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 PARGDS-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.
Cell-based therapy has emerged as a promising therapy for cardiovascular disease. Particularly, bone marrow (BM)-derived cells have been most extensively investigated and have shown encouraging results in preclinical studies. Clinical trials, however, have demonstrated split results in post-myocardial infarction cardiac repair. Mechanistically, transdifferentiation of BM-derived cells into cardiovascular tissue demonstrated by earlier studies is now known to play a minor role in functional recovery, and humoral and paracrine effects turned out to be main mechanisms responsible for tissue regeneration and functional recovery. With this advancement in the mechanistic insight of BM-derived cells, new efforts have been made to identify cell population, which can be readily isolated and obtained in sufficient quantity without mobilization and have higher therapeutic potential. Recently, haematopoietic CD31+ cells, which are more prevalent in bone marrow and peripheral blood, have been revealed to have angiogenic and vasculogenic activities and strong potential for therapeutic neovascularization in ischaemic tissues. This article will cover the recent advances in BM-derived cell-based therapy and implication of CD31+ cells.LINKED ARTICLES
This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2
Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. Objective: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. Methods and Results: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR + (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. Conclusions: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.
Background: Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines.
Methods: Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS.
Results and Conclusions: At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α –myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.
Since the successful generation of induced pluripotent stem cells (iPSC) from adult somatic cells using integrating-viral methods, various methods have been tried for iPSC generation using non-viral and non-integrating technique for clinical applications. Recently, various non-viral approaches such as protein, mRNA, microRNA, and small molecule transduction were developed to avoid genomic integration and generate stem cell-like cells from mouse and human fibroblasts. Despite these successes, there has been no successful generation of iPSC from bone marrow (BM)-derived hematopoietic cells derived using non-viral methods to date. Previous reports demonstrate the ability of polymeric micro and nanoparticles made from polyketals to deliver various molecules to macrophages. MicroRNA-loaded nanoparticles were created using the olyketal polymer PK3 (PK3-miR) and delivered to somatic cells for 6 days, resulting in the formation of colonies. Isolated cells from these colonies were assayed and substantial induction of the pluripotency markers Oct4, Sox2, and Nanog were detected. Moreover, colonies transferred to feeder layers also stained positive for pluripotency markers including SSEA-1. Here, we demonstrate successful activation of pluripotency-associated genes in mouse BM-mononuclear cells using embryonic stem cell (ESC)-specific microRNAs encapsulated in the acid sensitive polyketal PK3. These reprogramming results demonstrate that a polyketal-microRNA delivery vehicle can be used to generate various reprogrammed cells without permanent genetic manipulation in an efficient manner.
by
Leni Moldovan;
Mirela Anghelina;
Taylor Kantor;
Desiree Jones;
Eness Ramadan;
Yang Xiang;
Kun Huang;
Arunark Kolipaka;
William Malarkey;
Nima Ghasemzadeh;
Peter J. Mohler;
Arshed Quyyumi;
Nicanor I. Moldovan;
Young-sup Yoon
Background
Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs.
Methods and Results
We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow–derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media.
Conclusions
We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.
Objective
Myeloid lineage cells (MLCs) such as macrophages are known to play a key role in post-ischemic neovascularization. However, the role of MLC-derived reactive oxygen species (ROS) in this process and the chemical identity of the ROS remain unknown.
Methods and Results
Transgenic mice with MLC-specific over-expression of catalase (TgCat-MLC mice) were created on a C57BL/6 background. Macrophage catalase activity was increased 3.4-fold compared to wild-type mice. After femoral artery ligation, LASER Doppler perfusion imaging revealed impaired perfusion recovery in TgCat-MLC mice. This was associated with fewer collateral vessels, as assessed by micro CT angiography, and decreased capillary density. Impaired functional recovery of the ischemic limb was also evidenced by a 50% reduction in spontaneous running activity. The deficient neovascularization was associated with a blunted inflammatory response, characterized by decreased macrophage infiltration of ischemic tissues, and lower mRNA levels of inflammatory markers such as tumor necrosis factor-α, osteopontin, and matrix mettaloproteinase-9. In vitro macrophage migration was impaired in TgCat-MLC mice, suggesting a role for H2O2 in regulating the ability of macrophages to infiltrate ischemic tissues.
Conclusions
MLC-derived H2O2 plays a key role in promoting neovascularization in response to ischemia and is a necessary factor for the development of ischemia-induced inflammation.
Purpose of Review: Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) emerged as an important source of cells for cardiovascular regeneration. This review summarizes protocols for generating hPSC-ECs and provides an overview of the current state of the research in clinical application of hPSC-derived ECs. Recent Findings: Various systems were developed for differentiating hPSCs into the EC lineage. Stepwise two-dimensional systems are now well established, in which various growth factors, small molecules, and coating materials are used at specific developmental stages. Moreover, studies made significant advances in clinical applicability of hPSC-ECs by removing undefined components from the differentiation system, improving the differentiation efficiency, and proving their direct vascular incorporating effects, which contrast with adult stem cells and their therapeutic effects in vivo. Finally, by using biomaterial-mediated delivery, investigators improved the survival of hPSC-ECs to more than 10 months in ischemic tissues and described long-term behavior and safety of in vivo transplanted hPSC-ECs at the histological level. Summary: hPSC-derived ECs can be as a critical source of cells for treating advanced cardiovascular diseases. Over the past two decades, substantial improvement has been made in the differentiation systems and their clinical compatibility. In the near future, establishment of fully defined differentiation systems and proof of the advantages of biomaterial-mediated cell delivery, with some additional pre-clinical studies, will move this therapy into a vital option for treating those diseases that cannot be managed by currently available therapies.
Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.