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  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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  • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
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  • A61K2039/515—Animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1352—Mesenchymal stem cells
  • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)

Definitions

• a process for preventing restimulation of activated T cells by contacting activated T cells with mesenchymal stem cells in an amount effective to prevent and/or reduce a subsequent T cell response to a foreign antigen.
• the mesenchymal stem cells that are used may be autologous to the T cells and/or allogeneic to the T cells.
• one method of the present invention provides contacting the recipient of donor tissue with mesenchymal stem cells.
• the method involves administering mesenchymal stem cells to the recipient of donor tissue.
• the mesenchymal stem cells can be administered to the recipient before or at the same time as the transplant or subsequent to the transplant.
• the mesenchymal stem cells may be autologous or may be allogeneic to the recipient and can be obtained from the donor.
• the allogeneic mesenchymal stem cells can also be obtained from a source other than the donor and such source need not be matched either to the donor type or the recipient type.
• the mesenchymal stem cells are modified to express a molecule that induces cell death.
• the mesenchymal stem cells can be used to deliver to the immune system a molecule that induces apoptosis of activated T cells carrying a receptor for the molecule. This results in the deletion of activated T lymphocytes and in the suppression of an unwanted immune response to a transplant.
• allogeneic mesenchymal stem cells are modified to express a cell death molecule.
• the molecule can be exogenous or endogenous to the mesenchymal stem cells.
• the mesenchymal stem cells express the cell death molecule Fas ligand or TRAIL ligand.
• the mesenchymal stem cells can also be administered to the recipient as part of the transplant.
• the present invention provides a method for reducing or ameliorating an immune response by providing to the recipient donor tissue or organ that is perfused with or includes mesenchymal stem cells obtained from the donor of the organ or tissue or mesenchymal stem cells from a third party or mesenchymal stem cells autologous to the T cells.
• the mesenchymal stem cells ameliorate an immune response by the recipient's T cells against the foreign tissue when it is transplanted into the recipient.
• the mesenchymal stem cells can be obtained from the recipient, for example, prior to the transplant.
• the mesenchymal stem cells can be isolated and stored frozen until needed.
• the mesenchymal stem cells may also be culture- expanded to desired amounts and stored until needed.
• the mesenchymal stem cells are administered to the recipient in an amount effective to reduce or eliminate an ongoing adverse immune response caused by the donor transplant against the recipient (host).
• the presentation of the mesenchymal stem cells to the recipient undergoing an adverse immune response caused by a transplant inhibits the ongoing
• a further embodiment includes modifying the recipient's mesenchymal stem cells with a molecule that induces activated T cell death.
• Fig. 4 shows the secondary response of responder T cells activated by stimulator (allogeneic) PBMCs, and subsequently cultured with allogeneic MSCs (stimulator) and then exposed to autologous PBMCs, allogeneic PBMCs (stimulator or third party) or no cells.
• stimulator allogeneic
• Fig. 6 shows the suppression of a primary MLR in the canine model by MSCs.
• autol autologous;
• ident DLA identical litter mates;
• unrel unrelated.
• Fig. 7 shows the suppression of a primary MLR by non-adherent MSCs.
• Fig. 8 shows a schematic map of EGFP pOT24 plasmid used in Example 8.
• Fig. 9 shows the suppressive effect of MSC supernatants generated from human mesenchymal stem cells or human mesenchymal stem cell-suppressed mixed lymphocyte reaction cultures on a primary mixed lymphocyte reaction.
• Fig. 10 shows the suppressive effect of MSC supernatants generated from human mesenchymal stem cells or human mesenchymal stem cell-suppressed mixed lymphocyte reaction cultures on an ongoing mixed lymphocyte reaction.
• Fig. 11 shows the suppression by baboon mesenchymal stem cells of a mixed lymphocyte reaction between human responder T cells and human stimulator PBMC cells.
• Fig. 12 shows the suppression by baboon or human mesenchymal stem cells of a mixed lymphocyte reaction between human responder T cells and baboon stimulator PBMC cells (donor 5957).
• an allogeneic mesenchymal stem cell is obtained from a different individual of the same species as the recipient.
• Donor antigen refers to antigens expressed by the donor tissue to be transplanted into the recipient. Alloantigens are antigens which differ from antigens expressed by the recipient.
• Donor tissue, organs or cells to be transplanted is the transplant. As examples of transplants may be included skin, bone marrow, and solid organs such as heart, pancreas, kidney, lung and liver.
• an alloantigen is an antigen that is foreign to the recipent.
• the inventors have discovered that when mesenchymal stem cells are contacted with allogeneic T lymphocytes, in vitro, the allogeneic T cells do not proliferate. Normally, co-culturing cells from different individuals results in a T cell response, manifested by activation and proliferation of the T cells, known as a mixed lymphocyte reaction (MLR).
• MLR mixed lymphocyte reaction
• mesenchymal stem cells can suppress an MLR between allogeneic cells.
• Mesenchymal stem cells actively reduced the allogeneic T cell response in mixed lymphocyte reactions in a dose dependent manner.
• mesenchymal stem cells from different donors did not exhibit specificity of reduced response with regard to MHC type.
• mesenchymal stem cells did not need to be MHC matched to the target cell population in the mixed lymphocyte reaction in order to reduce the proliferative response of alloreactive T cells to mesenchymal stem cells.
• the mesenchymal stem cells can also be xenogeneic to the responder or stimulator cells or both.
• supernatants derived from mesenchymal stem cell cultures can suppress an MLR between allogeneic cells.
• supernatants derived from mesenchymal stem cell cultures also referred to herein as "MSC supernatant”
• MSC supernatant can be obtained from mesenchymal stem cells cultured alone or mesenchymal stem cells co-cultured with cell undergoing an immune response, i.e. T cells undergoing a mixed lymphocyte reaction.
• Mesenchymal stem cell supernatants actively reduced the allogeneic T cell response in mixed lymphocyte reactions in a dose dependent manner. As with mesenchymal stem cells, supernatants from mesenchymal stem cell cultures from different donors did not exhibit specificity of reduced response with regard to MHC type.
• a soluble factor or compound may be secreted into the mesenchymal stem cell culture medium that has a suppressive effect on mixed lymphocyte reactions.
• a stronger suppressive effect is seen using supernatants from MSCs exposed to a mixed lymphocyte reaction.
• the present invention provides a method of reducing, inhibiting or eliminating an immune response by administering allogeneic mesenchymal stem cells to a recipient of a donor tissue, organ or cells.
• the mesenchymal stem cells are administered to the recipient contemporaneously with the transplant.
• the mesenchymal stem cells can be administered prior to the administration of the transplant.
• the mesenchymal stem cells can be administered to the recipient about 3 to 7 days before transplantation of the donor tissue.
• the cells may be administered subsequent to the transplant.
• mesenchymal stem cells can be used to condition a recipient's immune system to donor or foreign tissue by administering to the recipient, prior to, or at the same time as transplantation of the donor tissue, mesenchymal stem cells in an amount effective to reduce or eliminate an immune response against the transplant by, for example, the recipient's T cells.
• the mesenchymal stem cells affect the T cells of the recipient such that the T cell response is reduced or eliminated when presented with donor or foreign tissue.
• host rejection of the transplant may be avoided or the severity thereof reduced.
• T lymphocytes that have already been exposed to antigenic stimulation i.e. are activated
• mesenchymal stem cells the T cells do not produce an immune response or produce a reduced immune response, to subsequent antigenic stimulation by allogeneic cells.
• mesenchymal stem cells induce a state of hyporesponsiveness of the T cells.
• activated T cells were made non-responsive to further allogeneic stimulation by exposure of preactivated T cells to mesenchymal stem cells.
• the mesenchymal stem cells can be autologous or allogeneic to the T cells.
• the present invention provides a method for treating a patient who is undergoing an adverse immune response to a transplant by administering mesenchymal stem cells to such patient in an amount effective to reduce or suppress the immune response.
• the mesenchymal stem cells are obtained from the tissue donor, the transplant recipient or a third party.
• the MSCs may be xenogeneic to the donor, the recipient or both.
• the mesenchymal stem cells may further be modified to express a cell death molecule to enhance the elimination of activated T cells.
• the cell death molecule may be
• mesenchymal stem cells which have been engineered to express the exogenous cell death molecule.
• the present invention provides a method to reduce or inhibit or eliminate an immune response by a donor transplant against a recipient thereof (graft versus host). Accordingly, the invention provides contacting a donor organ or tissue with mesenchymal stem cells prior to transplant. The mesenchymal stem cells ameliorate, inhibit or reduce an adverse response by the donor transplant against the recipient.
• the donor transplant prior to transplant the donor transplant is treated with allogeneic (recipient) tissue or cells which activate the T cells in the donor transplant.
• the donor transplant is then treated with mesenchymal stem cells, autologous or allogeneic, prior to transplant.
• the mesenchymal stem cells prevent restimulation, or induce hyporesponsiveness, of the T cells to subsequent antigenic stimulation.
• the mesenchymal stem cells may be further modified to express a cell death molecule such that activated T cells contacted with the mesenchymal stem cells will be eliminated.
• Subsequent treatment with the mesenchymal stem cells inhibits or eliminates further activation of the T cells in the marrow, thereby reducing or eliminating an adverse affect by the donor tissue, i.e. the therapy reduces or eliminates graft versus host response.
• a transplant recipient suffering from graft versus host disease may be treated to reduce or eliminate the severity thereof by administering to such recipient mesenchymal stem cells autologous or allogeneic to the donor, which allogeneic cells can be mesenchymal stem cells autologous to the recipient or third party mesenchymal stem cells, in an amount effective to reduce or eliminate a graft rejection of the host.
• the mesenchymal stem cells inhibit or suppress the activated T cells in the donor tissue from mounting an immune response against the recipient, thereby reducing or eliminating a graft versus host response.
• the donor tissue is exposed to mesenchymal stem cells such that the mesenchymal stem cells integrate into the organ graft itself prior to transplantation.
• mesenchymal stem cells are preferably allogeneic to the recipient and may be donor mesenchymal stem cells or mesenchymal stem cells obtained from other than the donor or recipient.
• mesenchymal stem cells autologous to the recipient may be used to suppress an immune response against the graft.
• the mesenchymal stem cells are engineered to express cell death molecules such that any alloreactive host T cells will be eliminated upon contact with these mesenchymal stem cells.
• the mesenchymal stem cells remaining in the local site would also suppress any subsequent T cell response that may occur.
• a "cell death molecule” is a molecule that interacts or binds with its cognate receptor on a stimulated T cell inducing T cell death or apoptosis.
• the Fas system has been shown to be involved in a number of cell functions in vivo including negative selection of thymocytes, maintaining immune privilege sites within the body, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity (Green and Ware, Proc Natl Acad Sci, 94(12):5986-90 (1997)).
• CTL cytotoxic T-lymphocyte
• TRAIL tumor necrosis factor receptor
• DR4 tumor necrosis factor receptor
• CD27 and CD70 proc Natl Acad Sci, 94:6346-6351 (1997)
• Fas expression is restricted to stimulated T cells and sites of immune privilege.
• TRAIL is detected in many normal tissues. Both Trail-ligand and CD27, but not Fas-ligand, are expressed on unmanipulated human mesenchymal stem cells.
• the mesenchymal stem cells of the present invention can be used in conjunction with current modes of treating donor tissue rejection or graft versus host disease.
• An advantage of such use is that by ameliorating the severity of the immune response in a transplant recipient, the amount of drug used in treatment and/or the frequency of administration of drug therapy can be reduced, resulting in alleviation of general immune suppression and unwanted side effects.
• mesenchymal stem cells of the present invention may be required, eliminating the need for chronic immunosuppressive drug therapy.
• multiple administrations of mesenchymal stem cells may be employed.
• the invention described herein provides for preventing or treating transplant rejection by administering the mesenchymal stem cells in a prophylactic or therapeutically effective amount for the prevention or treatment or amelioration of transplant rejection of an organ, tissue or cells from the same species, or a xenograft organ or tissue transplant and or graft versus host disease.
• Administration of a single dose of mesenchymal stem cells may be effective to reduce or eliminate the T cell response to tissue allogeneic to the T cells or to "non- self tissue, particularly in the case where the T lymphocytes retain their nonresponsive character (i.e., tolerance or anergy) to allogeneic cells after being separated from the mesenchymal stem cells.
• the dosage of the mesenchymal stem cells varies within wide limits and will, of course be fitted to the individual requirements in each particular case. In general, in the case of parenteral administration, it is customary to administer from about 0.01 to about 5 million cells per kilogram of recipient body weight. The number of cells used will depend on the weight and condition of the recipient, the number of or frequency of administrations, and other variables known to those of skill in the art.
• the mesenchymal stem cells can be administered by a route which is suitable for the tissue, organ or cells to be transplanted. They can be administered systemically, i.e., parenterally, by intravenous injection or can be targeted to a particular tissue or organ, such as bone marrow.
• the human mesenchymal stem cells can be administered via a subcutaneous implantation of cells or by injection of stem cell into connective tissue, for example muscle.
• the cells can be suspended in an appropriate diluent, at a concentration of from about 0.01 to about 5 x 10 6 cells/ ml.
• Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the cells and with the recipient, such as buffered saline solution or other suitable excipients.
• the composition for administration must be formulated, produced and stored according to standard methods complying with proper sterility and stability.
• mesenchymal stem cells can be isolated, preferably from bone marrow, purified, and expanded in culture, i.e. in vitro, to obtain sufficient numbers of cells for use in the methods described herein.
• Mesenchymal stem cells the formative pluripotent blast cells found in the bone, are normally present at very low frequencies in bone marrow (1 :100,000) and other mesenchymal tissues. See, Caplan and Haynesworth, U.S. Patent No. 5,486,359. Gene transduction of mesenchymal stem cells is disclosed in Gerson et al U.S. Patent No. 5,591,625.
• Example 1 Absence of Alloreactivity of Mesenchymal Stem Cells The mixed lymphocyte reaction measures the compatibility of the donor's surface antigens and is an indication of the likelihood of rejection of donor tissue.
• Cell surface antigens responsible for eliciting transplant rejection are class I and class II MHC antigens.
• T cells are alloreactive to foreign MHC antigens.
• Class I and II MHC molecules stimulate the mixed lymphocyte reaction.
• T A T cells from individual A
• the mPBMC ⁇ S were seeded at 20K and 100K.
• the cultures were pulsed with 3 H-thymidine for the last 18 hours of the culture period to measure T cell proliferation.
• the results shown in Figure 1 indicate that the T A cells recognized the PBMC B as being foreign. (See bars under "T A + mPBMC B ".) With more PBMC B s present, the more the T cells proliferated.
• hMSCs human mesenchymal stem cells
• T A human mesenchymal stem cells
• the cells were cultured in flat-bottom microtiter wells for a total of 7 days. Cultures were pulsed with 3 H-thymidine for the last 18 hours of the culture period to measure T cell proliferation. Two days prior to coculture with the T cells, the human mesenchymal stem cells were seeded into microtiter wells at the number given above (confluent) and treated with IFN- ⁇ (50 units/ml) to stimulate surface antigen expression on MSCs. Non-transduced hMSCs or hMSCs transduced with human B7-1 or human B7-2 costimulation molecules were incubated with the T cells. Control cells were transduced with Neo.
• Fig. 1 See Figure 1 "T A + transduced hMSCs" demonstrate that the T lymphocytes were nonresponsive (did not proliferate) to the human mesenchymal stem cells, i.e., they were not recognized as being foreign.
• MLR mixed lymphocyte reactions
• PBMC A 10 5 PBMCs from individual A
• PBMC B 10 5 target individual B's PBMCs
• the PBMC B s were irradiated with 3000 rads X irradiation to prevent their proliferation due to activation by PBMC A S. Thus, only PBMC A S would proliferate.
• PBMC A S and PBMC B s were mixed, a mixed lymphocyte reaction occurred wherein the PBMC A cells (responder cells) were activated by the surface antigens on the PBMC B s (stimulator cells).
• the cultures were incubated over an interval of 7 days and were pulsed with 3 H-thymidine during the final 18 hours.
• the PBMC A S proliferated giving counts of 40,000. See Figure 2, 1st bar, ("NONE" refers to no mesenchymal stem cells present.).
• PBMC A S and PBMC B s were mixed in the presence of mesenchymal stem cells, the mixed lymphocyte reaction was suppressed.
• 10 5 PBMC A S were mixed with 10 5 PBMC B s in microtiter plate wells coated with an adherent monolayer of human mesenchymal stem cells.
• the mesenchymal stem cells were plated in the wells in amounts ranging from 7500 to 22,500 mesenchymal stem cells per well.
• Two mesenchymal stem cell populations were tested: human mesenchymal stem cells were obtained from an individual B and human mesenchymal stem cells were obtained from an individual that did not match either individual A's or B's MHC type (a third party).
• the cultures were incubated over an interval of 7 days and were pulsed with H-thymidine during the final 18 hours.
• the MLR was suppressed. See Figure 2.
• the mesenchymal stem cells suppressed the mixed lymphocyte reaction.
• mesenchymal stem cells actively suppressed the mixed lymphocyte reaction when the cells were cultured together.
• T cells from donor 248 were primed by allogeneic PBMCs from donor 273 (d 273) for 7 days, then cultured for 3 additional days alone or in the presence of IFN- ⁇ -treated MSCs from the same donor (d273). Cells were then re- stimulated by the same donor (d273), autologous (d248) or "third party" (d244) PBMCs.
• PBMC Peripheral blood mononuclear cells
• T cells were activated by irradiated PBMCs (d 273).
• PBMCs used for stimulation were X-ray irradiated with 3,000 rad using Cabinet X ray system (Faxitron X ray, Buffalo Grove, IL).
• 2 x 10 7 responders were mixed with 2 x 10 7 stimulators in 20 mis assay medium in 10 cm tissue culture dishes. The cells were incubated at 37°C in 5% CO 2 atmosphere for 7 days.
• T cells activated in the 1° MLR were collected, washed once with MSC medium and re-suspended in assay medium at 10 6 /ml in 10 ml and were added to 10 cm tissue culture dishes containing autologous or allogeneic MSCs or medium alone, and incubated for an additional 3 days.
• T cells cultured with MSCs or media were collected, washed once with MSC media, and restimulated with irradiated PBMCs from the original donor, an unrelated donor or autologous PBMCs.
• 5 x 10 4 primed responders and 5 x 10 4 irradiated stimulators were incubated in 96-well plates. Assays were performed in triplicate. Cultures were pulsed with 1 ⁇ Ci of [ H] thymidine (Amersham) for 18 hours before harvesting. Cultures were collected using Harvester 96 (Tomtec), filters were analyzed using Microbeta Trilux liquid scintillation and luminescence counter (E.G.&G Wallac). Data are presented as mean cpm ⁇ SD of three replicates.
• T cells cultured alone showed an accelerated response to "same donor” re-stimulation with peak at day 2.
• “Third party” response was also accelerated, practically with the same kinetics as “same donor”, but with a lower maximum and a slightly delayed start. (Fig 3).
• T cells cultured on allogeneic MSCs subsequently showed no response either to "same donor” or "third party” PBMCs during 6 days of culture (Fig.4).
• T cells preactivated in the MLR for 7 days were incubated alone or with MSCs for an additional 3 days (1.0 x 10 6 /ml T cells, 10 ml/dish). After 3 days of incubation with MSCs, T cells were collected and re-stimulated with irradiated PBMC 273 (original donor), 413 (autologous), PBMC 10 (third party) or PHA (5 ⁇ g/ml) in the presence or absence of autologous (d413) PBMC. Cells were added at 5 x 10 4 /well, cultures were pulsed with [ 3 H]thymidine at indicated time points for an additional 18 hours. The results indicate that treatment of activated T cells with autologous (d413)
• Example 5 Suppression of Primary MLR by Canine MSCs
• Canine PBMCs were purified from peripheral blood by centrifugation on Ficoll-Paque gradient (1.077).
• Stimulator PBMCs were X-ray irradiated 2200 rad (7 min 70 kV).
• 10 5 irradiated stimulators were mixed with 10 5 responder PBMCs in 96- well plates in the presence or absence of pre-plated canine MSC (E647, 2 x 10 4 /well). Cultures were incubated for 6 days and pulsed with [ H]TdR (5Ci/mmol, l ⁇ Ci/well) for an additional 16 hours. Results are shown in Figures 6A-6D.
• E647 and E645 were litter mates ( DLA identical). The results showed that autologous as well as allogeneic MSCs suppressed the primary MLR.
• Example 6 Suppression of Primary MLR by Non- Adherent MSCs
• T cells from d273 (2 x 10 5 /well) were mixed with irradiated PBMCs from d244 (2 x 10 5 /well) and different numbers of MSCs.
• MSCs from dD244 or d273 were pre-treated with IFN- ⁇ (900 U/ml for 3 days) or left untreated, trypsinized on the day of experiment and added at the same time as T cells and PBMCs.
• Cultures were incubated for 7 days,[ 3 H]TdR (5Ci/mmol, 1 ⁇ Ci/well) was added for an additional 16- 18 hours. Results are shown in Figure 7 and demonstrate that non-adherent MSCs also suppressed a primary MLR.
• Juvenile baboons (papio anubis) were studied. Male and non-pregnant female baboons weighed 7-20 kg and were between 3-16 years of age. They were screened for tuberculosis papilloma virus, titered for cytomegalovirus (CMV), and tested with the primate viral screen consisting of testing for simian virus, and including fecal floatation and smears. Donor and recipient pairs were determined by major histocompatibility complex (MHC)-disparity through PCR typing. During the study period, the baboons were housed in an individual area beside a companion animal.
• MHC major histocompatibility complex
• Needle marrow aspirates were obtained from the iliac crest for isolation and culture-expansion of the MSCs.
• the marrow aspirate was obtained from an alternate side once a week for four consecutive weeks.
• the volume of the aspirate was determined by an estimate of 10% of the animal's blood volume. Blood volume
• cefazolin Prior to the procedure, 500 mg cefazolin was administered intramuscularly (IM) for perioperative antibacterial prophylaxis.
• Baboons were sedated and anesthetized for the procedure with ketamine at 10 mg/kg IM, and xylazine 1 mg/kg IM.
• the sites of needle insertion were scrubbed with povidone-iodine and then rinsed with alcohol.
• Aspirates were obtained from the iliac crest using a 16-gauge, 2-inch bone marrow needle. A syringe was attached to the needle, and suction was applied to remove the marrow.
• the analgesic Buprenorphine was given at 0.03 mg/kg IM Q12 x 2 doses.
• DPBS Phosphate Buffered Saline
• the media in the triple flasks was decanted, and the flasks were rinsed with 50 ml DPBS. After decanting the DPBS, 23 ml of 0.05% trypsin was added to each triple flask. The flasks were placed in a 37°C incubator for 3 minutes. After cell detachment, 23 ml complete medium was added to each flask. The cell suspensions were transferred to 50 ml conical tubes and the flasks were washed with 30 ml HBSS.
• the tubes were centrifuged at 2200 RPM for 5 minutes at RT.
• the harvested MSCs were formulated at approximately 10 x 10 6 cells per ml cryoprotectant solution consisting of 85% Plasma-Lyte A (Baxter IV Therapy), 10%)
• the final product was prepared at 115% of the dose required on infusion day.
• baboon Prior to surgery the baboon was given cefazolin at 500 mg IM as an antibacterial prophylaxis perioperatively. The baboon was sedated and anesthetized with ketamine at 10 mg/kg IM and intravenous Thiopental induction, a 1-2% isofluorane inhalational anesthetic. Skin was harvested from the anterior abdominal wall and placed on a pre-labeled moistened saline gauze pad. This skin was divided into two grafts; one was used as the third party control for another recipient baboon and one was used as an autologous control for this same animal. The animal was then placed in a prone position.
• the analgesic Buprenorphine was administered at Q12 x 2 doses and Ancef daily for 2 days. The animal was observed daily, and the grafts were photographed every other day beginning on post-graft day 7.
• Each baboon was sedated with ketamine lOmg/kg IM for examination. While sedated, two-three milliliters of marrow were obtained from the iliac crest by needle aspiration and collected in sodium heparin on days 4, 13, and 30, the end of study. A skin biopsy was harvested on the same day that marrow aspirates were obtained.
• EGFP-1 gene from the jellyfish Aequorea victoria (Clontech, CA). EGFP gene was cloned into retroviral vector pJM573-neo (resulting plasmid was named pOT-24).
• the plasmid pJM573-neo was derived from pN2 (Keller et. Al, 1985, Nature 318:149) with the following modifications: murine retroviral gag initiation site was substituted with an in-frame stop codon; 5'LTR and 3'LTR were constructed into the same cassette; neomycin phosphotransferase gene (neo) and an internal ribosomal entry site (IRES) were inserted into pN2.
• a schematic map of EGFP pOT24 plasmid is shown in Figure 8.
• Amphotropic retrovirus was prepared by transducing PA317 cells twice with ecotropic virus using a centrifugal transduction procedure followed by selection with G418 (0.5 mg/ml). Retroviral supernatant was collected. The titer of the pooled EGFP retrovirus on 3T3 cells was 1.2xl0 6 CFU/ml. GFP-retroviral supernatants were cryopreserved at -70°C.
• the washed mononuclear cells obtained at the Percoll interface were established in 10, T-185 flasks containing 30 ml of complete media and 10 x 10 6 cells/flask.
• the supernatant was removed and the cell pellets were resuspended in complete media.
• the cells were pooled, counted and examined for viability.
• Cells were plated into 15, T80 flasks containing 18 ml of complete medium and 0.4 x 10 6 cells per flask. On day 15 in culture, the first transduction was performed on 15 of the 18 flasks. The media was removed. Aliquots of the retroviral supernatant were thawed and polybrene was added to a final concentration of 8 ⁇ g/ml to make the transduction cocktail.
• the cell medium was replaced with 10 ml of the transduction cocktail, and the flasks were centrifuged at 3000 RPM for 1 hour at 32°C.
• the cells were harvested as described above and taken from PI to P2.
• Three x 10 6 cells were added to 100 ml of complete medium, and poured into triple-flasks (500 cm ).
• Fifteen triple-flasks were prepared with transduced cells and three were prepared with untransduced cells. Any remaining cells were cryopreserved.
• a freeze solution was prepared containing 10% DMSO and 90% FBS.
• Ten x 10 6 cells were resuspended in 1 ml of freezing solution. The vials were labeled and cryopreserved in a Nalgene Cryo container for a minimum of 4 hours at -70°C, and stored at -70°C.
• the washed mononuclear cells obtained at the Percoll interface were established in 15, T-75 flasks containing 20 ml of complete media and 12 x 10 6 cells/flask. On day 2 of culture, the media in the flasks and in the dishes was replaced entirely with fresh complete media. On day 6 of primary culture for cMSC, the first transduction was performed as described above. Three flasks were not transduced, and fresh media was replaced on day 6. On day 7 of culture, the media was replaced with fresh media.
• the transduced cMSC yield per flask for donors CAN-07-01 and CAN-07-02, harvested 4 days after passage 2 and plated at 3 x 10 6 per flask was 5.9 and 6.7 x 10 6
• the untransduced cMSC yield per flask was 8.4 and 7.5 x 10 6
• the transduced cMSC yield per flask for donors CAN-07-03 and CAN-07-04, harvested 4 days after passage 1 (different transduction and passage design) and plated at 3 x 10 6 per flask was 20.0 and 14.0 x 10
• the untransduced cMSC yield per flask was 25.3 and 18.0 x 10°.
• CFU Assays on cMSCfrom PO Cultures CFU colony assays were prepared at the time of primary culture establishment by plating 0.5 x 10 6 cells in triplicate in 100 mm dishes containing 10 ml complete media. The dishes were incubated at 37°C and 5% CO 2 . The media was replaced with fresh media each 2 to 4 days. On day 10 in culture, the CFU assay dishes were rinsed with HBSS twice, fixed with 1% gluteraldehyde for 15 minutes, rinsed with HBSS twice, and air dried. The cMSC in the dishes were then stained with 0.1 % crystal violet, rinsed with deionized water three times, and air dried. Colonies were counted to calculate the number of colonies forming per 10 6 cells plated.
• CFU assays plated on day of mononuclear cell isolation and culture establishment and harvested on day 10 yielded 56, 46.7, 114, and 72 colonies per 10 cells for dogs CAN-07-01, CAN-07-02, CAN-07-03, and CAN-07-04, respectively.
• the media in the triple flasks was decanted, and the flasks were rinsed with 50 ml DPBS. After decanting the DPBS, 23 ml of 0.25% trypsin was added to each triple flask. The flasks were placed in a 37°C incubator for 3 minutes. After cell detachment, 23 ml complete medium was added to each flask. The cell suspensions were transferred to 50 ml conical tubes and the flasks were washed with 30 ml HBSS. The tubes were centrifuged at 2200 RPM for 5 minutes at RT. The pellets containing the transduced or untransduced cells, respectively, were pooled and counted.
• Blood samples (2 ml) were obtained before (pre) and during the cMSC infusion at five and fifteen minutes after the start of the infusion, as well as 1- and 2- hour, and 1-, 2-, 3-, and 4-day time points.
• Cell lysates were prepared using the PuregeneTM (Gentra Systems, Inc.) DNA Isolation Kit for use in an Anti EGFP DNA PCR Elisa with digoxigenin incorporated into the product and a second step Anti- digoxigenin colorimetric assay to detect of the level of GFP marked cMSC in the bloodstream.
• Bone marrow to be used as the transplant graft was harvested from the DLA- identical littermate prior to TBI. Aspirates were obtained from each humerus using an 11 -gauge, 4-6 inch ball-top stainless steel marrow harvest needle, attached to polyvinyl tubing originating from a vacuum flask containing 100 ml Tissue Culture Medium 199 and 4 ml (4000 U) heparin. The marrow is passed through 300- and 200-um pore size, and stored at 4°C in a transfer pack container, labeled with the donor and recipient, until infusion later that day. The bone marrow total nucleated cell count (BM-TNC) of the marrow is corrected to exclude any nucleated cells which would be present in the volume of peripheral blood obtained during the marrow harvest.
• BM-TNC bone marrow total nucleated cell count
• the total nucleated cell count (TNC) of the bone marrow was corrected to exclude any TNC which would be present in the volume of peripheral blood obtained during the marrow harvest.
• Corrected doses of marrow were 4.3, 3.5, 3.1, and 2.0 (mean 3.2) x 10 8 TNC/kg to dogs CAN-07-01, CAN-07-02, CAN-07-03, and CAN- 07-04, respectively.
• Uncorrected bone marrow doses were 5.6, 4.2, 4.5, and 2.7 (mean 4.3) x lO 8 TNC/kg.
• the marrow was placed at room temperature.
• the marrow was infused intravenously through a butterfly needle inserted into the cephalic vein, by exerting pressure on the bag over 1 to 2 minutes.
• oral antibiotics neomycin sulfate and polymyxin sulfate
• these oral antibiotics were administered until absolute neutrophil counts reached 500/mm .
• the systemic antibiotic Baytril was administered intravenously twice daily and continued until absolute neutrophil counts reached 1 ,000/mm 3 consistently. Fluid and electrolytes lost as results of transient radiation toxicity were replaced by subcutaneous administration of 500 ml of Ringers Solution, twice daily until food and water were accepted.
• the tissues were trimmed to about 1 inch square pieces and placed into separate labeled 50 ml conical tubes filled with 10% Neutral Buffered Formalin (pH 6.8 - 7.2).
• the tissues were embedded in paraffin, sectioned and stained with Hematoxylin and Eosin. Bone marrow samples were stained with Periodic Acid Schiff s stain.
• Bone marrow aspirates obtained prior to necropsy were collected in 15 ml labeled tubes from the left and right humieri, femora, and iliac crests from each dog. A subset of the tissue samples were obtained during necropsy and trimmed to about 1/4 inch square pieces, wrapped in PBS-soaked gauze and placed separately in a labeled zip-lock bag. The bone marrow aspirates were held on ice.
• PBS Phosphate Buffered Saline
• the tubes were centrifuged and the supernatant was drained off and the pellets were allowed to dry for approximately 1 to 6 hrs.
• the DNA was allowed to hydrate overnight at room temperature and was subsequently stored at 4°C.
• Peripheral blood and bone marrow samples were first lysed with RBC lysis solution (Ammonium Chloride Buffer). DNA was then isolated from the lysates as described above. DNA was quantified by the addition of 998 ⁇ l deionized H 0 and 2 ⁇ l DNA from the sample into a cuvette and vortexed. A spectrophotometer was used to determine the optical density (OD). The OD was read at 260 and 280, and the concentration of DNA was calculated for ⁇ g/ml. The DNA concentration was adjusted to 1 ⁇ g/ml using deionized water.
• the anti-EGFP DNA PCR Elisa assay used in these studies detects infused cMSCs utilizing oligonucleotide primers specific for GFP.
• PCR-ELISA DIG labeling/detection
• PCR was performed in the presence of digoxigenin-labeled nucleotides to label the amplified product.
• 25 ⁇ l of the PCR product was denatured and allowed to hybridize in solution to 5'-biotinylated oligonucleotide probe at 37°C in streptavidin-coated microtiter plate.
• the bound probe-PCR product was detected by an anti-digoxigenin peroxidase conjugate and by use of the colorimetric substrate 2,2'Azinobis (3-ethylbenzthiazoline-sulfonic Acid) (ABTS). Titration standard curves were generated using transfected control cMSC to approximate concentration of DNA per quantity of DNA used in the assay. By first correlating with an internal standard for PCR of the DLA Class II genomic DNA, and then correlating DNA concentration to cell equivalents, and assuming one retrovirus integration event per transduced cell, an estimation of cell number can be obtained.
• the mean day to a threshold (to 3 consecutive values) of platelets to 10,000/mm 3 was 12.8 (range 1 1-17), to 50,000/mm 3 was 19.8 (range 16-25), and to 100,000/mm 3 23.0 (range 20-27).
• the mean day to a threshold value (to 3 consecutive days) of absolute neutrophil cells to 500/mm 3 was 9.3 (range 8-11), and to 1,000/mm 3 was 10.5 (range 9-13).
• Detectable DNA signal could be found within 1 hour of infusion and again at 2 days.
• One sample could be quantitatively measured at 3 days post infusion for GFP DNA. This timepoint is consistent with the previous observations in the autologous canine transplant study in which signal was found at 2 and 3 days
• T cells from donor 155 were purified from PBMC by negative immunomagnetic selection with anti-CD 19 and anti-CD 14 MicroBeads (Miltenyi Biotec).
• PBMC from donor 413 were X-ray irradiated with 3600 rad (12 min at 70 kV).
• MSCs from different donors (219, 459, 461 - all at passage 5) were added into the cultures at 1.2 xlO 5 cells/well for 3 additional days. In control cultures, the same volume of medium was added instead of MSCs.
• the same number of MSCs were plated alone, and cultured for 3 additional days. After 3 days of culture (on day 6 after initiation of the primary MLR), the cells were resuspended by pipetting, and 200 ⁇ l of the cell suspensions were transferred into a 96-well plate in triplicate, and pulsed with [H 3 ]TdR (5 Ci/mmol, 1 ⁇ Ci/well) for 18 hours to determine the level of proliferation. The remaining cells were centrifuged at 1250 rpm for 10 minutes, the supernatants were collected, aliquoted and kept frozen at -80°C.
• T cells from donor 155 were purified from PBMCs by negative immunomagnetic selection with anti-CD 19 and anti-CD 14 MicroBeads (Miltenyi Biotec). PBMCs from donor 413 or from donor 273 were X-ray irradiated with 3600 rad (12 min at 70 kV). In 96-well tissue culture plates T cells (1.5 x 10 5 /well) were mixed with PBMCs (1.5 x 10 5 /well) in the presence or absence of different supernatants that were added at the initiation of cultures at the dilutions (1/8, 1/32, 1/128, 1/512, 1/2048 and 1/8192).
• MLR +MSC459 (#3) MLR +MSC461, and MSCs alone, (#8) MSC219 alone (#9)
• Lymphoproliferation was determined on day 7 of culture by pulsing the cells with 3 H-thymidine for the final 18 hours prior to cell harvest for scintillation counting.
• the results illustrated in Figure 11 show that baboon MSCs suppressed robust human MLRs by greater than 50%.

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