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Immunomodulatory Effects Of Adipose-derived Stem Cells And Bone Marrow-derived Stem Cells In Rat Hind-limb Composite Tissue Allotransplantation
Sudheer K. Ravuri, PhD, Jonas Schnider, MD, Jan Plock, MD, Wakako Tsuji, MD, PhD, Meghan M. McLaughlin, BS, Peter J. Rubin, MD, Kacey G. Marra, PhD, Mario Solari, MD, Vijay S. Gorantla, MD, PhD.
University of Pittsburgh, Pittsburgh, PA, USA.
Background: Mesenchymal stem cells (MSCs) have broad range of applications in nerve repair. In vitro and local administration in vivo studies had well demonstrated enhanced nerve regeneration. However there is unmet need to develop and study systemic application of MSCs for immunomodulation and/or immunosuppression for improving functional outcomes after CTA. Hence this study was intended to investigate whether donor adipose-derived stem cells (ASCs) and bone marrow derived stem cells (BMSCs) have immunomodulatory effects, such as modulation of related cytokines and prolongation of allograft survival in a rodent hind limb model.
Purpose: To determine systemic effects on immune modulation and suppression by evaluating the cytokine and growth factor expression profiles of secreted and/or circulatory proteins in sera of hind limb allotransplanted rodent animals that were intravenously administered with rodent ASCs and BMSCs.
Materials and Methods: 29 sera were collected from hind-limb transplanted and control animals and subjected to rat model Panomics multiplex Luminex assay, that were separately and systemically injected via intravenous route with PKH26 (red) dye labeled Brown Norway rat ASCs at 1 million and 5 million per injection. Prior to that, rat ASCs and BMSCs were isolated as per laboratory optimized standard protocols. Cytokine and growth factor secretion profiles were demonstrated in sera bled at different time points (4, 6, 18 weeks). Sera was also bled from rejected and control (naïve) animals. 35ul of each neat sera with no preservative was analyzed on Affymetrix Panomics multiplex panel. Core facility optimized standard protocols were applied to analyze the samples in singles (represents 100 analysts).
Results: Graphical representation was made to correlate multiplex cytokine/growth factor targets embedded in panomics multiples luminex panel with error bars plotted +/- one SD. 12 vital cytokines/growth factors were up or down regulated in the analysis. RANKL, ICAM-1, IL-12, IFN-y, IL-13, IL-2, IP-10, Leptin, MIP-2, RANTES, VCAM, VEGF-A were predominantly changed in their expression profile. Groups injected with 1 million ASCs at 4 and 6 weeks showed increased levels in ICAM, but 3-fold reduction in 6 weeks when compared to 4 weeks. Graft rejected and control animals showed low levels of ICAM secretion. BMSCs injected animals showed similar results. IL-2 was increased by 27-fold in 4 weeks when compared to 6 weeks. However, 18 weeks long-term animals showed prolonged secretion upto 30-fold. Conversely BMSCs injected animals showed 42-fold increase in 10 weeks and 18 weeks long-term. Leptin was highly elevated upto 75-fold in 18 weeks long-term and rejected animals. RANKL increased upto 24-fold in 18 weeks long term with ASCs and 10 weeks in BMSCs, but 48-fold increase in BMSCs rejected animals. Poor or no secretion of VEGF-A, TNF-a, MIP-a, MCP-3, IL-4, IL-5, IL-6, IL-10, IL-17, G-CSF, GM-CSF was found in all group analyses.
Conclusion: Based on these preliminary results, it was concluded that the cytokine profiles of ASCs and BMSCs were similar to each other in immune modulation and indicative of rejection. Further analysis of molecular targets is in progress to correlate with Luminex data.
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