Sophia Kelaini

Direct reprogramming of adult cells: avoiding the pluripotent state

The procedure of using mature, fully differentiated cells and inducing them toward other cell types while bypassing an intermediate pluripotent state is termed direct reprogramming. Avoiding the pluripotent stage during cellular conversions can be achieved either through ectopic expression of lineage-specific factors (transdifferentiation) or a direct reprogramming process that involves partial reprogramming toward the pluripotent stage. Latest advances in the field seek to alleviate concerns that include teratoma formation or retroviral usage when it comes to delivering reprogramming factors to cells. They also seek to improve efficacy and efficiency of cellular conversion, both in vitro and in vivo. The final products of this reprogramming approach could be then directly implemented in regenerative and personalized medicine.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. Methods and Results: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen‐coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR‐199b is involved in EC differentiation. A step‐wise increase in expression of miR‐199 was detected during EC differentiation. Notably, miR‐199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA‐mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR‐199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR‐199b‐mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR‐199b also regulated VEGF expression and angiogenesis. Conclusions: This study indicates a novel role for miR‐199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405–1418

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

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). Stem Cells 2017 Stem Cells 2017;35:952–966

Regenerating the Cardiovascular System Through Cell Reprogramming; Current Approaches and a Look Into the Future

Cardiovascular disease (CVD), despite the advances of the medical field, remains one of the leading causes of mortality worldwide. Discovering novel treatments based on cell therapy or drugs is critical, and induced pluripotent stem cells (iPS Cells) technology has made it possible to design extensive disease-specific in vitro models. Elucidating the differentiation process challenged our previous knowledge of cell plasticity and capabilities and allows the concept of cell reprogramming technology to be established, which has inspired the creation of both in vitro and in vivo techniques. Patient-specific cell lines provide the opportunity of studying their pathophysiology in vitro, which can lead to novel drug development. At the same time, in vivo models have been designed where in situ transdifferentiation of cell populations into cardiomyocytes or endothelial cells (ECs) give hope toward effective cell therapies. Unfortunately, the efficiency as well as the concerns about the safety of all these methods make it exceedingly difficult to pass to the clinical trial phase. It is our opinion that creating an ex vivo model out of patient-specific cells will be one of the most important goals in the future to help surpass all these hindrances. Thus, in this review we aim to present the current state of research in reprogramming toward the cardiovascular systems regeneration, and showcase how the development and study of a multicellular 3D ex vivo model will improve our fighting chances.

Constitutively active SMAD2/3 facilitates efficient transcription factor-mediated cell conversion

The generation of induced pluripotent stem cells (iPSCs) is a promising and exciting tool for regenerative medicine. Since their first appearance in 2006 by Yamanaka et al , where adult mouse fibroblasts were reprogrammed using a cocktail of only four master transcription factors (TFs), Oct4 , Sox2 , Klf4 , and c - Myc reprogramming factors (1), iPSCs have served as valuable tools in studying disease. Indeed, even years after this groundbreaking discovery, cellular identity conversion of somatic cells through exogenous introduction of TFs towards iPSCs remains one of the most powerful tools (2) for disease modelling, drug screening, tissue engineering and transplantation therapies. Nevertheless, despite recent advances, the induction efficiency of these cells and subsequent differentiation to the desired cell types is, very low (3) and has a lengthy reprogramming process with substantial limitations to create and maintain functional cells. In a combined effort, laboratories led by Keisuke Kaji investigated a new approach of improving the efficiency of iPSCs generation by the reprogramming factors through the expression of constitutively active SMAD2/3 (4).

FSTL3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β-catenin Nuclear Translocation Through Inhibition of GSK3β Activity

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. Stem Cells 2018;36:1033–1044

RBPs Play Important Roles in Vascular Endothelial Dysfunction Under Diabetic Conditions

Diabetes is one of the major health care problems worldwide leading to huge suffering and burden to patients and society. Diabetes is also considered as a cardiovascular disorder because of the correlation between diabetes and an increased incidence of cardiovascular disease. Vascular endothelial cell dysfunction is a major mediator of diabetic vascular complications. It has been established that diabetes contributes to significant alteration of the gene expression profile of vascular endothelial cells. Post-transcriptional regulation by RNA binding proteins (RBPs) plays an important role in the alteration of gene expression profile under diabetic conditions. The review focuses on the roles and mechanisms of critical RBPs toward diabetic vascular endothelial dysfunction. Deeper understanding of the post- transcriptional regulation by RBPs could lead to new therapeutic strategies against diabetic manifestation in the future.

X-Box-Binding Protein 1 Splicing Induces an Autophagic Response in Endothelial Cells: Molecular Mechanisms in ECs and Atherosclerosis

Abstract Atherosclerosis is the leading cause of death in the developed world and involves the production of an atherosclerotic plaque in the artery wall, limiting blood flow and resulting in conditions such as peripheral artery disease, coronary heart disease, myocardial infarction, and stroke. Autophagy is a method of self-digestion, primarily a survival pathway for the cell, to remove and/or recycle old and damaged proteins in the cytoplasm. There is increasing evidence that autophagy takes place in severe atherosclerotic plaques implicating macrophages and vascular smooth muscle cells. In addition, oxidized low-density lipoprotein (Ox-LDL) can also trigger autophagy in endothelial cells (ECs) through LC3β/BECLIN-1, leading to the lysosome-mediated degradation of Ox-LDL. However the role of autophagy in atherosclerosis still remains shrouded in mystery, as it is still debated whether autophagy is a damaging or a protective mechanism or a balance of both is needed for normal cellular function. X-Box binding protein 1 (XBP1) mRNA splicing is involved in the regulation of autophagy in ECs through BECLIN-1 transcriptional activation. It has recently been shown that sustained activation of XBP1 results in EC apoptosis and development of atherosclerosis. More evidence has shown the importance of XBP1 in eliciting an autophagic response in ECs. Therefore, it seems that the threshold of the autophagic responses could be determined through the tight regulation of the expression and duration of splicing activation of molecules, such as XBP1s, in a cell-specific manner.

200 Dedifferentiated or Reborn Again? Elucidating The Chromatin Remodelling Mechanisms During Endothelial Cell Reprogramming for Cardiovascular Therapy

The vascular endothelium is central to cardiovascular homeostasis. Repair and regeneration of endothelial cells (ECs) has been an important research focus for a number of years. The recent ability to derive ECs through cell reprogramming has opened new avenues. Reprogramming somatic cells to ECs is in its infancy, but the road ahead looks very promising. A new reprogramming strategy has ruled out safety issues concerned with teratoma formation. Cells exposed to reprogramming factors for 4 days become epigenetically primed and have been defined as Partially induced Pluripotent Stem (PiPS) cells. They do not transverse pluripotency, and so do not form tumours. They have shown the ability to be differentiated into ECs by culture conditions. Efficiency of reprogramming has increased from 0.01% to 30–40% in the case of PiPS-ECs, but there is scope for improvement as the underlying mechanisms are still unclear. The role of epigenetics in reprogramming has come to the forefront recently and the ability to generate a homogenous and functional EC population will be best sought through chromatin remodelling mechanisms. A protein found to be crucial in 4-day reprogramming was SETSIP, or SET similar protein. SETSIP has high sequence homology to SET with an additional 10 amino acids at the N-terminus. SET plays roles in chromatin remodelling as a transcriptional regulator and roles in differentiation, apoptosis and cell cycle progression. The study aims to elucidate a robust and efficient protocol for the production of a homogenous and functional EC population for use in personalised cardiovascular medicine. SETSIP has been overexpressed and knocked out of early PiPS-ECs and iPS-ECs to observe the effect on EC reprogramming. Luciferase assays have been undertaken to understand the EC specific pathways regulated by SETSIP. Experiments have also been performed to establish the effect of treatment with VEGF on SETSIP expression and EC differentiation. Future work will involve the employment of CRISPR technology to create a SETSIP deficient cell line to observe the differentiation potential of the cells and phenotype of any derived ECs. SETSIP was found to translocate to the cell nuclei, and capable of regulating expression of important EC markers. The functional consequences of this were assessed in vitro and in vivo where SETSIP was found to be important for the formation of vascular tubules. Furthermore, epigenetic modulators such as CBP/p300 were identified as potential mediators of the gene regulatory effects of SETSIP in ECs. These results represent an important step forward in understanding the process of EC reprogramming for use in regenerative medicine. These findings provide knowledge of the intricate processes during EC reprogramming not only to support the scientific validity of the newly generated ECs but also to ensure the safety of bringing cellular reprogramming to the bedside of cardiovascular patients.

Induced Pluripotent Stem Cells and Vascular Disease

iPS cells have revolutionised the field of regenerative medicine and are considered powerful medical tools in the potential treatment of numerous diseases. They are also increasingly explored as management approaches for vascular and cardiovascular diseases as well as other associated disorders such as renal failure and aneurysms.