Quaking is a Key Regulator of Endothelial Cell Differentiation, Neovascularization and Angiogenesis

Abstract The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA-binding protein Quaking isoform 5 (QKI-5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA-binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient-specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI-5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI-5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3′ UTR of STAT3. Importantly, mouse iPS-ECs overexpressing QKI-5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI-5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI-5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI-5 is induced during EC differentiation from iPSCs. RNA binding protein QKI-5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI-5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm).</jats:p

Follistatin-Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β-Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase-3β Activity

Abstract The fight against vascular disease requires functional endothelial cells (ECs) which could be provided by differentiation of induced Pluripotent Stem Cells (iPS Cells) in great numbers for use in the clinic. However, the great promise of the generated ECs (iPS-ECs) in therapy is often restricted due to the challenge in iPS-ECs preserving their phenotype and function. We identified that Follistatin-Like 3 (FSTL3) is highly expressed in iPS-ECs, and, as such, we sought to clarify its possible role in retaining and improving iPS-ECs function and phenotype, which are crucial in increasing the cells’ potential as a therapeutic tool. We overexpressed FSTL3 in iPS-ECs and found that FSTL3 could induce and enhance endothelial features by facilitating β-catenin nuclear translocation through inhibition of glycogen synthase kinase-3β activity and induction of Endothelin-1. The angiogenic potential of FSTL3 was also confirmed both in vitro and in vivo. When iPS-ECs overexpressing FSTL3 were subcutaneously injected in in vivo angiogenic model or intramuscularly injected in a hind limb ischemia NOD.CB17-Prkdcscid/NcrCrl SCID mice model, FSTL3 significantly induced angiogenesis and blood flow recovery, respectively. This study, for the first time, demonstrates that FSTL3 can greatly enhance the function and maturity of iPS-ECs. It advances our understanding of iPS-ECs and identifies a novel pathway that can be applied in cell therapy. These findings could therefore help improve efficiency and generation of therapeutically relevant numbers of ECs for use in patient-specific cell-based therapies. In addition, it can be particularly useful toward the treatment of vascular diseases instigated by EC dysfunction.</jats:p

Cardiac organoids: a model to investigate the effect of diabetes on cardiac development and function

IntroductionDiabetes and associated cardiovascular diseases (CVDs) are a class of disorders affecting the heart or blood vessels. Despite progress in clinical research and therapy, CVDs still represent the leading cause of mortality and morbidity worldwide. The hallmarks of cardiac diseases include heart dysfunction and cardiomyocyte death, inflammation, fibrosis, scar tissue, hyperplasia, hypertrophy, and abnormal ventricular remodelling. The loss of cardiomyocytes is an irreversible process that leads to fibrosis and scar formation, which, in turn, induce heart failure with progressive and dramatic consequences. Both genetic and environmental factors pathologically contribute to the development of CVDs, but the precise causes that trigger cardiac diseases and their progression are still largely unknown. The lack of reliable human model systems for such diseases has hampered the unravelling of the underlying molecular mechanisms and cellular processes involved in heart diseases at their initial stage and during their progression. In this study we use induced pluripotent stem cells (iPSCs) from diabetic and non-diabetic donors to recapitulate an iPSC-driven cardiac model with the aim of underlining the potential of stem-cell biology-based approaches in the elucidation of the pathophysiology of cardiac disease.MethodsCardiomyocytes were generated from iPS cells from both diabetic (DiPSC-CMs) and non-diabetic donors (NDiPS-CMs) within a thirteen day differentiation protocol. Cardiac commitment was assessed by Flow cytometry, PCR analysis, and Immunofluorescence staining. Morphological features of the sarcomere arrangement and mitochondria size were assessed using TEM microscopy. Cardiac function was measured by assessing calcium flux using Flexstation. Beating qualities were assessed by Nikon 6D Life imaging.ResultsUpon differentiation both DiPSC-CMs and NDiPSC-CMs presented strong cardiac commitment as evident by the significant expression of cardiac markers evaluated by PCR and flow cytometry. iPS-CM from both donors presented no significant differences in the expression of cardiac markers brachyury and cardiac troponin as assessed by flow cytometry (p &gt; 0.7078) and sarcomere proteins a-Actinin, myosin light chain MLCA2 assessed by immunofluorescence staining. Cardiomyocytes derived from diabetic donors (DiPS-CMs) showed differences in the uptake of calcium when these compared to the nondiabetic counterparts (NDiPS-CMs). Calcium flux was measure by capturing fluorescence intensity of Fura-2 calcium dye by Flexstation (p &gt; 0.0156). TEM imaging and assessment of mitochondria between DiPSC-CMs and NDiPS-CMs showed differences in mitochondrial morphological features such as aspect ratio, perimeter and length (p &gt; 0.05) between our two groups indicating a morphological change that may underly be related to an underlying mitochondrial disorder associated to a metabolic dysfunction.<br/