Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways.

Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31+ and 78%-83% VE-cadherin+ ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE-cadherin+ ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919.

Medical Disruptions During Center-Based Cardiac Rehabilitation: A Necessary Appraisal for the Development of Emerging Remote and Virtual Care Models.

PURPOSE: The purpose of this study is to show that with remote and virtual cardiac rehabilitation (CR) care models rapidly emerging, CR core components must be maintained to prioritize safety and effectiveness. Currently, there is a paucity of data on medical disruptions in phase 2 center-based CR (cCR). This study aimed to characterize the frequency and types of unplanned medical disruptions. METHODS: We reviewed 5038 consecutive sessions from 251 patients enrolled in cCR program from October 2018 to September 2021. Quantification of events was normalized to sessions to control for multiple disruptions that occurred to a single patient. A multivariate logistical regression model was used to predict comorbid risk factors for disruptions. RESULTS: Fifty percent of patients experienced one or more disruptions during cCR. Glycemic events (71%) and blood pressure (12%) abnormalities accounted for most of these while symptomatic arrhythmias (8%) and chest pain (7%) were less frequent. Sixty-six percent of events occurred within the first 12 wk. The regression model showed that a diagnosis of diabetes mellitus was the strongest predictor for disruptions (OR = 2.66: 95% CI, 1.57-4.52; P < .0001). CONCLUSIONS: Medical disruptions were frequent during cCR, with glycemic events being most common and occurring early. A diagnosis of diabetes mellitus was a strong independent risk factor for events. This appraisal suggests that patients living with diabetes mellitus, particularly those on insulin, need to be the highest priority for monitoring and planning and suggests that a hybrid care model may be beneficial in this population.

Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.

Hemophilia A (HA) is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells (ECs) in a non-diseased human being, ECs hold great potential for development as a cell therapy for HA. We showed that HA patient-specific induced pluripotent stem cells (HA-iPSCs) could provide a renewable supply of ECs. The HA-iPSC-derived ECs were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene, and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA-iPSC-derived ECs were retained in the animals for at least 10-16 weeks and maintained their expression of FVIII, GFP, and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into HA mice, these transduced HA-iPSC-derived ECs significantly reduced blood loss in a tail-clip bleeding test and produced therapeutic plasma levels (11.2%-369.2%) of FVIII. Thus, our studies provide proof-of-concept that HA-iPSC-derived ECs can serve as a factory to deliver FVIII for the treatment of HA not only in adults but also in newborns.

Enhancing Retention of Human Bone Marrow Mesenchymal Stem Cells with Prosurvival Factors Promotes Angiogenesis in a Mouse Model of Limb Ischemia.

Mesenchymal stem/stromal cells (MSCs) offer great promise in the treatment of ischemic injuries, including stroke, heart infarction, and limb ischemia. However, poor cell survival after transplantation remains a major obstacle to achieve effective MSC therapies. To improve cell survival and retention, we transplanted human bone marrow MSCs with or without a specific prosurvival factor (PSF) cocktail consisting of IGF1, Bcl-XL, a caspase inhibitor, a mitochondrial pathway inhibitor, and Matrigel into the limbs of immune deficient mice, after induction of hindlimb ischemia. The PSF markedly prolonged the retention of the MSCs in the ischemic limb muscles as demonstrated by bioluminescence imaging. Using microcomputed tomography to image the limb muscle vasculature in the mice 9 weeks after the transplantation, we found that the mice transplanted with MSCs without PSF did not show a significant increase in the blood vessels in the ischemic limb compared with the nontransplanted control mice. In contrast, the mice transplanted with MSCs plus PSF showed a significant increase in the blood vessels, especially the larger and branching vessels, in the ischemic limb compared with the control mice that did not receive MSCs. Thus, we demonstrated that prolonged retention of MSCs using PSF effectively promoted angiogenesis in ischemic animal limbs. This study highlights the importance of enhancing cell survival in the development of effective MSC therapies to treat vascular diseases.

Potential long-term treatment of hemophilia A by neonatal co-transplantation of cord blood-derived endothelial colony-forming cells and placental mesenchymal stromal cells.

BACKGROUND: Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. METHODS: In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. RESULTS: In vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 µl to 155.78 ± 44.93 µl in a tail-clip assay. CONCLUSIONS: This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA.