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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;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/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. RESULT: 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.

The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling. However, hPSC-CMs differentiated in conventional two-dimensional systems are structurally and functionally immature. Moreover, these differentiation systems generate predominantly one type of cell. Since the heart includes not only CMs but other cell types, such monolayer cultures have limitations in simulating the native heart. Accordingly, three-dimensional (3D) cardiac tissues have been developed as a better platform by including various cardiac cell types and extracellular matrices. Two advances were made for 3D cardiac tissue generation. One type is engineered heart tissues (EHTs), which are constructed by 3D cell culture of cardiac cells using an engineering technology. This system provides a convenient real-time analysis of cardiac function, as well as a precise control of the input/output flow and mechanical/electrical stimulation. The other type is cardiac organoids, which are formed through self-organization of differentiating cardiac lineage cells from hPSCs. While mature cardiac organoids are more desirable, at present only primitive forms of organoids are available. In this review, we discuss various models of hEHTs and cardiac organoids emulating the human heart, focusing on their unique features, utility, and limitations.

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 via 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 into 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.

Cell-based therapy has emerged as a promising option for treating advanced ischemic cardiovascular disease by inducing vascular regeneration. However, clinical trials with adult cells turned out disappointing in general. As a newer approach, direct reprogramming has emerged to efficiently generate endothelial cells (ECs), which can promote neovascularization and vascular regeneration. This review provides recent updates on the direct endothelial reprogramming. In general, directly reprogrammed ECs can be generated by two approaches: one by transitioning through a plastic intermediate state and the other in a one-step transition without any intermediate states toward pluripotency. Moreover, the methods to deliver reprogramming factors and chemicals for the fate conversion are highlighted. Next, the therapeutic effects of the directly reprogrammed ECs on animal models are reviewed in detail. Other applications using directly reprogrammed ECs, such as tissue engineering and disease modeling, are also discussed. Lastly, the remaining questions and foremost challenges are addressed.

Introduction: Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. Regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse CMs via adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Myh6-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We measured CM proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart (fold increase of adult vs. prenatal: 32.1 ± 3.3, P < 0.01). Overexpression of CBX7 in neonatal mouse CMs reduced CM proliferation (% of Ki67 + CMs: Ad-Mock, 22.3 ± 1.7 vs. Ad-CBX7, 7.7 ± 0.7, P < 0.001). The haplodeficiency of CBX7 (Tnnt2-Cre;Cbx7 fl/+ ) enhanced proliferation of neonatal CMs in vivo (% of Ki67 + CMs: wild-type, 7.9 ± 0.9% vs. mutants, 24.7 ± 1.2%, P < 0.01), leading to increased myocardial wall thickness, cardiomegaly and neonatal lethality. Genetic deletion of CBX7 in CMs (Myh6-MCM;Cbx7 fl/fl ) at P1 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 ± 1.4% vs. tamoxifen, 18.3 ± 1.8%, P < 0.01), leading to cardiomegaly at 3 months. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was perinatally upregulated in the mouse hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased in the mouse hearts and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. This is the first study to demonstrate the role of CBX7 in regulation of CM proliferation and CBX7 could be an important target for cardiac regeneration.

Atherosclerosis is the primary cause of hardening and narrowing arteries, leading to cardiovascular disease accounting for the high mortality in the United States. For developing effective treatments for atherosclerosis, considerable efforts have been devoted to developing

SummaryCardiomyocyte (CM) proliferation notably decreases during the perinatal period. At present, regulatory mechanisms for this loss of proliferative capacity is poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Here, we report that CBX7 inhibits proliferation of perinatal CMs by controlling TARDBP/Rbm38 pathway. Gene expression profiling demonstrated that CBX7 expression in the heart was low during the prenatal period, abruptly increased during the perinatal period, and sustained constantly throughout the adulthood. CBX7, when overexpressed via adenoviral transduction in neonatal CMs, reduced proliferation and promoted multinucleation of the CMs. Mutant mice carrying targeted inhibition of CBX7 in CMs exhibited cardiomegaly with increased proliferation of CMs at postnatal stages. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was upregulated in the postnatal hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Together, this study provides a novel insight into the role of CBX7 in regulation of CM proliferation during the perinatal period.

The fabrication of functional tissues is essential for clinical applications such as disease treatment and drug discovery. Recent studies have revealed that the mechanical environments of tissues, determined by geometric cell patterns, material composition, or mechanical properties, play critical roles in ensuring proper tissue function. Here, we propose an acoustophoretic technique using surface acoustic waves to fabricate therapeutic vascular tissue containing a three-dimensional collateral distribution of vessels. Co-aligned human umbilical vein endothelial cells and human adipose stem cells that are arranged in a biodegradable catechol-conjugated hyaluronic acid hydrogel exhibit enhanced cell-cell contacts, gene expression, and secretion of angiogenic and anti-inflammatory paracrine factors. The therapeutic effects of the fabricated vessel constructs are demonstrated in experiments using an ischemia mouse model by exhibiting the remarkable recovery of damaged tissue. Our study can be referenced to fabricate various types of artificial tissues that mimic the original functions as well as structures., Engineering 3D tissues faces the challenge of adequate vascularisation for nutrient delivery and gas exchange deep inside the construct. Here the authors use surface acoustic waves to create an aligned array of blood vessels in a hyaluronic acid hydrogel and use it to improve function in a mouse hindlimb ischemia model.

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.

Atherosclerosis is a chronic inflammatory disease driven by lipid accumulation in arteries, leading to narrowing and thrombosis. It affects the heart, brain, and peripheral vessels and is the leading cause of mortality in the United States. Researchers have strived to design nanomaterials of various functions, ranging from non-invasive imaging contrast agents, targeted therapeutic delivery systems to multifunctional nanoagents able to target, diagnose, and treat atherosclerosis. Therefore, this review aims to summarize recent progress (2017-now) in the development of nanomaterials and their applications to improve atherosclerosis diagnosis and therapy during the preclinical and clinical stages of the disease.

CBX7 is a polycomb group protein, and despite being implicated in many diseases, its role in cell proliferation has been controversial: some groups described its pro-proliferative properties, but others illustrated its inhibitory effects on cell growth. To date, the reason for the divergent observations remains unknown. While several isoforms for CBX7 were reported, no studies investigated whether the divergent roles of CBX7 could be due to distinct functions of CBX7 isoforms. In this study, we newly identified mouse CBX7 transcript variant 1 (mCbx7v1), which is homologous to the human CBX7 gene (hCBX7v1) and is expressed in various mouse organs. We revealed that mCbx7v1 and hCBX7v1 encode a 36 kDa protein (p36CBX7) whereas mCbx7 and hCBX7v3 encode a 22 kDa protein (p22CBX7). This study further demonstrated that p36CBX7 was localized to the nucleus and endogenously expressed in proliferating cells whereas p22CBX7 was localized to the cytoplasm, induced by serum starvation in both human and mouse cells, and inhibited cell proliferation. Together, these data indicate that CBX7 isoforms are localized in different locations in a cell and play differing roles in cell proliferation. This varying function of CBX7 isoforms may help us understand the distinct function of CBX7 in various studies.

Tissue repair after myocardial infarction entails a vigorous angiogenic response involving as yet incompletely defined immune cell-endothelial cell interactions. We identify the monocyte and macrophage-derived cytokine meteorin-like (METRNL) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT receptor tyrosine kinase (KIT). METRNL’s angiogenic effects on cultured human endothelial cells were mediated through KIT and its downstream signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl-deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue injury.

Two new sources of ECs were generated from human induced pluripotent stem cells (hiPSCs), and by direct reprogramming of somatic cells without undergoing the stages of stem or progenitor cell. These two types of ECs will advance our understanding of EC biology and can become a novel therapeutic option for treating ischemic cardiovascular diseases.

Transplantations of various stem cells or their progeny have repeatedly improved cardiac performance in animal models of myocardial injury; however, the benefits observed in clinical trials have been generally less consistent. Some of the recognized challenges are poor engraftment of implanted cells and, in the case of human cardiomyocytes, functional immaturity and lack of electrical integration, leading to limited contribution to the heart’s contractile activity and increased arrhythmogenic risks. Advances in tissue and genetic engineering techniques are expected to improve the survival and integration of transplanted cells, and to support structural, functional, and bioenergetic recovery of the recipient hearts. Specifically, application of a prefabricated cardiac tissue patch to prevent dilation and to improve pumping efficiency of the infarcted heart offers a promising strategy for making stem cell therapy a clinical reality.

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.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.

Cardiovascular disease is presently the number one cause of death worldwide. Current stents used to treat cardiovascular disease have a litany of unacceptable shortcomings: adverse clinical events including restenosis, neointimal hyperplasia, thrombosis, inflammation, and poor re-endothelialization. We have developed a biocompatible, multifunctional, peptide amphiphile-based nanomatrix coating for stents. In this study, we evaluated the ability of the nanomatrix coated stent to simultaneously address the issues facing current stents under physiological flow conditions in vitro. We found that the nanomatrix coated stent could increase endothelial cell migration, adhesion, and proliferation (potential for re-endothelialization), discourage smooth muscle cell migration and adhesion (potential to reduce neointimal hyperplasia and restenosis), and decrease both platelet activation and adhesion (potential to prevent thrombosis) as well as monocyte adhesion (potential to attenuate inflammatory responses) under physiological flow conditions in vitro. These promising results demonstrate the potential clinical utility of this nanomatrix stent coating, and highlight the importance of biocompatibility, multifunctionality, and bioactivity in cardiovascular device design.

Background: Direct reprogramming of fibroblasts into cardiomyocytes (CMs), endothelial cells (ECs), or smooth muscle cells (SMCs) has emerged as a promising strategy for cell-based therapy. Moreover, tissue engineering has become a crucial means to enhance the effects of the cell therapy. However, simultaneous reprogramming of somatic cells into all three cell types or a tissue-like structure has not been investigated despite its significance. Here, we aimed to directly reprogram fibroblasts into a cardiovascular tissue containing all three cell types and determined their regenerative effects on infarcted hearts. Methods and results: Adult mouse fibroblasts were transfected with a pre-selected cardiac microRNA mimic, miR-208b-3p, and treated with ascorbic acid and BMP4 one day after the transfection for 9 days. At day 10, cells were reprogrammed into a tissue-like structure referred to as reprogrammed cardiovascular tissue (rCVT), which contains reprogrammed CM (16.9 ± 8.0% positive for TNNT2)-, EC (28.4 ± 3.5% positive for PECAM1)-, and SMC (12.0 ± 2.9% positive for SMTN)-like cells, and 14 identified mouse proteins of extracellular matrix. Sarcomeric striations, calcium oscillations and action potentials were shown in reprogrammed CMs. Reprogrammed ECs took up Dil-conjugated acetylated low density lipoprotein and reacted with DAF-FM diacetate, suggesting production of nitric oxide by the cells. Therapeutic effects of rCVT were determined on a mouse model of myocardial infarction with echocardiography and histological analysis over 4 months. Implantation of this rCVT onto the infarcted mouse heart reduced regional cardiac strains and improved cardiac function. Histological examination showed migration of reprogrammed cells from rCVT into the infarcted hearts. Migrated rECs and rSMCs contributed to vessel formation in collaboration with host vascular cells. Migrated rCMs initially displayed immature characteristics but became mature over time and formed gap junctions with host CMs. Conclusions: This study demonstrates that a combination of miR-208b-3p, AA and BMP4 can directly reprogrammed single adult somatic cells into a cardiac tissue-like structure containing CMs, ECs, and SMCs, and that rCVT is effective for cardiac repair.

Rationale: For therapeutic neovascularization through cell therapy to treat cardiovascular diseases, recently, we successfully generated reprogrammed ECs, directly from human dermal fibroblasts (HDF) with the ETV2 transcription factor. Although functional ECs were generated, ETV2 was delivered lentiviral vector, which is clinically unsuitable. Objective: In this study, we aimed to generate clinically compatible rECs, by employing an adenoviral vector to minimize genetic integration. Furthermore, to prolong the cell retention and improve cell survival, we applied synthetic biomaterial, polypeptide amphiphile (PA) nanomatrix gel and tested for experimental cell therapy in animal models. Methods and Results: After we transduced HDFs with Ad- ETV2 (Ad- ETV2 -HDF), EC gene expression was measured by qRT-PCR and flow cytometry. We found that Ad- ETV2- HDFs displayed a cobblestone-like morphology and showed notable induction of EC genes (CDH5, KDR, PECAM1). Flow cytometry analysis showed peak expression of CDH5 and KDR at D6 and continuous increase of PECAM1 over 9 days. Also, Ad- ETV2 -HDFs took up acetylated LDL and bound lectin, suggesting that these cells possess functional EC characteristics. To enrich cells showing EC characters, Ad- ETV 2-HDFs were sorted for KDR by FACS at day 6. qRT-PCR showed substantial enrichment of EC-specific gene expression in KDR + cells. Only KDR + cells (reprogrammed ECs, rECs) formed tube-like structures on Matrigel. In addition, immunostaining confirmed high expression of EC marker proteins in rECs. Next, we tested the functionality of rECs in hindlimb ischemia mouse model. At 4 weeks after injection of 4 x 10 5 rECs into the ischemic hindlimb, we observed rECs incorporated into host blood vessels or resided perivascular regions. Furthermore, we found more rECs in the tissues and were directly incorporated into the vessels when they were encapsulated in PA nanomatrix gel compared to bare rEC-injected mice. Conclusions: Together, these data strongly support the generation of Ad- ETV2 -induced rECs (Ad-rECs) and enhanced retention of rECs by PA nanomatrix gel in tissue. With this study, we can develop advanced concept and techniques for clinical application of rECs and establish a new platform for future cell therapy.

Attempts to regenerate cardiac vessels in order to treat ischemic myocardial disease have continued to be an old goal in the field of gene therapy. However, most studies that have applied proangiogenic factors have shown that the effects were limited. We examined the therapeutic effects of the ETV2 gene, a factor induced by postnatal injury, which is involved in the differentiation and specification of endothelial cells in the developmental stages in animal models of ischemic myocardial infarction via the adenoviral expression system. The ETV2 gene, using adenoviral vectors, was efficiently delivered in normal heart and myocardial infarction models and induced gene expression. In the myocardial infarction model of the control group, the function of the heart deteriorated after induction over time, but deterioration in the adenoviral ETV2-treated myocardial infarction group was delayed. To confirm whether this effect was due to angiogenesis, the expression of proangiogenic factors was examined, and the expression of vascular endothelial growth factor and angiopoietin were significantly increased in the group treated with ETV2. This result was reproduced via ETV2 gene expression at the cellular level. In order to confirm the effects of angiogenesis more deeply, we confirmed that the dividing vascular endothelial cells expressed Ki67. As a result, it was confirmed that a significant number of Ki67-positive cells were present in the functional blood vessel region in the group treated with ETV2. Taken together, the results suggest that angiogenesis plays a role in regenerating cardiac vessels when the ETV2 gene is introduced into the myocardial infarction model, which may be considered as a candidate for treatment of ischemic heart disease.

Current systems generating lymphatic endothelial cell (LEC) from human pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival in vivo . Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel. hPSCs were cultured with GSK3-β inhibitor for 3 days to induce differentiation into the mesodermal lineage. The mesodermal cells were cultured for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN + FLT4 + cells showed highly purified and fully functional LEC characteristics in vitro. These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs/PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. This study demonstrated for the first time that PA-RGDS encapsulation can substantially improve lymphedema repair in mouse tail through enhancement of cell survival and lymphatic neovascularizationThis engineered hPSC-LEC therapy represents a novel option for treating lymphedema.

Two new sources of ECs were generated from human induced pluripotent stem cells (hiPSCs), and by direct reprogramming of somatic cells without undergoing the stages of stem or progenitor cell. These two types of ECs will advance our understanding of EC biology and can become a novel therapeutic option for treating ischemic cardiovascular diseases.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide an excellent platform to study human brain development and diseases in complex 3D tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. Here, we engineered hESCs to ectopically express human ETS variant 2 (hETV2) to create in vitro vasculature in hCOs, namely vhCOs (vascularized hCOs). hETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vascularization resulted in enhanced functional maturation of organoids. We found that vhCOs acquired several blood-brain barrier (BBB) characteristics, including an increase in the expression of tight junctions, nutrient transporters, and trans-endothelial electrical resistance. Finally, hETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature that resemble the early prenatal brain, and present a robust model to study brain disease in vitro.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges., In this Comment, Ngan Huang et al. discuss recent advances in cardiovascular tissue engineering and some of the main challenges that remain in translating these advances to the clinic. The authors propose future direction for the field to focus research efforts.

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.

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.

Background 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). Methods 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. Results Culture of UCB mononuclear cells yielded two distinct types of cells by morphology after 2weeks 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. Conclusion Late EPC and MSC are effective for infarct repair, apparently mediated through common humoral mechanisms.

Summary 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, Ca2+ 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., Graphical Abstract, Highlights • Molecular beacon (MB)-based method was developed to purify ventricular CMs from ESCs • Ventricular CM-specific MBs targeting Irx4 mRNA were successfully generated • About 98% of the CMs sorted via Irx4-MB displayed ventricular CM-like phenotypes • Irx4-MB-based purified CMs continuously maintained ventricular CM characteristics, In this article, Yoon and colleagues demonstrate that vCMs can be purified from differentiating ESCs using MBs targeting mRNA encoding Irx4, a transcription factor specific for vCMs. Purified IRX4-MB-positive cells displayed vCM-like action potentials and continuously maintained their vCM characteristics. This study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) emerged as an important source of cells for cardiovascular regeneration. This review summarizes protocols for generating hPSC-ECs and provides an overview of the current state of the research in clinical application of hPSC-derived ECs. Various systems were developed for differentiating hPSCs into the EC lineage. Stepwise two-dimensional systems are now well established, in which various growth factors, small molecules, and coating materials are used at specific developmental stages. Moreover, studies made significant advances in clinical applicability of hPSC-ECs by removing undefined components from the differentiation system, improving the differentiation efficiency, and proving their direct vascular incorporating effects, which contrast with adult stem cells and their therapeutic effects in vivo. Finally, by using biomaterial-mediated delivery, investigators improved the survival of hPSC-ECs to more than 10 months in ischemic tissues and described long-term behavior and safety of in vivo transplanted hPSC-ECs at the histological level. hPSC-derived ECs can be as a critical source of cells for treating advanced cardiovascular diseases. Over the past two decades, substantial improvement has been made in the differentiation systems and their clinical compatibility. In the near future, establishment of fully defined differentiation systems and proof of the advantages of biomaterial-mediated cell delivery, with some additional pre-clinical studies, will move this therapy into a vital option for treating those diseases that cannot be managed by currently available therapies.

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.

The purpose of this study was to investigate the effects of diabetes on mesenchymal stem cells (MSCs) in terms of their angiogenic and therapeutic potential for repairing tissue ischemia. We culture-isolated MSCs from streptozotocin-induced diabetic rats (D-MSCs) and compared their proliferation, differentiation, and angiogenic effects with those from normal rats (N-MSCs). The angiogenic effects of MSCs were evaluated by real-time PCR, in vitro tube formation assay, and transplantation of the MSCs into a hindlimb ischemia model followed by laser Doppler perfusion imaging. The number of MSCs derived from diabetic rats was smaller, and their proliferation rate was slower than N-MSCs. Upon induction of differentiation, the osteogenic and angiogenic differentiation of D-MSCs were aberrant compared to N-MSCs. The expression of angiogenic factors was lower in D-MSCs than N-MSCs. D-MSCs cocultured with endothelial cells resulted in decreased tube formation compared to N-MSCs. D-MSCs were ineffective to improve hindlimb ischemia and showed lower capillary density and angiogenic gene expression in ischemic limbs than N-MSCs. D-MSCs have defective proliferation and angiogenic activities and are ineffective for repairing hindlimb ischemia. Newer measures are needed before MSCs can be employed as a source for autologous cell therapy.

BackgroundCell 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.ObjectivesThis 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.MethodsCD31+ 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.ResultsThe 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.ConclusionsShort-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.

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.