A novel role of CD73-IFNγ signalling axis in human mesenchymal stromal cell mediated inflammatory macrophage suppression.

Introduction: Immunomodulation is the predominant mechanism via which Mesenchymal stromal cells (MSCs) mediate their therapeutic benefits. However, inconsistent success in numerous clinical trials warrants a better understating of the molecular mechanisms regulating their immunomodulatory properties. CD73, an ecto-5'-nucleotidase is abundantly expressed by MSCs, however its precise role in regulating their immunomodulatory properties is still elusive. The present study explored the role of CD73 in Interferon-gamma (IFNγ) sensing and in turn their ability to suppress "inflammatory" M1 macrophages. Materials and methods: CD73 knockdown MSCs (CD73-KDN) were initially assessed for expression of immunoregulatory molecules and IFNγ sensing ability by analysing expression of IFNγ signalling downstream targets such as pSTAT-1, Interferon-Stimulated Genes (ISG) and Indoleamine 2,3-dioxygnease (IDO), a prototypic IFNγ-induced immunomodulator. Next CD73-KDN MSCs were co-cultured with inflammatory M1 macrophages and evaluated for their ability to suppress them. To delineate the contributory role of CD73 and IFNγ signalling downstream target IDO, they were overexpressed independently in CD73-KDN MSCs and re-evaluated for their ability to suppress M1 macrophages. Results: CD73-KDN MSCs exhibited reduced expression of immunoregulatory molecules and were refractory to IFNγ signalling as indicated by attenuated expression of pSTAT-1, Interferon-Stimulated Genes (ISG) and Indoleamine 2,3-dioxygnease (IDO) upon IFNγ exposure. Since sensing of inflammation is critical for MSC mediated immunomodulation, CD73-KDN MSCs were functionally evaluated for their ability to immune-modulate "inflammatory" M1 macrophages wherein they failed to suppress M1 macrophages. Interestingly, ectopic expression of either CD73 or IFNγ signalling target IDO1 in CD73-KDN MSCs restored their ability to suppress M1 macrophages, establishing the importance of CD73-IFNγ signalling axis in MSC-mediated inflammatory macrophage suppression. Conclusion: The present study uncovers the unexplored role of CD73-IFNγ axis in MSC-mediated M1 macrophage suppression. MSC-educated macrophages are the actual immune-modulators at MSC transplant sites, thus CD73 can serve as a key immune-potency marker for benchmarking therapeutically relevant MSCs.

Deubiquitinase USP1 influences the dedifferentiation of mouse pancreatic ß-cells.

Loss of insulin-secreting ß-cells in diabetes may be either due to apoptosis or dedifferentiation of ß-cell mass. The ubiquitin-proteasome system comprising E3 ligase and deubiquitinases (DUBs) controls several aspects of ß-cell functions. In this study, screening for key DUBs identified USP1 to be specifically involved in dedifferentiation process. Inhibition of USP1 either by genetic intervention or small-molecule inhibitor ML323 restored epithelial phenotype of ß-cells, but not with inhibition of other DUBs. In absence of dedifferentiation cues, overexpression of USP1 was sufficient to induce dedifferentiation in ß-cells; mechanistic insight showed USP1 to mediate its effect via modulating the expression of inhibitor of differentiation (ID) 2. In an in vivo streptozotocin (STZ)-induced dedifferentiation mouse model system, administering ML323 alleviated hyperglycemic state. Overall, this study identifies USP1 to be involved in dedifferentiation of ß-cells and its inhibition may have a therapeutic application of reducing ß-cell loss during diabetes.

Advancement in Understanding the Concept of Epithelial to Mesenchymal Transition in Pancreatic ß-Cells: Implication in Diabetes.

Distinct molecular processes are engaged during histogenesis, and Epithelial to Mesenchymal Transition (EMT) is one of the key evolutionarily conserved processes that facilitates organ development. Molecular pathways governing EMT are embedded within developmental programs and operate in cells of different tissues. Among varied cell types, EMT in pancreatic ß-cells is of greater interest as the existence of EMT in these cells is highly debated. Although in vitro generation of human islet-derived mesenchymal progenitor cells has been proven beyond doubt, the existence of EMT in pancreatic ß-cells in vivo remains enigmatic. Understanding the in-depth process of EMT in in vivo human ß-cells is challenged by the limitations of lineage-tracing studies, which are otherwise feasible in mice. Exploring EMT of ß-cells would greatly facilitate the generation of clinically relevant ß-cells either by enhancing long-term in vitro culture of endogenous islets or by differentiation of pluripotent stem cells to mature ß-cells. This review is an update on the recent progress in understanding the EMT process of ß-cells and how the investigations have helped to resolve the mystery of the existence of EMT in pancreatic ß-cells.

Dynamics of Ubiquitination in Differentiation and Dedifferentiation of Pancreatic ß-cells: Putative Target for Diabetes.

Impairment in the function of insulin-producing pancreatic ß-cells is a hallmark of both type 1 and 2 diabetes (T1D/T2D). Despite over a century of effort, there is still no precise treatment regimen available for acute diabetes. Enhancing the endogenous ß-cells either by protecting them from apoptosis or dedifferentiation is a classic alternative to retaining the ß-cell pool. Recent reports have acknowledged the protein homeostasis mediated by the ubiquitin-proteasome system as one of the essential components in maintaining the ß-cell pool. Degradation of the targeted substrate by the proteasome is majorly regulated by the ubiquitination status of the targeted protein dictated by E3 ligases and deubiquitinase enzymes. Imbalance in the function of these enzymes results in the malfunction of ß-cells and, subsequently, hyperglycemia. Ubiquitination involves the covalent attachment of one or more ubiquitin moieties to the target protein by E3 ubiquitin ligases and deubiquitinases (DUBs) are the enzymes that antagonize the action of E3 ligases. Knowing different E3 ligases and deubiquitinases in the process of differentiation and dedifferentiation of ß-cells probably paves the way for designing novel modulators that enhance either the differentiation or abate the dedifferentiation process. In this review, we will discuss the importance of the balanced ubiquitination process, an understanding of which would facilitate the restraining of ß-cells from exhaustion.

Mitochondrial Superoxide Dismutase Specifies Early Neural Commitment by Modulating Mitochondrial Dynamics.

Studies revealing molecular mechanisms underlying neural specification have majorly focused on the role played by different transcription factors, but less on non-nuclear components. Earlier, we reported mitochondrial superoxide dismutase (SOD2) to be essential for self-renewal and pluripotency of mouse embryonic stem cells (mESCs). In the present study, we found SOD2 to be specifically required for neural lineage, but not the meso- or endoderm specification. Temporally, SOD2 regulated early neural genes, but not the matured genes, by modulating mitochondrial dynamics-specifically by enhancing the mitochondrial fusion protein Mitofusin 2 (MFN2). Bio-complementation strategy further confirmed SOD2 to enhance mitochondrial fusion process independent of its antioxidant activity. Over-expression of SOD2 along with OCT4, but neither alone, transdifferentiated mouse fibroblasts to neural progenitor-like colonies, conclusively proving the neurogenic potential of SOD2. In conclusion, our findings accredit a novel role for SOD2 in early neural lineage specification.

Does altering proteasomal activity and trafficking reduce the arborization mediated specific vulnerability of SNpc dopaminergic neurons of Parkinson's disease?

Parkinson's disease (PD) is a late-onset degenerative neuronal disorder and stands second among the neurological disorders with 1% of the total world population being affected. The disease originates majorly due to compromised function of the dopaminergic (DA) neurons in the Substantia Nigra pars compacta (SNpc), but not the ventral tegmental area (VTA) region of the midbrain. The differential susceptibility for degeneration is majorly attributed to morphological, molecular, and electrophysiological heterogeneity existing in DA neurons of SNpc and VTA. Long-range axonal arborization and a higher number of synapses in SNpc DA neurons make it more vulnerable compared to VTA DA neurons. Studies have shown that a decrease in such axonal arborization places DA neurons at decreased risk in PD. The two well established underlying mechanisms are a) As arborization is an energy-demanding process, increased redistribution of mitochondria to the axonal terminals occurs to satisfy the bioenergetic requirement b) The stabilization of axon-promoting factors at the axonal tip is an essential component for enhancing the arborization process. Interfering with any of these two processes would probably alleviate the degeneration of SNpc DA neurons. To accomplish the decreased stability of arborizing factors and thereby increase the resilience of SNpc DA neurons, we hypothesize the activation of anterograde transport-dependent recruitment of proteasomes to axon terminals as one of the most favorable approaches. Understanding this putative avenue of enhancing proteasomal activity and migration to the axonal tip could provide insight into the progression of neurodegeneration in PD and possibly offer a novel therapeutic strategy.

Cyclosporine A-Mediated IL-6 Expression Promotes Neural Induction in Pluripotent Stem Cells.

Differentiation of pluripotent stem cells (PSCs) to neural lineages has gathered huge attention in both basic research and regenerative medicine. The major hurdle lies in the efficiency of differentiation and identification of small molecules that facilitate neurogenesis would partly circumvent this limitation. The small molecule Cyclosporine A (CsA), a commonly used immunosuppressive drug, has been shown to enhance in vivo neurogenesis. To extend the information to in vitro neurogenesis, we examined the effect of CsA on neural differentiation of PSCs. We found CsA to increase the expression of neural progenitor genes during early neural differentiation. Gene silencing approach revealed CsA-mediated neural induction to be dependent on blocking the Ca2+-activated phosphatase calcineurin (Cn) signaling. Similar observation with FK506, an independent inhibitor of Cn, further strengthened the necessity of blocking Cn for enhanced neurogenesis. Surprisingly, mechanistic insight revealed Cn-inhibition dependent upregulation of IL-6 protein to be necessary for CsA-mediated neurogenesis. Together, these findings provide a comprehensive understanding of the role of CsA in neurogenesis, thus suggesting a method for obtaining large numbers of neural progenitors from PSCs for possible transplantation.

Novel role of mitochondrial manganese superoxide dismutase in STAT3 dependent pluripotency of mouse embryonic stem cells.

Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.

Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study.

AIMS AND OBJECTIVES: Isolation, characterization and differentiation of dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED). METHODS: The pulp tissue was digested in collagenase and cultured in DMEM Dulbecco's Modified Eagle's Media). The stem cells were identified and isolated. Surface characterization of cells was done with flow cytometer using surface markers. An immuno cytochemistry analysis was done. Differentiation potential was analyzed using various differentiation markers. RESULTS: Flow cytometry analyses for various CD markers showed similar results for both DPSCs and SHED. The cells showed positive expression for pluripotent, ectodermal and mesodermal markers. Cells differentiated into osteoblasts and adipocytes. CONCLUSION: The study demonstrated that stem cells existed in deciduous and permanent pulp tissue. The stem cells present in pulp tissue can be isolated, cultivated and expanded in vitro. Both DPSCs and SHED show almost a similar expression pattern profile for variety of antigens tested.