TGF2 in culture supernatants was not detected, as measured by ELISA (data not shown), consistent with the low gene transcript value we have reported. dose of busulfan (5 mg/kg) was given on day +5. Seven days after skin transplantation, ASCs (3 106) were infused i.v. with or without donor bone marrow cells (BMCs; 5 105). ASC+BMC coinfusion with minimal conditioning led to stable lymphoid and myeloid macrochimerism, deletion of alloreactive T cells, expansion of regulatory T cells, and long-term allograft survival ( 200 days). ASCs constitutively produced high levels of anti-inflammatory/immunoregulatory factors such as prostaglandin E2, indoleamine 2,3-dioxygenase, APO-1/Fas (CD95), and programmed cell IFN-alphaA death-1 ligand-2. These findings serve as a foundation for developing a translational advanced VCA protocol, embodying both ASCs and low-dose donor BMCs, in nonhuman primates, with the goal of enhancing functional outcomes and eliminating the complications associated with long-term immunosuppression. test. A value of .05 was considered significant for all tests (GraphPad Software, Inc., San Diego, CA, http://www.graphpad.com). Results T-Cell Immunosuppressive Capacity of ASCs Human ASCs added to murine mixed lymphocyte (MLR) cultures at the initiation of culture suppressed murine T-cell proliferation in a dose-dependent manner NAN-190 hydrobromide (Fig. 1A). As shown in Figure 1B, CD4+ T cells from ASC MLR cocultures did not divide (with a low expression of Ki-67, a molecule expressed during cell NAN-190 hydrobromide cycle progression)  and remained phenotypically na?ve, with low expression of CD25 compared with CD4+ T cells from control MLR cultures. Furthermore, soluble factors from day 3 ASC/MLR and ASC IFN-treated culture supernatants demonstrated potent inhibition of alloantigen-driven proliferation (Fig. 1C). We, and others, have demonstrated that ASCs express and produce PGE2 and indoleamine 2,3-dioxygenase (IDO) constitutively [28, 35C38]. In addition, the immunomodulatory effects of ASCs are associated with expressed mRNA transcripts for TNFRSF6 (FAS), HGF, PD-L1, and PD-L2 and lower transcript numbers for membrane HLA-G1 (mHLA-G1) (Fig. 1D). Consistent with the findings of Crop et al. , under MLR proinflammatory conditions and IFN NAN-190 hydrobromide stimulation, we determined that ASCs expressed marked increases of IDO (218.5-fold) and mHLA-G (11.2-fold) and modest increases of PD-L1 (4.1-fold) and PD-L2 (5.1-fold) gene transcripts. Protein expression validation NAN-190 hydrobromide of mRNA transcript results was performed using ELISA, cell surface staining, and intracellular staining for IDO using flow cytometric analysis (Fig. 2E). TGF2 in culture supernatants was not detected, as measured by ELISA (data not shown), consistent with the low gene transcript value we have reported. The HGF levels were not assayed. Open in a separate window Figure 1. Human ASCs inhibit murine allo-MLR lymphocyte proliferation. (A): Na?ve C57BL/6 splenocytes (as responder cells) were cultured 1:1 with irradiated (30 Gy) na?ve BALB/c stimulatory cells. ASCs were added to the MLR at the onset of culture at the indicated responder/ASC ratios. After 4 days, the quadruplicate replicate cocultures were pulsed for 18 hours with bromodeoxyuridine (BrdU). A specific enzyme-linked immunosorbent assay (ELISA) kit was used to measure BrdU incorporation into newly synthesized DNA, expressed as the mean absorbance SD. The results are expressed as the mean percentage of alloproliferation of control cultures. ?, .05, significant difference compared with control NAN-190 hydrobromide group. (B): Effect of ASC coculture on the expression of Ki-67 and CD25 on CD4+ responder T cells. A representative staining of gated CD4+ T cells from control MLR cultures and ASC-treated MLR cultures. (C): Supernatants from allo-MLR/ASC cocultures inhibited MLR lymphocyte proliferation. Na?ve C57BL/6 splenocytes (as responders cells) were cultured 1:2 with irradiated (30 Gy) na?ve BALB/c stimulatory cells. Supernatants (10% final plating dilution) from day 3 allo-MLR cultures, allo-MLR/ASC cocultures, or IFN treated (10 ng/ml) was added to the MLR at the onset of culture. Cell proliferation.