Search

Current dogma dictates that, during adulthood, endothelial cells (ECs) are locked in an immutable stable homeostatic state. By contrast, herein we show that maintenance of EC fate and function are linked and active processes, which depend on the constitutive cooperativity of only two ETS transcription factors (TFs), ERG and Fli1. Although deletion of either ERG or Fli1 manifests subtle vascular dysfunction, their combined genetic deletion in adult ECs results in acute vasculopathy and multi-organ failure, due to loss of EC fate and integrity, hyperinflammation and spontaneous thrombosis, leading to death. ERG and Fli1 co-deficiency causes rapid transcriptional silencing of pan and organotypic vascular core genes, with dysregulation of inflammation and coagulation pathways. Vascular hyperinflammation leads to impaired hematopoiesis with myeloid skewing. Accordingly, enforced ERG and FLI1 expression in adult human mesenchymal stromal cells activates vascular programs and functionality, enabling in vivo engraftment of a perfusable vascular network. Genome-wide association study analysis identified vascular diseases that are associated with FLI1/ERG mutations. Constitutive expression of ERG and Fli1 upholds EC fate, physiological function and resilience in adult vasculature, whereas their functional loss can contribute to systemic human diseases.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3–/– bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium., Barcia Durán et al. show that the tyrosine kinase gene Jak3 is expressed in multiple endothelial cell types in the mouse, including in non-hematopoietic organs, with particularly high expression in the bone marrow. Using mice lacking Jak3, they show that Jak3 expressed in the bone marrow niche is important for maintaining long-term repopulating hematopoietic stem cells.

Significance Human PSC-derived iBMECs have been generated to study disease mechanisms and drug development for neurological disorders. However, their full transcriptomic characterization is unclear, which could result in inaccurate physiological studies and development of treatments with ineffective clinical outcomes. Utilizing a comprehensive transcriptomic metaanalysis validated by physiological studies, we find that many current protocols used to generate iBMECs produce a homogenous epithelial cell population. Overexpression of ETS transcription factors reprogram these cells into phenotypic endothelial cells (rECs) which recapitulate certain vascular functions, albeit lacking expression of some organotypic transporter genes and high electrical resistance in vitro. Nevertheless, they represent a crucial step toward the generation of an in vitromodel suitable for physiological and pharmaceutical studies of the blood–brain barrier., Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood–brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

The menisci transmit load by increasing the contact area and decreasing peak contact stresses on the articular surfaces. Meniscal lesions are among the most common orthopedic injuries, and resulting meniscectomies are associated with adverse polycaprolactone contact mechanics changes and, ultimately, an increased likelihood of osteoarthritis. Meniscus scaffolds were fabricated by 3D-printing a network of circumferential and radial filaments of resorbable polymer (poly(desaminotyrosyl-tyrosine dodecyl ester dodecanoate)) and infused with collagen-hyaluronan. The scaffold demonstrated an instantaneous compressive modulus (1.66 ± 0.44 MPa) comparable to native meniscus (1.52 ± 0.59 MPa). The scaffold aggregate modulus (1.33 ± 0.51 MPa) was within 2% of the native value (1.31 ± 0.36 MPa). In tension, the scaffold displayed a comparable stiffness to native tissue (127.6-97.1 N/mm) and an ultimate load of 33% of the native value. Suture pull-out load of scaffolds (83.1 ± 10.0 N) was within 10% of native values (91.5 ± 15.4 N). Contact stress analysis demonstrated the scaffold reduced peak contact stress by 60-67% and increased contact area by 38%, relative to partial meniscectomy. This is the first meniscal scaffold to match both the axial compressive properties and the circumferential tensile stiffness of the native meniscus. The improvement of joint contact mechanics, relative to partial meniscectomy alone, motivates further investigation using a large animal model. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2457-2465, 2019.

The ability to generate haematopoietic stem cells in vitro would have immeasurable impact upon many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach to convert adult murine endothelial cells (ECs) to haematopoietic stem cells (HSCs), termed reprogrammed endothelial cells into haematopoietic stem and progenitor cells (rEC-HSPCs). The conversion is achieved without cells transitioning through a pluripotent state and comprises three phases: induction, specification, and expansion. Adult ECs are first isolated from Runx1-IRES-GFP; Rosa26-rtTa mice and maintained in culture under EC growth factor stimulation and Tgfβ inhibition. In the first (induction) phase of conversion (days 0–8), four transcription factors—FosB, Gfi1, Runx1, and Spi1 (FGRS)—are expressed transiently, which results in endogenous Runx1 expression. During the second (specification) phase (days 8–20), endogenous Runx1(+) FGRS-transduced ECs commit to a haematopoietic fate and no longer require exogenous FGRS expression. Finally, the vascular niche drives robust proliferation of rEC-HSPCs during the expansion phase (days 20–28). The resulting converted cells possess a transcriptomic signature and long-term self-renewal capacity indistinguishable from those of adult HSCs. In this protocol we also describe functional in vitro and in vivo assays that can be used to demonstrate that rEC-HSPCs are competent for clonal engraftment and possess multi-lineage reconstitution potential, including antigen-dependent adaptive immune function. This approach thus provides a tractable strategy to interrogate the generation of engraftable haematopoietic cells, advancing the mechanistic understanding of haematopoietic development and HSC self-renewal.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders., The kidney is vascularized with highly specialized and zonated endothelial cells that are essential for its filtration function. Here, Barry et al. provide a single-cell RNA sequencing analysis of the kidney vasculature that highlights its transcriptional heterogeneity and uncovers pathways important for its development and function.

Brain microvascular endothelial cells (BMECs) possess unique properties underlying the blood-brain-barrier (BBB), that are crucial for homeostatic brain functions and interactions with the immune system. Modulation of BBB function is essential for treatment of neurological diseases and effective tumor targeting. Studies to-date have been hampered by the lack of physiological models using cultivated human BMECs that sustain BBB properties. Recently, differentiation of induced pluripotent stem cells (iPSCs) into cells with BBB-like properties has been reported, providing a robust in vitro model for drug screening and mechanistic understanding of neurological diseases. However, the precise identity of these iBMECs remains unclear. Employing single-cell RNA sequencing, bioinformatic analysis and immunofluorescence for several pathways, transcription factors (TFs), and surface markers, we examined the molecular and functional properties of iBMECs differentiated either in the absence or presence of retinoic acid. We found that iBMECs lack both endothelial-lineage genes and ETS TFs that are essential for the establishment and maintenance of EC identity. Moreover, iBMECs fail to respond to angiogenic stimuli and form lumenized vessels in vivo. We demonstrate that human iBMECs are not barrier-forming ECs but rather EpCAM+ neuroectodermal epithelial cells (NE-EpiCs) that form tight junctions resembling those present in BBB-forming BMECs. Finally, overexpression of ETS TFs (ETV2, FLI1, and ERG) reprograms NE-EpiCs to become more like the BBB-forming ECs. Thus, although directed differentiation of human iBMECs primarily gives rise to epithelial cells, overexpression of several ETS TFs can divert them toward a vascular BBB in vitro.

The menisci are fibrocartilaginous tissues that are crucial to the load-sharing and stability of the knee, and when injured, these properties are compromised. Meniscus replacement scaffolds have utilized the circumferential alignment of fibers to recapitulate the microstructure of the native meniscus; however, specific consideration of size, shape, and morphology has been largely overlooked. The purpose of this study was to personalize the fiber-reinforcement network of a meniscus reconstruction scaffold. Human cadaveric menisci were measured for a host of tissue (length, width) and subtissue (regional widths, root locations) properties, which all showed considerable variability between donors. Next, the asymmetrical fiber network was optimized to minimize the error between the dimensions of measured menisci and predicted fiber networks, providing a 51.0% decrease (p = 0.0091) in root-mean-square (RMS) error. Finally, a separate set of human cadaveric knees was obtained, and donor-specific fiber-reinforced scaffolds were fabricated. Under cyclic loading for load-distribution analysis, in situ implantation of personalized scaffolds following total meniscectomy restored contact area (253.0 mm2 to 488.9 mm2, p = 0.0060) and decreased contact stress (1.96 MPa to 1.03 MPa, p = 0.0025) to near-native values (597.4 mm2 and 0.83 MPa). Clinical use of personalized meniscus devices that restore physiologic contact stress distributions may prevent the development of post-traumatic osteoarthritis following meniscal injury.

During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously harnessed this vestigial identity to achieve the direct conversion of adult endothelial cells (ECs) into long-term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs (Lis, R. et al., Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature, 545:439-445. 2017). We first reprogrammed adult human endothelium using lentiviral vectors encoding FOSB, GFI1, SPI1, and RUNX1 (FGRS) under constitutive promoters and an inductive vascular niche (Sandler V. et al., Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014). The resulting rEC-HSPCs maintained exogenous expression of the four transcription factors for over 20 weeks post-transplantation into immuno-compromised NSG mice. Constitutive expression of Spi1, however, has been shown to hinder lymphoid differentiation in vivo by blocking T lymphopoiesis (Anderson, M. et al. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16:285-296. 2002). In addition, mice of the NSG strain cannot educate native B or T cells to maturity. Our system of constitutive exogenous FGRS expression was therefore unable to confer transplanted immuno-compromised mice the ability to generate an adaptive immune response. Here, we obtained human rEC-HSPCs making use of (i) doxycycline-inducible vectors to temporarily overexpress FGRS and (ii) transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin) for transplantation assays. We show that human rEC-HSPCs engraft primary- and secondary-transplanted mice for over a year at levels of up to 20% in the spleen or bone marrow. Engrafted cells differentiate into all blood lineages including phenotypically and functionally mature T cells in the absence of exogenous FGRS expression in vivo. Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week post-LCMV infection. Functional and phenotypic analyses are presented in juxtaposition with experiments using cord blood-transplanted mice. These results demonstrate that our present direct conversion strategy generates bona fide human hematopoietic stem cells from adult endothelial cells. Disclosures Rafii: Angiocrine Bioscience: Equity Ownership.

Purpose Ischemia/reperfusion (I/R) during partial nephrectomy (PN) contributes to acute kidney injury (AKI), which is inaccurately assessed using existent clinical markers of renal function. We evaluated I/R-related changes in expression in hypoxia inducible factor 1α (HIF-1α) and toll-like receptor 4 (TLR4), within kidney tissue and peripheral blood leukocytes (PBL) in a porcine model of PN. Materials and Methods Three adult pigs each underwent unilateral renal hilar cross clamping for 180 min followed by a 15 min reperfusion. The contralateral kidney served as control. Biopsies of clamped kidneys were obtained at baseline (time 0), every 60 min during the hypoxic phase, and post-reperfusion. Control kidneys were biopsied once at 180 min. Peripheral blood was sampled at time 0, every 30 min during the hypoxic phase, and post-reperfusion. HIF-1α and TLR4 expression in kidney tissue and PBL were analyzed by Western blotting. I/R-related histological changes were assessed. Results Expression of HIF-1α in clamped kidneys and PBL was below detection level at baseline, rising to detectable levels after 60 min of hypoxia, and continuing to rise throughout the hypoxic and reperfusion phases. Expression of TLR-4 in clamped kidneys followed a similar trend with initial detection after 30–60 min of hypoxia. Control kidneys exhibited no change in HIF-1α or TLR-4 expression. I/R-related histologic changes were minimal, primarily mild tubular dilatation. Conclusions In a porcine model of PN, HIF-1α and TLR4 exhibited robust, I/R-related increases in expression in kidney tissue and PBL. Further studies investigating these molecules as potential markers of AKI are warranted.