The aim of this study was to establish an innovative, less-toxic stem cell-based therapy, which will extend allograft survival without the need for life-long immunosuppression. We have developed and tested two alternative therapies of CD90+/CD90+ and CD90+/MSC Di-Chimeric Cells (DCC) for induction of tolerance in Vascularized Composite Allograft (VCA) and Heart transplantation. DCC present both the donor and recipient characteristics and diverse molecular and secretion profiles, which determine their intercellular cross-talk, and as such may elicit different tolerogenic properties when applied as the supportive therapy in the context of VCA or solid organ- heart transplantation. Under Aim 1 of this grant application, we evaluated the molecular profile of DCC of hematopoietic CD90+ cells and mesenchymal stem cells (MSC) origin and determined their pro-tolerogenic cytokine mRNA expression profiles following cell fusion. The bone marrow of ACI and Lewis rats was used for the selection of CD90+ HSC and MSC isolation. Next, the parent cell lines were assessed for: cell lines purity, surface markers expression, and cell viability. The in vitro T-cell suppression and regulatory T-cell (Treg) induction assays were assessed by flow cytometry. Cytokine mRNA expression profiles were assessed in real time-PCR. Under Aim 2, we assessed in vivo the tolerogenic effect of the supportive DCC therapy using a new generation of created hematopoietic and MSC Di-Chimeric Cells in VCA and heart allograft transplant models. The outcomes measures included evaluation of VCA and heart allograft survival up to 100 days, assessment of heart allograft histology at day 7, and day 100 post-transplant. At the study end-point, the peripheral blood, lymphoid organs, and liver were harvested for assessment of chimerism by flow cytometry. Confirmation of the DCC engraftment combined with ex vivo assessment of cytokine expression and in vivo monitoring of allograft survival would allow determining a preferential DCC therapy for the potential clinical application for tolerance induction in VCA and solid organ transplantation

Specific Aim 1: To evaluate molecular and secretion profiles of CD90+/CD90+ DCC and CD90+/MSC DCC and to determine pro-tolerogenic cytokine secretion profiles following cell fusion.

Specific Aim 2: To assess in vivo the tolerogenic effect of the supportive therapy of new generation of CD90+/CD90+ DCC and CD90+/MSC DCC in the VCA and heart transplantation models

1. We have successfully confirmed, the feasibility of ex vivo creation of two new chimeric cell lines of hematopoietic (CD90+/CD90) and hematopoietic/MSC (CD90+/MSC) origin. Flow cytometry and confocal microscopy confirmed DCC creation by presence of overlapping PKH26/ PKH67 fluorescence dye. DCC purity was assessed between 85-95% after fusion procedures.
2. Phenotype assessment of the created DCC confirmed markers characteristic for the parent cells. Flow cytometry analysis of the surface marker expression specific for the parent cells before fusion and for both DCC lines after fusion confirmed that CD90+/CD90+ DCC expressed HSC specific markers (CD90, CD45, and CD34) and CD90+/MSC DCC expressed both HSC (CD90, CD45, and CD34) and MSC (CD29, CD73) specific markers. We have also confirmed the maintenance of hematopoietic and mixed (hematopoietic and mesenchymal) phenotype in the cultured DCC cells after fusion.
3. Assessment of allogeneic responses of DCC lines by mixed lymphocyte reactivity (MLR) assay revealed that both DCC lines evoked decreased T-cell proliferation when compared to the allogenic parent cells confirming tolerogenic properties of DCC.
4. Assessment of regulatory T-cells induction by DCC by fluorescence-based lymphocyte reactivity assay revealed that DCC of CD90+/MSC origin induced higher level of regulatory T-cells in vitro, comparable to the MSC controls, indicating maintenance of immunomodulatory characteristics of DCC after fusion.
5. Evaluation of transcriptional levels of cytokines revealed undetectable levels of transcripts IL4 and IFNG. CD90+/CD90+ DCC expressed higher level of IL10 transcripts implying their immunomodulatory properties.
6. Total chimerism assessed at study end-point by FC for CD90+/CD90+ DCC was above 2.00 % in bone marrow compartment, thymus, and spleen and high level of chimerism of 9.24% was found in liver, indicating tolerogenic properties of CD90+/CD90+ DCC therapy. In contrast in the saline control group no chimerism was detected whereas in MSC control and CD90+/MSC DCC chimerism level was assessed below 1%.
7. We have successfully performed allogenic VCA transplants as well as heterotopic heart allograft transplants, and tested survival under novel 7-day IS protocol of anti-TCR alpha/beta antibody and tacrolimus supported by intraosseous delivery of DCC (CD90+/CD90+ or CD90+/MSC). DCC therapy (CD90+/CD90+) significantly extended VCA and heart allograft survival (up to 100 days) under 7- day IS protocol of anti-TCR alpha/beta antibody and tacrolimus combined with DCC. In contrast saline and MSC controls without IS rejected transplants between 7-11 days. Pathology assessment of heart allografts revealed decreased fibrosis and inflammatory response as well as lack of calcifications in DCC therapy groups when compared with controls. 8. Both DCC therapies expressed tolerogenic properties and extended significantly VCA and heart allograft survival under short 7 day IS protocol. These findings are encouraging and further studies are needed to assess the potential clinical application of DCC therapies in VCA and heart transplantation.