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Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

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Cytochalasin B (CB), administered i.p. to C57B1/6 mice in a single dose as a suspension in carboxymethylcellulose 2%/Tween 20 1%, inhibits in a dose-dependent and time-dependent manner the ability of spleen cells to respond to allogeneic P815 mastocytoma tumor cells in vitro. Spleen cells from CB-treated animals sensitized to X-irradiated P815 cells in 4-day cultures at a 50:1 responder:stimulator ratio and tested for specific cytotoxicity against 51Cr-labelled P815 target cells showed strong inhibition 3 h after CB treatment at a dose of 50 mg/kg. A dose of 25 mg/kg showed measureable but not statistically significant inhibition at 3 h, whereas 10 mg/kg produced only slight inhibition, and 5 mg/kg and 2 mg/kg were noninhibitory. None of the doses produced significant suppression 19 h or 72 h after CB treatment. Addition to the sensitization cultures of human recombinant interleukin-2 (rhIL-2) at 350 BRMP units/ml completely restored tumor lytic capacity. C57B1/1 mice treated with CB 50 mg/kg, i.p. and challenged i.p. with 3 x 10(7) allogeneic P815 mastocytoma cells showed a brief, time-dependent, statistically significant abrogation of allogeneic responsiveness consistent with transient reversible immunosuppression within 3-12 h following CB treatment. No such inhibition of host allogeneic responsiveness in vivo was observed when CB was administered 24 h prior to, simultaneously with, or 1, 2, or 4 days after tumor challenge. Thus CB at the highest tolerated i.p. dose in vivo causes only a transient inhibition of anti-allo-responsiveness measured in culture, and rhIL-2 used in vitro restores lytic capacity. The anti-allo effect of CB is also seen to be transient directly in vivo since allogeneic tumor outgrowth is permitted for only a brief period following administration of CB. These results indicate that the use of CB in vivo in anti-tumor chemotherapy protocols will not be complicated by profound or prolonged immunosuppressive effects.