MESENCHYMAL STEM CELLS AND USES THEREFOR

Abstract

Compositions and methods of promoting wound healing in a human by administering to the human mesenchymal stem cells in an effective amount.

Claims

1.-20. (canceled)

21. A method for reducing or limiting inflammation in a human comprising administering genetically unmanipulated mesenchymal stem cells to the human, wherein the administration mediates a shift in the T cell population in the human from pro-inflammatory TH1 cells to anti-inflammatory TH2 cells.

22. The method of claim 21, wherein the inflammation is in the brain or gut.

23. The method of claim 21, wherein the human has inflammatory bowel disease.

24. The method of claim 21, wherein the human has an inflammatory bowel disease, and the mesenchymal stem cells promote increased secretion of interleukin 10.

25. The method of claim 21, wherein the human has an inflammatory bowel disease, and the mesenchymal stem cells promote generation of T.sub.reg cells.

26. The method of claim 21, wherein the mesenchymal stem cells are allogeneic.

27. The method of claim 21, wherein the mesenchymal stem cells are autologous.

28. The method of claim 21, wherein the mesenchymal stem cells are administered by intravenous, intraarterial, or intraperitoneal administration.

29. The method of claim 21, wherein the mesenchymal stem cells are administered as a cell suspension in a pharmaceutically acceptable liquid medium.

30. The method of claim 21, wherein the mesenchymal stem cells were harvested from a mesenchymal stem cell containing tissue, isolated, and expanded in culture.

31. The method of claim 23, wherein the mesenchymal stem cells are allogeneic.

32. The method of claim 23, wherein the mesenchymal stem cells are autologous.

33. The method of claim 23, wherein the mesenchymal stem cells are administered by intravenous, intraarterial, or intraperitoneal administration.

34. The method of claim 23, wherein the mesenchymal stem cells are administered as a cell suspension in a pharmaceutically acceptable liquid medium.

35. The method of claim 23, wherein the mesenchymal stem cells were harvested from a mesenchymal stem cell containing tissue, isolated, and expanded in culture.

36. The method of claim 24, wherein the mesenchymal stem cells are allogeneic.

37. The method of claim 24, wherein the mesenchymal stem cells are autologous.

38. The method of claim 24, wherein the mesenchymal stem cells are administered by intravenous, intraarterial, or intraperitoneal administration.

39. The method of claim 25, wherein the mesenchymal stem cells are allogeneic.

40. The method of claim 25, wherein the mesenchymal stem cells are autologous.

Description

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0086] The invention now will be described with respect to the drawings, wherein:

[0087] FIGS. 1A-1C MSCs modulate dendritic cell functions. (A) Flow cytometric analysis of mature monocytic DC1 cells using antibodies against HLA-DR and CD11c and of plasmacytoid DC2 cells using antibodies against HLA-DR and CD123 (IL-3 receptor). (---): isotype control; (—): FITC/PE conjugated antibodies. (B) MSCs inhibit TNF-α secretion (primary y-axis) and increase IL-10 secretion (secondary y-axis) from activated DC1 and DC2 respectively. (C) MSCs cultured with mature DC1 cells inhibit IFN-γ secretion (primary y-axis) by T cells and increase IL-4 levels (secondary y-axis) as compared to MSC or DC alone. The decreased production of pro-inflammatory IFN-γ and increased production of anti-inflammatory IL-4 in the presence of MSCs indicated a shift in the T cell population towards an anti-inflammatory phenotype.

[0088] FIGS. 2A-2C MSCs inhibit pro-inflammatory effector T cell function. (A) Flow cytometric analysis of T.sub.Reg cell numbers (in %) by staining PBMCs or non-adherent fraction in MSC+PBMC culture (MSC+PBMC) with FITC-conjugated CD4 (x-axis) and PE conjugated CD25 (y-axis) antibodies. Gates were set based on isotype control antibodies as background. Graphs are representative of 5 independent experiments. (B) T.sub.H1 cells generated in presence of MSCs secreted reduced levels of IFN-γ (primary y-axis) and T.sub.H2 cells generated in presence of MSCs secreted increased amounts of IL-4 (secondary y-axis) in cell culture supernatants. (C) MSCs inhibit IFN-γ secretion from purified NK cells cultured for 0, 24, or 48 hours in a 24-well plate. Data shown are mean±SD cytokine secretion in one experiment and are representative of 3 independent experiments.

[0089] FIGS. 3A-3B MSCs lead to increased numbers of T.sub.reg cell population and increased GITR expression. (A) A CD.sup.4+ CD25.sup.+ T.sub.reg cell population from PBMC or MSC+PBMC (MSC to PBMC ratio 1:10) cultures (cultured without any further stimulation for 3 days) was isolated using a 2-step magnetic isolation procedure. These cells were irradiated (to block any further proliferation) and used as stimulators in a mixed lymphocyte reaction (MLR), where responders were allogeneic PBMCs (stimulator to responder ratio 1:100) in the presence of phytohemagglutinin (PHA) (2.5 mg/ml). The cells were cultured for 48 hours, following which .sup.3H thymidime was added, and incorporated radioactivity was counted after 24 hours. The results showed that the T.sub.reg population generated in the presence of MSCs (lane 3) was similar functionally to the T.sub.reg cells generated in the absence of MSCs (lane 2). (B) PBMCs were cultured for 3 days in the absence (top plot) or presence (bottom plot) of MSCs (MSC to PBMC ratio 1:10), following which the non-adherent fraction was harvested and immunostained with FITC-labeled GITR and PE-labeled CD4. Results show a greater than twofold increase in GITR expression in cells cultured in the presence of MSCs.

[0090] FIGS. 4A-4D MSCs produce PGE.sub.2 and blocking PGE.sub.2 reverses MSC-mediated immunomodulatory effects. (A) PGE.sub.2 secretion (mean±SD) in culture supernatants obtained from MSCs cultured in the presence or absence of PGE.sub.2 blockers NS-398 or indomethacin (Indometh.) at various concentrations. Inhibitor concentrations are in μM and data presented are values obtained after 24 hour culture (B) COX-1 and COX-2 expression in MSCs and PBMCs using real-time RT-PCR. MSCs expressed significantly higher levels of COX-2 as compared to PBMCs, and when MSCs were cultured in presence of PBMCs, there was a >3-fold increase in COX-2 expression in MSCs. Representative data from 1 of 3 independent experiments is shown. The MSC +PBMC cultures were setup in a trans-well chamber plate where MSCs were plated onto the bottom chamber and PBMCs onto the top chamber. (C) Presence of PGE.sub.2 blockers indomethacin (Ind.) or NS-398 increases TNF-α secretion from activated DCs () and IFN-γ secretion from T.sub.H1 cells () as compared to controls. Data were calculated as % change from cultures generated in absence of MSCs and PGE.sub.2 inhibitors (D) Presence of PGE.sub.2 blockers indomethacin (Indo) and NS-398 during MSC-PBMC co-culture (1:10) reverses MSC-mediated anti-proliferative effects on PHA-treated PBMCs. Data shown are from one experiment and are representative of 3 independent experiments.

[0091] FIG. 5 Constitutive MSC cytokine secretion is elevated in the presence of allogeneic PBMCs. Using previously characterized human MSCs, the levels of the cytokines IL-6 and VEGF, lipid mediator PGE.sub.2, and matrix metalloproteinase 1 (pro-MMP-1) in culture supernatant of MSCs cultured for 24 hours in the presence (hatched bars) or absence (open bars) of PBMCs (MSC to PBMC ratio 1:10) were analyzed. The MSCs produced IL-6, VEGF, and PGE.sub.2 constituitively, and the levels of these factors increased upon co-culture with PBMCs, thereby suggesting that MSCs may play a role in modulating immune functions in an inflammatory setting.

[0092] FIG. 6 MSCs inhibit mitogen-induced T-cell proliferation in a dose-dependent manner. Increasing numbers of allogeneic PBMCs were incubated with constant numbers of MSCs (2,000 cells/well) plated on a 96-well plate in the presence or absence of PHA (2.5 mg /ml) for 72 hours, and .sup.3H thymidine incorporation determined (in counts per minute, or cpm). There was a dose-dependent inhibition of the proliferation of PHA-treated PBMCs in the presence of MSCs. Representative results from 1 of 3 independent experiments are shown. Similar results were reported by LeBlanc, et al., Scand J. Immunol., Vol, 57, pg. 11 (2003)

[0093] FIG. 7 Schematic diagram of proposed MSC mechanism of action. MSCs mediate their immuno-modulatory effects by affecting cells from both the innate (DCs-pathways 2-4: and NK-pathway 6) and adaptive (T-pathways 1 and 5 and B-pathway 7) immune systems. In response to an invading pathogen, immature DCs migrate to the site of potential entry, mature and acquire an ability to prime naïve T cells (by means of antigen specific and co-stimulatory signals) to become protective effector T cells (cell-mediated T.sub.H1 or humoral T.sub.H2 immunity). During MSC-DC interaction, MSCs, by means of direct cell-cell contact or via secreted factor, may alter the outcome of immune response by limiting the ability of DCs to mount a cell-mediated response (pathway 2) or by promoting the ability to mount a humoral response (pathway 4) Also, when mature effector T cells are present, MSCs may interact with them to skew the balance of T.sub.H1 (pathway 1) responses towards T.sub.H2 responses (pathway 5), and probably towards an increased IgE producing B cell activity (pathway 7), desirable outcomes for suppression of GvHD and autoimmune disease symptoms. MSCs in their ability to result in an increased generation of T.sub.Reg population (pathway 3) may result in a tolerant phenotype and may aid a recipient host by dampening bystander inflammation in their local micro-environment, Dashed line (----) represents proposed mechanism.

EXAMPLES

[0094] The invention now will be described with respect to the following examples; it is to be understood, however, that the scope of the present invention is not to be limited thereby.

Example 1

Materials and Methods

Culture of Human MSCs

[0095] Human MSCs were cultured as described by Pittenger et al., Science, Vol. 284, pg. 143 (1999). Briefly, marrow samples were collected from the iliac crest of anonymous donors following informed consent by Poietics Technologies, Div of Cambrex Biosciences. MSCs were cultured in complete Dulbecco's Modified Eagle's Medium-Low Glucose (Life Technologies, Carlsbad, Calif.) containing 1% antibiotic-antimyotic solution (Invitrogen, Carlsbad, Calif.) and 10% fetal bovine serum (FBS, JRH BioSciences, Lenexa, Kans.). MSCs grew as an adherent monolayer and were detached with trypsin/EDTA (0.05% trypsin at 37° C. for 3 minutes). All MSCs used were previously characterized for multilineage potential and retained the capacity to differentiate into mesenchymal lineages (chondrocytic, adipogenic, and osteogenic) (Pittenger, et al., Science. Vol. 284, pg. 143 (1999)).

Isolation of Dendritic Cells

[0096] Peripheral blood mononuclear cells (PBMCs) were obtained from Poietics Technologies, Div of Cambrex Biosciences (Walkersville, Md.). Precursors of dendritic cells (DCs) of monocytic lineage (CD1c.sup.+) were positively selected from PBMCs using a 2-step magnetic separation method according to Dzionek, et al, J. Immunol., Vol. 165, pg. 6037 (2000), Briefly, CD1c expressing B cells were magnetically depleted of CD19″ cells using magnetic beads, followed by labeling the B-cell depleted fraction with biotin-labeled CD1c (BDCA1.sup.+) and anti-biotin antibodies and separating them from the unlabeled cell fraction utilizing magnetic columns according to the manufacturer's instructions (Miltenyi Biotech, Auburn, Calif.). Precursors of DCs of plasmacytoid lineage were isolated from PBMCs by immuno-magnetic sorting of positively labeled antibody coated cells (BDCA2.sup.+) (Miltenyi Biotech, Auburn, Calif.).

MSC⋅DC culture

[0097] In most experiments, human MSCs and DCs were cultured in equal numbers for various time periods and cell culture supernatant collected and stored at −80° C. until further evaluation. In selected experiments, MSCs were cultured with mature DC1 or DC2 cells (1:1 MSC:DC ratio) for 3 days, and then the combined cultures (MSCs and DCs) were irradiated to prevent any proliferation. Next, antibody purified, naïve, allogeneic T cells (CD4.sup.+,CD45RA.sup.+) were added to the irradiated MSCsiDCs and cultured for an additional 6 days. The non-adherent cell fraction (purified T cells) was then collected from the cultures, washed twice and re-stimulated with PHA for another 24 hours, following which cell culture supernatants were harvested and analyzed for secreted IFN-γ and 1L-4 by ELISA.

Isolation of NK Cells

[0098] Purified populations of NK cells were obtained by depleting non-NK cells that are magnetically labeled with a cocktail of biotin-conjugated monoclonal antibodies (anti-CD3, -CD14, -CD19, -CD 36 and anti-IgE antibodies) as a primary reagent and anti-biotin monoclonal antibodies conjugated to Microbeads as secondary labeling reagent, The magnetically labeled non-NK cells were retained in MACS (Millenyi Biotech, Auburn, Calif.) columns in a magnetic field, while NK cells passed through and were collected.

Isolation of T.SUB.Reg .Cell Population

[0099] The T.sub.Reg cell population was isolated using a 2-step isolation procedure. First non-CD4.sup.+ T cells were indirectly magnetically labeled with a cocktail of biotin labeled antibodies and anti-biotin microbeads, The labeled cells were then depleted by separation over a MACS column (Miltenyi Biotech, Auburn, Calif.). Next, CD4.sup.+CD25.sup.+ cells were directly labeled with CD25 microbeads and isolated by positive selection from the pre-enriched CD4 T cell fraction. The magnetically labeled CD4.sup.+CD25 T cells were retained on the column and eluted after removal of the column from the magnetic field.

[0100] In order to determine whether the increased CD4+CD25+ population generated in the presence of MSCs were suppressive in nature, CD4+CD25+ T.sub.reg cell populations were isolated from PBMC or MSC+PBMC (MSC to PBMC ratio 1:10) cultures (cultured without any further stimulation for 3 days) using a 2-step magnetic isolation procedure. These cells were irradiated to block any further proliferation and used as stimulators in a mixed lymphocyte reaction (MLR), where responders were allogeneic PBMCs (stimulator to responder ratio 1:100) in the presence of PHA (2.5 μg/ml). The culture was carried out for 48 hours, following which .sup.3H thymidine was added. Incorporated radioactivity was counted after 24 hours.

[0101] PBMCs were cultured in the absence or presence of MSCs (MSC to PBMC ratio 1:10), following which the non-adherent fraction was harvested and immunostained with FITC-labeled glucocorticoid-induced TNF receptor, or GITR, and PE-labeled CD4.

Generation of T.sub.H1/T.sub.H2 Cells

[0102] Peripheral blood mononuclear cells (PBMCs) were plated at 2×10.sup.6 cells /ml for 45 min. at 37° C. in order to remove monocytes. Non-adherent fraction was incubated in the presence of plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies under T.sub.H1 (IL-2 (4 ng/ml)+IL-12 (5 ng/ml)+anti-IL-4 (1 μg/ml)) orT.sub.H2 (IL-2 (4 ng/ml)+IL-4 (4 ng/ml)+anti-IFN-γ (1 μg/ml)) conditions for 3 days in the presence or absence of MSCs. The cells were washed and then re-stimulated with PHA (2.5 μg/ml) for another 24 or 48 hours, following which levels of IFN-γ and IL-4 were measured in culture supernatants by ELISA (R&D Systems, Minneapolis, Minn.).

Analysis of Levels of VEGF, PGE.SUB.2 .and Pro-MIMP-1 in Culture Supernatant of MSCs

[0103] Using previously characterized human MSCs, the levels of Interleukin-6 (IL-6). VEGF, lipid mediator prostaglandin E.sub.2 (PGE.sub.2), and matrix metalloproteinase 1 (pro-MMP-1) were analyzed in culture supernatant of MSCs cultured for 24 hours in the presence or absence of PBMCs (MSC to PBMC ratio 1:10).

Proliferation of PBMCs

[0104] Purified PBMCs were prepared by centrifuging leukopack (Cambrex, Walkersville, Md.) on Ficoll-Hypaque (Lymphoprep, Oslo, Norway), Separated cells were cultured (in triplicates) in the presence or absence of MSCs (plated 3-4 hours prior to PBMC addition to allow them to settle) for 48 hours in presence of the mitogen PHA (Sigma Chemicals, St. Louis, Mo.). In selected experiments, PBMCs were resuspended in medium containing PGE.sub.2 inhibitors indomethacin (Sigma Chemicals, St. Louis, Mo.) or NS-938 (Cayman Chemicals, Ann Arbor, Mich.). (.sup.3H)-thymidine was added (20 μl in a 200 μl culture) and the cells harvested after an additional 24 hour culture using an automatic harvester. The effects of MSCs or PGE.sub.2 blockers were calculated as the percentage of the control response (100%) in presence of PHA.

Quantitative RT-PCR

[0105] Total RNA from cell pellets were prepared using a commercially available kit (Qiagen, Valencia, Calif.) and according to the manufacturer's instructions. Contaminating genomic DNA was removed using the DNA-free kit (Ambion, Austin, Tex.). Quantitative RT-PCR was performed on a MJ Research Opticon detection system (South San Francisco, Calif.) using OuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, Calif.) with primers at concentration of 0.5 μM. Relative changes in expression levels in cells cultured under different conditions were calculated by the difference in Ct values (crossing point) using β-actin as internal control. The sequence for COX-1 and COX-2 specific primers were: COX-1:5′-CCG GAT GCC AGT CAG GAT GAT G-3′(forward) (SEQ ID NO:1), 5′-CTA GAC AGC CAG ATG CTG ACA G-3′ (reverse) (SEQ ID NO:2); COX-2: 5′-ATC TAC CCT CCT CAA GTC CC-3′(forward) (SEQ ID NO:3), 5′-TAC CAG AAG GGC AGG ATA CAG-3′ (reverse) (SEQ ID NO:4).

[0106] Increasing numbers of allogeneic PBMCs were incubated with constant numbers of MSCs (2,000 cells/well) plated on a 96 well plate in the presence of PHA (2.5 μg/ml) for 72 hours, and .sup.3H thymidine incorporation (counts per minute, cpm) was determined. The PBMCs and MSCs were cultured at ratios of MSC:PBMC of 1:1, 1:3, 1:10, 1:30, and 1:81.

Results

[0107] In the present studies, the interaction of human MSCs with isolated immune cell populations, including dendritic cells (DC1 and DC2), effector T cells (T.sub.H1 and T.sub.H2) and NK cells was examined, The interaction of MSCs with each immune cell type had specific consequences, suggesting that MSCs may modulate several steps in the immune response process. The production of secreted factor(s) that modulate and may be responsible for MSC immuno-modulatory effects was evaluated and prostaglandin synthesis was implicated.

[0108] Myeloid (DC1) and plasmacytoid (DC2) precursor dendritic cells were isolated by immuno-magnetic sorting of BDCA1.sup.+ and BDCA2.sup.+ cells respectively and matured by incubation with GM-CSF and IL-4 (1×10.sup.3 IU/ml and 1×10.sup.3 IU/ml, respectively) for DC1 cells, or IL-3 (10 ng/ml) for DC2 cells, Using flow cytometry, DC1 cells were HLA-DR.sup.+ and CD11c.sup.+, whereas DC2 cells were HLA-DR.sup.+ and CD123.sup.+ (FIG. 1A). In the presence of the inflammatory agent bacterial lipopolysaccharide (LPS, ng/ml), DC1 cells produced moderate levels of TNF-α but when MSCs were present (ratios examined 1:1 and 1:10), there was >50% reduction in TNF-α secretion (FIG. 1B), On the other hand, DC2 cells produced IL-10 in the presence of LFS and its levels were increased greater than 2-fold upon MSC:DC2 co-culture (1:1) (FIG. 1B), Therefore, the MSCs modified the cytokine profile of activated DCs in culture towards a more tolerogenic phenotype. Additionally, activated DCs, when cultured with MSCs, were able to reduce IFN-γ and increase IL-4 levels secreted by naïve CD4.sup.+ T cells (FIG. 1C) suggesting a MSC-mediated shift from pro-inflammatory to anti-inflammatory T cell phenotype.

[0109] As increased IL-10 secretion plays a role in generation of regulatory cells (Kingsley, et al., J. Immunol., Vol. 168, pg. 1080 (2002)), T-regulatory cells (T.sub.Reg) were quantified by flow cytometry in co-cultures of PBMCs and MSCs. Upon culture of PBMCs with MSCs for 3-5 days, there was an increase in T.sub.Reg cell numbers as determined by staining of PBMCs with anti-CD4 and anti-CD25 antibodies (FIG. 2A), further supporting a MSC-induced tolerogenic response. The CD4.sup.+CD25.sup.+ T.sub.Reg cell population, generated in presence of MSCs expressed increased levels of gluocorticoid-induced TNF receptor (GTR), a cell surface receptor expressed on T.sub.Reg cell populations, and was suppressive in nature as it suppressed allogeneic T cell proliferation (FIG. 3A,B). Next, MSCs were investigated as to their direct ability to affect T cell differentiation, Using antibody selected purified T cells (CD4.sup.+ Th cells). IFN-γ producing T.sub.H1 and IL-4 producing T.sub.H2 cells were generated in presence or absence of MSCs. When MSCs were present during differentiation, there was reduced IFN-γ secretion by T.sub.H1 cells and increased IL-4 secretion by T.sub.H2 cells (FIG. 2B). No significant change in IFN-γ or IL-4 levels were seen when MSCs were added to the culture after Th cells had differentiated (at 3 days) into effector T.sub.H1 or T.sub.H2 types (data not shown). These experiments suggest that MSG's can affect effector T cell differentiation directly and alter the T cell cytokine secretion towards a humoral phenotype.

[0110] Similarly, when MSCs were cultured with purified NK cells (CD3-, CD14-, CD19-, CD36.sup.−) at a ratio 1:1 for different time periods (0-48 hrs), there was decreased IFN-γ secretion in the culture supernatant (FIG. 2C), thereby suggesting that MSCs can modulate NK cell functions also.

[0111] Previous work has indicated that MSCs modify T-cell functions by soluble factor(s) (LeBlanc, et al., Exp. Hematol., Vol. 31, pg. 890 (2003); Tse, et al., Transplantation, Vol. 75, pg, 389 (2003). It was observed that the MSCs secreted several factors, including IL-6, prostaglandin E.sub.2, VEGF and proMMP-1 constitutively, and the levels of each increased upon culture with PBMCs (FIG. 5). In order to investigate MSC-derived factors leading to inhibition of TNF-α and increase of IL-10 production by DCs, the potential role of prostaglandin E.sub.2 was investigated, as it has been shown to inhibit TNF-α production by activated DCs (Vassiliou, et al., Cell, Immunol., Vol. 223, pg. 120 (2003)). Conditioned media from MSC culture (24 hour culture of 0.5×10.sup.6 cells/ml) contained approx. 1000 μg/ml of PGE.sub.2 (FIG. 4A), There was no detectable presence of known inducers of PGE.sub.2 secretion, e.g., TNF-α, IFN-γ or 1β (data not shown) in the culture supernatant indicating a constitutive secretion of PGE.sub.2 by MSCs. The PGE.sub.2 secretion by hMSCs was inhibited 60-90% in the presence of known inhibitors of PGE.sub.2 production, NS-398 (5 μM) and indomethacin (4 μM) (FIG. 4A). As the release of PGE.sub.2 secretion occurs as a result of enzymatic activity of constitutively active cycloxygenase enzyme 1 (COX-1) and inducible cycloxygenase enzyme 2 (COX-2) (Harris, et al., Trends lmmunol., Vol. 23, pg. 144 (2002)) the mRNA expression for COX-1 and COX-2 in MSCs and PBMCs using trans-well culture system was analyzed, MSCs expressed significantly higher levels of COX-2 as compared to PBMCs and the expression levels increase >3-fold upon co-culture of MSCs and PBMCs (MSC to PBrvIC ratio 1:10) for 24 hours (FIG. 46). Modest changes in COX-1 levels were seen suggesting that the increase in PGE.sub.2 secretion upon MSC-PBMC co-culture (FIG. 5) is mediated by COX-2 up-regulation. To investigate whether the immunomodulatory effects of MSC on DCs and T-cells were mediated by PGE.sub.2, MSCs were cultured with activated dendritic cells (DC1) or T.sub.H1 cells in the presence of PGE.sub.2 inhibitors NS-398 or indomethacin. The presence of NS-3.98 or indomethacin increased TNF-α secretion by DC1s, and IFN-γ secretion from T.sub.H1 cells (FIG. 4C), respectively, suggesting that MSC effects on immune cell types may be mediated by secreted PGE.sub.2. Recent studies have shown that MSCs inhibit T-cell proliferation induced by various stimuli (DeNicola, et al., Blood, Vol. 99, pg. 3838 (2002); LeBlanc, et al., Scand. J. Immunol., Vol. 57, pg. 11 (2003)). It was observed that MSCs inhibit mitogen-induced T cell proliferation in a dose-dependent manner (FIG. 6) and when PGE.sub.2 inhibitors NS 398 (5 μM) or indomethacin (4 μM) were present, there was a >70% increase in CH) thymidine incorporation by PHA-treated PBMCs in MSC containing cultures as compared to controls without inhibitors (FIG. 4D).

[0112] In summary, a model of MSC interaction with other immune cell types (FIG. 7) is proposed. When mature T cells are present, MSCs may interact with them directly and inhibit the pro-inflammatory IFN-γ production (pathway 1) and promote regulatory T cell phenotype (pathway 3) and anti-inflammatory T.sub.H2 cells (pathway 5) Further, MSCs can alter the outcome of the T cell immune response through DCs by secreting FGE.sub.2, inhibiting pro-inflammatory DCI cells (pathway 2) and promoting anti-inflammatory DC2 cells (pathway 4) or regulatory DCs (pathway 3). A shift towards T.sub.H2 immunity in turn, suggests a change in B cell activity towards increased generation of IgE/IgG1 subtype antibodies (pathway 7). MSCs, by their ability to inhibit IFN-γ secretion from NK cells likely modify NK cell function (pathway 6). This model of MSC Immune cell interactions is consistent with the experimentation performed in several other laboratories (LeBlanc, et al., Exp. Hematol., Vol. 31, pg. 890 (2003); Tse, et Transplantation, Vol. 75, pg. 389 (2003); DiNicola, et al., Blood, Vol. 99, pg. 3838 (2002)). Further examination of the proposed mechanisms is underway and animal studies are now necessary to examine the in vivo effects of MSC administration

Example 2

[0113] Mesenchymal stem cells were given to a 33-year-old female patient suffering from severe Grade IV gastrointestinal graft-versus-host disease (GVHD). The patient was refractory to all other GVHD treatments. Endoscopic views of the patient's colon showed areas of ulceration and inflammation prior to treatment. Histology of the patient's colon showed that the graft-versus-host disease had destroyed the vast majority of the patient's intestinal crypts, prior to treatment.

[0114] The patient was given an intravenous infusion of allogeneic mesenchymal stem cells in 50 ml of Plasma Lyte A in an amount of 3×10 cells per kilogram of body weight.

[0115] The patient was evaluated at two weeks post-infusion. At two weeks post-infusion, an endoscopic view of the patient's colon showed that the areas of inflammation and ulceration visible prior to treatment were resolved. In addition, a biopsy of the patient's colon showed significant regeneration of intestinal crypts. Thus, the administration of the mesenchymal stem cells to the patient resulted in a significant reduction in the inflammatory component of gastrointestinal graft-versus-host disease, and resulted in the regeneration of new functional intestinal tissue.

[0116] The disclosures of all patents, publications, including published patent applications, depository accession numbers, and database accession numbers are hereby incorporated by reference to the same extent as if each patent, publication, depository accession number, and database accession number were specifically and individually incorporated by reference.

[0117] It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.