Generation of Red Blood Cells from Human Pluripotent Stem Cells-An Update.

Red blood cell (RBC) transfusion is a lifesaving medical procedure that can treat patients with anemia and hemoglobin disorders. However, the shortage of blood supply and risks of transfusion-transmitted infection and immune incompatibility present a challenge for transfusion. The in vitro generation of RBCs or erythrocytes holds great promise for transfusion medicine and novel cell-based therapies. While hematopoietic stem cells and progenitors derived from peripheral blood, cord blood, and bone marrow can give rise to erythrocytes, the use of human pluripotent stem cells (hPSCs) has also provided an important opportunity to obtain erythrocytes. These hPSCs include both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). As hESCs carry ethical and political controversies, hiPSCs can be a more universal source for RBC generation. In this review, we first discuss the key concepts and mechanisms of erythropoiesis. Thereafter, we summarize different methodologies to differentiate hPSCs into erythrocytes with an emphasis on the key features of human definitive erythroid lineage cells. Finally, we address the current limitations and future directions of clinical applications using hiPSC-derived erythrocytes.

Polycomb Group Protein CBX7 Represses Cardiomyocyte Proliferation Through Modulation of the TARDBP/RBM38 Axis.

BACKGROUND: Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown. METHODS: We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-Cre;Cbx7fl/+ and Myh6-MCM;Cbx7fl/fl, respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques. RESULTS: We explored Cbx7 expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of Cbx7 increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes. CONCLUSIONS: Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration.

Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Reduced angiovasculogenic and increased inflammatory profiles of cord blood cells in severe but not mild preeclampsia.

Preeclampsia (PE) is a prevalent pregnancy disorder that leads to high maternal and fetal morbidity and mortality. While defective vascular development and angiogenesis in placenta are known as crucial pathological findings, its pathophysiological mechanism remains elusive. To better understand the effects of PE on angio-vasculogenesis and inflammatory networks in the fetus and to identify their biological signatures, we investigated the quantitative and functional characteristics of cord blood-derived mononuclear cells (CB-MNCs) and CD31-positive MNCs. Flow cytometry analysis demonstrated that the CB-MNCs from the severe PE group had significantly decreased number of cells expressing CD3, CD11b, CD14, CD19, KDR, and CD31 compared with the normal group. Quantitative real time PCR (qRT-PCR) shows down-regulation of the major angiogenic factor VEGFA in MNCs and CD31+ MNCs in severe PE. The major inflammatory cytokines IL1 was highly upregulated in CD31+ CB-MNCs in the severe PE patients. Mild PE patients, however, did not display any significant difference in expression of all measured angiogenic genes and most inflammatory genes. These findings show distinct angiogenic and inflammatory signatures from severe PE, and they may play a significant role in the pathogenesis of vascular defects in placenta of severe PE.

Enhanced Therapeutic and Long-Term Dynamic Vascularization Effects of Human Pluripotent Stem Cell-Derived Endothelial Cells Encapsulated in a Nanomatrix Gel.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. METHODS: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3ß inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5+ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. RESULTS: The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. CONCLUSIONS: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.

Direct Reprogramming of Human Dermal Fibroblasts Into Endothelial Cells Using ER71/ETV2.

RATIONALE: Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. OBJECTIVE: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. METHODS AND RESULTS: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. CONCLUSIONS: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.

Non-genetic Purification of Ventricular Cardiomyocytes from Differentiating Embryonic Stem Cells through Molecular Beacons Targeting IRX-4.

Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.

Generation of pure lymphatic endothelial cells from human pluripotent stem cells and their therapeutic effects on wound repair.

Human pluripotent stem cells (hPSCs) have emerged as an important source for cell therapy. However, to date, no studies demonstrated generation of purified hPSC-derived lymphatic endothelial cells (LECs) and tested their therapeutic potential in disease models. Here we sought to differentiate hPSCs into the LEC lineage, purify them with LEC markers, and evaluate their therapeutic effects. We found that an OP9-assisted culture system reinforced by addition of VEGF-A, VEGF-C, and EGF most efficiently generated LECs, which were then isolated via FACS-sorting with LYVE-1 and PODOPLANIN. These hPSC-derived LYVE-1(+)PODOPLANIN(+)cells showed a pure committed LEC phenotype, formed new lymphatic vessels, and expressed lymphangiogenic factors at high levels. These hPSC-derived LECs enhanced wound healing through lymphangiogenesis and lymphvasculogenesis. Here we report, for the first time, that LECs can be selectively isolated from differentiating hPSCs, and that these cells are potent for lymphatic vessel formation in vivo and wound healing. This system and the purified hPSC-derived LECs can serve as a new platform for studying LEC development as well as for cell therapy.

Enhanced therapeutic neovascularization by CD31-expressing cells and embryonic stem cell-derived endothelial cells engineered with chitosan hydrogel containing VEGF-releasing microtubes.

Various stem cells and their progeny have been used therapeutically for vascular regeneration. One of the major hurdles for cell-based therapy is low cell retention in vivo, and to improve cell survival several biomaterials have been used to encapsulate cells before transplantation. Vascular regeneration involves new blood vessel formation which consists of two processes, vasculogenesis and angiogenesis. While embryonic stem cell (ESC)-derived endothelial cells (ESC-ECs) have clearer vasculogenic potency, adult cells exert their effects mainly through paracrine angiogenic activities. While these two cells have seemingly complementary advantages, there have not been any studies to date combining these two cell types for vascular regeneration. We have developed a novel chitosan-based hydrogel construct that encapsulates both CD31-expressing BM-mononuclear cells (BM-CD31(+) cells) and ESC-ECs, and is loaded with VEGF-releasing microtubes. This cell construct showed high cell survival and minimal cytotoxicity in vitro. When implanted into a mouse model of hindlimb ischemia, it induced robust cell retention, neovascularization through vasculogenesis and angiogenesis, and efficiently induced recovery of blood flow in ischemic hindlimbs. This chitosan-based hydrogel encapsulating mixed adult and embryonic cell derivatives and containing VEGF can serve as a novel platform for treating various cardiovascular diseases.

Cell therapy with embryonic stem cell-derived cardiomyocytes encapsulated in injectable nanomatrix gel enhances cell engraftment and promotes cardiac repair.

A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.