Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells.

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Photopolymerizable Hydrogel for Enhanced Intramyocardial Vascular Progenitor Cell Delivery and Post-Myocardial Infarction Healing.

Cell transplantation success for myocardial infarction (MI) treatment is often hindered by low engraftment due to washout effects during myocardial contraction. A clinically viable biomaterial that enhances cell retention can optimize intramyocardial cell delivery. In this study, a therapeutic cell delivery method is developed for MI treatment utilizing a photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. Human vascular progenitor cells, capable of forming functional vasculatures upon transplantation, are combined with an in situ photopolymerization approach and injected into the infarcted zones of mouse hearts. This strategy substantially improves acute cell retention and promotes long-term post-MI cardiac healing, including stabilized cardiac functions, preserved viable myocardium, and reduced cardiac fibrosis. Additionally, engrafted vascular cells polarize recruited bone marrow-derived neutrophils toward a non-inflammatory phenotype via transforming growth factor beta (TGFß) signaling, fostering a pro-regenerative microenvironment. Neutrophil depletion negates the therapeutic benefits generated by cell delivery in ischemic hearts, highlighting the essential role of non-inflammatory, pro-regenerative neutrophils in cardiac remodeling. In conclusion, this GelMA hydrogel-based intramyocardial vascular cell delivery approach holds promise for enhancing the treatment of acute myocardial infarction.

Transcriptomic and proteomic pathways of diabetic and non-diabetic mitochondrial transplantation.

Reduced mitochondrial function increases myocardial susceptibility to ischemia-reperfusion injury (IRI) in diabetic hearts. Mitochondrial transplantation (MT) ameliorates IRI, however, the cardioprotective effects of MT may be limited using diabetic mitochondria. Zucker Diabetic Fatty (ZDF) rats were subjected to temporary myocardial RI and then received either vehicle alone or vehicle containing mitochondria isolated from either diabetic ZDF or non-diabetic Zucker lean (ZL) rats. The ZDF rats were allowed to recover for 2 h or 28 days. MT using either ZDF- or ZL-mitochondria provided sustained reduction in infarct size and was associated with overlapping upregulation of pathways associated with muscle contraction, development, organization, and anti-apoptosis. MT using either ZDF- or ZL-mitochondria also significantly preserved myocardial function, however, ZL- mitochondria provided a more robust long-term preservation of myocardial function through the mitochondria dependent upregulation of pathways for cardiac and muscle metabolism and development. MT using either diabetic or non-diabetic mitochondria decreased infarct size and preserved functional recovery, however, the cardioprotection afforded by MT was attenuated in hearts receiving diabetic compared to non-diabetic MT.

Engineered immunomodulatory accessory cells improve experimental allogeneic islet transplantation without immunosuppression.

Islet transplantation has been established as a viable treatment modality for type 1 diabetes. However, the side effects of the systemic immunosuppression required for patients often outweigh its benefits. Here, we engineer programmed death ligand-1 and cytotoxic T lymphocyte antigen 4 immunoglobulin fusion protein-modified mesenchymal stromal cells (MSCs) as accessory cells for islet cotransplantation. The engineered MSCs (eMSCs) improved the outcome of both syngeneic and allogeneic islet transplantation in diabetic mice and resulted in allograft survival for up to 100 days without any systemic immunosuppression. Immunophenotyping revealed reduced infiltration of CD4+ or CD8+ T effector cells and increased infiltration of T regulatory cells within the allografts cotransplanted with eMSCs compared to controls. The results suggest that the eMSCs can induce local immunomodulation and may be applicable in clinical islet transplantation to reduce or minimize the need of systemic immunosuppression and ameliorate its negative impact.

Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2.

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(ß)-two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection-but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/ß in the treatment of patients with COVID-19.

A Biphasic Osteovascular Biomimetic Scaffold for Rapid and Self-Sustained Endochondral Ossification.

Regeneration of large bones remains a challenge in surgery. Recent developmental engineering efforts aim to recapitulate endochondral ossification (EO), a critical step in bone formation. However, this process entails the condensation of mesenchymal stem cells (MSCs) into cartilaginous templates, which requires long-term cultures and is challenging to scale up. Here, a biomimetic scaffold is developed that allows rapid and self-sustained EO without initial hypertrophic chondrogenesis. The design comprises a porous chondroitin sulfate cryogel decorated with whitlockite calcium phosphate nanoparticles, and a soft hydrogel occupying the porous space. This composite scaffold enables human endothelial colony-forming cells (ECFCs) and MSCs to rapidly assemble into osteovascular niches in immunodeficient mice. These niches contain ECFC-lined blood vessels and perivascular MSCs that differentiate into RUNX2+ OSX+ pre-osteoblasts after one week in vivo. Subsequently, multiple ossification centers are formed, leading to de novo bone tissue formation by eight weeks, including mature human OCN+ OPN+ osteoblasts, collagen-rich mineralized extracellular matrix, hydroxyapatite, osteoclast activity, and gradual mechanical competence. The early establishment of blood vessels is essential, and grafts that do not contain ECFCs fail to produce osteovascular niches and ossification centers. The findings suggest a novel bioengineering approach to recapitulate EO in the context of human bone regeneration.

Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Abnormal Flow Conditions Promote Endocardial Fibroelastosis Via Endothelial-to-Mesenchymal Transition, Which Is Responsive to Losartan Treatment.

Endocardial fibroelastosis (EFE) is defined by fibrotic tissue on the endocardium and forms partly through aberrant endothelial-to-mesenchymal transition. However, the pathologic triggers are still unknown. In this study, we showed that abnormal flow induces EFE partly through endothelial-to-mesenchymal transition in a rodent model, and that losartan can abrogate EFE development. Furthermore, we translated our findings to human endocardial endothelial cells, and showed that laminar flow promotes the suppression of genes associated with mesenchymal differentiation. These findings emphasize the role of flow in promoting EFE in endocardial endothelial cells and provide a novel potential therapy to treat this highly morbid condition.

Interleukin-8 Receptors CXCR1 and CXCR2 Are Not Expressed by Endothelial Colony-forming Cells.

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and - 2 in ECFCs.Graphical Abstract.

Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA.

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated 13 diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

Bioengineering hemophilia A-specific microvascular grafts for delivery of full-length factor VIII into the bloodstream.

Hemophilia A (HA) is a bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatments are based on regular infusions of FVIII concentrates throughout a patient's life. Alternatively, viral gene therapies that directly deliver F8 in vivo have shown preliminary successes. However, hurdles remain, including lack of infection specificity and the inability to deliver the full-length version of F8 due to restricted viral cargo sizes. Here, we developed an alternative nonviral ex vivo gene-therapy approach that enables the overexpression of full-length F8 in patients' endothelial cells (ECs). We first generated HA patient-specific induced pluripotent stem cells (HA-iPSCs) from urine epithelial cells and genetically modified them using a piggyBac DNA transposon system to insert multiple copies of full-length F8. We subsequently differentiated the modified HA-iPSCs into competent ECs with high efficiency, and demonstrated that the cells (termed HA-FLF8-iECs) were capable of producing high levels of FVIII. Importantly, following subcutaneous implantation into immunodeficient hemophilic (SCID-f8ko) mice, we demonstrated that HA-FLF8-iECs were able to self-assemble into vascular networks, and that the newly formed microvessels had the capacity to deliver functional FVIII directly into the bloodstream of the mice, effectively correcting the clotting deficiency. Moreover, our implant maintains cellular confinement, which reduces potential safety concerns and allows effective monitoring and reversibility. We envision that this proof-of-concept study could become the basis for a novel autologous ex vivo gene-therapy approach to treat HA.

DKK3 (Dikkopf-3) Transdifferentiates Fibroblasts Into Functional Endothelial Cells-Brief Report.

Objective- To determine the role of a cytokine-like protein DKK3 (dikkopf-3) in directly transdifferentiating fibroblasts into endothelial cells (ECs) and the underlying mechanisms. Approach and Results- DKK3 overexpression in human fibroblasts under defined conditions for 4 days led to a notable change in cell morphology and progenitor gene expression. It was revealed that these cells went through mesenchymal-to-epithelial transition and subsequently expressed KDR (kinase insert domain receptor) at high levels. Further culture in EC defined media led to differentiation of these progenitors into functional ECs capable of angiogenesis both in vitro and in vivo, which was regulated by the VEGF (vascular endothelial growth factor)/miR (microRNA)-125a-5p/Stat3 (signal transducer and activator of transcription factor 3) axis. More importantly, fibroblast-derived ECs showed the ability to form a patent endothelium-like monolayer in tissue-engineered vascular grafts ex vivo. Conclusions- These data demonstrate that DKK3 is capable of directly differentiating human fibroblasts to functional ECs under defined media and provides a novel potential strategy for endothelial regeneration.

Plasticity of vascular resident mesenchymal stromal cells during vascular remodeling.

Vascular remodeling is a complex and dynamic pathological process engaging many different cell types that reside within the vasculature. Mesenchymal stromal/stem cells (MSCs) refer to a heterogeneous cell population with the plasticity to differentiate toward multiple mesodermal lineages. Various types of MSC have been identified within the vascular wall that actively contribute to the vascular remodeling process such as atherosclerosis. With the advances of genetic mouse models, recent findings demonstrated the crucial roles of MSCs in the progression of vascular diseases. This review aims to provide an overview on the current knowledge of the characteristics and behavior of vascular resident MSCs under quiescence and remodeling conditions, which may lead to the development of novel therapeutic approaches for cardiovascular diseases.

Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts.

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-ß1 (TGFß1), human umbilical cord-derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFß1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.

Adventitial Sca1+ Cells Transduced With ETV2 Are Committed to the Endothelial Fate and Improve Vascular Remodeling After Injury.

OBJECTIVE: Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. APPROACH AND RESULTS: We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control. CONCLUSIONS: These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.

Transdifferentiated Human Vascular Smooth Muscle Cells are a New Potential Cell Source for Endothelial Regeneration.

Endothelial dysfunction is widely implicated in cardiovascular pathological changes and development of vascular disease. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great interest to explore alternative cell sources capable of generating functional ECs. Vascular smooth muscle cell (SMC) composes the majority of the vascular wall and retains phenotypic plasticity in response to various stimuli. The aim of this study is to test the feasibility of the conversion of SMC into functional EC through the use of reprogramming factors. Human SMCs are first dedifferentiated for 4 days to achieve a vascular progenitor state expressing CD34, by introducing transcription factors OCT4, SOX2, KLF4 and c-MYC. These SMC-derived progenitors are then differentiated along the endothelial lineage. The SMC-converted ECs exhibit typical endothelial markers expression and endothelial functions in vitro, in vivo and in disease model. Further comprehensive analysis indicates that mesenchymal-to-epithelial transition is requisite to initiate SMCs reprogramming into vascular progenitors and that members of the Notch signalling pathway regulate further differentiation of the progenitors into endothelial lineage. Together, we provide the first evidence of the feasibility of the conversion of human SMCs towards endothelial lineage through an intermediate vascular progenitor state induced by reprogramming.

Mesenchymal stem cells and vascular regeneration.

In recent years, MSCs have emerged as a promising therapeutic cell type in regenerative medicine. They hold great promise for treating cardiovascular diseases, such as myocardial infarction and limb ischemia. MSCs may be utilized in both cell-based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits to patients. The efficacy of MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Increasing our understanding of the mechanisms of MSC involvement in vascular regeneration will be beneficial in boosting present therapeutic approaches and developing novel ones to treat cardiovascular diseases. In this review, we aim to summarize current progress in characterizing the in vivo identity of MSCs, to discuss mechanisms involved in cell-based therapy utilizing MSCs, and to explore current and future strategies for vascular regeneration.

Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage.

Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)-HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW-HA-stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2-induced HA synthesis and organization drives ESC-SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225-1238.