A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation.

The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

Pioneer factor ETV2 safeguards endothelial cell specification by recruiting the repressor REST to restrict alternative lineage commitment.

Mechanisms of cell fate specification remain a central question for developmental biology and regenerative medicine. The pioneer factor ETV2 is a master regulator for the endothelial cell (EC) lineage specification. Here, we studied mechanisms of ETV2-driven fate specification using a highly efficient system in which ETV2 directs human induced pluripotent stem cell-derived mesodermal progenitors to form ECs over two days. By applying CUT&RUN, single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses, we characterized the transcriptomic profiles, chromatin landscapes, dynamic cis-regulatory elements (CREs), and molecular features of EC cell differentiation mediated by ETV2. This defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. Induced ETV2 expression both directed specification of endothelial progenitors and suppressed acquisition of alternative fates. Functional screening and candidate validation revealed cofactors essential for efficient EC specification, including the transcriptional activator GABPA. Surprisingly, the transcriptional repressor REST was also necessary for efficient EC specification. ETV2 recruited REST to occupy and repress non-EC lineage genes. Collectively, our study provides an unparalleled molecular analysis of EC specification at single-cell resolution and identifies the important role of pioneer factors to recruit repressors that suppress commitment to alternative lineages.

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.

Engineering large and geometrically controlled vascularized nerve tissue in collagen hydrogels to restore large-sized volumetric muscle loss.

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

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.

Results of an international survey about methods used to isolate human endothelial colony-forming cells: guidance from the SSC on Vascular Biology of the ISTH.

BACKGROUND: Assessment of endothelial colony-forming cell (ECFC) number and vasculogenic properties is crucial for exploring vascular diseases and regeneration strategies. A previous survey of the Scientific and Standardization Committee on Vascular Biology of the International Society on Thrombosis and Haemostasis clarified key methodological points but highlighted a lack of standardization associated with ECFC culture. OBJECTIVES: The aim of this study was to provide expert consensus guidance on ECFC isolation and culture. METHODS: We surveyed 21 experts from 10 different countries using a questionnaire proposed during the 2019 International Society on Thrombosis and Haemostasis Congress in Melbourne (Australia) to attain a consensus on ECFC isolation and culture. RESULTS: We report here the consolidated results of the questionnaire. There was agreement on several general statements, mainly the technical aspects of ECFC isolation and cell culture. In contrast, on the points concerning the definition of a colony of ECFCs, the quantification of ECFCs, and the estimation of their age (in days or number of passages), the expert opinions were widely dispersed. CONCLUSION: Our survey clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of ECFC isolation and culture procedures for clinical laboratory practice and robustness of results. To this end, we propose a standardized protocol for the isolation and expansion of ECFCs from umbilical cord and adult peripheral blood.

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.

Human Endothelial Colony-Forming Cells.

Endothelial colony-forming cells (ECFCs) are progenitor cells that can give rise to colonies of highly proliferative vascular endothelial cells (ECs) with clonal expansion and in vivo blood vessel-forming potential. More than two decades ago, the identification of ECFCs in human peripheral blood created tremendous opportunities as having a clinically accessible source of autologous ECs could facilitate meaningful therapies with the potential to impact multiple vascular diseases. Nevertheless, until recently, the field of endothelial progenitor cells has been plagued with ambiguities and controversies, and reaching a consensus on the definition of ECFCs has not been straightforward. Moreover, although the basic phenotypical and functional characteristics of cultured ECFCs are now well established, some fundamental questions such as the origin of ECFCs and their physiological roles in health and disease remain incompletely understood. Here, I highlight some critical studies that have shaped our current understanding of ECFCs in humans. Insights into the biological attributes of ECFCs are essential for facilitating the clinical translation of their therapeutic potential.

Mechanical strain triggers endothelial-to-mesenchymal transition of the endocardium in the immature heart.

BACKGROUND: Endothelial-to-mesenchymal-transition (EndMT) plays a major role in cardiac fibrosis, including endocardial fibroelastosis but the stimuli are still unknown. We developed an endothelial cell (EC) culture and a whole heart model to test whether mechanical strain triggers TGF-ß-mediated EndMT. METHODS: Isolated ECs were exposed to 10% uniaxial static stretch for 8 h (stretch) and TGF-ß-mediated EndMT was determined using the TGF-ß-inhibitor SB431542 (stretch + TGF-ß-inhibitor), BMP-7 (stretch + BMP-7) or losartan (stretch + losartan), and isolated mature and immature rats were exposed to stretch through a weight on the apex of the left ventricle. Immunohistochemical staining for double-staining with endothelial markers (VE-cadherin, PECAM1) and mesenchymal markers (αSMA) or transcription factors (SLUG/SNAIL) positive nuclei was indicative of EndMT. RESULTS: Stretch-induced EndMT in ECs expressed as double-stained ECs/total ECs (cells: 46 ± 13%; heart: 15.9 ± 2%) compared to controls (cells: 7 ± 2%; heart: 3.1 ± 0.1; p < 0.05), but only immature hearts showed endocardial EndMT. Inhibition of TGF-ß decreased the number of double-stained cells significantly, comparable to controls (cells/heart: control: 7 ± 2%/3.1 ± 0.1%, stretch: 46 ± 13%/15 ± 2%, stretch + BMP-7: 7 ± 2%/2.9 ± 0.1%, stretch + TGF-ß-inhibitor (heart only): 5.2 ± 1.3%, stretch + losartan (heart only): 0.89 ± 0.1%; p < 0.001 versus stretch). CONCLUSIONS: Endocardial EndMT is an age-dependent consequence of increased strain triggered by TGF- ß activation. Local inhibition through either rebalancing TGF-ß/BMP or with losartan was effective to block EndMT. IMPACT: Mechanical strain imposed on the immature LV induces endocardial fibroelastosis (EFE) formation through TGF-ß-mediated activation of endothelial-to-mesenchymal transition (EndMT) in endocardial endothelial cells but has no effect in mature hearts. Local inhibition through either rebalancing the TGF-ß/BMP pathway or with losartan blocks EndMT. Inhibition of endocardial EndMT with clinically applicable treatments may lead to a better outcome for congenital heart defects associated with EFE.

A Safe, Fibrosis-Mitigating, and Scalable Encapsulation Device Supports Long-Term Function of Insulin-Producing Cells.

Encapsulation and transplantation of insulin-producing cells offer a promising curative treatment for type 1 diabetes (T1D) without immunosuppression. However, biomaterials used to encapsulate cells often elicit foreign body responses, leading to cellular overgrowth and deposition of fibrotic tissue, which in turn diminishes mass transfer to and from transplanted cells. Meanwhile, the encapsulation device must be safe, scalable, and ideally retrievable to meet clinical requirements. Here, a durable and safe nanofibrous device coated with a thin and uniform, fibrosis-mitigating, zwitterionically modified alginate hydrogel for encapsulation of islets and stem cell-derived beta (SC-ß) cells is reported. The device with a configuration that has cells encapsulated within the cylindrical wall, allowing scale-up in both radial and longitudinal directions without sacrificing mass transfer, is designed. Due to its facile mass transfer and low level of fibrotic reactions, the device supports long-term cell engraftment, correcting diabetes in C57BL6/J mice with rat islets for up to 399 days and SCID-beige mice with human SC-ß cells for up to 238 days. The scalability and retrievability in dogs are further demonstrated. These results suggest the potential of this new device for cell therapies to treat T1D and other diseases.

A Zwitterionic Polyurethane Nanoporous Device with Low Foreign-Body Response for Islet Encapsulation.

Encapsulation of insulin-producing cells is a promising strategy for treatment of type 1 diabetes. However, engineering an encapsulation device that is both safe (i.e., no cell escape and no breakage) and functional (i.e., low foreign-body response (FBR) and high mass transfer) remains a challenge. Here, a family of zwitterionic polyurethanes (ZPU) with sulfobetaine groups in the polymer backbone is developed, which are fabricated into encapsulation devices with tunable nanoporous structures via electrospinning. The ZPU encapsulation device is hydrophilic and fouling-resistant, exhibits robust mechanical properties, and prevents cell escape while still allowing efficient mass transfer. The ZPU device also induces a much lower FBR or cellular overgrowth upon intraperitoneal implantation in C57BL/6 mice for up to 6 months compared to devices made of similar polyurethane without the zwitterionic modification. The therapeutic potential of the ZPU device is shown for islet encapsulation and diabetes correction in mice for ≈3 months is demonstrated. As a proof of concept, the scalability and retrievability of the ZPU device in pigs and dogs are further demonstrated. Collectively, these attributes make ZPU devices attractive candidates for cell encapsulation therapies.

Targeting Nanoparticles to Bioengineered Human Vascular Networks.

Pharmacotherapy of vascular anomalies has limited efficacy and potentially limiting toxicity. Targeted nanoparticle (NP) drug delivery systems have the potential to accumulate within tissues where the vasculature is impaired, potentially leading to high drug levels (increased efficacy) in the diseased tissue and less in off-target sites (less toxicity). Here, we investigate whether NPs can be used to enhance drug delivery to bioengineered human vascular networks (hVNs) that are a model of human vascular anomalies. We demonstrate that intravenously injected phototargeted NPs enhanced accumulation of NPs and the drug within hVNs. With phototargeting we demonstrate 17 times more NP accumulation within hVNs than was detected in hVNs without phototargeting. With phototargeting there was 10-fold more NP accumulation within hVNs than in any other organ. Phototargeting resulted in a 6-fold increase in drug accumulation (doxorubicin) within hVNs in comparison to animals injected with the free drug. Nanoparticulate approaches have the potential to markedly improve drug delivery to vascular anomalies.

Local Immunomodulatory Strategies to Prevent Allo-Rejection in Transplantation of Insulin-Producing Cells.

Islet transplantation has shown promise as a curative therapy for type 1 diabetes (T1D). However, the side effects of systemic immunosuppression and limited long-term viability of engrafted islets, together with the scarcity of donor organs, highlight an urgent need for the development of new, improved, and safer cell-replacement strategies. Induction of local immunotolerance to prevent allo-rejection against islets and stem cell derived ß cells has the potential to improve graft function and broaden the applicability of cellular therapy while minimizing adverse effects of systemic immunosuppression. In this mini review, recent developments in non-encapsulation, local immunomodulatory approaches for T1D cell replacement therapies, including islet/ß cell modification, immunomodulatory biomaterial platforms, and co-transplantation of immunomodulatory cells are discussed. Key advantages and remaining challenges in translating such technologies to clinical settings are identified. Although many of the studies discussed are preliminary, the growing interest in the field has led to the exploration of new combinatorial strategies involving cellular engineering, immunotherapy, and novel biomaterials. Such interdisciplinary research will undoubtedly accelerate the development of therapies that can benefit the whole T1D population.

A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes.

Transplantation of stem cell-derived ß (SC-ß) cells represents a promising therapy for type 1 diabetes (T1D). However, the delivery, maintenance, and retrieval of these cells remain a challenge. Here, we report the design of a safe and functional device composed of a highly porous, durable nanofibrous skin and an immunoprotective hydrogel core. The device consists of electrospun medical-grade thermoplastic silicone-polycarbonate-urethane and is soft but tough (~15 megapascal at a rupture strain of >2). Tuning the nanofiber size to less than ~500 nanometers prevented cell penetration while maintaining maximum mass transfer and decreased cellular overgrowth on blank (cell-free) devices to as low as a single-cell layer (~3 micrometers thick) when implanted in the peritoneal cavity of mice. We confirmed device safety, indicated as continuous containment of proliferative cells within the device for 5 months. Encapsulating syngeneic, allogeneic, or xenogeneic rodent islets within the device corrected chemically induced diabetes in mice and cells remained functional for up to 200 days. The function of human SC-ß cells was supported by the device, and it reversed diabetes within 1 week of implantation in immunodeficient and immunocompetent mice, for up to 120 and 60 days, respectively. We demonstrated the scalability and retrievability of the device in dogs and observed viable human SC-ß cells despite xenogeneic immune responses. The nanofibrous device design may therefore provide a translatable solution to the balance between safety and functionality in developing stem cell-based therapies for T1D.

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.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

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.

In Vivo Vascular Network Forming Assay.

The capability of forming functional blood vessel networks is critical for the characterization of endothelial cells. In this chapter, we will review a modified in vivo vascular network forming assay by replacing traditional mouse tumor-derived Matrigel with a well-defined collagen-fibrin hydrogel. The assay is reliable and does not require special equipment, surgical procedure, or a skilled person to perform. Moreover, investigators can modify this method on-demand for testing different cell sources, perturbation of gene functions, growth factors, and pharmaceutical molecules, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.