U.S. patent application number 13/810878 was filed with the patent office on 2013-12-12 for high telomerase activity bone marrow mesenchymal stem cells, methods of producing the same and pharmaceuticals and treatment methods based thereon.
This patent application is currently assigned to University of Southern California. The applicant listed for this patent is Kentaro Akiyama, Chider Chen, Songtao Shi. Invention is credited to Kentaro Akiyama, Chider Chen, Songtao Shi.
Application Number | 20130330300 13/810878 |
Document ID | / |
Family ID | 45497448 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130330300 |
Kind Code |
A1 |
Shi; Songtao ; et
al. |
December 12, 2013 |
High Telomerase Activity Bone Marrow Mesenchymal Stem Cells,
Methods of Producing the Same and Pharmaceuticals and Treatment
Methods Based Thereon
Abstract
Disclosed are isolated human bone marrow mesenchymal stem cells
having high telomerase activity (tBMMSCs). Also disclosed are
isolated human CD34.sup.+ bone marrow mesenchymal stem cells. Also
disclosed are bone marrow mesenchymal stem cells treated with a
telomerase induction agent.
Inventors: |
Shi; Songtao; (Los Angeles,
CA) ; Akiyama; Kentaro; (Los Angeles, CA) ;
Chen; Chider; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shi; Songtao
Akiyama; Kentaro
Chen; Chider |
Los Angeles
Los Angeles
Los Angeles |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
University of Southern
California
Los Angeles
CA
|
Family ID: |
45497448 |
Appl. No.: |
13/810878 |
Filed: |
July 20, 2011 |
PCT Filed: |
July 20, 2011 |
PCT NO: |
PCT/US11/44731 |
371 Date: |
August 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61366095 |
Jul 20, 2010 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/372 |
Current CPC
Class: |
C12N 5/0663 20130101;
A61K 35/28 20130101; A61K 31/60 20130101; C12N 2501/02 20130101;
A61K 31/60 20130101; A61K 2035/122 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/93.7 ;
435/372 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
Contract No R01DE17449 awarded by the National Institute of Dental
and Craniofacial Research/National Institute for Health. The
government has certain rights in the invention.
Claims
1. Isolated human bone marrow mesenchymal stem cells having high
telomerase activity (tBMMSCs).
2. The isolated human bone marrow mesenchymal stem cells according
to claim 1, wherein said cells are SSEA4.sup.+.
3. Isolated human CD34.sup.+ bone marrow mesenchymal stem
cells.
4. The isolated human CD34.sup.+ bone marrow mesenchymal stem cells
according to claim 3, wherein the stem cells are CD73.sup.+
5. The isolated human CD34.sup.+ bone marrow mesenchymal stem cells
according to claim 3, wherein said stem cells are CD105.sup.+
6. The isolated human CD34.sup.+ bone marrow mesenchymal stem cells
according to claim 3, wherein the stem cells fail to form CFU-F in
plastic cultures, but adhere on mesenchymal stem cell-produced
extra-cellular matrix.
7. The isolated human CD34.sup.+ bone marrow mesenchymal stem cells
according to claim 3, where said stem cells are capable of
differentiating into osteoblasts, adipocytes, and chondrocytes.
8. Isolated aspirin treated bone marrow mesenchymal stem cells.
9. A method of increasing telomerase activity in CD34.sup.- human
bone marrow mesenchymal stem cells comprising: contacting human
bone marrow messenchymal stem cells with an effective amount of a
telomerase inducing agent.
10. The method of claim 7, wherein the telomerase inducing agent is
aspirin.
11. A method of isolating high telomerase bone marrow mesenchymal
stem cells comprising: culturing a sample of bone marrow derived
all nuclear cells on a plastic substrate; removing cells that do
not adhere to the substrate; culturing any cells in the medium on
an BMMSC-ECM coated medium; collecting colonies forming attached
cells on the BMMSC-ECM medium.
12. A method of treating systemic lupus erythematosus, comprising
administering to the patient a therapeutically effective amount of
isolated high telomerase human bone marrow mesenchymal stem cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/366,095, filed Jul. 20, 2010, the entire
contents of which are incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates in general to bone marrow
mesenchymal stem cells, and more specifically to a subset of novel
BMMSCs having high telomerase activity, pharmaceutical compositions
comprising the BMMSCs, immunomodulation methods using the BMMSCs,
and treatment methods for systemic lupus erythemetosis by
administration of the BMMSCs.
BACKGROUND OF THE INVENTION
[0004] Bone marrow mesenchymal stem cells (BMMSCs) are hierarchical
postnatal stem cells capable of self-renewing and differentiating
into osteoblasts, chondrocytes, adipocytes, and neural cells
(Bianco et al., 2001; Friedenstein et al., 1974; Owen et al., 1988;
Pittenger et al., 1999; Prockop et al., 1997).
[0005] Due to the heterogeneity of the BMMSCs, there is no single,
unique marker allowing for BMMSC isolation, rather an array of cell
molecules are utilized to profile BMMSCs. It is widely accepted
that BMMSCs express SH2 (CD105), SH3/SH4 (CD73), integrin
.beta..sub.1 (CD29), CD44, Thy-1 (CD90), CD71, vascular cell
adhesion molecule-1 (CD106), activated leukocyte cell adhesion
molecule (CD166), STRO-1, GD2, melanoma cell adhesion molecule
(CD146), Octamer-4 (Oct4), and stage-specific embryonic antigen-4
(SSEA4) (Conget et al., 1999; Galmiche et al., 1993; Gronthos et
al., 2003; Haynesworth et al., 1992; Martinez et al., 2007;
Pittenger et al., 1999; Sacchetti et al., 2007; Shi et al., 2003;
Simmons et al., 1991; Sordi et al., 2005). It is generally believed
that BMMSCs are negative for hematopoietic cell markers such as
CD14 and CD34 with a very low level of telomerase activity (Conget
et al., 1999; Covas et al., 2008; Galmiche et al., 1993;
Haynesworth et al., 1992; Martinez et al., 2007; Pittenger et al.,
1995; Sacchetti et al., 2008; Shi et al., 2002, 2003; Sordi et al.,
2005). Recent studies have implied that mouse BMMSCs might express
the hematopoietic surface molecules, CD45 (Chen et al., 2007) and
CD34 (Copland et al., 2008).
[0006] BMMSCs are considered to be progenitors of osteoblasts with
the capacity to regenerate bone and marrow components in vivo.
These findings have led to extensive studies using BMMSCs for
mineralized tissue engineering. The clinical evidence appears to
support the notion that BMMSC implantation is able to improve
cell-based skeletal tissue regeneration (Kwan et al., 2008; Panetta
et al., 2009). Recently, evidence has accumulated that BMMSCs
produce a variety of cytokines and display profound
immunomodulatory properties (Nauta et al., 2007; Uccelli et al.,
2007, 2008), perhaps by inhibiting the proliferation and function
of several major immune cells such as natural killer (NK) cells,
dendritic cells, T and B lymphocytes (Aggarwal and Pittenger, 2005;
Nauta et al., 2007; Uccelli et al., 2007, 2008). These unique
properties make BMMSCs of great interest for clinical applications
in treating immune disorders (Nauta and Fibbe, 2007; Bernardo et
al., 2009).
[0007] BMMSCs are thought to be derived from bone marrow stromal
compartment, initially appearing as adherent, single colony
clusters (colony-forming unit-fibroblasts [CFU-F]), and
subsequently proliferating on culture dishes (Friedenstein et al.,
1980). Adherent BMMSCs are able to proliferate and undergo
osteogenic differentiation, providing the first evidence of CFU-F
as precursors for osteoblastic lineage (Friedenstein et al., 1980).
For over 40 years, the adherent CFU-F assay has been used as an
effective approach to identify and select BMMSCs. To date, the
CFU-F assay has been considered to be one of the gold standards for
BMMSC isolation and expansion (Clarke et al., 1989; Friedenstein et
al., 1970).
SUMMARY OF THE INVENTION
[0008] Bone marrow mesenchymal stem cells (BMMSCs) are a
heterogeneous population of postnatal precursor cells with the
capacity of differentiating into multiple cell types and offering
alternative treatments for a variety of diseases. We have shown
that the standard adherent CFU-F assay collects the majority of
BMMSCs, but distinct subpopulations of BMMSCs are sustained in the
culture suspension.
[0009] One aspect of the present invention is directed to novel
subsets of BMMSCs with enhanced therapeutic potential.
[0010] Another aspect of the present invention is directed to
methods of collecting and isolating the novel BMMSCs of the present
invention.
[0011] Another aspect of the present invention is directed to
methods for inducing the conversion of regular BMMSCs into more
therapeutically potent BMMSCs.
[0012] Another aspect of the present invention includes isolated
human bone marrow mesenchymal stem cells having high telomerase
activity. High telomerase activity is most broadly defined as a
population of BMMSCs that have higher telomerase activity than
Regular BMMSCs, but preferably the isolated subset of human BMMSCs
has a telomerase activity of at least two times higher than regular
BMMSCs. In a preferred embodiment, at least about 6%, and more
preferably at least 20% of the cells of the isolated human bone
marrow mesenchymal stem cells of the invention are CD34.sup.+.
[0013] The isolated human bone marrow mesenchymal stem cells
according to the present invention include: (1) isolated BMMSCs
derived from non-adherent cells in the plastic culture (hereinafter
referred to as "tBMMSCs"); (2) isolated CD34.sup.+ BMMSCs,
preferably, CD34.sup.+/CD73.sup.+ BMMSCs; and (3) Human CD34.sup.-
BMMSCs that have been treated with a telomerase induction agent
(e.g. TAT-BMMSCs).
[0014] Another aspect of the present invention inventions is
directed to pharmaceutical compositions comprising the isolated
human bone marrow mesenchymal stem cells according to the present
invention. Additionally, the pharmaceutical composition may further
comprise a carrier.
[0015] Another aspect of the present invention is directed to the
separation and isolation of tBMMSCs from a heterogenous population
of postnatal precursor cells. tBMMSCs are capable of adhering to
extracellular cell matrix (ECM)-coated dishes and showing
mesenchymal stem cell characteristics with distinction to
hematopoietic cells as evidenced by co-expression of CD73 or CD105
with CD34, forming single colony cluster on ECM, and fail to
differentiate into hematopoietic cell lineage.
[0016] Another aspect of the present invention is a method of
converting regular CD34- BMMSCs to tBMMSCs by treating BMMSCs with
telomerase, including aspirin and it's related compounds with
similar chemical structure.
[0017] Another aspect of the present invention is directed to
methods of modulating the immune system. The methods of the present
invention involve administering to a patient in need thereof an
effective amount of the isolated human bone marrow mesenchymal stem
cells according to the present invention.
[0018] Another aspect of the present invention is directed to
treatment methods for systemic lupus erythematosus (SLE) via,
without being limited by theory, high levels of nitric oxide (NO)
production. The treatment methods include administering to a
patient in need thereof an effective amount of the isolated human
bone marrow mesenchymal stem cells according to the present
invention. This high NO production in the isolated human bone
marrow mesenchymal stem cells according to the present invention,
for example tBMMSCs, is positively regulated by telomerase activity
coupling with the Wnt/beta-catenin signaling. Furthermore, we show
that telomerase activator-induced tBMMSCs also exhibit
significantly improved immunomodulatory function, suggesting a
feasibility of inducing immuno-activated BMMSCs to improve
cell-based therapies for immune disorders.
[0019] These and other aspects of the present invention are
described with reference to the figures, description, examples, and
other disclosures as described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows that tBMMSCs are capable of attaching on
ECM-coated culture dish. (A) Hypothetic model indicates that bone
marrow all nuclear cells (ANCs) were seeded at 15.times.10.sup.6
into 10 cm culture dishes and incubated for 2 days in the regular
culture medium at 37.degree. C. with 5% CO2, and subsequently
non-attached cells from culture suspension were transplanted into
immunocompromised mice subcutaneously using hydroxyapatite
tricalcium phosphate (HA) as a carrier for 8 weeks. Newly formed
bone (B) by osteoblasts (open arrows) and associated connective
tissue (C) were detected in this non-attached cell transplants by
H&E staining. Original magnification; .times.200. Bar=100
.mu.m. (B) Hypothetic model of isolating tBMMSCs. Primary ANCs were
seeded at 15.times.10.sup.6 into 10 cm culture dishes, BMMSCs
usually attach on plastic dishes within 2 days, however, a small
portion of BMMSCs in primary ANCs failed to attach to the culture
dishes and remain in the cell suspension. The cell suspensions
containing putative non-attached BMMSCs were collected and
transferred to the cultured dishes coated with ECM produced by
BMMSCs with generating single colony clusters (CFU-F). These
ECM-attached BMMSCs (tBMMSCs) were sub-cultured on regular plastic
culture dishes for additional experiments. (C) The number of
plastic attached CFU-F generated from 1.5.times.10.sup.6 whole bone
marrow ANCs is more than 7 folds high than that derived from
BMMSC-ECM adherent tBMMSCs. (D) Flow cytometric analysis indicates
that tBMMSCs express high levels of mesenchymal stem cell markers
CD73 (81.8%), Sca-1 (87.74%), Oct4 (40.7%), and SSEA4 (24.56%)
compared to regular BMMSCs (CD73: 70.8%, Sca-1: 52.16%, Oct4:
14.08%). However, it appears that tBMMSCs and BMMSCs express
similar level of SSEA-4. (E) Proliferation rates of SSEA4.sup.+
tBMMSCs and regular BMMSCs were assessed by BrdU incorporation
assay for 24 hrs. The number of positive cells was indicated as a
percentage to the total number of each population. The percentage
of positive cells is significantly increased in tBMMSCs when
compared to control group. (F) tBMMSCs exhibit a significant
increase in population doublings when compared to regular
BMMSCs.
[0021] FIG. 2 shows that tBMMSCs express CD34 and possess high
telomerase activity. (A) Flow cytometric analysis showed that
regular BMMSCs fail to express CD34, but positive for CD45 antibody
staining (21.35%). However, tBMMSCs express both CD34 (23.37%) and
CD45 (31.22%). (B) Flow cytometric analysis also showed that
CD34.sup.+ tBMMSCs were positive anti CD73 (13.8%) and Oct4
(13.41%) antibody staining. None staining groups were used as
negative controls. (C, D) Western blot analysis indicates that
tBMMSCs express CD34 and mesenchymal surface molecules CD73 and
CD105. In contrast, regular BMMSCs only express CD73 and CD105 (C).
tBMMSCs express CD34 at passage 1-5 (D). .beta.-actin was used as a
sample loading control. BMC: whole bone marrow ANC. (E, F)
Immunocytostaining confirms that tBMMSCs are double positive for
CD34/CD73 (triangle, E) and CD34/CD105 (triangles, F). Regular
BMMSCs are negative for CD34 antibody staining and only positive
for anti CD73 (E) and CD 105 (F) antibody staining. Bar=100 .mu.m.
(G) tBMMSCs have significant high level of telomerase activity than
BMMSCs. HEK293T cells (293T) were used as positive control and heat
inactive HEK293T cells (H.I.) were used as negative control
measured by a Telo TAGGG Telomerase PCR ELISA kit. (H) Western blot
verifies that tBMMSCs express telomerase reverse transcriptase
(TERT) and BMMSCs are negative for anti TERT antibody staining. (I)
There are 3.77% cells are double positive for anti CD34 and CD73
antibody staining in whole bone marrow ANCs, these
CD34.sup.+/CD73.sup.+ cells can be sorted out from bone marrow
using flow cytometric sorter. (J) CD34.sup.+/CD73.sup.+ cells form
CFU-F on BMMSC-ECM cultures at frequency similar to tBMMSCs. (K)
CD34.sup.+/CD73.sup.+ BMMSCs show higher telomerase activity than
regular BMMSCs. HEK293T cells were used as positive control (293T)
and heat inactive HEK293T cells were used as negative control
(H.I.) measured by a Telo TAGGG Telomerase PCR ELISA kit. (L)
CD34.sup.+/CD73.sup.+ BMMSCs also show a significant NO production
when compared to regular BMMSCs. The results were representative of
five independent experiments. Scale bars=50 .mu.m. ***P<0.001.
The graph bar represents mean.+-.SD.
[0022] FIG. 3 shows that aspirin treatment elevates CD34 expression
in BMMSCs. (A) Flow cytometric analysis indicated that
aspirin-treated BMMSC (TAT-BMMSC) exhibits positive expression of
CD34 when compared to the negative CD34 expression in regular BMMSC
(BMMSC). The expression levels of CD45 in TAT-BMMSC were lower than
that in BMMSCs and tBMMSC. (B) TAT-BMMSCs express significant high
levels of Scal1, Oct4 and CD34 when compared to BMMSCs, but at much
lower level than tBMMSC. However, TAT-BMMSC expresses much lower
level of CD45 compared to tBMMSC and regular BMMSCs. (C) Western
blot analysis showed that tBMMSCs and aspirin-treated BMMSCs
express CD34, but BMMSCs fail to express to CD34. The results were
representative of five independent experiments. **P<0.01;
***P<0.005. The graph bar represents mean.+-.SD.
[0023] FIG. 4 shows Hematopoietic differentiation of tBMMSCs. (A)
BMMSCs, tBMMSCs and aspirin treated BMMSCs were cultured onto 35 mm
low attach culture dish (2.times.10.sup.4/dish) under hematopoietic
differentiation medium with or without erythropoietin (EPO; 3 U/mL)
for 7 days. Whole bone marrow cells and linage negative bone marrow
cells (Linage- cells) were used as positive controls. The results
were representative of five independent experiments. (B) Mice
received either regular BMMSCs (BMMSC, n=5) or PBS without cells
(Control, n=8) failed to survive over 14 days. The whole bone
marrow cell infusion group (Whole BM cells, n=3) is a positive
control group with survival over 110 days after irradiation.
tBMMSCs can extend life span of lethal dose irradiated mice
(tBMMSC, n=10). Kaplan-meier survival curves.
[0024] FIG. 5 shows that tBMMSCs show up-regulated immunomodulatory
properties. (A) NO levels in the supernatant of tBMMSC and regular
BMMSC culture (each 0.2.times.10.sup.6/well on 24-well plate) were
significantly higher in INF-.gamma. (25 ng/ml)/IL-1.beta. (5
ng/ml)-treated tBMMSC group than in regular BMMSCs. (B-J) Anti-CD3
and anti-CD28 (each 1 .mu.g/ml) antibodies-activated spleen (SP)
cells (1.times.10.sup.6/chamber) in the upper chambers were
co-cultured with or without tBMMSCs or regular BMMSCs
(1.times.10.sup.5/chamber) in the bottom chamber using a transwell
system. Three days after the co-culture, cell viability of the
activated SP cells was assayed using a cell counting kit-8 (B-D).
tBMMSC-coculture showed a significant reduction on cell viability
of activated SP cells compared to the cells cultured without BMMSCs
(BMMSC-) and with regular BMMSCs (B). The effects of reducing
spleen cell viability by tBMMSCs, but not regular BMMSCs, were
abolished in general NOS inhibitor L-NMMA (1 mM)-treated (C) and
iNOS specific inhibitor 1400W (0.2 mM)-treated (D) groups. Three
days after the co-culture, the activated SP cells in the upper
chamber were stained to detect apoptotic cells as described in
Materials and Methods (E-J). Both tBMMSCs and regular BMMSCs were
capable of inducing significant amount of Annexin V (+) early
apoptotic cells (E) and Annexin V (+) 7AAD (+) late apoptotic and
dead cells (H) compared to negative control groups (BMMSC-). It
appeared that tBMMSCs have a significant effect than regular BMMSC
in induction of early (E) and late (H) apoptotic cells. Both L-NMMA
and 1400W were able to abolish tBMMSC and BMMSC induced Annexin V
(+) (F, I) and Annexin V (+) 7AAD (+) cells (G, J). It appeared
that 1400W treatment has more significant inhibition on
tBMMSC-induced early apoptosis of activated SP cells (G). (K-M)
Activated CD4.sup.+CD25- T-cells (1.times.10.sup.6/well) and
tBMMSCs or regular BMMSCs (each 0.1.times.10.sup.6/well) were
co-cultured in the presence of TGF.beta.1 (2 ng/ml) and IL-2 (2
ng/ml) with or without NOS inhibitor for 3 days. The floating cells
were stained for CD4.sup.+CD8.sup.-CD25.sup.+FoxP3.sup.+ regulatory
T cells (Treg). tBMMSCs showed a significant effect in
up-regulating Foxp3.sup.+ regulatory T cells (Treg) (K). However,
L-NMMA and 1400W treatments resulted in a abolishing of
tBMMSC-induced up-regulation of Treg (L, M). The results were
representative of, at least, three independent experiments.
*P<0.05; **P<0.01; ***P<0.001. The graph bar represents
mean.+-.SD.
[0025] FIG. 6 shows that tBMMSCs showed superior therapeutic effect
on SLE-like MRL/lpr mice. (A) A hypothetic model showing that
tBMMSCs or regular BMMSCs from C3H/HeJ mice were infused into the
tail vein of 10-week-old MRL/lpr mice (0.1.times.10.sup.6 cells/10
g of mouse body weight). (B) tBMMSC and BMMSC treatment recover
SLE-induced basal membrane disorder and mesangium cell over-growth
in glomerular (G) (H&E staining). (C) Urine protein levels were
assessed at 2 weeks post BMMSC infusion. Both tBMMSCs and BMMSCs
were capable of reducing urine protein levels compared to MRL/lpr
group. However, tBMMSCs offered a more significant reduction of
urine protein levels compared to regular BMMSCs. (D, E) ELISA
quantified that levels of anti dsDNA IgG and IgM antibodies were
significantly increased in the peripheral blood of MRL/lpr mice
when compared to the undetectable level (N.D.) in controls (C3H).
tBMMSC and BMMSC treatments were able to reduce levels of anti
dsDNA IgG and IgM, but tBMMSCs show superior treatment effect than
BMMSC in reducing dsDNA IgG level (D). (F) tBMMSC and BMMSC
treatments were able to significantly reduce anti nuclear antibody
(ANA) in MRL/lpr mice, which was significantly increased compared
to the control (n=6). But tBMMSC showed better effect in reducing
ANA levels compared to BMMSC treatment. (G) tBMMSC and BMMSC
treatments were able to increase albumin level compared to the
level in MRL/lpr mice), which were significantly decreased compared
to the control (n=6). tBMMSC treatments show more effective in
elevating albumin level in serum when compared to BMMSC-treated
group. (H) Flow cytometric analysis showed that the number of
CD25.sup.+Foxp3.sup.+ Tregs in CD4.sup.+ T lymphocytes of MRL/lpr
peripheral blood was reduced as compared to the control). BMMSC and
tBMMSC treatments elevated the number of Tregs. It appeared that
tBMMSCs induced a more significant elevation of Treg levels than
BMMSCs. (I) Flow cytometry revealed that MRL/lpr mice had
significantly increased level of CD4.sup.+IL17.sup.+IFNg.sup.- T
lymphocytes (Th17 cells) in spleen compared to control group. The
Th17 cells were markedly decreased in BMMSC and tBMMSC treated
groups. tBMMSC treatment induced a more significant reduction of
Th17 cells than BMMSCs. The results were representative of six
independent experiments. *P<0.05; **P<0.01; ***P<0.001.
The graph bar represents mean.+-.SD.
[0026] FIG. 7 shows that Nitric oxide production by BMMSCs is
governed by telomerase and Wnt/beta-catenin signaling. (A, B)
tBMMSCs were cultured with telomerase inhibitor III (1 .mu.M) for
one week. Telomerase activity (A) and NO production (B) were
significantly reduced in telomerase inhibitor treatment group. 293T
cells and heat-inactivated samples were used as positive and
negative control, respectively. (C-E) Regular BMMSCs were cultured
with aspirin or Telomerase inhibitor III (Telo I, 1 .mu.M) for one
week. Aspirin can elevate telomerase activity (C), telomerase
reverse transcriptase (TERT) expression (D) and NO production (E)
in BMMSCs. In contrast, Telomerase inhibitor III reduces telomerase
activity (C) and NO production (E). (F) In aspirin treatment group,
a Wnt inhibitor, DKK1 (DKK, 10 ng/ml), was added to the BMMSC
cultures for three days (DKK-TAT), which led to a significantly
reduction of NO levels compared to aspirin (TAT) group. (G) Western
blot analysis showed that DKK1 can reduce active .beta.-catenin
levels. Aspirin (TAT) treatment can partially block DKK1-induced
down-regulation of activated beta-catenin expression. (H) DKK1
treatment was able to abolish aspirin (TAT)-induced telomerase
activity in BMMSCs (DKK-TAT). (I) When BMMSCs were cultured with
Chiron, activator of beta catenin signaling, at 1 and 10 .mu.M for
1 week. NO production in BMMSCs was significantly increased in a
dose dependent manner as measured by Total NO/Nitrite/Nitrate kit.
(J) Western blot analysis confirmed that Chiron treatment induces
up-regulated expression of active beta-catenin in BMMSCs. (K)
Chiron treatment is able to induce a high telomerase activity,
which is blocked by telomerase inhibitor III (Telo i-Chiron) when
used prior to the Chiron induction. 293T cell and heat inactivated
sample were used as positive and negative control respectively. (L)
Chiron induced high NO production can be blocked by telomerase
inhibitor III treatment. The results were representative of five
independent experiments. *P<0.05; **P<0.01; ***P<0.001.
The graph bar represents mean.+-.SD.
[0027] FIG. 8 shows that aspirin-treated BMMSCs showed improved
therapeutic effect on SLE-like MRL/lpr mice. (A) Urine protein
levels were assessed at 2 weeks post BMMSC infusion. Both BMMSCs
and aspirin (TAT) treated BMMSCs (TAT-BMMSC) were capable of
reducing urine protein levels compared to MRL/lpr group. However,
TAT-BMMSC offered a more significant reduction of urine protein
levels compared to regular BMMSCs when 0.1.times.10.sup.6 or
0.01.times.10.sup.6 cells were systemically infused. It appeared
that 0.01.times.10.sup.6 BMMSCs failed to reduce urine protein
level compared to MRL/lpr mice. (B, C) ELISA quantified that levels
of anti dsDNA IgG and IgM antibodies were significantly increased
in the peripheral blood of MRL/lpr mice when compared to the
controls (C3H). TAT-BMMSC and BMMSC treatments were able to reduce
levels of anti dsDNA IgG and IgM, but TAT-BMMSC show superior
treatment effect than BMMSC in reducing dsDNA IgG and IgM levels.
TAT-BMMSC of 0.01.times.10.sup.6 cell infusion group was able to
significantly reduce the levels of anti dsDNA IgG and IgM. (D)
TAT-BMMSC and BMMSC treatments were able to significantly reduce
anti nuclear antibody (ANA) in MRL/lpr mice, which was
significantly increased compared to the control (C3H). But
TAT-BMMSC of 0.01.times.10.sup.6 cell infusion group showed better
effect in reducing ANA levels compared to BMMSC treatment. (E)
ELISA analysis showed that TAT-BMMSC and BMMSC treatments were able
to reduce serum IL17 levels compared to the high level in MRL/lpr
mice. However, TAT-BMMSC of 0.01.times.10.sup.6 cell infusion group
showed more effective in reducing IL17 level in serum when compared
to BMMSC-treated group. (F) Flow cytometry revealed that MRL/lpr
mice had significantly increased level of
CD4.sup.+IL17.sup.+IFNg.sup.- T lymphocytes (Th17 cells) in spleen
compared to control group (C3H). The Th17 cells were markedly
decreased in TAT-BMMSC and BMMSC treated groups. TAT-BMMSC
treatment induced a more significant reduction of Th17 cells than
BMMSC groups. (G) Flow cytometric analysis showed that the number
of CD25.sup.+Foxp3.sup.+ Tregs in CD4.sup.+ T lymphocytes of
MRL/lpr peripheral blood was reduced as compared to the control
(C3H). TAT-BMMSC and BMMSC treatments elevated the number of Tregs.
It appeared that TAT-BMMSCs induced a more significant elevation of
Treg levels than BMMSCs when 0.01.times.10.sup.6 cells were
systemically infused. The results were representative of six
independent experiments. *P<0.05; **P<0.01; ***P<0.001.
The graph bar represents mean.+-.SD.
[0028] FIG. 9 shows that human bone marrow contains tBMMSCs. (A)
human tBMMSCs (htBMMSC) showed significantly high level telomerase
activity than BMMSCs (hBMMSC) as measured by Telo TAGGG Telomerase
PCR ELISA kit. 293T cells and heat inactive (H.I.) were used as
positive and negative controls, respectively. (B) htBMMSCs produce
high level of NO than that of hBMMSCs as assessed by Total
NO/Nitrite/Nitrate kit. (C) Kynurenine production was significantly
increased in htBMMSC compare to hBMMSC (p<0.005). (D) When
hBMMSC or htBMMSC were co-cultured with active T cell, the
kynurenine level in co-culture system was dramatically increased
with more significantly increase in htBMMSC group compare to hBMMSC
group. (E) Annexin V and 7AAD double positive apoptotic cell
numbers in active T cells were increased when co-cultured with
hBMMSC or htBMMSC. However, apoptotic cell rate was significantly
increased in htBMMSC group compared to hBMMSC group. The results
were representative of three independent experiments. *P<0.05;
**P<0.01; ***p<0.005. The graph bar represents
mean.+-.SD.
[0029] FIG. 10 shows the number of CFU-F in BMMSC cultures. Primary
ANCs were seeded at 1.times.106 into 6 cm normal plastic culture
dishes (Plastic) or the culture dishes coated with ECM produced by
BMMSCs (ECM) for 14 days. The CFU-F number was significantly
increased in BMMSCs cultured in ECM coated dishes. The results were
representative of five independent experiments. ***P<0.001. The
graph bar represents mean.+-.SD.
[0030] FIG. 11 shows the multipotent differentiation of tBMMSCs.
(a) Alizarin Red S and alkaline phosphatase (ALP) staining showed
that tBMMSCs were similar to regular BMMSCs in osteogenic
differentiation in vitro. (b) tBMMSCs or regular BMMSCs
(4.times.106 cells/transplant) were transplanted into
immunocompromised mice using HA/TCP (HA) as a carrier for 8 weeks.
Bone formation was detected in tBMMSC and BMMSC transplants,
evidenced by H&E staining. HA; hydroxyapatite tricalcium
phosphate, B; bone, M; bone marrow, C; connective tissue; Original
magnification; X 200. Bar=50 .mu.m. (c) tBMMSCs are capable of
forming Oil Red O positive cells and expression of PPAR.gamma.2 and
LPL mRNA as seen in regular BMMSCs by Oil Red O staining and RT-PCR
analysis, respectively. Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was used as an internal control. The results were
representative of five independent experiments. Scale bars=100
.mu.m. 1: negative control, 2: BMMSC, 3: tBMMSC. (d) Chondrogenic
differentiation was assessed by Alcian blue staining for acidic
sulfated mucosubstances, Pollak's Trichrome staining for collagen,
and immunohystochemical staining for collagen type II. tBMMSCs were
able to differentiate into chondrocytes as observed in regular
BMMSCs. Bar=50 .mu.m. The results were representative of three
independent experiments. The graph bar represents mean.+-.SD.
[0031] FIG. 12 shows the NO level in tBMMSCs. BMMSCs and tBMMSCs
(2.times.105/well) were cultured for 3 days and treated with L-NMMA
(1 mM) or 1400W (0.2 mM) for 3 days. (a) The collected culture
supernatant was used to measure NO level. The results were
representative of five independent experiments. (b) Western blot
analysis showed that iNOS expression was inhibited by LNMMA and
1400W. *P<0.05; ***P<0.001. The graph bar represents
mean.+-.SD.
[0032] FIG. 13 shows the osteoclast activity in tBMMSC-treated
MRL/lpr mice. (a) TRAP staining indicated the increased number of
TRAP positive cells in epiphysis of the distal femurs of MRL/lpr
mice as compared to the control (C3H). tBMMSC and BMMSC infusion
resulted in a significant reduced number of TRAP positive cells. It
appears that tBMMSC group shows more significant reduction of
number of TRAP positive cells than BMMSC group. (b, c) ELISA
revealed that MRL/lpr mice have increased levels of soluble RANKL
(sRANKL) (b) and C-terminal telopeptides of type I collagen (CTX)
(c) in serum as compared to the controls. tBMMSC and BMMSC infusion
can significantly reduce levels of sRANKL (B) and CTX (C), but
tBMMSC group showed a more effective in reduce levels of sRANKL (b)
and CTX (c). The results were representative of five independent
experiments. *P<0.05; **P<0.01; ***P<0.001. The graph bar
represents mean.+-.SD.
[0033] FIG. 14 shows that the immunomodulatory properties of BMMSCs
are regulated by telomerase. SP cells (1.times.106/chamber),
activated with anti CD3 (5 .mu.g/mL) and CD28 (2 .mu.g/mL)
antibodies, were co-cultured with or without BMMSCs
(0.2.times.106/chamber) using a Trans-well system (Corning) for 3
days. BMMSCs were treated with TAT analog (TAT, 3 .mu.M) for 3 days
prior to the co-culture. Cell viability of SP cells was measured
using a cell counting kit-8 (Dojindo Molecular Technoloies,
Gaithersburg, Md.). Apoptotic cells were stained with Annexin V-PE
apoptosis detection kit I (BD Bioscience) and analyzed with
FACSCalibur (BD Bioscience). TAT analog-treated BMMSCs (TAT-BMMSC)
could significantly reduce activated SP cell viability (a) and
enhance early (b) and late (c) apoptosis of activated SP cells
compared to regular BMMSCs. The results were representative of five
independent experiments. *P<0.05; **P<0.01; ***P<0.001.
The graph bar represents mean.+-.SD.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
[0034] BMMSCs: bone marrow mesenchymal stem cells;
[0035] CFU-F: colony-forming units fibroblastic;
[0036] ECM: extracellular cell matrix;
[0037] Oct4: Octamer-4;
[0038] SSEA4: stage specific embryonic antigen-4;
[0039] SLE: systemic lupus erythematosus;
[0040] HA/TCP: hydroxyapatite tricalcium phosphate;
[0041] Tregs: CD4.sup.+CD25.sup.+Foxp3.sup.+ regulatory T
cells;
[0042] ANCs: all nuclear cells
Definitions
[0043] Unless otherwise indicated herein, all terms used herein
have the meanings that the terms would have to those skilled in the
art of the present invention. Practitioners are particularly
directed to current textbooks for definitions and terms of the art.
It is to be understood, however, that this invention is not limited
to the particular methodology, protocols, and reagents described,
as these may vary.
[0044] Individual cells and cell populations will be referred to
herein by use of a `+` or a `-` symbol to indicate whether a
certain cell or cell population expresses or lacks a specific
marker, e.g. a CD molecule. When used in connection with a single
cell, the use of a `+` or a `-` symbol indicates whether that cell
expresses or lacks the specific marker. For example, a "CD34+",
CD31-" cell is one that expresses CD34, but not CD31. When used in
connection with cell populations, the use of a `+` or a `-` symbol
to indicate whether a certain cell population, or a portion
thereof, expresses or lacks the specific marker.
[0045] As used herein, so-called "regular BMMSCs" are BMMSCs
appearing as adherent, single colony clusters (colony-forming
unit-fibroblasts [CFU-F]) on regular plastic culture, and
subsequently proliferating on culture dishes (Friedenstein et al.,
1980).
[0046] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented.
[0047] An "therapeutically effective amount" of tBMMCs is an amount
sufficient to carry out a specifically stated purpose. An
"effective amount" may be determined empirically and in a routine
manner in relation to the stated purpose.
[0048] A "Carrier" or "Carriers" as used herein include
pharmaceutically acceptable carriers, excipients, or stabilizers
which are nontoxic to the cell or mammal being exposed thereto at
the dosages and concentrations employed. The physiologically
acceptable carrier may be a sterile aqueous pH buffered solution.
Examples of physiologically acceptable carriers include buffers
such as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid; low molecular weight (less than about 10
residues) polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants.
[0049] One aspect of the present invention is directed to unique
subsets of isolated human bone marrow mesenchymal stem cells having
high telomerase activity. High telomerase activity is most broadly
defined as a population of BMMSCs that have higher telomerase
activity than Regular BMMSCs, but preferably the isolated subset of
human BMMSCs has a telomerase activity of at least two times higher
than regular BMMSCs. Isolated human BMMSCs having high telomerase
activity according to the present invention are generally
characterized by having an increased expression of CD34 relative to
regular BMMSCs. Preferably, at least about 6%, and more preferably
at least 20% of the cells of the isolated human bone marrow
mesenchymal stem cells of the invention are CD34.sup.+. In its
broadest sense, the term "isolated" when used in connection with a
population of cells of interest, means that the population of cells
is at least partially isolated from other cell types or other
cellular material with which it naturally occurs in the tissue of
origin (e.g., bone marrow). In another embodiment, the isolated
stem cells also are substantially free of soluble, naturally
occurring molecules.
[0050] The isolated human bone marrow mesenchymal stem cells
according to the present invention include: (1) isolated human
BMMSCs derived from non-adherent cells in the plastic culture
(hereinafter referred to as "tBMMSCs"); (2) isolated CD34.sup.+
BMMSCs, preferably, CD34.sup.+/CD73.sup.+ BMMSCs; and (3) Human
CD34- BMMSCs that have been treated with a telomerase induction
agent (e.g. TAT-BMMSCs). Unless otherwise specifically stated, all
BMMSCs in the present invention are human BMMSCs.
[0051] The human bone marrow useable in connection with the present
invention may generally be obtained from within human bone.
Preferably, the bone marrow is post natal bone marrow. All
nucleated cells of the bone marrow are typically used. Most
preferably, bone marrow derived all nuclear cells (ANCs) from
femurs and tibias are used as described herein.
[0052] Specific cell types described and identified herein may be
isolated from collected cells employing techniques known by those
skilled in the art, such as for example, but not limited to density
gradient centrifugation, magnet cell separation, flow cytometry,
affinity cell separation or differential adhesion techniques. In a
preferred embodiment, the stem cells of the present invention can
be purified by, for example, flow cytometry (e.g., FACS analysis),
as discussed below. The high telomerase BMMSCs described herein
will undergo ex vivo expansion according to known methods for
BMMSCs to enrich cell numbers for tissue regeneration or systemic
therapies.
[0053] Isolated tBMMSCs
[0054] tBMMSCs are generally isolated from a heterogenous
population of postnatal precursor cells. Isolated tBMMSCs are
generally characterized as human BMMSCs that fail to form single
colony clusters (CFU-F) in plastic cultures but are capable of
adhering on mesenchymal stem cell-produced ECM and exhibit
increased expression of telomerase relative to regular human
BMMSCs. tBMMSCs show mesenchymal stem cell characteristics with
distinction to hematopoietic cells as evidenced by co-expression of
CD73 or CD105 with CD34. tBMMSCs fail to differentiate into
hematopoietic cell lineage.
[0055] Another aspect of the present invention is directed to a
method of isolating tBMMSCs comprising: culturing a sample of bone
marrow derived all nuclear cells on a plastic substrate; removing
cells that do not adhere to the plastic substrate; culturing the
removed cells on a BMMSC-ECM coated medium; and collecting colonies
forming attached cells on the BMMSC-ECM medium.
[0056] More specifically, tBMMSCs may be produced and isolated as
follows: Primary ANCs are seeded on plastic substrate, for example
plastic culture dishes. tBMMSCs in primary ANCs fail to attach to
the culture dishes and remain in the cell suspension. The cell
suspensions containing putative non-attached tBMMSCs are collected
and transferred to cultured dishes coated with Extracellular matrix
(ECM) produced by BMMSCs, resulting in the generation of single
colony clusters (CFU-F). These ECM-attached BMMSCs (tBMMSCs) are
sub-cultured according to known methods on regular plastic culture.
Typical flow cytometric analysis indicates that tBMMSCs express
high levels of mesenchymal stem cell markers CD73 (e.g. about 80%),
Sca-1 (e.g. about 90%), and Oct4 (e.g. about 40%) compared to
regular BMMSCs (CD73: e.g. about 70%, Sca-1: about 50%, Oct4: about
14%). However, it appears that tBMMSCs and BMMSCs express similar
level of SSEA-4.
[0057] tBMMSCs express CD34 and possess high telomerase activity
relative to regular BMMSCs. As described herein, regular BMMSCs
fail to express CD34, but are positive for CD45 (about 20%).
However, tBMMSCs express both CD34 (about 25%) and CD45 (about
30%). Western blot analysis indicates that tBMMSCs express CD34 and
mesenchymal surface molecules CD73 and CD105. In contrast, regular
BMMSCs only express CD73 and CD105. tBMMSCs also have significantly
higher levels of telomerase activity than regular BMMSCs.
[0058] To ensure purity of tBMMSCs, it is preferred to isolate and
substantially purify tBMMSCs that express a marker known to be
expressed in regular BMMSCs selected from the group consisting of
STRO-1, CD29, CD73, CD90, CD105, CD146, Octamer-4 (Oct4), and
stage-specific embryonic antigen-4 (SSEA4). In a preferred
embodiment, SSEA4.sup.+ tBMMSCs may be isolated and purified by
techniques generally known to those of ordinary skill, such as
immune FACS. A sample of tBMMSCs stem cells is "substantially pure"
when it is at least 80%, or at least 90%, or at least 95%, and, in
certain cases, at least 99% free of cells other than cells of
interest. Thus, for example, a sample of SSEA4.sup.+ tBMMSCs stem
cells is "substantially pure" when it is at least 80%, or at least
90%, or at least 95%, and, in certain cases, at least 99% free of
cells other than SSEA4.sup.+ tBMMSCs. Purity can be measured by any
appropriate method, for example, by fluorescence-activated cell
sorting (FACS), or other assays which distinguish cell types.
[0059] Isolated CD34.sup.+ Human BMMSCs
[0060] CD34.sup.+ BMMSCs are distinct from regular BMMSCs in terms
of having elevated telomerase activity and high levels of the
earlier mesenchymal stem cell marker, Oct4, along with increased
immunomodulatory function. The mechanism that may contribute to the
up-regulated immunomodulatory function is associated with high NO
production in tBMMSCs (Ren et al., 2008) and NO-driven high Treg
level (Niedbala et al., 2007), which appears to be governed by
telomerase activity coupled with Wnt/beta-catenin signaling.
Without being limited to theory, this is believed to be the reason
that tBMMSCs have a superior therapeutic effect in treating SLE
mice.
[0061] Isolated CD34.sup.+ BMMSCs fail to form CFU-F in plastic
cultures but are capable of adhering on mesenchymal stem
cell-produced ECM and differentiating into osteoblasts, adipocytes,
and chondrocytes from both C3H/HeJ and C57BL/6J mice. CD34.sup.+
BMMSCs coexpress mesenchymal stem cell markers CD73 and CD105.
Furthermore, CD34.sup.+ BMMSCs are distinct from HSC due to the
fact that they are not able to differentiate into hematopoietic
cell lineage in vitro and fail to rescue lethal dose irradiated
mice.
[0062] Preferably, the isolated human BMSSCs are double positive
for CD34 and at least one other marker known to be expressed in
regular BMMSCs selected from the group consisting of STRO-1, CD29,
CD73, CD90, CD105, CD146, Octamer-4 (Oct4), and stage-specific
embryonic antigen-4 (SSEA4). Preferably, the BMMSCs are both
CD34.sup.+ and CD73.sup.+. Preferably, Isolated CD34.sup.+ BMMSCs
are substantially pure. A sample of CD34.sup.+ BMMSCs is
"substantially pure" when it is at least 80%, or at least 90%, or
at least 95%, and, in certain cases, at least 99% free of cells
other than cells of interest. Thus, for example, a sample
(population) of CD34.sup.+ BMMSCs is "substantially pure" when it
is at least 80%, or at least 90%, or at least 95%, and, in certain
cases, at least 99% free of cells other than CD34.sup.+ BMMSCs.
Purity can be measured by any appropriate method, for example, by
fluorescence-activated cell sorting (FACS), or other assays which
distinguish cell types.
[0063] As described herein, about 4% of human BMMSC's cells are
double positive for CD34 and CD73 in whole bone marrow ANCs. These
CD34.sup.+/CD73.sup.+ cells can be sorted out and isolated from
bone marrow using conventional techniques, such as a flow
cytometric sorter. The use of flow cytometry to isolate
CD34.sup.+/CD73.sup.+ BMMSCs from whole bone marrow offers a
practical approach to isolate and collect tBMMSC for clinical
therapeutic use. CD34.sup.+/CD73.sup.+ cells can be sorted out and
isolated from tBMMSCs and from regular BMMSCs that have been
treated with a telomerase induction agent as described herein.
Preferably, the CD34.sup.+/CD73.sup.+ BMMSCs are "substantially
pure." A group of CD34.sup.+/CD73.sup.+ BMMSCs are "substantially
pure" when it is at least 80%, or at least 90%, or at least 95%,
and, in certain cases, at least 99% free of cells other than
CD34.sup.+/CD73.sup.+ BMMSCs.
[0064] CD34.sup.+/CD73.sup.+ cells form CFU-F on BMMSC-ECM cultures
at frequency similar to tBMMSCs. CD34.sup.+/CD73.sup.+ BMMSCs also
show higher telomerase activity than regular BMMSCs.
CD34.sup.+/CD73.sup.+ BMMSCs also show a significant increase in NO
production compared to regular BMMSCs.
[0065] CD34.sup.- BMMSCs Treated with a Telomerase Induction
Agent.
[0066] Another aspect of the present invention directed to a method
of increasing telomerase activity in CD34.sup.- human bone marrow
mesenchymal stem cells comprising: contacting human bone marrow
messenchymal stem cells with an effective amount of a telomerase
inducing agent. The CD34- BMMSCs may, for example, be regular
BMMSCs. As defined herein, a group of BMMSCs is CD34.sup.- if less
than about 1% of the group is CD34.sup.+.
[0067] The telomerase activity of the CD34.sup.- BMMSCs can be
increased by adding an effective amount of telomerase induction
agent to the culture medium. One preferred telomerase induction
agent is aspirin, but structural and functional analogues of
aspirin may be substituted. The culture conditions may be
appropriately determined by those of ordinary skill by measurement
of the telomerase activity levels as described herein. When aspirin
is used, it is preferably added into culture medium at about 2
.mu.g/ml to about 50 .mu.g/ml for about 1 week. Culture under these
conditions results in significantly increased level of telomerase
activity in BMMSCs was achieved.
[0068] In one specific embodiment, regular human BMMSC are treated
with a telomerase induction agent to become BMMSCs having high
telomerase activity with improved immunomodulatory function.
Specifically, when aspirin is added into culture medium at 2.5
.mu.g/ml or 50 .mu.g/ml for 1 week, significantly increased level
of telomerase activity in BMMSCs was achieved. The resulting BMMSCs
are referred to herein as TAT-BMMSCs. TAT-BMMSCs exhibits positive
expression of CD34 when compared to the negative CD34 expression in
regular BMMSCs. The expression levels of CD45 in TAT-BMMSC were
lower than that in BMMSCs and tBMMSC. TAT-BMMSCs express
significant high levels of Scal1, Oct4 and CD34 when compared to
BMMSCs, but at much lower level than tBMMSC. However, TAT-BMMSC
expresses much lower level of CD45 compared to tBMMSC and regular
BMMSCs. Western blot analysis showed that tBMMSCs and
aspirin-treated BMMSCs express CD34, but BMMSCs fail to express to
CD34.
[0069] Therapeutic Applications of High Telomerase Activity
BMMSCs
[0070] Another aspect of the present invention is directed to using
the BMMSCs of the present invention in the treatment of one or more
disorders.
[0071] Another aspect of the present inventions is directed to a
method of immunomodulation comprising administering to a patient in
need thereof a therapeutically effective amount of isolated human
bone marrow mesenchymal stem cells of the present invention.
[0072] Another aspect of the present invention is directed to a
method of increasing the NO concentration in vivo, comprising
administering to a patient in need thereof a therapeutically
effective amount of the isolated human bone marrow mesenchymal stem
cells of the present invention. NO is a gaseous biological mediator
with important roles in affecting macrophage and T cell function
(Sato et al., 2007; Bogdan et al., 2001). iNOS is induced by
IFN.gamma., TNF.alpha., IL-1.alpha., or IL-1.beta. in BMMSCs, and
iNOS.sup.-/- mice show a reduced ability to suppress T cell
functions (Ren et al., 2008). It has been reported that active
endothelial NOS along with estrogen receptor cooperatively
regulates human telomerase revere transcriptase (hTERT) expression
in the endothelium (Grasselli et al., 2008). We describe herein the
functional role of high telomerase activity in improving
immunomodulatory activity of BMMSCs via elevation of approximately
10 .mu.M NO production and approximately 5% up-regulation of Treg.
Telomerase-enhanced NO production is also associated with
Wnt/.beta.-catenin signaling, in which Wnt inhibitor DKK1 can block
telomerase activator-induced telomerase activity and the associated
NO production in BMMSCs. Furthermore, Wnt activator Chiron is able
to promote telomerase activity and NO production in BMMSCs.
Pre-treatment with telomerase inhibitor can partially abolish
Wnt-activator-induced telomerase activity. These data suggest that
telomerase coupled with Wnt/beta-catenin signaling to promote NO
production. Therefore, in addition to the functional role in
participating in the Wnt/beta-catenin signaling pathway (Park et
al., 2009), telomerase also collaborates with Wnt/beta-catenin
signaling to modulate NO production. Both telomerase and
Wnt/beta-catenin activators can induce a high NO production in
regular BMMSCs leading to an improved reduction of activated SP
cell viability. But only telomerase activator treatment is capable
of enhancing apoptosis of activated SP cells. It is possible that
other immunomodulatory factors may also contribute to elevated
immunomodulation of tBMMSC.
[0073] Another aspect of the present invention is directed to the
treatment of systemic lupus erythematosus comprising administering
to a patient in need thereof a therapeutically effective amount of
the isolated human bone marrow mesenchymal stem cells of the
present invention.
[0074] As used herein, the term an "effective amount" of the BMMSCs
of the present invention, when used in connection with a method, is
an amount of the BMMSCs sufficient to carry out a specifically
stated purpose. In general, an "effective amount" in reference to
treatment of a disease or disorder may be determined empirically by
reference to the data and standards disclosed herein and in a
routine manner in relation to the stated purpose. An effective
amount is preferably given in a single dose to the patient;
however, the effective amount may be delivered to the patient as a
number of doses over a period of time. As describe herein, the
dosage of 0.1.times.10.sup.6 cells/10 g body weight are sufficient
to treat SLE mice in case of regular BMMSC. By using high
telomerase activity BMMSCs, the dosage can be reduced to
0.01.times.10.sup.6 cells/10 g body weight with therapeutic effect.
Those of ordinary skill can apply this to treatment of humans using
known models relating mouse to human dosages and using known
techniques for optimization of dosages.
[0075] The present invention further includes a pharmaceutical
composition comprising an effective amount of pharmaceutical
composition comprising isolated bone marrow mesenchymal stem cells
having high telomerase activity in a carrier medium. The
pharmaceutical compositions of the present invention are used for
administration of the isolated bone marrow mesenchymal stem cells
having high telomerase activity for treatment in accordance with
any of the methods described herein.
[0076] In the methods described herein, the BMMSCs of the present
invention should be compatible with the patient and be administered
in a therapeutically effective amount of the BMMSCs. The
therapeutically effective amount can range from the maximum number
of cells that is safely received by the patient to the minimum
number of cells necessary for to achieve the intended effect. One
of ordinary skill in the art can determine and optimize effective
amounts according to known techniques to effectuate the intended
purpose of the treatment.
[0077] The therapeutically effective amount of the BMMSCs can be
suspended in a pharmaceutically acceptable carrier. Such a carrier
may include but is not limited to a suitable culture medium plus 1%
serum albumin, saline, buffered saline, dextrose, water, and
combinations thereof. The formulation should suit the mode of
administration.
[0078] In a preferred embodiment, the BMMSC preparation or
composition is formulated for systemic administration to human
beings in accordance with procedures for pharmaceutical
formulations knows to those of ordinary skill. Typically,
compositions for systemic administration are solutions in sterile
isotonic aqueous buffer. The ingredients may be supplied either
separately or mixed together in unit dosage form, for example, as a
cryopreserved concentrate in a hermetically sealed container such
as an ampoule indicating the quantity of active agent.
[0079] A variety of means for administering cells to subjects will,
in view of this specification, be apparent to those of skill in the
art. Such methods include may include systemic administration or
injection of the cells into a target site in a subject. Cells may
be inserted into a delivery device which facilitates introduction
by injection or implantation into the subjects. Such delivery
devices may include tubes, e.g., catheters, for injecting cells and
fluids into the body of a recipient subject. In a preferred
embodiment, the tubes additionally have a needle, e.g., a syringe,
through which the cells of the invention can be introduced into the
subject at a desired location. The cells may be prepared for
delivery in a variety of different forms. For example, the cells
may be suspended in a solution or gel. Cells may be mixed with a
pharmaceutically acceptable carrier or diluent in which the cells
of the invention remain viable. Pharmaceutically acceptable
carriers and diluents include saline, aqueous buffer solutions,
solvents and/or dispersion media. The use of such carriers and
diluents is well known in the art. The solution is preferably
sterile and fluid, and will often be isotonic. Preferably, the
solution is stable under the conditions of manufacture and storage
and preserved against the contaminating action of microorganisms
such as bacteria and fungi through the use of, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like.
[0080] Modes of administration of the isolated human BMMSCs include
but are not limited to systemic intravenous or intra-arterial
injection and injection directly into the tissue at the intended
site of activity. The preparation can be administered by any
convenient route, for example by infusion or bolus injection and
can be administered together with other biologically active agents.
Administration is preferably systemic. It may be advantageous,
under certain conditions, to use a site of administration close to
or nearest the intended site of activity. When the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0081] Administration of the BMMSCs of this invention may be done
in combination with one or more further therapeutic agents
including simultaneous (concurrent) and consecutive administration
in any order.
EXAMPLES
[0082] The following examples are provided in order to demonstrate
and further illustrate certain embodiments and aspects of the
present invention and are not to be construed as limiting the scope
thereof. While such examples are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be utilized. Indeed, those of ordinary skill in the
art can readily envision and produce further embodiments, based on
the teachings herein, without undue experimentation.
Experimental Methods
[0083] Animals.
[0084] Female C3H/HeJ, C57BL/6J, and C3MRL-Fas.sup.lpr/J mice were
purchased from Jackson Lab. Female immunocompromised mice (Beige
nude/nude XIDIII) were purchased from Harlan. All animal
experiments were performed under the institutionally approved
protocols for the use of animal research (USC #10874 and
10941).
[0085] Antibodies.
[0086] Anti Oct4, SSEA4, active .beta. catenin and .beta. catenin
were purchased from Millipore. Anti Sca-1-PE, CD34-PE, CD34-FITC,
CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3.epsilon. and
CD28 antibodies were purchased from BD Bioscience. Anti CD105-PE,
Foxp3-PE, IL17-PE, and IFN.gamma.-APC antibodies were purchased
from eBioscience. Unconjugated anti CD34, CD73, and CD105, and anti
TERT were purchased from Santa Cruz Biosciences. Anti .beta. actin
antibody was purchased from Sigma.
[0087] Isolation of Mouse Bone Marrow Mesenchymal Stem Cells
(BMMSCs).
[0088] The single suspension of bone marrow derived all nuclear
cells (ANCs) from femurs and tibias were seeded at
15.times.10.sup.6 into 100 mm culture dishes (Corning) under
37.degree. C. at 5% CO.sub.2 condition. Non-adherent cells were
removed after 48 hours and attached cells were maintained for 16
days in alpha minimum essential medium (.alpha.-MEM, Invitrogen)
supplemented with 20% fetal bovine serum (FBS, Equitech-bio), 2 mM
L-glutamine, 55 .mu.M 2-mercaptoethanol, 100 U/ml penicillin, and
100 .mu.g/ml streptomycin (Invitrogen). Colonies-forming attached
cells were passed once for further experimental use.
[0089] Preparation of Extracellular Matrix (ECM) Coated Dishes.
[0090] ECM coated dishes were prepared as described in Chen et al.
(2007). Briefly, 100% confluence of BMMSCs was cultured in culture
medium with 100 nM L-ascorbic acid phosphate (Wako Pure Chemical).
After 2 weeks, cultures were washed with PBS and incubated with
0.005% Triton X-100 (Sigma) for 5-10 min at room temperature to
remove cells. The ECM was treated with DNase I (100 units/ml;
Sigma) for 1 h at 37.degree. C. The ECM was washed with PBS three
times and stored in 2 ml of PBS containing 100 U/ml penicillin, 100
.mu.g/ml streptomycin, and 0.25 .mu.g/ml fungizone (Invitrogen) at
4.degree. C.
[0091] Isolation of tBMMSCs.
[0092] Bone marrow-derived ANCs were seeded at 15.times.10.sup.6
into 100 mm culture dishes and cultured for 48 hrs. The culture
supernatant were collected and centrifuged to obtain putative
non-attached BMMSCs. The cells were re-seeded at indicated numbers
on ECM-coated dishes. After 48 hrs, the floating cells in the
cultures were removed with PBS and the attached cells on ECM were
maintained for additional 14 days. Colonies-forming attached cells
were passed once and sub-cultured on regular plastic culture dishes
for further experiments. For some stem cell characterization
analysis, we collected SSEA4 positive tBMMSCs using
FACS.sup.Calibur flow cytometer (BD Bioscience) and expanded in the
cultures.
[0093] Colony Forming Unit-Fibroblastic (CFU-F) Assay.
[0094] One million cells of ANCs from bone marrow were seeded on
T25 cell culture flask (Nunc). After 16 days, the cultures were
washed by PBS and stained with 1% toluidine blue solution in 2%
paraformaldehyde (PFA). The cell cluster that has more than 50
cells was counted as a colony under microscopy. The colony number
was counted in five independent samples per each experimental
group.
[0095] Cell Proliferation Assay.
[0096] The proliferation of BMMSC and tBMMSC was performed by
bromodeoxyuridine (BrdU) incorporation assay. Each cell population
(1.times.10.sup.4 cells/well) were seeded on 2-well chamber slides
(Nunc) and cultured for 3 days. The cultures were incubated with
BrdU solution (1:100) (Invitrogen) for 20 hours, and stained with a
BrdU staining kit (Invitrogen). BrdU-positive and total cell
numbers were counted in ten images per subject. The BrdU assay was
repeated in 5 independent samples for each experimental group.
[0097] Population Doubling Assay.
[0098] 0.5.times.10.sup.6 cells of BMMSCs and pBMMSCs were seeded
on 60 mm culture dishes at the first passage. Upon reaching
confluence, the cells were passaged at the same cell density. The
population doubling was calculated at every passage according to
the equation: log.sub.2 (number of harvested cells/number of seeded
cells). The finite population doublings were determined by
cumulative addition of total numbers generated from each passage
until the cells ceased dividing.
[0099] Flow Cytometric Analysis of Mesenchymal Stem Cell Surface
Molecules.
[0100] BMMSCs or pBMMSCs (0.2.times.10.sup.6) were incubated with 1
.quadrature.g of PE conjugated antibodies or isotype-matched
control IgGs (Southern Biotech) at 4.degree. C. for 45 min. Samples
were analyzed by FACScalibur flow cytometer (BD Bioscience). For
dual color analysis, the cells were treated with PE conjugated and
FITC conjugated antibodies or isotype-matched control IgGs (each 1
.quadrature.g). The cells were analyzed on FACS.sup.Calibur (BD
Bioscience).
[0101] Immunofluorescent Microscopy.
[0102] The cells subcultured on 8-well chamber slides (Nunc)
(2.times.10.sup.3/well) were fixed with 4% PFA. The samples were
incubated with the specific or isotype-matched mouse antibodies
(1:200) overnight at 4.degree. C., and treated with
Rhodamine-conjugated secondary antibodies (1:300, Jackson
ImmunoResearch; Southern Biotechnology). Finally, they were mounted
by Vectashield mounting medium containing
4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).
[0103] Isolation of CD34.sup.+CD73.sup.+ Double Positive Cells.
[0104] Bone marrow derived ANCs were stained with anti CD34-FITC
and anti CD73-PE antibodies for 30 min on ice under dark condition.
After wash with PBS, cells were re-suspended into OPTI-MEM
(Invitrogen) supplement with 2% FBS and antibiotics (100 U/ml
penicillin and 100 .mu.g/ml streptomycin) and sorted by MOFLO XDP
Cell Sorter (BECKMAN Coulter). The sorted double positive cells
were seeded on ECM coated 60 mm dish at density of
1.times.10.sup.6/dish and cultured for further experiments.
[0105] In Vivo Bone Formation Assay.
[0106] 4.0.times.10.sup.6 of cells were mixed with
hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders (40
mg, Zimmer Inc.) and subcutaneously transplanted into 8 weeks old
immunocompromised mice. After 8 weeks, the transplants were
harvested, fixed in 4% PFA and then decalcified with 5% EDTA (pH
7.4), followed by paraffin embedding. The paraffin sections were
stained with hematoxylin and eosin (H&E) and analyzed by an NIH
Image-J. The newly-formed mineralized tissue area from five fields
was calculated and shown as a percentage to total tissue area.
[0107] In Vitro Osteogenic Differentiation Assay.
[0108] BMMSCs and tBMMSCs were cultured under osteogenic culture
condition containing 2 mM .beta.-glycerophosphate (Sigma), 100
.mu.M L-ascorbic acid 2-phosphate and 10 nM dexamethasone (Sigma).
After induction, the cultures were stained with alizarin red or
alkaline phosphatase.
[0109] In Vitro Adipogenic Differentiation Assay.
[0110] For adipogenic induction, 500 nM isobutylmethylxanthine, 60
.mu.M indomethacin, 500 nM hydrocortisone, 10 .mu.g/ml insulin
(Sigma), 100 nM L-ascorbic acid phosphate were added into the
culture medium. After 10 days, the cultured cells were stained with
Oil Red-O and positive cells were quantified by using an NIH
Image-J. Total RNA was also isolated from cultures after 10 days
induction for further experiments.
[0111] Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Analysis.
[0112] Extraction of total RNA and RT-PCR were performed according
to standard procedures.
[0113] Western Blotting Analysis.
[0114] 20 mg of protein were used and SDS-PAGE and western blotting
were performed according to standard procedures. .beta.-actin on
the same membrane served as the loading control.
[0115] Inhibitor Treatment.
[0116] tBMMSCs and BMMSCs were treated with 1 mM L-NMMA (Cayman
Chemical) or 0.2 mM 1400 W (Cayman Chemical) to inhibit total NOS
or iNOS, respectively. Aspirin 50 .mu.g/ml (TAT) and telomerase
inhibitor III (1 .mu.M; EMD Chemicals) were used to activate and
suppress telomerase activity in cultured BMMSCs, respectively.
CHIRON 99021 (1 or 10 .mu.M; Chiron Corporation) and Dickkopf 1
(DKK1, 10 ng/ml, R&D Systems) were used as an activator and
inhibitor to regulate .beta. catenin levels in BMMSCs.
[0117] Measurement of Telomerase Activity.
[0118] The Telomerase activity was measured using TeloTAGGG
Telomerase PCR ELISA kit (Roche).
[0119] Measurement of Nitric Oxide Production.
[0120] BMMSCs (0.2.times.10.sup.6/well) were cultured on 24-well
plates with or without cytokines (IFN.gamma., 25 ng/ml; IL-1.beta.,
5 ng/ml, R&D Systems) and chemicals (L-NMMA, 1 mM; 1400W, 0.2
mM; aspirin, 50 .mu.g/ml; Telomerase inhibitor III, 1 .mu.M; CHIRON
99021, 1 or 10 .mu.M; DKK1, 10 ng/ml) at indicated concentration
and days. The same chemical concentration was also used in
combination treatment such as DKK and aspirin or Telomerase
inhibitor and CHIRON99021. The supernatant from each culture was
collected and measured nitric oxide concentration using Total
Nitric Oxide and Nitrate/Nitrite Parameter Assay kit (R&D
Systems) according to manufacturer's instruction.
[0121] Cell Apoptosis and Cell Survival Assay.
[0122] Transwell system (Corning) was used for co-culture
experiments. 0.2.times.10.sup.6 of tBMMSCs or BMMSCs were seeded on
each lower chamber. In the upper chambers, activated splenocytes
(1.times.10.sup.6/chamber), which were pre-stimulated with
plate-bounded anti CD3.epsilon. antibody (5 .mu.g/ml) and soluble
anti CD28 antibody (2 .mu.g/ml) for 3 days, were loaded. Both
chambers were filled with a complete medium containing Dulbecco's
Modified Eagle Medium (DMEM, Lonza) with 10% heat-inactivated FBS,
50 .mu.M 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate
(Sigma), 1% non-essential amino acid (Cambrex), 2 mM L-glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin. To measure the
splenocyte viability, cell counting kit-8 (Dojindo Molecular
Technoloies) were used. For apoptosis of splenocyte analysis,
Annexin V-PE apoptosis detection kit I (BD Bioscience) were used
and analyzed on FACS.sup.Calibur (BD Bioscience).
[0123] In Vitro CD4.sup.+CD25.sup.+Foxp3.sup.+ Tregs Induction.
[0124] CD4.sup.+CD25.sup.- T-lymphocytes (1.times.10.sup.6/well),
collected by CD4.sup.+CD25.sup.+ regulatory T-cell Isolation kit
(Miltenyi Biotec), were pre-stimulated with plate bounded anti
CD3.epsilon. antibody (5 .mu.g/ml) and soluble anti CD28 antibody
(2 .mu.g/ml) for 3 days. These activated T-lymphocytes were loaded
on 0.2.times.10.sup.6 of BMMSC or tBMMSC cultures with recombinant
human TFG.beta.1 (2 .mu.g/ml) (R&D Systems) and recombinant
mouse IL2 (2 .mu.g/ml) (R&D Systems). After 3 days, cells in
suspension were collected and stained with anti CD4-PerCP, anti
CD8a-FITC, anti CD25-APC antibodies (each 1 .mu.g) for 45 min on
ice under dark condition. And then cells were stained with anti
Foxp3-PE antibody (1 .mu.g) using Foxp3 staining buffer kit
(eBioscience) for cell fixation and permeabilization. The cells
were analyzed on FACS.sup.Calibur (BD Bioscience).
[0125] Allogenic Mouse tBMMSC Transplantation into MRL/Lpr
Mice.
[0126] Under general anesthesia, C3H/HeJ-derived BMMSCs or tBMMSCs
(0.1.times.10.sup.6 cells/10 g body weight) were infused into
MRL/lpr mice via tail vein at 10 weeks old age (n=6). In control
group, MRL/lpr mice received PBS (n=5). All mice were sacrificed at
12 weeks old age for further analysis. The protein concentration in
urine was measured using Bio-Rad Protein Assay (Bio-Rad). The
number of white blood cells from peripheral blood was measured by
Coulter LH-750 (BECKMAN Coulter).
[0127] Measurement of Autoantibodies, Albumin, sRANKL and CTX.
[0128] Peripheral blood serum samples were collected from mice.
Autoantibodies, albumin, sRANKL and CTX were analyzed by
enzyme-linked immunosorbent assay (ELISA) method using commercial
available kits (anti-dsDNA antibodies, ANA, and albumin, alpha
diagnostic; sRANKL, R&D Systems; CTX, Nordic Bioscience
Diagnostics A/S) according to their manufactures' instructions. The
results were averaged in each group. The intra-group differences
were calculated between the mean values.
[0129] TRAP Staining.
[0130] Deparaffinized sections were re-fixed with a mixture of 50%
ethanol and 50% acetone for 10 min. TRAP-staining solutions were
freshly made (1.6% naphthol AS-BI phosphate in
N,N-dimethylformamide and 0.14% fast red-violet LB diazonium salt,
0.097% tartaric acid and 0.04% MgCl.sub.2 in 0.2 M sodium acetate
buffer, pH 5.0) and mixed in 1:10. The sections were incubated in
the solution for 10 min at 37.degree. C. under shield and
counterstained with toluidine blue. All regents for TRAP staining
were purchased from Sigma.
[0131] Histometry.
[0132] Area of trabecular bone was measured on bone sections with
H&E staining. To quantify osteoclast activity in the bones,
number of mature osteoclasts was determined by TRAP positive cells
attached on the bone surface. Each number of cells and area were
measured from five representative images per each sample using an
NIH Image-J, followed by calculating the means. The data were
average the means in each experimental group. The results were
shown as each indicated percentage.
[0133] Rescue Lethal Dose Irradiated Mice.
[0134] In each group, 1.times.10.sup.6 cells in 50 ml PBS or PBS
alone as control were injected into the tail vein of recipient mice
at 1 day post lethal irradiation (8.5 Gy per mouse). The survival
date of each mouse was recorded and analyzed.
[0135] Statistics.
[0136] Student's t-test was used to analyze statistic difference.
The p values less than 0.05 were considered significant.
Example 1
A Subset of BMMSCs Fails to Adhere to Plastic Culture Dishes, but
Attaches to ECM-Coated Culture Dishes
[0137] To determine whether a subset of BMMSCs remains in culture
suspension, we seeded 15.times.10.sup.6 bone marrow all nuclear
cells (ANCs) under regular plastic culture conditions for 2 days
and subsequently transplanted all non-attached cells into
immunocompromised mice subcutaneously using hydroxyapatite
tricalcium phosphate (HA/TCP) as a carrier. At 8 weeks
post-transplantation, newly formed bone was identified in the
transplants by H&E staining (FIG. 1A), suggesting that the
BMMSC culture suspension may contain cells with a capacity of
differentiating into bone forming cells in vivo. Added evidence
indicated that extracellular matrix (ECM) produced by BMMSCs
(BMMSC-ECM) can adhere higher numbers of CFU-F when compared to
plastic cultures (FIG. 10; Chen et al., 2007). Thus, we collected
culture medium at 2 days post-primary CFU-F culture and loaded the
medium onto BMMSC-ECM-coated dishes (FIG. 1B). A subset of BMMSCs
(named tBMMSCs) in the suspension was able to adhere to the
BMMSC-ECM and form CFU-F (FIG. 1B), at a lower incidence compared
to the number of CFU-F generated from regular BMMSCs (FIG. 1C).
tBMMSCs were found to express mesenchymal stem cell associated
markers (CD73, stem cell antigen 1 [Sca-1], Octamer 4 [Oct4], and
stage specific antigen 4 [SSEA4]) as evidenced by flow cytometric
analysis (FIG. 1D). When compared with regular BMMSCs, tBMMSCs
expressed significantly higher levels of Sca-1 (87.74% vs. 52.16%
in BMMSCs) and Oct4 (40.7% vs. 14.08% in BMMSCs), both earlier
progenitor surface molecules for mesenchymal stem cells. In order
to characterize stem cell properties of tBMMSCs, we collected SSEA4
positive tBMMSCs and assessed their proliferation rate by
bromodeoxyuridine (BrdU) labeling. We found that tBMMSCs had a
significantly elevated BrdU uptake rate compared to regular BMMSCs
(FIG. 1E). In addition, we used a continuous cell culture assay to
indicate that SSEA4.sup.+ tBMMSCs acquired a significantly
increased number of population doubling (FIG. 1F). These data imply
that tBMMSCs are distinct from regular BMMSCs in terms of
attachment, proliferation, and self-renewal.
[0138] To examine the multipotent differentiation potential, we
revealed that tBMMSCs are analogous to BMMSCs in expression of
alkaline phosphatase (ALP), mineralized nodule accumulation under
the osteogenic inductive cultures, and bone regeneration when
transplanted into immunocompromised mice using HA/TCP as a carrier
(FIGS. 11A, 11B). Furthermore, we showed that tBMMSCs were similar
to regular BMMSCs in forming Oil red O positive cells under
adipogenic inductive conditions, expression of adipogenic genes
peroxisome proliferator-activated receptor gamma 2 (PPAR.gamma.2)
and lipoprotein lipase (LPL), and differentiating into chondrocytes
under the chondrogenic inductive conditions with expression of
proteoglycan, trichrome positive collagen, and type II collagen
(FIGS. 11C, 11D). These data confirm that tBMMSCs are a novel
subset of non-adherent BMMSCs.
Example 2
tBMMSCs Express Telomerase and CD34, but are Distinct from
Hematopoietic Stem Cells
[0139] In order to characterize tBMMSCs, flow cytometric analysis
was used to examine whether tBMMSC expressed hematopoietic cell
markers. We found that 17.4% of tBMMSCs, but not regular BMMSCs,
expressed CD34, a HSC and endothelial cell marker (FIG. 2A). BMMSCs
(21.25%) and tBMMSCs (31.22%) expressed CD45, another hematopoietic
marker, at passage 2 (FIG. 2A). Both BMMSCs and tBMMSCs were
negative to CD11b antibody staining (data not shown), excluding
that tBMMSCs are derived from monocyte/macrophage lineage cells.
Importantly, CD34.sup.+ tBMMSCs co-expressed BMMSC associated
markers (CD73 or Oct4), as evidenced by flow cytometric analysis
(FIG. 2B). Western blot analysis confirmed that tBMMSCs expressed
CD34, CD73, and CD105 (FIG. 2C), and regular BMMSCs expressed CD73
and CD105, but lacked expression of CD34 (FIG. 2C). tBMMSCs show a
continued expression of CD34 from passage 1 to 5, however, the
expression levels appear reduced after passage 3 (FIG. 2D). To
further verify CD34 expression in tBMMSCs, we used double
immunocytostaining to show that tBMMSCs co-express CD34 with
mesenchymal markers CD73 and CD105 (FIGS. 2E, 2F) and regular
BMMSCs are negative for anti-CD34 antibody staining (FIGS. 2E, 2F).
More interestingly, we found that tBMMSCs possessed significantly
higher levels of telomerase activity compared to regular BMMSCs by
PCR-ELISA assay and Western blot analysis (FIGS. 2G, 2H),
implicating that tBMMSCs may be a primitive subpopulation of
BMMSCs.
[0140] Next, we used flow cytometry to sort CD34 and CD73
double-positive cells from bone marrow ANCs and recovered 3.77%
double-positive cells (FIG. 2I). These CD34 and CD73
double-positive cells exhibit mesenchymal stem cell
characteristics, including forming single colony clusters (FIG. 2J)
and differentiating into osteogenic and adipogenic cells (data not
shown), indicating a feasible approach of directly isolating
tBMMSC-like cells from bone marrow. CD34.sup.+/CD73.sup.+ BMMSCs
are analogous to tBMMSCs in terms of having higher level of
telomerase activity and high NO production when compared to regular
BMMSCs (FIGS. 2K, 2L).
[0141] To exclude potential HSC contamination in tBMMSCs, we used
aspirin (TAT) to elevate telomerase level in regular CD34.sup.-
BMMSCs (FIG. 7C). After the aspirin treatment, BMMSCs exhibit
higher levels of Sca-1 and Oct4 expression when compared to BMMSCs,
but at a lower level than tBMMSCs (FIG. 3A, 3B). Importantly,
aspirin (TAT)-treated CD34- BMMSCs acquire a positive CD34
expression (FIG. 3A). Western blot analysis confirmed that aspirin
(TAT)-treated BMMSCs express CD34, but at a lower level than
tBMMSCs (FIG. 3C). These data suggest that CD34 expression in
BMMSCs is not due to HSC contamination.
[0142] It is generally believed that CD34 expression is associated
with HSCs and endothelial populations. HSCs can differentiate into
hematopoietic cell lineage and rescue lethal dose-irradiated
subjects. Thus, we use hematopoietic differentiation medium to
treat tBMMSCs, aspirin (TAT)-treated BMMSCs and regular BMMSCs and
find all of these cells fail to differentiate into hematopoietic
cell lineage as seen in bone marrow cells and linage cells served
as positive controls capable of forming colony clusters (FIG. 4A).
Next, we infused tBMMSC systemically to rescue lethal
dose-irradiated mice and found that tBMMSCs, but not regular
BMMSCs, can extend the lifespan of lethal dose-irradiated mice
(FIG. 4B). However, tBMMSCs failed to rescue lethal dose-irradiated
mice, as shown in bone marrow group (FIG. 4B). These experimental
evidences further indicate that CD34 expression in tBMMSCs is not
due to HSC contamination.
Example 3
tBMMSCs Possess Superior Immunomodulatory Functions Via High Nitric
Oxide (NO) Production
[0143] Recently, immunomodulatory properties were identified as an
important stem cell characteristic of BMMSCs, leading to utilize
systemic infused BMMSCs to treat a variety of immune diseases
(Nauta et al., 2007; Uccelli et al., 2007, 2008). Here we found
that tBMMSCs exhibited a significant increased capacity for NO
production compared to regular BMMSCs when treated with interferon
gamma (IFN.gamma.) and interleukin 1 beta (IL-1.beta.) (FIG. 5A).
It is known that NO plays a critical role in BMMSC-mediated
immunosuppression (Ren et al., 2008), therefore, we assessed the
functional role of high NO production in tBMMSC-associated
immunomodulatory properties. Spleen (SP) cells were activated with
stimulation of anti-CD3 and anti-CD28 antibodies for 3 days and
then co-cultured with tBMMSCs or regular BMMSCs in the presence of
the general nitric oxide synthase (NOS) inhibitor,
NG-monomethyl-L-arginine (L-NMMA), or the inducible NOS (iNOS)
inhibitor, 1400W, using a Transwell culture system. The efficacy of
L-NMMA and 1400W to inhibit NO production in BMMSCs was verified
(FIG. 12). Although both tBMMSCs and regular BMMSCs were capable of
inhibiting cell viability of activated SP cells, tBMMSCs showed a
marked inhibition of SP cell viability over that of regular BMMSCs
(FIG. 5B). Interestingly, both L-NMMA and 1400W were able to
significantly block tBMMSC, but not regular BMMSC, induced cell
viability of activated SP cells (FIGS. 5C, 5D). Furthermore, flow
cytometric analysis indicated that both tBMMSCs and regular BMMSCs
induce apoptosis of activated SP cells in the Transwell culture
system, including early apoptotic cells (FIG. 5E) and late
apoptotic and dead cells (FIG. 5H). However, tBMMSCs show an
elevated capacity in inducing activated SP cell apoptosis compared
to regular BMMSCs (FIGS. 5E, 5H). When L-NMMA and 1400W were added
to the cultures, the number of early and late apoptotic SP cells
was significantly reduced in both tBMMSC and regular BMMSC groups
(FIGS. 5F, 5G, 5I, 5J). Treatment with 1400W resulted in a
significantly greater inhibition of early apoptotic SP cells in
tBMMSC group compared to the regular BMMSC group (FIG. 5G). These
data suggest that NO production is required for BMMSC-mediated
immunomodulation.
[0144] Next, we co-cultured naive-T-cells with tBMMSCs or regular
BMMSCs in the presence of IL-2 and transforming growth factor beta
1 (TGF-.beta.1). We found that tBMMSCs showed a significant
up-regulation of CD4.sup.+CD25.sup.+Foxp3.sup.+ regulatory T cell
(Tregs) levels when compared to regular BMMSCs (FIG. 5K). Both
L-NMMA and 1400W were able to inhibit BMMSC- and tBMMSC-induced
up-regulation of Tregs, as shown by flow cytometric analysis (FIGS.
5L, 5M). The regulatory effect on Tregs was more significant in the
tBMMSC group compared to the BMMSC group (FIGS. 5L, 5M). These data
further verified the role of NO in tBMMSC-induced immunomodulatory
effect.
Example 4
tBMMSCs Transplantation Improves Multiple Organ Function in MRL/Lpr
Mice
[0145] In order to examine in vivo immunomodulatory properties of
tBMMSCs, we infused allogenic tBMMSCs and BMMSCs into MRL/lpr mice
at 10 weeks of age and analyzed treatment response at 12 weeks of
age (FIG. 6A). We found that both tBMMSCs and BMMSCs were capable
of improving SLE-induced glomerular basal membrane disorder (FIG.
6B) and reducing the urine protein level (FIG. 6C). It appeared
that tBMMSCs were superior compared to BMMSCs in terms of reducing
the overall urine protein levels (FIG. 6C). As expected, MRL/lpr
mice showed remarkable increase in the levels of autoantibodies,
including anti-double strand DNA (dsDNA) IgG and IgM antibodies
(FIGS. 6D, 6E), and anti-nuclear antibody (ANA; FIG. 6F) in the
peripheral blood. Although tBMMSC and BMMSC infusion showed
significant decreased levels of dsDNA IgG, IgM antibodies and ANA
in peripheral blood (FIGS. 6D-F), tBMMSCs showed superior
therapeutic effect in reducing dsDNA IgG antibody and ANA levels
when compared to BMMSC group (FIGS. 6D, 6F). Additionally,
decreased serum albumin levels in MRL/lpr mice were recovered by
tBMMSC and BMMSC infusion (FIG. 6G), but tBMMSC treatment results
in more significant recovery than BMMSC treatment (FIG. 6G).
[0146] Next, we used flow cytometric analysis to reveal that tBMMSC
show more effectiveness in recovering the decreased level of
CD4.sup.+CD25.sup.+Foxp3.sup.+ Tregs and increased number of
CD4.sup.+IL17.sup.+IFN.gamma..sup.- T-lymphocytes in peripheral
blood when compared to BMMSCs (FIG. 6H, 6I). Furthermore, we showed
that tBMMSCs are superior to BMMSCs in terms of reducing increased
number of tartrate-resistant acid phosphatase (TRAP) positive
osteoclasts in the distal femur epiphysis of MRL/lpr mice (FIG.
13A), elevated serum levels of soluble runt-related NF-.kappa.B
ligand (sRANKL), a critical factor for osteoclastogenesis, (FIG.
13B) and bone resorption marker C-terminal telopeptides of type I
collagen (CTX, FIG. 13C). These data suggest that tBMMSCs show
superior therapeutic effect for SLE disorders compared to
BMMSCs.
Example 5
NO Production in BMMSCs is Modulated by Telomerase Activity Coupled
with Wnt/.beta.-Catenin Signaling
[0147] Since elevated NO production telomerase activity were
observed in tBMMSCs, it is important to elucidate whether
telomerase activity governs NO production in tBMMSCs. We found that
telomerase inhibitor III is effective in inhibiting telomerase
activity along with reducing NO production in tBMMSCs (FIGS. 7A,
7B). Similar effects of the telomerase inhibitor were also found in
regular BMMSCs (FIG. 7C, 7E). In contrast, aspirin (TAT) treatment
leads to a significantly elevated telomerase activity, telomerase
reverse transcriptase (TERT) expression and NO production in BMMSCs
(FIG. 7C-E). These data imply that telomerase activity may be
associated with NO production in BMMSCs.
[0148] Recently, it was reported that telomerase directly modulates
Wnt/beta-catenin signaling by serving as a cofactor in a
beta-catenin transcriptional complex (Park et al., 2009). Thus, we
assessed whether telomerase activity-associated NO production in
BMMSCs could be down-regulated by the Wnt inhibitor, Dickkopf 1
(DKK1). Interestingly, we found that DKK1 was able to significantly
block aspirin-induced NO production in BMMSCs when added to the
cultures prior to the aspirin (TAT) treatment (FIG. 7F). The
efficacy of DKK1 in reducing activated .beta.-catenin level was
confirmed by Western blot analysis (FIG. 7G). Moreover, we found
that aspirin (TAT) was able to partially block DKK1-induced
down-regulation of activated beta-catenin (FIG. 7G). In order to
examine the mechanism by which DKK1 inhibits aspirin (TAT)-induced
NO production, we showed that DKK1 is capable of blocking aspirin
(TAT)-induced telomerase activity (FIG. 7H). These data indicate
that telomerase-driven NO production is coupled with
Wnt/.beta.-catenin signaling.
[0149] Next, we determined whether Wnt/beta-catenin signaling
affected NO production in BMMSCs. We used Chiron 99021 (Chiron) to
treat BMMSCs for 7 days and showed elevation of active beta-catenin
in a dose-dependent manner (FIG. 7I), confirming efficacy of Chiron
as a Wnt/beta-catenin activator. We then showed that Chiron
treatment up-regulated NO production in BMMSCs in a dose-dependent
manner (FIG. 7J), along with an elevated telomerase activity in
BMMSCs (FIG. 7K). Further, we showed that Chiron-induced telomerase
activity and NO production could be blocked by 3 days of
pre-treatment with telomerase inhibitor III (FIGS. 7K, 7L). These
findings suggest that telomerase and Wnt/beta-catenin
collaboratively enhance telomerase activity and induce NO
production in BMMSCs.
Example 6
Aspirin Treatment Generates Immunomodulatory Activated BMMSCs
[0150] In order to determine whether telomerase affects
immunomodulatory properties of regular BMMSCs, we showed that
aspirin (TAT) is able to promote BMMSC-induced reduction of
activated SP cell viability and elevation of early and late
apoptosis of activated SP cells (FIGS. 14A-14C). These data suggest
a potential of inducing telomerase activity in regular BMMSC to
improve their immunomodulatory functions, as seen in tBMMSCs.
[0151] In order to confirm therapeutic effect of aspirin
(TAT)-treated BMMSCs (TAT-BMMSC), we infused either
0.1.times.10.sup.6 or 0.01.times.10.sup.6TAT-BMMSC into MRL/lpr
mice at 10 weeks of age and analyzed treatment response at 12 weeks
of age. We found that both aspirin treated TAT-BMMSC and BMMSC were
capable of reducing the urine protein level when compared to
MRL/lpr mice (FIG. 8A). TAT-BMMSC were more effective in reducing
the overall urine protein levels at both 0.1.times.10.sup.6 and
0.01.times.10.sup.6 groups when compared to BMMSC. It appeared that
infusion of 0.01.times.10.sup.6 BMMSCs fail to significantly reduce
urine protein levels (FIG. 8A). Although TAT-BMMSC and BMMSC
infusion showed significant decreased levels of dsDNA IgG, IgM
antibodies and ANA in peripheral blood (FIGS. 8B-D), TAT-BMMSC
showed superior therapeutic effect in reducing dsDNA IgG and IgM
antibodies and ANA levels when compared to BMMSC group at both
0.1.times.10.sup.6 and 0.01.times.10.sup.6 groups (FIGS. 8B-D).
Additionally, ELISA and flow cytometric analysis revealed that
TAT-BMMSC show more effectiveness in reducing serum IL17 levels in
0.01.times.10.sup.6 group (FIG. 8E) and number of
CD4.sup.+IL17.sup.+IFN.gamma..sup.- T-lymphocytes in both
0.1.times.10.sup.6 and 0.01.times.10.sup.6 groups (FIG. 8F) and
elevating the level of CD4.sup.+CD25.sup.+Foxp3.sup.+ Tregs in
0.01.times.10.sup.6 group when compared to BMMSCs (FIG. 8G). These
data indicate that the number of BMMSCs in immuno-therapy could be
significantly reduced with ex vivo telomerase activator
treatment.
Example 7
[0152] Human bone marrow contain tBMMSCs and aspirin treatment can
induce regular human BMMSC to become tBMMSCs with improved
immunomodulatory function. When aspirin was added into culture
medium at 2.5 .mu.g/ml or 50 .mu.g/ml for 1 week, there is a
significantly increased level of telomerase activity in BMMSCs.
[0153] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not intended to be
limiting, with the true scope and spirit being indicated by the
following claims. Those skilled in the art will recognize, or be
able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
[0154] All references cited herein, including but not limited to
patents, patent applications, and non-patent literature, are hereby
incorporated by reference herein in their entirety.
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