Lee et al., Circ Res, 2017

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. We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. 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. 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.

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Han et al., Cell Transplant, 2016

Recent evidence has suggested that diabetic neuropathy (DN) is pathophysiologically related to both impaired angiogenesis and a deficiency of neurotrophic factors in the nerves. It is widely known that vascular and neural growths are intimately associated. Mesenchymal stem cells (MSCs) promote angiogenesis in ischemic diseases and have neuroprotective effects, particularly on Schwann cells. Accordingly, we investigated whether DN could be improved by local transplantation of MSCs by augmenting angiogenesis and neural regeneration such as remyelination. In sciatic nerves of streptozotocin (STZ)-induced diabetic rats, motor and sensory nerve conduction velocities (NCVs) and capillary density were reduced, and axonal atrophy and demyelination were observed. After injection of bone marrow-derived MSCs (BM-MSCs) into hindlimb muscles, NCVs were restored to near-normal levels. Histological examination demonstrated that injected MSCs were preferentially and durably engrafted in the sciatic nerves, and a portion of the engrafted MSCs were distinctively localized close to vasa nervora of sciatic nerves. Furthermore, vasa nervora increased in density, and the ultrastructure of myelinated fibers in nerves was observed to be restored. Real-time RT-PCR experiments showed that gene expression of multiple factors involved in angiogenesis, neural function, and myelination were increased in the MSC-injected nerves. These findings suggest that MSC transplantation improved DN through direct peripheral nerve angiogenesis, neurotrophic effects, and restoration of myelination.

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Alexander et al., ACS Appl Mater Interfaces

Inflammatory responses play a critical role in tissue-implant interactions, often limiting current implant utility. This is particularly true for cardiovascular devices. Existing stent technology does little to avoid or mitigate inflammation or to influence the vasomotion of the artery after implantation. We have developed a novel endothelium-mimicking nanomatrix composed of peptide amphiphiles that enhances endothelialization while decreasing both smooth muscle cell proliferation and platelet adhesion. Here, we evaluated whether the nanomatrix could prevent inflammatory responses under static and physiological flow conditions. We found that the nanomatrix reduced monocyte adhesion to endothelial cells and expression of monocyte inflammatory genes (TNF-α, MCP-1, IL-1β, and IL-6). Furthermore, the nitric-oxide releasing nanomatrix dramatically attenuated TNF-α-stimulated inflammatory responses as demonstrated by significantly reduced monocyte adhesion and inflammatory gene expression in both static and physiological flow conditions. These effects were abolished by addition of a nitric oxide scavenger. Finally, the nanomatrix stimulated vasodilation in intact rat mesenteric arterioles after constriction with phenylephrine, demonstrating the bioavailability and bioactivity of the nanomatrix, as well as exhibiting highly desired release kinetics. These results demonstrate the clinical potential of this nanomatrix by both preventing inflammatory responses and promoting vasodilation, critical improvements in stent and cardiovascular device technology.

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Kim et al., Int J Cardiol, 2016

This study sought to systematically investigate the derivation of late outgrowth endothelial progenitor cells (late EPC) and mesenchymal stem cells (MSC) from umbilical cord blood (UCB) and to examine their therapeutic effects on myocardial infarction (MI). The expression of angiogenic genes was determined by qRT-PCR. Myocardial infarction (MI) was induced in rats, and cells were directly transplanted into the border regions of ischemic heart tissue. Culture of UCB mononuclear cells yielded two distinct types of cells by morphology after 2 weeks in the same culture conditions. These cells were identified as late EPC and MSC, and each was intramyocardially injected into rat hearts after induction of MI. Echocardiography and histologic analyses demonstrated that both EPC and MSC improved cardiac function and enhanced vascularization, although fibrosis was reduced only in the EPC transplanted hearts. Different paracrine factors were enriched in EPC and MSC. However, once injected into the hearts, they induced similar types of paracrine factors in the heart. Transplanted EPC or MSC were mostly localized at the perivascular areas. This study demonstrated that EPC and MSC can be simultaneously derived from UCB under the same initial culture conditions, and that common paracrine factors are involved in the repair of MI. Late EPC and MSC are effective for infarct repair, apparently mediated through common humoral mechanisms.

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Ban et al., Stem Cell Reports, 2015

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.

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Lee et al., Sci Rep, 2015

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.

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Lee et al., Biomaterials, 2015

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.

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Kim et al., J Am Coll Cardiol, 2014

Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31(+) cells are a promising cell source owing to their high angiovasculogenic and paracrine activities. This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31(+) cells, and to determine whether these cultured CD31(+) cells are effective for cardiac and vascular repair. CD31(+) cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms. The CD31(+) cells cultured in endothelial cell medium (EC-CD31(+) cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31(+) cells. When implanted into mouse MI or HLI models, EC-CD31(+) cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31(+) cells. Histologically, injected EC-CD31(+) cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year. Short-term cultured EC-CD31(+) cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31(+) cells may be a promising source for treating ischemic cardiovascular diseases.

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Wile et al., Nat Protoc, 2014

Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

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Ban et al., ACS Nano, 2014

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.

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