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Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells., (© 2021. The Author(s).)

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates. NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation., (© 2021 by The American Society of Hematology.)

Over the last 20 years, significant progress has been made in the development of immunodeficient mouse models that now represents the gold standard tool in stem cell biology research. The latest major improvement has been the use of biomaterials in these xenogeneic mouse models to generate human "bone marrow like" tissues, which not only provides a more relevant xenograft model but can also potentially enable us to delineate the interactions that are specific between human bone marrow cells. There are a number of biomaterials and strategies to create humanized niches in immunodeficient mouse models, and the methods can also differ significantly among various research institutes. Here, we describe a protocol to create a humanized 3D collagen-based scaffold human niche in immunodeficient mouse model(s). This humanized in vivo model provides a powerful technique for understanding the human BM microenvironment and the role it plays in the regulation of normal as well as malignant hematopoiesis.

Aims: To explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration and the possibility of cotransplantation hUCMSCs and endothelial cells (ECs) for angiogenesis and pulp regeneration in vivo., Materials and Methods: hUCMSCs and human umbilical vein endothelial cells (HUVECs) were cocultured for matrigel angiogenesis assay in vitro and Matrigel plug assay in vivo. Next, we used the transwell coculture system to coculture hUCMSCs and HUVECs in vitro for RNA- sequencing (RNA-seq). Last, encapsulated hUCMSCs and HUVECs in scaffolds were injected into the root segments, and transplanted into immunodeficient mice for dental pulp regeneration., Key Findings: In vitro Matrigel angiogenesis assay and in vivo Matrigel plug assay indicated that cocultured hUCMSCs and HUVECs promote vascular formation of HUVECs, especially in 1:5 (hUCMSCs:HUVECs) coculture group. The RNA-seq result indicated that cocultured HUVECs exhibited high Hif-1 signaling pathway activity. We performed the cell transfection assay to knock down HIF1A-AS2 in HUVECs and then coculture with hUCMSCs, and the expression of VEGFA, HIF1A and PECAM1 were reduced. In pulp regeneration assay, Cotransplantation of hUCMSCs and HUVECs (1,5) group showed pulp-like tissue regeneration., Significance: Cocultured hUCMSCs and HUVECs can promote vascular formation of HUVECs, and the optimal coculture ration is 1:5 (hUCMSCs:HUVECs). hUCMSCs promote angiogenesis of HUVECs through the long noncoding RNA HIF1A-AS2-activation of the Hif-1 signaling pathway. Cotransplantation of hUCMSCs and HUVECs can regenerate dental pulp-like tissue in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia., Methods and Results: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography., Conclusion: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.

Islet transplantation has been demonstrated to be a promising therapy for type 1 diabetes mellitus. Although it is a minimally invasive operating procedure and provides easy access for graft monitoring, subcutaneous transplantation of the islet only has limited therapeutic outcomes, owing to the poor capacity of skin tissue to foster revascularization in a short period. Herein, 3D cell spheroids of clinically accessible umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells are formed and employed for codelivery with β cells subcutaneously. The 3D stem cell spheroids, which can secrete multiple proangiogenic and prosurvival growth factors, induce robust angiogenesis and prevent β cell graft death, as indicated by the results of in vivo bioluminescent tracking and histological analysis. These experimental data highlight the efficacy of the 3D stem cell spheroids that are fabricated using translationally applicable cell types in promoting the survival and function of subcutaneously transplanted β cells., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Aims: Allogeneic human umbilical mesenchymal stem cells (alloUMSC) are convenient cell source for stem cell-based therapy. However, immune rejection is a major obstacle for clinical application of alloUMSC for cardiac repair after myocardial infarction (MI). The immune rejection is due to the presence of human leukocyte antigen (HLA) class I molecule which is increased during MI. The aim of this study was to knockout HLA light chain β 2 -microglobulin (B2M) in UMSC to enhance stem cell engraftment and survival after transplantation., Methods and Results: We developed an innovative strategy using CRISPR/Cas9 to generate UMSC with B2M deletion (B2M - UMSC). AlloUMSC injection induced CD8 + T cell-mediated immune rejection in immune competent rats, whereas no CD8 + T cell-mediated killing against B2M - UMSC was observed even when the cells were treated with IFN-γ. Moreover, we demonstrate that UMSC-derived exosomes can inhibit cardiac fibrosis and restore cardiac function, and exosomes derived from B2M - UMSC are more efficient than those derived from UMSC, indicating that the beneficial effect of exosomes can be enhanced by modulating exosome's imprinting. Mechanistically, microRNA sequencing identifies miR-24 as a major component of the exosomes from B2M - UMSCs. Bioinformatics analysis identifies Bim as a putative target of miR-24. Loss-of-function studies at the cellular level and gain-of-function approaches in exosomes show that the beneficial effects of B2M - UMSCs are mediated by the exosome/miR-24/Bim pathway., Conclusion: Our findings demonstrate that modulation of exosome's imprinting via B2M knockout is an efficient strategy to prevent the immune rejection of alloUMSCs. This study paved the way to the development of new strategies for tissue repair and regeneration without the need for HLA matching.

Background: Hemangioma (Hem) is a benign tumor commonly seen in infancy with a relative high morbidity. Human umbilical vein endothelial cell (HUVEC)-derived extracellular vesicles (EVs) are actively participated in Hem. Therefore, this study is designed to figure out the underlying mechanism of HUVEC-derived EVs in Hem., Methods: Initially, EVs were separated from HUVECs and identified. HUVEC-derived EVs in normoxia or hypoxia were then cultivated with Hem endothelial cells (HemECs) to test the proliferation, apoptosis, and migration of HemECs. Microarray analysis was performed to select microRNAs (miRs) with differential expression. miR-210 in hypoxia-induced HUVECs was silenced, and the relevant EVs were extracted and then co-cultured with HemECs to perform biological effect experiments. Then, the target relation between miR-210 and homeobox A9 (HOXA9) was identified by the dual luciferase reporter gene assay and RNA immunoprecipitation assay. Moreover, xenograft transplantation was also applied to confirm the in vitro experiments., Results: Hypoxia-induced HUVECs promoted release of EVs, which were absorbed by HemECs. Hypoxia-induced HUVEC-EVs promoted HemEC proliferation and migration and inhibited apoptosis. miR-210 from the hypoxia-induced HUVEC-EVs was highly expressed and promoted HemEC growth. Silencing miR-210 expression in the hypoxia-induced HUVEC-EVs suppresses Hem development in vivo. In addition, miR-210 targeted HOXA9., Conclusion: Silencing miR-210 in HUVEC-derived EVs could suppress Hem by targeting HOXA9. This investigation may provide novel insights for Hem treatment., (© 2020 S. Karger AG, Basel.)

Osteoarthritis (OA) is a degenerative condition of the temporomandibular joint (TMJ) characterised by chronic inflammation and damage to joint structures. Because of the complexity of TMJ-OA, only symptomatic treatments are currently available. Recent reports have shown that many of stem cells can exert anti-inflammatory and tissue-regenerating effects. In this study, we investigated the potential cartilage-regenerating and anti-inflammatory effects of human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs) for the treatment of TMJ-OA. hUCM-MSC lines, isolated from different donors, which showed different activities in vitro. Using a selected cell line, we used different concentrations of hUCM-MSCs to assess therapeutic effects in a rabbit model of monosodium iodoacetate-induced TMJ-OA. Compared with the untreated control group, the potential regenerative result and anti-inflammatory effects of hUCM-MSCs were evident at all the tested concentrations in rabbits with induced TMJ-OA. The median dose of hUCM-MSCs showed the prominent cartilage protective effect and further cartilage regeneration potential. This effect occurred via upregulated expression of growth factors, extracellular matrix markers, and anti-inflammatory cytokines, and reduced expression of pro-inflammatory cytokines. The anti-inflammatory effect of hUCM-MSCs was comparable to that of dexamethasone (DEX). However, only hUCM-MSCs showed potential chondrogenesis effects in this study. In conclusion, our results indicate that hUCM-MSCs may be an effective treatment option for the treatment of TMJ-OA.

Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VMs grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ (phospholipase C) and ERK (extracellular signal-regulated kinase) activity.

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.-Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.

Rationale: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models., Objective: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation., Methods and Results: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins., Conclusions: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.

The aim of this study was to develop an effective cell sheet translocation method using a cell adhesive and temperature-responsive hydroxybutyl chitosan hydrogel (HBC). The polydopamine (PD)-coated HBC hydrogels were prepared by the dopamine self-polymerization on the surface of HBC hydrogel with different coating time, termed as P30, P60 and P120, respectively. Gelling property of HBC was not affected by PD coating. The PD-coated HBC hydrogels promoted the attachment and proliferation of mouse fibroblast cells (L929) and human umbilical vein endothelial cells (HUVECs), and allowed formation of monolayer cell sheet. In vitro translocation of HUVECs sheet could be obtained successively through phase transition of PD coated HBC hydrogel from gel to sol, and the cells sheet transferred from P30 hydrogel to a round cell coverglass maintained relatively complete monolayer and normal cell morphology. The results showed that P30 hydrogel has the potential to be used for cell transplantation therapy., (Copyright © 2018. Published by Elsevier B.V.)

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.

Manipulation of human T cell functioning by delivery of macromolecules such as DNA, RNA, or protein is limited, unless the human T cells have been stimulated or electropermeabilized. To achieve successful adaptation and survival of a grafted organ, the alloreactive T cells that induce graft rejection must be regulated. Corticosteroids, calcineurin inhibitors, and mTOR inhibitors, which are systemic immunosuppressants, are currently used for transplantation, with significant side effects. In this study, we demonstrated that a cell-permeable peptide (CPP), dNP2, could efficiently deliver proteins into human CD4 and CD8 T cells. We confirmed regulatory functioning of the cytoplasmic domain of CTLA-4 conjugated with dNP2 (dNP2-ctCTLA-4) in human T cell activation, proliferation, and chemokine receptor expression. We utilized a human skin allograft system in SCID/beige mice to examine whether dNP2-ctCTLA-4 could inhibit allograft rejection by controlling T cell responses. The grafted skin tissue inflammation, allogeneic T cell infiltration, and blood cytokine level was markedly reduced by dNP2-ctCTLA-4, resulting in successful transplantation. In addition, it also inhibited T cell alloresponses against microvessels formed form Bcl-2-transduced human umbilical vein endothelial cells implanted into Balb/c Rag1 -/- /IL-2Rγ -/- double knockout (DKO) mice, assessed as reduced T cell infiltration and granzyme B expression. These results collectively suggest that dNP2 peptide conjugation offers a valuable tool for delivering macromolecules like proteins into human T cells, and dNP2-ctCTLA-4 is a novel agent that shows potential in controlling human T cell responses to allow successful adaptation of grafted tissues., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Therapy targeting the neurovascular unit may provide effective neuroprotection against neonatal hypoxia-ischemia (HI). We hypothesized that the peripheral injection of hypoxia-preconditioned human umbilical vein endothelial cells (HUVECs) following HI protects against neurovascular damage and provides long-term neuroprotection in a postpartum (P) day-7 rat pup model. Compared with normoxic HUVECs, hypoxic HUVECs showed enhanced migration and angiogenesis in vitro and had augmented migration effects into the brain when administered intraperitoneally in vivo after HI. Moreover, 24 and 72 h post-HI, the hypoxic HUVECs group but not the normoxic HUVECs or culture-medium groups had significantly higher preservation of microvessels and neurons, and attenuation of blood-brain barrier damage than the normal-saline group. Compared to control or normal-saline groups, only the hypoxic HUVECs group had no impaired foot steps and showed a significant reduction of brain area loss at P42. Next-generation sequencing showed hypoxia-induced upregulation and downregulation of 209 and 215 genes in HUVECs, respectively. Upstream regulator analysis by ingenuity pathway analysis (IPA) identified hypoxia-inducible factor 1-alpha as the key predicted activated transcription regulator. After hypoxia, 12 genes (ADAMTS1, EFNA1, HIF1A, LOX, MEOX2, SELE, VEGFA, VEGFC, CX3CL1, HMMR, SDC, and SERPINE) associated with migration and/or angiogenesis were regulated in HUVECs. In addition, 6 genes (VEGFA, VEGFC, NTN4, TGFA, SERPINE1, and CX3CL1) involved in the survival of endothelial and neuronal cells were also markedly altered in hypoxic HUVECs. Thus, cell therapy by using hypoxic HUVECs that enhance migration and neurovascular protection may provide an effective therapeutic strategy for treating neonatal asphyxia.

Mitochondria contribute to key processes of cellular function, while mitochondrial dysfunction is implicated in metabolic disorders, neurodegenerative diseases, and cardiovascular diseases, in which angiogenesis - the formation of new blood capillaries - is dysregulated. The Hippo signaling transducer, Yes-associated protein (YAP1) binds to the TEA domain (TEAD1) transcription factor and controls angiogenesis. YAP1 also regulates glucose metabolism through peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), a major player controlling mitochondrial biogenesis. However, the role of YAP1-TEAD1-PGC1α signaling in mitochondrial structure, cellular metabolism, and angiogenesis in endothelial cells (ECs) remains unclear. We now find that knockdown of TEAD1 decreases the expression of PGC1α and suppresses mitochondrial biogenesis, glycolysis, and oxygen consumption in ECs. A YAP1 mutant construct, YAP1S127A, which stimulates binding of YAP1 to TEAD1, upregulates the expression of PGC1α, induces mitochondrial biogenesis, and increases oxygen consumption and glycolytic flux in ECs; in contrast, YAP1S94A, which fails to bind to TEAD1, attenuates these effects. PGC1α knockdown inhibits YAP1S127A-induced EC sprouting in vitro and vascular morphogenesis in the fibrin gel subcutaneously implanted on mice, while overexpression of PGC1α reverses vascular morphogenesis suppressed by YAP1S94A. These results suggest that YAP1-TEAD1 signaling induces mitochondrial biogenesis in ECs and stimulates angiogenesis through PGC1α. Modulation of YAP1-TEAD1-PGC1α signaling in ECs may provide a novel intervention for angiogenesis-related diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin ® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

Small-diameter vascular grafts are needed for the treatment of coronary artery diseases in the case of limited accessibility of the autologous vessels. Synthetic scaffolds have many disadvantages so in recent years vascular constructs (VCs) made from cellularized natural scaffolds was seen to be very promising but number of studies comprising this area is very limited. In our study, our aim is to generate fully natural triple-layered VC that constitutes all the layers of blood vessel with vascular cells. CD146+ perivascular cells (PCs) were isolated from human umbilical cord vein (HUCV) and differentiated into smooth muscle cells (SMCs) and fibroblasts. They were then combined with collagen type I/elastin/dermatan sulfate and collagen type I/fibrin to form tunica media and tunica adventitia respectively. HUCV endothelial cells (ECs) were seeded on the construct by cell sheet engineering method after fibronectin and heparin coating. Characterization of the VC was performed by immunolabeling, histochemical staining and electron microscopy (SEM and TEM). Differentiated cells were identified by means of immunofluorescent (IF) labeling. SEM and TEM analysis of VCs revealed the presence of three histologic tunicae. Collagen and elastic fibers were observed within the ECM by histochemical staining. The vascular endothelial growth factor receptor expressing ECs in tunica intima; α-SMA expressing SMCs in tunica media and; the tenascin expressing fibroblasts in tunica adventitia were detected by IF labeling. In conclusion, by combining natural scaffolds and vascular cells differentiated from CD146+ PCs, VCs can be generated layer by layer. This study will provide a preliminary blood vessel model for generation of fully natural small-diameter vascular grafts., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1788-1798, 2018., (© 2017 Wiley Periodicals, Inc.)

Emerging evidence suggests that vascular endothelial growth facto (VEGF) is important in the treatment of various ischemic and cardiovascular diseases. However, it often suffers from high cost and easy deactivation with a short half-life. Here, we describe a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF promoting the binding of the signaling protein to its VEGF receptor 2 (VEGFR2), activating receptor phosphorylation and pro-angiogenic related genes expression, and further stimulating downstream VEGF-dependent endothelial cell viability, migration, tube formation and rat aortic rings outgrowth. Interestingly, the obvious recruitment of mural cells were occurred to stabilize the sprouted microvessels. In addition, the pro-angiogenic potential of 26SCS composited VEGF was confirmed in vivo using the chick embryo chorioallantoic membrane (CAM) assay with an extensive perfusable vascular network. A longer monitoring was administered subcutaneously to mice in a biocompatible gelatin sponge and showed that VEGF with 26SCS had the capability to efficiently enhance neovascularization. These findings highlight that 26SCS, the semi-synthetic natural polymer, may be a promising coagent with VEGF for vascular therapy., Statement of Significance: Vascular endothelial growth factor (VEGF) is crucial for facilitating angiogenesis to supply oxygen and nutrient during wound healing and tissue regeneration. However, appropriate use of VEGF is an ongoing challenge due to its rapidly clearance and severe side effects at higher dosage. In this study, we described a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF, which could significantly promote its binding capacity to VEGF receptor 2 and further stimulate the angiogenic behavior of endothelial cells. We further confirmed that 26SCS was spatially combined with VEGF in a "lying manner", and this spatial arrangement was more conducive to exposure of the receptor binding domain of VEGF. Additionally, it also promoted in vivo angiogenesis in a chicken chorioallantoic membrane assay and mouse subcutaneous implant model. This strategy may afford a new avenue to enhance pro-angiogenic capacity of VEGF., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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 3β 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., (© 2017 American Heart Association, Inc.)

BACKGROUND The use of decellularized biological scaffolds for the reconstruction of small-diameter vascular grafts remains a challenge in tissue engineering. Thrombogenicity is an important cause of obstruction in these vessels due to decellularization. Seeding of the decellularized vascular constructs with endothelial cells is therefore a prerequisite for the prevention of thrombosis. The aim of this study was to seed decellularized baboon arteries with endothelial cells and to compare the thrombogenicity to that of decellularized arteries after circulation of blood. MATERIAL AND METHODS Carotid, radial, and femoral arteries (12 arteries in total) were harvested from 2 Papio ursinus baboons. Ten arteries were decellularized. Normal morphology was confirmed in the control vessels. The effect of re-endothelialization was studied in the vessel scaffolds using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Decellularization resulted in vessel scaffolds with well-preserved extracellular matrix and intact basal membranes. Six of the decellularized vessel scaffolds were seeded with viable human umbilical vein endothelial cells (HUVEC). Luminal endothelialization was established after 7 days in a bioreactor and SEM confirmed confluency. Two control, 4 decellularized, and 6 decellularized re-endothelialized vessel scaffolds were studied in an in vitro flow chamber using baboon blood. RESULTS The decellularized arteries showed an absence of endothelial lining, and an intact basement membrane. The seeding process produced a complete endothelial layer on the surfaces of the arteries. After perfusion with whole blood, no thrombi were formed in the control arteries and re-endothelialized vessels. Widespread platelet activation and adhesion occurred in the decellularized vessels despite a relatively intact basal membrane. CONCLUSIONS This study supports the development of re-endothelialized tissue engineered small-vessel conduits.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-β1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732., (© 2017 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Biomaterials are only used as carriers of cells in the conventional tissue engineering. Considering the multi-cell environment and active cell-biomaterial interactions in tissue regeneration process, in this study, structural signals of aligned electrospun nanofibers and chemical signals of bioglass (BG) ionic products in cell culture medium are simultaneously applied to activate fibroblast-endothelial co-cultured cells in order to obtain an improved skin tissue engineering construct. Results demonstrate that the combined biomaterial signals synergistically activate fibroblast-endothelial co-culture skin tissue engineering constructs through promotion of paracrine effects and stimulation of gap junctional communication between cells, which results in enhanced vascularization and extracellular matrix protein synthesis in the constructs. Structural signals of aligned electrospun nanofibers play an important role in stimulating both of paracrine and gap junctional communication while chemical signals of BG ionic products mainly enhance paracrine effects. In vivo experiments reveal that the activated skin tissue engineering constructs significantly enhance wound healing as compared to control. This study indicates the advantages of synergistic effects between different bioactive signals of biomaterials can be taken to activate communication between different types of cells for obtaining tissue engineering constructs with improved functions., Statement of Significance: Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in co-culture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background and Objective: Chronic periodontitis causes damage to tooth-supporting tissues, resulting in tooth loss in adults. Recently, cell-sheet-based approaches have been studied to overcome the limitations of conventional cytotherapeutic procedures for periodontal regeneration. The purpose of the present study was to investigate the regenerative potential of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs) in three-dimensional (3D) cell sheet constructs for periodontal regeneration in vivo., Material and Methods: PDLSCs, HUVECs or co-cultures of both cells were seeded onto temperature-responsive culture dishes, and intact cell sheets were fabricated. Cell sheets were wrapped around the prepared human roots in three different combinations and implanted subcutaneously into immunodeficient mice., Results: Histological evaluation revealed that after 2, 4 and 8 wk of implantation, periodontal ligament-like tissue arrangements were observed around the implanted roots in experimental groups compared with controls. Vascular lumens were also observed in periodontal compartments of HUVEC-containing groups. Periodontal ligament regeneration, cementogenesis and osteogenesis were evident in the experimental groups at both weeks 4 and 8, as shown by immunostaining for periostin and bone sialoprotein. Human cells in the transplanted cell sheets were stained by immunohistochemistry for the presence of human mitochondria., Conclusions: The 3D cell sheet-based approach may be potentially beneficial and is thus encouraged for future regenerative periodontal therapy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

Objective: CD98 regulates integrin signaling and is critical for tumor cell proliferation. It is also expressed on endothelial cells (EC), but its role in angiogenesis is unclear., Approach and Results: We used specific genetic targeting and antibody blockade approaches to examine the function of CD98 in EC proliferation, blood vessel growth, and tumor angiogenesis. It is upregulated on angiogenic ECs, and EC-specific deletion of CD98 in mice inhibited tumor growth, retinal angiogenesis, and EC proliferation. Reconstitution with CD98 mutants showed that integrin and CD98 interaction is necessary for EC survival and growth. Moreover, anti-CD98 treatment inhibited vessel formation and reversed EC-assisted tumor growth., Conclusions: Our findings demonstrate a requirement for CD98 in EC growth and suggest that CD98-specific reagents could have a dual anticancer effect: directly by inhibiting tumor cell proliferation and indirectly by preventing tumor angiogenesis., (© 2016 American Heart Association, Inc.)

Vascular-derived endothelial cell (EC) network prefabrication in three-dimensional (3D) tissue constructs before transplantation is useful for inducing functional anastomosis with the host vasculature. However, the clinical application of ECs is limited by cell isolation from the existing vasculature, because of the requirement for invasive biopsies and difficulty in obtaining a sufficient number of cells. Endothelial colony-forming cells (ECFCs), which are a subtype of endothelial progenitor cells in the blood, have a strong proliferative and vasculogenic potential. This study attempted to fabricate prevascular 3D cell-dense tissue constructs using cord blood-derived ECFCs and evaluate the in vivo angiogenic potential of these constructs. Human umbilical vascular endothelial cells (HUVECs) were also used in comparison with ECFCs, which were sandwiched between two human dermal-derived fibroblast (FB) sheets using a fibrin-coated cell-sheet manipulator. The inserted ECFCs in double-layered FB sheets were cultured for 3 days, resulting in the formation of network structures similar to those of HUVECs. Additionally, when ECFCs were sandwiched with three FB sheets, a lumen structure was found in the triple-layered cell-sheet constructs at 3 days after co-culture. These constructs containing ECFCs were transplanted into the subcutaneous tissue of immune-deficient rats. One week after transplantation, ECFC-lined functional microvessels containing rat erythrocytes were observed in the same manner as transplanted HUVEC-positive grafts. These results suggest that ECFCs might become an alternative cell source for fabricating a prevascular structure in 3D cell-dense tissue constructs for clinical application. Copyright © 2013 John Wiley & Sons, Ltd., (Copyright © 2013 John Wiley & Sons, Ltd.)

The present study used a previously developed three-dimensional Gelatin/Hydroxyapatite (Gel/HA) homogeneous nanocomposite scaffold with porosity of 82% and interconnecting pores ranging from 300 to 500 μm. Cell-seeded scaffolds were used to evaluate bone regeneration of rat critical-size calvarial defect. Totally, 36 male Wistar rats were randomly divided into four experimental groups, including blank defect (defects without any graft), blank scaffold (defects filled with Gel/HA scaffold without cells), and two groups of cell-seeded scaffolds (defects filled with either Gel/HA scaffold seeded with osteoblast-like and endothelial cells or osteoblast-like cell-seeded constructs). After 1, 4, and 12 weeks of scaffold implantation, rats were sacrificed and the calvaria were harvested for histological, immunohistochemical and histomorphometric analysis. In vitro tests showed that scaffolds were nontoxic to cells and promoted ideal cellular attachment. In vivo investigation on scaffold revealed that blank calvarial defects indicated incomplete tissue coverage and little evidence of bone healing. However, blank scaffold and cell-seeded scaffolds significantly promoted osteoconduction and ostegogenesis. Taken together, pre-seeded Gel/HA nanocomposite scaffold with osteoblasts and endothelial cells presented an effective combination to improve osteogenesis in the engineered bone implant. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1770-1778, 2016., (© 2016 Wiley Periodicals, Inc.)

Background: In plastic surgery, skin flap is an important approach to reconstructive wound repairs. The rat dorsal skin flap is a clinically relevant and popular animal model to investigate and evaluate flap survival and necrosis. Nonetheless, flap survival is often unstable with unpredictable outcomes, regardless of previous attempts at design modification., Methods & Results: In the present study, we report a novel flap chamber that provides stable and reproducible outcomes by separating the dorsal skin flap from its surrounding skin by in situ immobilization. The flap chamber blocks circulation that disturbs flap ischemia from both basal and lateral sides of the flap tissue. Demarcation of skin necrosis is macroscopically evident on the flap and supported by distinct changes in histological architecture under microscopic examination. The utility of the novel skin flap chamber is further proven by applying it to the examination of flap survival in streptozotocin-induced diabetic rats with an increase in skin necrosis. The flap chamber also affords size modifications where a narrower flap chamber increases ischemia and provides manipulable therapeutic windows for studying cell therapies. Accordingly, intradermal injection of endothelial cells 3 days before flap ischemia significantly increases the survival of skin flaps., Conclusions: The novel flap chamber not only may stabilize the skin flap and provide reproducible outcomes that overcome the shortfalls of the traditional ischemic flap but also may afford size modifications that support research designs and test therapeutic approaches to regenerative repair.

There are increasing numbers of patients underwent partial nephrectomy, and recovery of disturbed renal function is imperative post partial nephrectomy. We previously have demonstrated the decellularized (DC) scaffolds could mediate the residual kidney regeneration and thus improve disturbed renal function after partial nephrectomy. However, the cellular changes including the angiogenesis in the implanted DC scaffold has not yet been elaborated. In this study, we observed that the scaffold promoted the proliferation of human umbilical vein endothelial cells (HUVEC) that adhered to the DC scaffold in vitro. We next examined the pathological changes of the implanted DC graft in vivo, and found a decreased volume of the scaffold and a dramatic angiogenesis within the scaffold. The average microvessel density (aMVD) increased at the early stage, while decreased at the later stage post transplantation. Expression level of vascular endothelial growth factor (VEGF) showed similar dynamic changes. In addition, many endothelial cells (ECs) and endothelial progenitor cells (EPCs) were distributed in the region which contained active angiogenesis in the scaffold. However, the implanted graft became fibrosis and the angiogenesis degraded at final stage roughly 8 weeks post transplantation. Our data indicate that DC scaffold can be vascularized in vivo and possible mechanisms are discussed., Competing Interests: The authors declare no competing financial interests.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.

Mesenchymal stem cell (MSC) is a promising tool for the therapy of immune disorders. However, their efficacy and mechanisms in treating allergic skin disorders are less verified. We sought to investigate the therapeutic efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) against murine atopic dermatitis (AD) and to explore distinct mechanisms that regulate their efficacy. AD was induced in mice by the topical application of Dermatophagoides farinae. Naïve or activated-hUCB-MSCs were administered to mice, and clinical severity was determined. The subcutaneous administration of nucleotide-binding oligomerization domain 2 (NOD2)-activated hUCB-MSCs exhibited prominent protective effects against AD, and suppressed the infiltration and degranulation of mast cells (MCs). A β-hexosaminidase assay was performed to evaluate the effect of hUCB-MSCs on MC degranulation. NOD2-activated MSCs reduced the MC degranulation via NOD2-cyclooxygenase-2 signaling. In contrast to bone marrow-derived MSCs, hUCB-MSCs exerted a cell-to-cell contact-independent suppressive effect on MC degranulation through the higher production of prostaglandin E2 (PGE2 ). Additionally, transforming growth factor (TGF)-β1 production from hUCB-MSCs in response to interleukin-4 contributed to the attenuation of MC degranulation by downregulating FcεRI expression in MCs. In conclusion, the subcutaneous application of NOD2-activated hUCB-MSCs can efficiently ameliorate AD, and MSC-derived PGE2 and TGF-β1 are required for the inhibition of MC degranulation., (© 2015 AlphaMed Press.)

The aim of this study was to compare the effect of implantation site (i.e., subcutaneous, SQ vs. intramuscular, IM) on bone forming capacity of cell-based and growth factor-based scaffolds in athymic nude rats after an implantation period of 8 weeks. Cell-based scaffolds consisted of porous hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds seeded with either human adipose tissue-derived mesenchymal stem cells (AT-MSCs) only or both AT-MSCs and human umbilical vein endothelial cells (HUVECs), which were precultured in osteogenic medium for 7 days. Growth factor-based scaffolds consisted of porous HA/TCP scaffolds with 20 µg preadsorbed bone morphogenetic protein-2 (BMP-2). Histological and histomorphometrical analysis were used to assess bone formation. A differentiation experiment was performed in parallel to compare the in vitro osteogenic capacity of cell-based scaffolds. The results showed that cell-based scaffolds showed evident osteogenic differentiation in vitro, with only marginal differences between AT-MSCs only and AT-MSCs/HUVECs. In vivo, none of the cell-based scaffolds showed bone formation, irrespective of the site of implantation. In contrast, all growth factor-based scaffolds showed bone formation at both implantation sites without differences in the amount of formed bone. In conclusion, the results of this study demonstrated that the bone forming capacity of HA/TCP scaffolds with pre-adsorbed BMP-2 was equal at different ectopic implantation sites. Further, despite obvious in vitro osteogenic differentiation of AT-MSCs and AT-MSCS/HUVECs on HA/TCP scaffolds, no bone formation of these cell-based scaffolds was observed in vivo. This indicates further investigation on bone formation mechanisms of AT-MSCs is needed before AT-MSCs can be used as a cytotherapeutic treatment in clinics., (© 2014 Wiley Periodicals, Inc.)

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.

Volume stability and growth of tissue engineered adipose tissue equivalents using adipose-derived stem cells (ASCs) rely strongly on angiogenesis and neovascularization to support the maintenance of cells. An attractive cellular approach is based on coimplantation of endothelial cells to create a vascular network. Endothelial progenitor cells (EPCs) are a promising cell population, since they can be easily isolated from autologous human peripheral blood and thus represent a clinically feasible option. We have previously shown in in vitro and semi-in vivo studies that ASCs exert beneficial effects on EPCs in terms of enhanced tube formation and formation of blood vessels, respectively. In this study, we investigated the in vivo effects of coimplantation on endothelial cell-mediated neovascularization and ASC-mediated adipose tissue formation. For this purpose, human ASCs and human EPCs (or HUVECs as direct comparison to EPCs) were suspended alone or in coculture in fibrin and subcutaneously injected into the back of athymic nude mice and explanted after 1, 3 or 6months. Our results show that monocultures of EPCs or HUVECs were not able to perform vasculogenesis and constructs exhibited complete resorption already after 1month. However, a remarkable difference between EPCs and HUVECs was detected when coimplanted with ASCs. While coimplanted HUVECs gave rise to a stable neovasculature which was characterized by perfusion with erythrocytes, coimplanted EPCs showed no ability to form vascular structures. In the case of HUVEC-derived neovasculature, coimplanted ASCs displayed perivascular properties by stabilizing these neovessels. However, formation of human adipose tissue was independent of coimplanted endothelial cells. Our results indicate that HUVECs are superior to EPCs in terms of promoting in vivo neovascularization and recruiting perivascular cells for vessel stabilization when coimplanted with ASCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell sheets harvested from temperature-responsive cell culture dishes (TRDs) has attracted considerable attention as effective tools for reconstructing the lost functions of tissues and organs in the regenerative medicine field. However, because of their thinness, handling problems sometimes arise when transferring cell sheets from a TRD to a target surface. In this study, we developed a facile cell transfer method referred to as in situ gelation by using both gelatin hydrogel and a support membrane. Gelation and low-temperature processes were simultaneously performed on TRD. Confluent cultured cells were efficiently harvested from TRD in less than 5 min by decreasing the incubation temperature to 20°C. Harvested cells were found to maintain their cell viability, extracellular matrix, and original shape, thus allowing transfer of the cells to another surface with a short incubation time at 37°C. This method is applicable for various cell types regardless of the formation of tight cell-cell junctions. In addition, because of the high flexibility of the gelatin-coated membrane, cells were efficiently transferred to the surface of a mouse subcutis and liver. When compared with conventional cell sheet manipulation methods, the interaction between the cell surface and membrane was reinforced by the uniformly formed gelatin gel layer without using a special device. Therefore, the in situ gelation method is a promising technique for cell sheet-based tissue engineering and regenerative medicine., (© 2014 Wiley Periodicals, Inc.)

Background and Purpose: We investigated the neurorestorative effects and underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in Type one diabetes mellitus (T1DM) rats., Methods: Type one diabetes mellitus rats were subjected to middle cerebral artery occlusion (MCAo) and 24 h later were treated with: (1) phosphate-buffered-saline; (2) HUCBCs. Brain endothelial cells (MBECs) were cultured and capillary tube formation was measured., Results: Human umbilical cord blood cells treatment significantly improved functional outcome and promoted white matter (WM) remodeling, as identified by Bielschowsky silver, Luxol fast blue and SMI-31 expression, increased oligodendrocyte progenitor cell and oligodendrocyte density after stroke in T1DM rats. HUCBC also promoted vascular remodeling, evident from enhanced vascular and arterial density and increased artery diameter, and decreased blood-brain barrier leakage. HUCBC treatment also increased Angiopoietin-1 and decreased receptor for advanced glycation end-products (RAGE) expression compared to T1DM-MCAo control. In vitro analysis of MBECs demonstrated that Ang1 inversely regulated RAGE expression. HUCBC and Ang1 significantly increased capillary tube formation and decreased inflammatory factor expression, while anti-Ang1 attenuated HUCBC-induced tube formation and antiinflammatory effects., Conclusion: Human umbilical cord blood cells is an effective neurorestorative therapy in T1DM-MCAo rats and the enhanced vascular and WM remodeling and associated functional recovery after stroke may be attributed to increasing Angiopoietin-1 and decreasing RAGE., (© 2014 John Wiley & Sons Ltd.)

Evidence suggests that apoptosis contributes significantly to cell death after cerebral ischemia. Our recent studies that utilized human umbilical cord blood-derived mesenchymal stem cells (hUCBSCs) demonstrated the potential of hUCBSCs to inhibit neuronal apoptosis in a rat model of CNS injury. Therefore, we hypothesize that intravenous administration of hUCBSCs after focal cerebral ischemia would reduce brain damage by inhibiting apoptosis and downregulating the upregulated apoptotic pathway molecules. Male Sprague-Dawley rats were obtained and randomly assigned to various groups. After the animals reached a desired weight, they were subjected to a 2 h middle cerebral artery occlusion (MCAO) procedure followed by 7 days of reperfusion. The hUCBSCs were obtained, cultured, and intravenously injected (0.25 × 10(6) cells or 1 × 10(6) cells) via the tail vein to separate groups of animals 24 h post-MCAO procedure. We performed various techniques including PCR microarray, hematoxylin and eosin, and TUNEL staining in addition to immunoblot and immunofluorescence analysis in order to investigate the effect of our treatment on regulation of apoptosis after focal cerebral ischemia. Most of the apoptotic pathway molecules which were upregulated after focal cerebral ischemia were downregulated after hUCBSCs treatment. Further, the staining techniques revealed a prominent reduction in brain damage and the extent of apoptosis at even the lowest dose of hUCBSCs tested in the present study. In conclusion, our treatment with hUCBSCs after cerebral ischemia in the rodent reduces brain damage by inhibiting apoptosis and downregulating the apoptotic pathway molecules.

Background and Objective: Recently, porcine acellular dermal matrix (PADM) has been proposed as a possible alternative to autogenous grafts in periodontal plastic surgery. The aim of the present study was to investigate the in vitro responses of four different oral cell lines cultured on a novel PADM. Furthermore, tissue reaction to PADM was evaluated histologically after subcutaneous implantation in mice., Material and Methods: Human gingival fibroblasts (HGF), human osteoblast-like cells, human umbilical vein endothelial cells and human oral keratinocytes (HOK) were cultured and transferred on to the PADM. A tissue culture polystyrene surface served as the control. The viability of all tested cell lines on PADM was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay and PrestoBlue(®) reagent. The ToxiLight(®) assay was performed to analyze the effect of PADM on adenylate kinase release. PADM was implanted into nude mice subcutaneously and subjected to histological analysis after 21 d., Results: Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assays, all tested cell lines cultured on PADM demonstrated a significant increase of viability compared to the control group (each p < 0.001) with the exception of HGF and HOK after 3 d (each p > 0.05). According to the PrestoBlue(®) analysis, all cell lines demonstrated a significant increase of viability compared to the control group at the particular points of measurement after 18 h (HGF p < 0.01; human osteoblast-like cells, human umbilical vein endothelial cells, HOK each p < 0.001). No significant cytotoxic effects of PADM on the tested cell lines could be observed, as assessed by changes in adenylate kinase release. Subcutaneous implantation of PADM into nude mice demonstrated good integration with surrounding tissues and significant revascularization of its collagen structure., Conclusion: Overall, the results suggest that PADM is a promising substitute for autogenous soft tissue grafts in periodontal surgery., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth., Methods and Results: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension., Conclusions: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage., (© 2014 American Heart Association, Inc.)

Objective: It is well established that angiogenesis is a complex and coordinated multistep process. However, there remains a lack of information about the genes that regulate individual stages of vessel formation. Here, we aimed to define the role of human interferon-induced transmembrane protein 1 (IFITM1) during blood vessel formation., Approach and Results: We identified IFITM1 in a microarray screen for genes differentially regulated by endothelial cells (ECs) during an in vitro angiogenesis assay and found that IFITM1 expression was strongly induced as ECs sprouted and formed lumens. We showed by immunohistochemistry that human IFITM1 was expressed by stable blood vessels in multiple organs. siRNA-mediated knockdown of IFITM1 expression spared EC sprouting but completely disrupted lumen formation, in both in vitro and in an in vivo xeno-transplant model. ECs lacking IFITM1 underwent early stages of lumenogenesis (ie, intracellular vacuole formation) but failed to mature or expand lumens. Coimmunoprecipitation studies confirmed occludin as an IFITM1 binding partner in ECs, and immunocytochemistry showed a lack of occludin at endothelial tight junctions in the absence of IFITM1. Finally, time-lapse video microscopy revealed that IFITM1 is required for the formation of stable cell-cell contacts during endothelial lumen formation., Conclusions: IFITM1 is essential for the formation of functional blood vessels and stabilizes EC-EC interactions during endothelial lumen formation by regulating tight junction assembly.

Aims: We elucidated the therapeutic potential of human umbilical vein endothelial cells (HUVECs) for ameliorating progressive heart failure in a myocardial infarction (MI) rat model., Main Methods: MI was induced by ligation of left anterior descending artery, and HUVEC was transplanted 1week after MI. Cardiac function was evaluated by echocardiography, and histological analyses were performed., Key Findings: Phosphate-buffered saline (MI-V, n=5) or HUVEC (MI-HV, n=5) were injected into the border zone and infarcted area 7days after ligation of the left coronary artery in rats. The MI-HV group showed attenuation of left ventricular (LV) remodeling compared with the MI-V group. In the infarcted myocardium, a few of injected HUVEC was retained up to 28days. The ratios of matrix metalloproteinase (MMP)-2 or MMP-9 to tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-3 were decreased in the MI-HV group compared with the MI-V group. In vivo zymography analysis showed that HUVEC transplantation decreased the activities of MMP-2 and MMP-9. In immunohistochemistry, decreased MMP-2 and increased TIMP-1 and TIMP-3 expression were observed at 48h after HUVEC transplantation. These effects on MMP/TIMP balance were inhibited by L-NAME administration (an eNOS inhibitor, 10mg/kg). NOS inhibition decreased the protein expressions of TIMP-1 and TIMP-3 but did not change the protein expressions of MMP-2 and MMP-9., Significance: Our data suggest that altered balance between MMP and TIMP by HUVEC transplantation contributed to attenuation of ventricular remodeling after MI via eNOS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although a number of growth factors and receptors are known to control tumor angiogenesis, relatively little is known about the mechanism by which these factors influence the directional endothelial cell migration required for cancer microvessel formation. Recently, it has been shown that the focal adhesion protein paxillin is required for directional migration of fibroblasts in vitro. Here, we show that paxillin knockdown enhances endothelial cell migration in vitro and stimulates angiogenesis during normal development and in response to tumor angiogenic factors in vivo. Paxillin produces these effects by decreasing expression of neuropilin 2 (NRP2). Moreover, soluble factors secreted by tumors that stimulate vascular ingrowth, including vascular endothelial growth factor (VEGF), also decrease endothelial cell expression of paxillin and NRP2, and overexpression of NRP2 reverses these effects. These results suggest that the VEGF-paxillin-NRP2 pathway could represent a new therapeutic target for cancer and other angiogenesis-related diseases.

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvβ3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥ 6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.