Adipose tissue-derived human mesenchymal stromal cells can better suppress complement lysis, engraft and inhibit acute graft-versus-host disease in mice.

BACKGROUND: Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transplantation of immunosuppressive human mesenchymal stromal cells (hMSCs) can protect against aGvHD post-HSCT; however, their efficacy is limited by poor engraftment and survival. Moreover, infused MSCs can be damaged by activated complement, yet strategies to minimise complement injury of hMSCs and improve their survival are limited. METHODS: Human MSCs were derived from bone marrow (BM), adipose tissue (AT) and umbilical cord (UC). In vitro immunomodulatory potential was determined by co-culture experiments between hMSCs and immune cells implicated in aGvHD disease progression. BM-, AT- and UC-hMSCs were tested for their abilities to protect aGvHD in a mouse model of this disease. Survival and clinical symptoms were monitored, and target tissues of aGvHD were examined by histopathology and qPCR. Transplanted cell survival was evaluated by cell tracing and by qPCR. The transcriptome of BM-, AT- and UC-hMSCs was profiled by RNA-sequencing. Focused experiments were performed to compare the expression of complement inhibitors and the abilities of hMSCs to resist complement lysis. RESULTS: Human MSCs derived from three tissues divergently protected against aGvHD in vivo. AT-hMSCs preferentially suppressed complement in vitro and in vivo, resisted complement lysis and survived better after transplantation when compared to BM- and UC-hMSCs. AT-hMSCs also prolonged survival and improved the symptoms and pathological features of aGvHD. We found that complement-decay accelerating factor (CD55), an inhibitor of complement, is elevated in AT-hMSCs and contributed to reduced complement activation. We further report that atorvastatin and erlotinib could upregulate CD55 and suppress complement in all three types of hMSCs. CONCLUSION: CD55, by suppressing complement, contributes to the improved protection of AT-hMSCs against aGvHD. The use of AT-hMSCs or the upregulation of CD55 by small molecules thus represents promising new strategies to promote hMSC survival to improve the efficacy of transplantation therapy. As complement injury is a barrier to all types of hMSC therapy, our findings are of broad significance to enhance the use of hMSCs for the treatment of a wide range of disorders.

Integrated transcriptomic and regulatory network analyses identify microRNA-200c as a novel repressor of human pluripotent stem cell-derived cardiomyocyte differentiation and maturation.

Aims: MicroRNAs (miRNAs) are crucial for the post-transcriptional control of protein-encoding genes and together with transcription factors (TFs) regulate gene expression; however, the regulatory activities of miRNAs during cardiac development are only partially understood. In this study, we tested the hypothesis that integrative computational approaches could identify miRNAs that experimentally could be shown to regulate cardiomyogenesis. Methods and results: We integrated expression profiles with bioinformatics analyses of miRNA and TF regulatory programs to identify candidate miRNAs involved with cardiac development. Expression profiling showed that miR-200c, which is not normally detected in adult heart, is progressively down-regulated both during cardiac development and in vitro differentiation of human embryonic stem cells (hESCs) to cardiomyocytes (CMs). We employed computational methodologies to predict target genes of both miR-200c and five key cardiac TFs to identify co-regulated gene networks. The inferred cardiac networks revealed that the cooperative action of miR-200c with these five key TFs, including three (GATA4, SRF and TBX5) targeted by miR-200c, should modulate key processes and pathways necessary for CM development and function. Experimentally, over-expression (OE) of miR-200c in hESC-CMs reduced the mRNA levels of GATA4, SRF and TBX5. Cardiac expression of Ca2+, K+ and Na+ ion channel genes (CACNA1C, KCNJ2 and SCN5A) were also significantly altered by knockdown or OE of miR-200c. Luciferase reporter assays validated miR-200c binding sites on the 3' untranslated region of CACNA1C. In hESC-CMs, elevated miR-200c increased beating frequency, and repressed both Ca2+ influx, mediated by the L-type Ca2+ channel and Ca2+ transients. Conclusions: Our analyses demonstrate that miR-200c represses hESC-CM differentiation and maturation. The integrative computation and experimental approaches described here, when applied more broadly, will enhance our understanding of the interplays between miRNAs and TFs in controlling cardiac development and disease processes.

Generation of Induced Pluripotent Stem Cells from Patients with COL3A1 Mutations and Differentiation to Smooth Muscle Cells for ECM-Surfaceome Analyses.

Use of experimentally derived induced pluripotent stem cells (iPSCs) has led to the development of cell models for differentiation, drug testing and understanding disease pathogenesis. For these models to be informative, reprogrammed cell lines need to be adequately characterized and shown to preserve all of the critical characteristics of pluripotency and differentiation. Here, we report a detailed protocol for the generation of iPSCs from human fibroblasts containing mutations in COL3A1 using a Sendai virus mediated integration-free reprogramming approach. We describe how to characterize the putative iPSCs in vivo and in vitro to ensure potency and differentiation potential. As an example of how these mutations may affect cell surface and extracellular matrix (ECM) interactions, we provide protocols for the differentiation of these cells into smooth muscle cells to illustrate how different cell types may display cell autonomous differences in collagen receptors that may affect their phenotype. These cells, when applied to mechanical model systems (see Chapter 18 by Bose et al.) facilitate an assessment of stiffness and stress-strain relationships useful for understanding how extracellular matrix dysfunction and its interactions with surface proteins contribute to disease processes.