BACKGROUND Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility due to 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. Further, 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 GSK3β inhibitor. Next, VEGF, EGF, and bFGF 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 hindlimb ischemia model using Laser Doppler perfusion imaging, bioluminescence imaging, real-time RT-PCR, and histological analysis. RESULTS The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and pro-angiogenic activities. When injected into ischemic hindlimbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared to media-, PA-RGDS-, or HUVEC-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. CONCLUSION We generated highly enriched hPSC-ECs via a clinically compatible system. Further, 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.

Current cardiovascular therapies are limited by loss of endothelium, restenosis, and thrombosis. The goal of this study is to develop a biomimetic hybrid nanomatrix that combines unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but they are hampered by lack of surface bioactivity to control cellular behavior. It is hypothesized that PAs can self-assemble onto the surface of ePCL nanofibers and endow them with characteristic properties of native endothelium. PAs, which comprise hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell adhesive ligands (YIGSR) or ploylysine (KKKKK) nitric oxide (NO) donors. Two different PAs (PA-YIGSR and PA-KKKKK) were successfully synthesized and mixed in a 90:10 (YK) ratio to obtain PA-YK. PA-YK was reacted with pure NO to develop PA-YK-NO, which was then self-assembled onto ePCL nanofibers to generate a hybrid nanomatrix, ePCL-PA-YK-NO. Uniform coating of self-assembled PA nanofibers on ePCL was confirmed by TEM. Successful NO release from ePCL-PA-YK-NO was observed. ePCL-YK and ePCL-PA-YK-NO showed significantly increased adhesion of human umbilical vein endothelial cells (HUVECs). Also, ePCL-PA-YK-NO showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL-PA-YK-NO also displayed significantly reduced platelet adhesion when compared to ePCL, ePCL-PA-YK, and collagen control. These results indicate that this hybrid nanomatrix has great potential applications in cardiovascular implants.Corresponding author: Dr. Ho-Wook Jun, Assistant Professor, 1825 University Boulevard, Shelby 806, Birmingham, AL, 35211. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Electrospinning has been garnering a lot of attention recently [7][8][9][10][11], due to its ability to fabricate highly interconnected, non-woven fibers with diameters in the nanoscale ranges, which are structurally similar to nanofibrillar extracellular matrix (ECM) proteins [12]. Due to their ability to physically resemble natural ECM protein structure, several studies have been conducted into using electrospun materials as cardiovascular devices such as vascular grafts [13][14][15][16][17]. An important feature of electrospinning is its ability to deposit these nanofibers on a rotating mandrel to form a tubular structure, which is essential for vascular grafts [18,19], and it is also possible to generate scaff...

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.

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