregation,
Compaction, and Fusion
[0087] Isolated partially based on their plastic adherence, hMSCs
are highly sensitive to biomechanical cues and substrate
elasticity, which regulate hMSC lineage commitment and
differentiation by modulating cytoskeletal tension and RhoA
activation (Kilian et al. 2010; McBeath et al. 2004). The
tension-mediated signaling is manifested in cytoskeletal
contractility, in which reorganization of actin microfilaments
plays a major role in hMSC realignment on topographical features
and response to mechanical stimuli (Zhao et al. 2010; Engler et al.
2006). However, these studies have focused on the biomechanical
cues on planar surfaces with primary impact on cell morphology and
spreading (Kilian et al. 2010). In 3D setting, actin mediated
contractility not only influences cell rounding, cell-cell
contacts, and compaction, but also leads to biomechanical
polarization that is known to play important roles in tissue
organization and morphogenesis (Amack and Manning 2012). While
studies have shown that 3D aggregation profoundly influence hMSC
biological properties, the role of actin-mediated contractility in
mediating the biomechanical properties of 3D aggregates such as
fusion and spreading is yet to be fully understood.
[0088] Self-assembly of multicellular aggregates involves
sequential steps of cell rounding, initial cell-cell contact,
cadherin accumulation, and aggregate compaction, and requires
reestablishment of the balance between surface and cortical
tensions mediated by actin (Sart et al. 2014). According to the DAH
by Steinberg (Steinberg 1962a; Steinberg 1962b), cadherins play a
fundamental role in cell sorting and aggregation during tissue
development and its perturbation interrupts limb development in
vivo and influences hMSC multi-lineage differentiation in vitro
(Oberlender and Tuan 1994; Shin et al. 2000; Yeh et al. 2012).
However, this physics-based reasoning has been challenged by recent
experimental evidence that adhesive molecules at the cortices of
adhering cells not only mechanically couple the neighboring cells
but also initiate local reorganization of actomyosin machinery that
lead to "mechanical polarization" of initially identical cells at
the boundary of a cell colony or aggregate (Amack and Manning 2012;
Maitre et al. 2012). As a result, surface tension of aggregates is
influenced by actomyosin-driven cell cortical contractility in
multicellular aggregates (Krieg et al. 2008; Manning et al. 2010).
Indeed, our results demonstrate that actomyosin contractility plays
a crucial role in regulating hMSC aggregation and that disruption
of actin filaments or inhibiting the phosphorylation of myosin
light chain by Y-27632 prevented aggregate formation and reduced
aggregate compaction in a dose-dependent manner.
[0089] Actin contractility also plays a major role in mediating
aggregate fusion and spreading on rigid adherent surface in which
disruption of actin by cytoD abolishes spreading and fusion of
adjacent aggregates. In contrast, hMSC aggregates treated with
nocodazole exhibited comparable contractility with control
aggregates and readily spread on adherent surface and fuse with
neighboring aggregates, confirming actomyosin-mediated
contractility as the main force in aggregate compaction, spreading,
and fusion. These results are in agreement with the tensegrity
model of cell structure and support the notion that microtubules
act as noncompressive structures of cytoskeleton to resist
contractile tension of the actin and its inhibition has limited
impact on aggregate compaction (Ingber 2003). It is of interest to
note that the induction of osteogenic differentiation significantly
reduced hMSC-OS aggregate spreading on adherent surfaces and fusion
with neighboring aggregates. Osteogenic differentiation of hMSCs is
accompanied by remarkable changes in cytoskeletal organization from
a large number of thin, parallel actin microfilament bundles to a
few thick actin filament bundles and corresponding changes in cell
biomechanical properties (Rodriguez et al. 2004; Titushkin and Cho
2007). These changes are thought to be induced by osteoinductive
components such as dexamethasone which is known to increase cell
stiffness by influencing polymerization of actin microfilaments
(Puig et al. 2009). Indeed, when placed in close contact, hMSC and
OD aggregates displayed markedly different fusion properties with
hMSC aggregates spread and enveloped OD aggregates (FIG. 14). On
the other hand, hMSC aggregates maintained in growth media have
comparable fusion properties even after extended culture up to 21
days (FIG. 15). Recent study suggests that chondrogenic induction
by TGF-.beta. leads to hMSC condensation and boundary formation
(Bhumiratana et al. 2014). Together, these results suggest that
hMSC phenotypic differentiation determines 3D aggregates'
biomechanical properties and influences their self-assembly into
functional units through cell fusion and sorting (Sart et al. 2014;
Jakab et al. 2010).
Example 6
Caspase-Dependent Functional Activation
[0090] Caspase-dependent mechanism has been suggested as a major
mechanism in functional activation associated with 3D hMSC
aggregation but the origin of the apoptotic signal remains unclear.
Aggregates of undifferentiated hMSCs experience considerable
compaction and a marked decrease in relative amount of cytoplasm
and cell volume and an increase in packing density (Bartosh et al.
2013). As such, aggregate compaction has been suggested as a
contributing factor to self-activate caspase-dependent signaling
and secretion of trophic factors (Bartosh et al. 2013; Kelm et al.
2012). However, reducing aggregate compaction by cytoD and Y-27632
treatments did not lead to increased cell viability nor reduced
caspase 3/7 expression. While the actin modulators are pleiotropic
and have cytotoxic effects on cell viability (Gourlay and Ayscough
2005), their limited reduction on aggregate caspase 3/7 expression
in contrast to the significantly increased cell viability by
caspase inhibitor (FIG. 9A) suggests an independent mediator for
caspase activation in 3D aggregates.
[0091] Apart from biomechanical forces, impediment of oxygen
transport and the resultant hypoxia region in the aggregates have
been cited as a cellular stressor for the increase in caspase
expression and subsequent functional activation (Bartosh et al.
2010). However, the comparable levels of size-independent ATP/cell
in aggregates of different sizes provide direct evidence that
oxygen diffusion is not a limiting factor in energy metabolism in
the aggregates. In prior studies, immunostaining of the apoptotic
activity in 3D hMSC aggregates revealed even distribution of
apoptotic cells (Kelm et al. 2012) and that functional enhancement
was also observed in aggregates below the threshold of oxygen
transport (Zimmermann and Mcdevitt 2014; Ylostalo et al. 2012). In
addition, oxygen is not limiting for electron transport in
mitochondria until levels are extremely low; even at an O.sub.2
concentration of 25 .mu.M, electron transport is reduced only by
33% (Chandel et al. 1996). Thus, oxygen gradient is unlikely a
direct contributing factor to the elevated apoptosis.
[0092] The comparable ATP/cell levels in aggregates of different
sizes and their significant reduction compared to adherent cells
suggest the changes in metabolic machinery following aggregation
independent from substrate gradients due to diffusion. The drastic
changes in cellular organization and creation of cellular and
extracellular microenvironment during multicellular assembly of
hMSCs 3D aggregates require effective reconfiguration of metabolic
network for cell survival and function. hMSCs have been shown to
use both glycolysis and OXPHO for ATP generation, exhibit metabolic
flexibility for survival in an ischemic environment, and undergo
coordinated changes of mitochondrial biogenesis during osteogenic
differentiation (Chen et al. 2008b; Mylotte et al. 2008). The
significant reduction of ATP/cell and the altered mitochondrial
potential upon aggregation suggest changes in mitochondrial
function that may directly contribute to the increased apoptosis.
In contrast to mitochondrial transformation which usually occurs
during cell differentiation or carcinogenesis, mitochondrial
plasticity, known as mitoplasticity, plays an important role for
the maintenance of cellular energy homeostasis and influences cell
fate in response to energy imbalance due to accidental
environmental changes in energy demand or supply (Jose et al.
2013). The drastic changes in actin dynamics during 3D aggregation
may play a role in the altered mitochondria function in the
aggregates as actin cytoskeleton influences mitochondrial
organization, short-range movement, and function in mammalian cells
(Sheng and Cai 2012; Quintero et al. 2009). One way that actin
organization and dynamics in the cell influence mitochondrial
network is by changes in the availability of polymerizable actin,
thereby influencing morphology, connectivity, and ATP production of
the mitochondrial network (Yu et al. 2010; Jayashankar and Rafelski
2014). In addition, the actin cytoskeleton is also an important
physiological regulator of ROS release from mitochondria and that a
reduction in actin dynamics leads to reduced mitochondria membrane
potential, suggesting its key role in the upstream action of
apoptosis pathways (Gourlay and Ayscough 2005). Apart from actin,
mitochondrial properties in the aggregates may also be influenced
and/or mitigated by mitogenic growth factors. Indeed, recent
studies have shown that media composition and the presence of
mitogenic factors in media significantly influence cell
proliferation and secretory functions in the aggregates (Zimmermann
and Mcdevitt 2014; Alimperti et al. 2014; Ylostalo et al. 2014).
Thus, further study is warranted to decipher the exact contribution
of actin-mediated biomechanical changes and metabolic adaption and
apoptosis in hMSC aggregate's functional activation.
Example 7
Aggregation as a Preconditioning Strategy for hMSC Functional
Activation
[0093] Spontaneous multicellular aggregation appears to select
cells that are small in size, have higher CXCR-4 expression, higher
migration, and higher resistance to ischemic stress in vitro,
suggesting the potential of aggregation as a preconditioning
strategy for enhancing hMSC therapeutic properties (FIGS. 12A-12H).
IL-6 is a cytokine that not only involved in inflammation but also
has many regenerative and anti-inflammatory functions (Scheller et
al. 2011), whereas PGE-2 is an important component of hMSC
secretome that modulates both innate and adaptive immune responses
(Aggarwal and Pittenger 2005). Our results confirmed prior
observation and demonstrated that the enhanced secretion of IL-6
and PGE-2 is dependent on hMSC stress response and elevated
apoptosis activity because the attenuation of apoptosis
significantly reduced both PGE-2 and IL-6 secretions.
[0094] The results of present study demonstrate that actin-mediated
contractility regulates aggregates biomechanical properties such as
compaction, fusion, and spreading, and that 3D aggregation alters
mitochondrial properties and induces cellular stress response and
functional activation. The results also show that functional
activation is regulated by caspase activation during aggregation
but independent from actin-mediated compaction. Prior studies have
shown that hMSC in vitro aging after extensive passaging altered
mitochondrial morphology and decreased antioxidant capacities with
reduced actin dynamic and migratory properties (Kasper et al. 2009;
Geissler et al. 2012). Thus, the drastic changes in cytoskeleton
during 3D aggregation may select a subset of hMSC that have dynamic
actin turnover characteristic of MSC from young donor and enable
them to effectively adapt during aggregate compaction. It is also
possible that formation of 3D aggregates reverts hMSC back to an
early phenotype as occurred when neural stem cells formed 3D
neurospheres (Pastrana et al. 2011). Nevertheless, the results of
current study support that notation that 3D aggregation
significantly influences hMSC properties and can be used as a
non-genetic preconditioning method to enhance the function and
therapeutic outcome of culture-expanded hMSC in a wide range of
diseases.
Example 8
Functional Activation of hBMSC by 3D Aggregation
[0095] 5,000 hBMSCs seeded in 96-well ultralow attachment (ULA)
plates spontaneously aggregated into 3D spheroids overnight. The
hBMSC aggregates were able to fuse and maintained their ability to
adhere to glass. cytochalasin D treatment of hBMSC aggregates
abrogated their fusion and adhesion properties and disrupted the
mechanically polarized outer layer as revealed by histology (FIG.
16F) suggesting that these properties are mediated by actin. hBMSC
3D aggregation significantly up-regulated CXCR-4 expression,
migration towards SDF-1, and increased their resistance to in vitro
ischemia (e.g., 2 hours in serum-free media under 2% O.sub.2). hASC
3D aggregation enhanced expression of anti-inflammatory cytokines
and growth factors (IL-10, HGF, STC-1, PGE-2, and IL-6). The hASC
aggregates underwent compaction with a 40% reduction in cell number
within 3 days, however hypoxia preconditioning (HPC) (i.e.,
expansion at 2% O.sub.2 for 3 days before aggregation)
significantly increased cell viability (FIGS. 16A-16E).
Example 9
Microcarrier Coating and Derivation of hBMSC Aggregates
[0096] FIG. 17 shows the procedure for coating of polystyrene
microcarriers (SoloHill, Ann Arbor, Mich.) with
poly(N-isopropylacrylamide) (PNIPAM)-based thermo-responsive film
(Maitre et al. 2012). Alternating PSS-co-PNIPAM
(Styrenesulfonate:Isopropylacrylamide=50:50 mol %) and
PAH-co-PNIPAM (Allylamine:Isopropylacrylamide=50:50 mol %) were
coated on the microcarriers, and the coating was confirmed by X-ray
photoelectron spectroscopy and tested in the bioreactor. After
seeding on the TRM, hBMSCs were expanded in the spinner flask for 7
days at 37.degree. C. for a five-fold expansion and then moved to
20.degree. C. to initiate cell detachment under agitation. After 2
hours at 20.degree. C., hBMSC detachment is completed and the
vessel returned to 37.degree. C. for overnight culture to derive 3D
aggregates. The results demonstrate the feasibility of integrated
hBMSC expansion and derivation of 3D aggregates in a single
bioreactor system (FIG. 18).
[0097] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
REFERENCES
[0098] Aggarwal, S. and M. F. Pittenger, Human mesenchymal stem
cells modulate allogeneic immune cell responses. Blood 105, 1815,
2005. [0099] Alimperti, S., P. Lei, Y. Wen, J. Tian, A. M.
Campbell, and S. T. Andreadis, Serum-free spheroid suspension
culture maintains mesenchymal stem cell proliferation and
differentiation potential. Biotechnology progress 30(4):974-983,
2014. [0100] Amack, J. D. and M. L. Manning, Knowing the
boundaries: extending the differential adhesion hypothesis in
embryonic cell sorting. Science 338, 212, 2012. [0101] Bara, J. J.,
R. G. Richards, M. Alini, and M. J. Stoddart, Concise review: Bone
marrow-derived mesenchymal stem cells change phenotype following in
vitro culture: Implications for basic research and the clinic. Stem
Cells 32(7):1713-1723, 2014. [0102] Baraniak, P. R., M. T. Cooke,
R. Saeed, M. A. Kinney, K. M. Fridley, and T. C. McDevitt,
Stiffening of human mesenchymal stem cell spheroid
microenvironments induced by incorporation of gelatin
microparticles. Journal of the Mechanical Behavior of Biomedical
Materials 11, 63, 2012. [0103] Bartosh, T. J., J. H. Ylostalo, A.
Mohammadipoor, N. Bazhanov, K. Coble, K. Claypool, R. H. Lee, H.
Choi, and D. J. Prockop, Aggregation of human mesenchymal stromal
cells (MSCs) into 3D spheroids enhances their antiinflammatory
properties. Proc Natl Acad Sci USA 107, 13724, 2010. [0104]
Bartosh, T. J., J. H. Ylostalo, N. Bazhanov, J. Kuhlman, and D. J.
Prockop, Dynamic compaction of human mesenchymal stem/precursor
cells into spheres self-activates caspase-dependent IL1 signaling
to enhance secretion of modulators of inflammation and immunity
(PGE2, TSG6, and STC1). Stem Cells 31, 2443, 2013. [0105] Bhang, S.
H., S. W. Cho, W. G. La, T. J. Lee, H. S. Yang, A. Y. Sun, S. H.
Baek, J. W. Rhie, and B. S. Kim, Angiogenesis in ischemic tissue
produced by spheroid grafting of human adipose-derived stromal
cells. Biomaterials 32, 2734, 2011. [0106] Bhumiratana, S., Eton,
R. E., Oungoulian, S. R., Wan, L. Q., Ateshian, G. A., &
Vunjak-Novakovic, G., Large, stratified, and mechanically
functional human cartilage grown in vitro by mesenchymal
condensation. Proceedings of the National Academy of Sciences, 111,
6940, 2014. [0107] Chandel, N. S., G. R. Budinger, and P. T.
Schumacker, Molecular oxygen modulates cytochrome c oxidase
function. J Biol Chem 271, 18672, 1996. [0108] Chavakis E, Dimmeler
S. Homing of Progenitor Cells to Ischemic Tissues. Antioxid Redox
Sign. 2011; 15:967-80. [0109] Chen, J. H., A. R. Baydoun, R. X. Xu,
L. Z. Deng, X. B. Liu, W. Q. Zhu, L. H. Shi, X. F. Cong, S. S. Hu,
and X. Chen, Lysophosphatidic acid protects mesenchymal stem cells
against hypoxia and serum deprivation-induced apoptosis. Stem Cells
26, 135, 2008a. [0110] Chen, C. T., Y. R. V. Shih, T. K. Kuo, O. K.
Lee, and Y. H. Wei, Coordinated changes of mitochondrial biogenesis
and antioxidant enzymes during osteogenic differentiation of human
mesenchymal stem cells. Stem Cells 26, 960, 2008b. [0111] Chen, G.
K., Z. G. Hou, D. R. Gulbranson, and J. A. Thomson, Actin-Myosin
contractility is responsible for the reduced viability of
dissociated human embryonic stem cells. Cell Stem Cell 7, 240,
2010. [0112] Copland, I. B. and J. Galipeau, Death and inflammation
following somatic cell transplantation. Seminars in Immunopathology
33, 535, 2011. [0113] Dalby, M. J., N. Gadegaard, R. Tare, A.
Andar, M. O. Riehle, P. Herzyk, C. D. W. Wilkinson, and R. O. C.
Oreffo, The control of human mesenchymal cell differentiation using
nanoscale symmetry and disorder. Nature Materials 6, 997, 2007.
[0114] dos Santos F F, Andrade P Z, da Silva C L, Cabral J M.
Bioreactor design for clinical-grade expansion of stem cells.
Biotechnol J. 2013; 8:644-54. [0115] Elmore, S., Apoptosis: A
review of programmed cell death. Toxicologic Pathology 35, 495,
2007. [0116] Engler, A. J., S. Sen, H. L. Sweeney, and D. E.
Discher, Matrix elasticity directs stem cell lineage specification.
Cell 126, 677, 2006. [0117] Fletcher, D. A. and D. Mullins, Cell
mechanics and the cytoskeleton. Nature 463, 485, 2010. [0118]
Geissler, S., M. Textor, J. Kuhnisch, D. Konnig, O. Klein, A. Ode,
T. Pfitzner, J. Adjaye, G. Kasper, and G. N. Duda, Functional
comparison of chronological and in vitro aging: differential role
of the cytoskeleton and mitochondria in mesenchymal stromal cells.
Plos One 7(12):e52700, 2012. [0119] Gonzalez-Rodriguez, D., K.
Guevorkian, S. Douezan, and F. Brochard-Wyart, Soft Matter Models
of Developing Tissues and Tumors. Science 338, 910, 2012. [0120]
Gourlay, C. W. and K. R. Ayscough, The actin cytoskeleton: a key
regulator of apoptosis and ageing? Nat Rev Mol Cell Biol 6, 583,
2005. [0121] Grayson W L, Ma T, Bunnell B. Human mesenchymal stem
cells tissue development in 3D PET matrices. Biotechnol Progr.
2004; 20:905-12. [0122] Grayson W L, Zhao F, Bunnell B, Ma T.
Hypoxia enhances proliferation and tissue formation of human
mesenchymal stem cells. Biochem Bioph Res Comm. 2007; 358:948-53.
[0123] Guo et al. (2014) Three-Dimensional Spheroid-Cultured
Mesenchymal Stem Cells Devoid of Embolism Attenuate Brain Stroke
Injury After Intra-Arterial Injection. Stem Cells and Development,
DOI: D01: 10.1089/scd.201 3.0338 [0124] Hildebrandt, C., H. Buth,
and H. Thielecke, A scaffold-free in vitro model for osteogenesis
of human mesenchymal stem cells. Tissue Cell 43, 91, 2011. [0125]
Ingber, D. E., Tensegrity I. Cell structure and hierarchical
systems biology. J Cell Sci 116, 1157, 2003. [0126] Ivascu, A. and
M. Kubbies, Diversity of cell-mediated adhesions in breast cancer
spheroids. International Journal of Oncology 31, 1403, 2007. [0127]
Jakab, K., Norotte, C., Marga, F., Murphy, K., Vunjak-Novakovic,
G., & Forgacs, G., Tissue engineering by self-assembly and
bio-printing of living cells. Biofabrication, 2, 022001, 2010.
[0128] Jayashankar, V. and S. M. Rafelski, Integrating
mitochondrial organization and dynamics with cellular architecture.
Current Opinion in Cell Biology 26, 34, 2014. [0129] Jose, C., S.
Melser, G. Benard, and R. Rossignol, Mitoplasticity: adaptation
biology of the mitochondrion to the cellular redox state in
physiology and carcinogenesis. Antioxidants & Redox Signaling
18, 808, 2013. [0130] Kasper, G., L. Mao, S. Geissler, A.
Draycheva, J. Trippens, J. Kuhnisch, M. Tschirschmann, K. Kaspar,
C. Perka, G. N. Duda, and J. Klose, Insights into mesenchymal stem
cell aging: involvement of antioxidant defense and actin
cytoskeleton. Stem Cells 27, 1288, 2009. [0131] Kelm, J. M., M.
Breitbach, G. Fischer, B. Odermatt, I. Agarkova, B. K. Fleischmann,
and S. P. Hoerstrup, 3D microtissue formation of undifferentiated
bone marrow mesenchymal stem cells leads to elevated apoptosis.
Tissue Eng Part A 18, 692, 2012. [0132] Kilian, K. A., B. Bugarija,
B. T. Lahn, and M. Mrksich, Geometric cues for directing the
differentiation of mesenchymal stem cells. Proc Natl Acad Sci USA
107, 4872, 2010. [0133] Kim, J. and T. Ma, Bioreactor strategy in
bone tissue engineering: pre-culture and osteogenic differentiation
under two flow configurations. Tissue Eng Part A
18(21-22):2354-2364, 2012. [0134] Kim J. and T. Ma, Autocrine
Fibroblast Growth Factor 2-Mediated Interactions between Human
Mesenchymal Stem Cells and the Extracellular Matrix under Varying
Oxygen Tension. J Cell Biochem. 114, 716-27, 2013a. [0135] Kim, J.
and T. Ma, Endogenous extracellular matrices enhance human
mesenchymal stem cell aggregate formation and survival. Biotechnol
Prog 29(2):441-451, 2013b. [0136] Krieg, M., Y. Arboleda-Estudillo,
P. H. Puech, J. Kafer, F. Graner, D. J. Muller, and C. P.
Heisenberg, Tensile forces govern germ-layer organization in
zebrafish. Nature Cell Biology 10, 429, 2008. [0137] Lee E J, Park
S J, Kang S K, Kim G H, Kang H J, Lee S W, et al. Spherical Bullet
Formation via E-cadherin Promotes Therapeutic Potency of
Mesenchymal Stem Cells Derived From Human Umbilical Cord Blood for
Myocardial Infarction. Molecular Therapy. 2012; 20:1424-33. [0138]
Lee R H, Pulin A A, Seo M J, Kota D J, Ylostalo J, Larson B L, et
al. Intravenous hMSCs Improve Myocardial Infarction in Mice because
Cells Embolized in Lung Are Activated to Secrete the
Anti-inflammatory Protein TSG-6. Cell Stem Cell. 2009; 5:54-63.
[0139] Li W Y, Choi Y J, Lee P H, Huh K, Kang Y M, Kim H S, et al.
Mesenchymal Stem Cells for Ischemic Stroke: Changes in Effects
After Ex Vivo Culturing. Cell Transplant. 2008; 17:1045-59. [0140]
Liao T, Moussallem M D, Kim J, Schlenoffd J B, Ma T.
N-Isopropylacrylamide-Based Thermoresponsive Polyelectrolyte
Multilayer Films for Human Mesenchymal Stem Cell Expansion.
Biotechnol. Prog., 2010, 26, 1705-1713. [0141] Lin, R. Z. and H. Y.
Chang, Recent advances in three-dimensional multicellular spheroid
culture for biomedical research. Biotechnology Journal 3, 1172,
2008. [0142] Lin, R. Z., L. F. Chou, C. C. M. Chien, and H. Y.
Chang, Dynamic analysis of hepatoma spheroid formation: roles of
E-cadherin and beta 1-integrin. Cell and Tissue Research 324, 411,
2006. [0143] Liu H, Xue W, Ge G, Luo X, Li Y, Xiang H, et al.
Hypoxic preconditioning advances CXCR4 and CXCR7 expression by
activating HIF-1alpha in MSCs. Biochem Biophys Res Commun. 2010;
401:509-15. [0144] Maitre, J. L., H. Berthoumieux, S. F. G. Krens,
G. Salbreux, F. Julicher, E. Paluch, and C. P. Heisenberg, Adhesion
functions in cell sorting by mechanically coupling the cortices of
adhering cells. Science 338, 253, 2012. [0145] Manning, M. L., R.
A. Foty, M. S. Steinberg, and E. M. Schoetz, Coaction of
intercellular adhesion and cortical tension specifies tissue
surface tension. Proc Natl Acad Sci USA 107, 12517, 2010. [0146]
McBeath, R., D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S.
Chen, Cell shape, cytoskeletal tension, and RhoA regulate stem cell
lineage commitment. Developmental Cell 6, 483, 2004. [0147]
Moolenaar, W. H., Lysophosphatidic acid, a multifunctional
phospholipid messenger. Journal of Biological Chemistry 270, 12949,
1995. [0148] Moussallem M D, Olenych S G, Scott S L, Keller T C,
3rd, Schlenoff J B. Smooth muscle cell phenotype modulation and
contraction on native and cross-linked polyelectrolyte multilayers.
Biomacromolecules. 2009; 10:3062-8. [0149] Munoz, N., J. Kim, Y.
Liu, T. M. Logan, and T. Ma, Gas chromatography-mass spectrometry
analysis of human mesenchymal stem cell metabolism during
proliferation and osteogenic differentiation under different oxygen
tensions. Journal of Biotechnology 169, 95, 2014. [0150] Mylotte,
L. A., A. M. Duffy, M. Murphy, T. O'Brien, A. Samali, F. Barry, and
E. Szegezdi, Metabolic flexibility permits mesenchymal stem cell
survival in an ischemic environment. Stem Cells 26, 1325, 2008.
[0151] Nelson, C. M., R. P. Jean, J. L. Tan, W. F. Liu, N. J.
Sniadecki, A. A. Spector, and C. S. Chen, Emergent patterns of
growth controlled by multicellular form and mechanics. Proc Natl
Acad Sci USA 102, 11594, 2005. [0152] Numasawa Y, Kimura T, Miyoshi
S, Nishiyama N, Hida N, Tsuji H, et al. Treatment of human
mesenchymal stem cells with angiotensin receptor blocker improved
efficiency of cardiomyogenic transdifferentiation and improved
cardiac function via angiogenesis. Stem Cells. 2011; 29:1405-14.
[0153] Oberlender, S. A. and R. S. Tuan, Expression and functional
involvement of N-cadherin in embryonic limb chondrogenesis.
Development 120, 177, 1994. [0154] Olenych S G, Moussallem M D,
Salloum D S, Schlenoff J B, Keller T C. Fibronectin and cell
attachment to cell and protein resistant polyelectrolyte surfaces.
Biomacromolecules. 2005; 6:3252-8. [0155] Parekkadan B, Milwid J M.
Mesenchymal Stem Cells as Therapeutics. Annu Rev Biomed Eng. 2010;
12:87-117. [0156] Pastrana, E., V. Silva-Vargas, and F. Doetsch,
Eyes wide open: a critical review of sphere-formation as an assay
for stem cells. Cell Stem Cell 8, 486, 2011. [0157] Potapova, I.
A., P. R. Brink, I. S. Cohen, and S. V. Doronin, Culturing of human
mesenchymal stem cells as three-dimensional aggregates induces
functional expression of CXCR4 that regulates adhesion to
endothelial cells. J Biol Chem 283, 13100, 2008. [0158] Prockop, D.
J., D. J. Kota, N. Bazhanov, and R. L. Reger, Evolving paradigms
for repair of tissues by adult stem/progenitor cells (MSCs). J Cell
Mol Med 14, 2190, 2010. [0159] Puig, F., N. Gavara, R. Sunyer, A.
Carreras, R. Farre, and D. Navajas, Stiffening and contraction
induced by dexamethasone in alveolar epithelial cells. Experimental
Mechanics 49, 47, 2009. [0160] Quintero, O. A., M. M. DiVito, R. C.
Adikes, M. B. Kortan, L. B. Case, A. J. Lier, N. S. Panaretos, S.
Q. Slater, M. Rengarajan, M. Feliu, and R. E. Cheney, Human Myo19
Is a Novel myosin that associates with mitochondria. Current
Biology 19, 2008, 2009. [0161] Rivron, N. C., E. J. Vrij, J.
Rouwkema, S. Le Gac, A. van den Berg, R. K. Truckenmuller, and C.
A. van Blitterswijk, Tissue deformation spatially modulates VEGF
signaling and angiogenesis. Proc Natl Acad Sci USA 109, 6886, 2012.
[0162] Rodriguez, J. P., M. Gonzalez, S. Rios, and V. Cambiazo,
Cytoskeletal organization of human mesenchymal stem cells (MSC)
changes during their osteogenic differentiation. Journal of
Cellular Biochemistry 93, 721, 2004. [0163] Rombouts W J C,
Ploemacher R E. Primary murine MSC show highly efficient homing to
the bone marrow but lose homing ability following culture.
Leukemia. 2003; 17:160-70. [0164] Russell, K. C., D. G. Phinney, M.
R. Lacey, B. L. Barrilleaux, K. E. Meyertholen, and K. C. O'Connor,
In vitro high-capacity assay to quantify the clonal heterogeneity
in trilineage potential of mesenchymal stem cells reveals a complex
hierarchy of lineage commitment. Stem Cells 28, 788, 2010. [0165]
Sart, S., A. C. Tsai, Y. Li, and T. Ma, Three-dimensional
aggregates of mesenchymal stem cells: cellular mechanisms,
biological properties, and applications. Tissue Engineering Part B,
Reviews, 20(5):365-380, 2014. [0166] Scheller, J., A. Chalaris, D.
Schmidt-Arras, and S. Rose-John, The pro- and anti-inflammatory
properties of the cytokine interleukin-6 Biochim Biophys Acta 1813,
878, 2011. [0167] Sepulveda, J. C., M. Tome, M. E. Fernandez, M.
Delgado, J. Campisi, A. Bernad, and M. A. Gonzalez, Cell senescence
abrogates the therapeutic potential of human mesenchymal stem cells
in the lethal endotoxemia model. Stem Cells 32(7):1865-1877, 2014.
[0168] Sharma S, Raju R, Sui S G, Hu W S. Stem cell culture
engineering--process scale up and beyond. Biotechnol J. 2011;
6:1317-29. [0169] Sheng, Z. H. and Q. Cai, Mitochondrial transport
in neurons: impact on synaptic homeostasis and neurodegeneration.
Nature Reviews Neuroscience 13, 77, 2012. [0170] Shin, C. S., F.
Lecanda, S. Sheikh, L. Weitzmann, S. L. Cheng, and R. Civitelli,
Relative abundance of different cadherins defines differentiation
of mesenchymal precursors into osteogenic, myogenic, or adipogenic
pathways. Journal of Cellular Biochemistry 78, 566, 2000. [0171]
Shinmura D, Togashi I, Miyoshi S, Nishiyama N, Hida N, Tsuji H, et
al. Pretreatment of human mesenchymal stem cells with pioglitazone
improved efficiency of cardiomyogenic transdifferentiation and
cardiac function. Stem Cells. 2011; 29:357-66.
[0172] Steinberg, M. S., On the mechanism of tissue reconstruction
by dissociated cells. I. Population kinetics, differential
adhesiveness. and the absence of directed migration. Proc Natl Acad
Sci USA 48, 1577, 1962a. [0173] Steinberg, M. S., On the mechanism
of tissue reconstruction by dissociated cells, Iii. Free energy
relations and the reorganization of fused, heteronomic tissue
fragments. Proc Natl Acad Sci USA 48, 1769, 1962b. [0174] Stroncek,
D. F., M. Sabatino, J. Ren, L. England, S. A. Kuznetsov, H. G.
Klein, and P. G. Robey, Establishing a bone marrow stromal cell
transplant program at the national institutes of health clinical
center. Tissue engineering Part B, Reviews 2014. DOI:
10.1089/ten.TEB.2013.0529 [0175] Sutlu T, Stellan B, Gilljam M,
Quezada H C, Nahi H, Gahrton G, et al. Clinical-grade, large-scale,
feeder-free expansion of highly active human natural killer cells
for adoptive immunotherapy using an automated bioreactor.
Cytotherapy. 2010; 12:1044-55. [0176] Tigyi, G., D. L. Dyer, and R.
Miledi, Lysophosphatidic acid possesses dual-action in
cll-proliferation. Proc Natl Acad Sci USA 91, 1908, 1994. [0177]
Titushkin, I. and M. Cho, Modulation of cellular mechanics during
osteogenic differentiation of human mesenchymal stem cells.
Biophysical Journal 93, 3693, 2007. [0178] Toma, C., W. R. Wagner,
S. Bowry, A. Schwartz, and F. Villanueva, Fate Of culture-expanded
mesenchymal stem cells in the microvasculature in vivo observations
of cell kinetics. Circulation Research 104, 398, 2009. [0179]
Wagner, W., P. Horn, M. Castoldi, A. Diehlmann, S. Bork, R.
Saffrich, V. Benes, J. Blake, S. Pfister, V. Eckstein, and A. D.
Ho, Replicative senescence of mesenchymal stem cells: a continuous
and organized process. Plos One 3(5):e2213, 2008. [0180] Wei L,
Fraser J L, Lu Z Y, Hu X, Yu S P. Transplantation of hypoxia
preconditioned bone marrow mesenchymal stem cells enhances
angiogenesis and neurogenesis after cerebral ischemia in rats.
Neurobiology of Disease. 2012; 46:635-45. [0181] Whitfield, M. J.,
W. C. J. Lee, and K. J. Van Vliet, Onset of heterogeneity in
culture-expanded bone marrow stromal cells. Stem Cell Res 11, 1365,
2013. [0182] Yeh, H. Y., B. H. Liu, and S. H. Hsu, The
calcium-dependent regulation of spheroid formation and
cardiomyogenic differentiation for MSCs on chitosan membranes.
Biomaterials 33, 8943, 2012. [0183] Ylostalo, J. H., Bartosh, T.
J., Tiblow, A., & Prockop, D. J., Unique characteristics of
human mesenchymal stromal/progenitor cells pre-activated in
3-dimensional cultures under different conditions. Cytotherapy,
16(11):1486-1500, 2014. [0184] Ylostalo, J. H., T. J. Bartosh, K.
Coble, and D. J. Prockop, Human Mesenchymal stem/stromal cells
cultured as spheroids are self-activated to produce prostaglandin
E2 that directs stimulated macrophages into an anti-inflammatory
phenotype. Stem Cells 30, 2283, 2012. [0185] Yu, Y. S., R.
Dumollard, A. Rossbach, F. A. Lai, and K. Swann, Redistribution of
mitochondria leads to bursts of ATP production during spontaneous
mouse oocyte maturation. Journal of Cellular Physiology 224, 672,
2010. [0186] Zhang, Q. Z., A. L. Nguyen, S. H. Shi, C. Hill, P.
Wilder-Smith, T. B. Krasieva, and A. D. Le, Three-dimensional
spheroid culture of human gingiva-derived mesenchymal stem cells
enhances mitigation of chemotherapy-induced oral mucositis. Stem
Cells and Development 21, 937, 2012. [0187] Zhao F, Ma T. Perfusion
bioreactor system for human mesenchymal stem cell tissue
engineering: Dynamic cell seeding and construct development.
Biotechnol Bioeng. 2005; 91:482-93. [0188] Zhao, F., J. J.
Veldhuis, Y. J. Duan, Y. Yang, N. Christoforou, T. Ma, and K. W.
Leong, [0189] Low oxygen tension and synthetic nanogratings improve
the uniformity and stemness of human mesenchymal stem cell layer.
Molecular Therapy 18, 1010, 2010. [0190] Zimmermann, J. A. and T.
C. Mcdevitt, Pre-conditioning mesenchymal stromal cell spheroids
for immunomodulatory paracrine factor secretion. Cytotherapy 16,
331, 2014.
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