Shahin Rafii

Plasticity between cell lineages is a fundamental but poorly understood property of regenerative tissues. In the gut tube, small intestine absorbs nutrients whereas colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screen, we identified MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrain colonic plasticity. MTA2 loss in adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-binds with HNF4A, an activating pan-intestine transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic and gain on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through a HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages; their release unleashes plasticity.

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Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFβ1 and using allogenic bone marrow–derived …

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and current therapy only treats symptoms but cannot stop progression of this debilitating disease. Current efforts have been directed at blocking lung inflammation to stop lung destruction, but the long-term benefit of this approach has been limited. Emerging data points to the centrality of the lung microvasculature to the pathogenesis of COPD but little is known of how vascular damage leads to progressive lung destruction. Defining the mechanism (s) of vascular pathogenesis of COPD provides a therapeutic path forward to reduce the morbidity and mortality for millions of patients with COPD. Endothelial cells (ECs) have increasingly been found to function not only as vascular conduits, but also as coordinators of organ homeostasis, regeneration, and repair by supplying tissue-specific growth factors, known as angiocrine …

Bone marrow endothelial cells deliver oxygen and nutrients and regulate bone formation and haematopoiesis in the surrounding microenvironment. A new study identifies a subtype of capillary that occurs exclusively in the epiphysis and displays unique characteristics that have a role in balancing osteogenesis and haematopoiesis.

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA …

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated …

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of …

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient–derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct “education signatures.” These …

Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with …

IntroductionDe novo generation of hematopoietic stem and progenitor cells (HSPCs) for therapeutic use has been a long-standing goal of regenerative medicine. During development, the first definitive hematopoietic stem cells (HSCs) derive from a small number of specialized vascular endothelial cells (EC), named hemogenic endothelial cells (HECs), located in the Aorta-Gonado-Mesonephrose (AGM). The process of HEC conversion to HSPCs is known as endothelial-to-hematopoietic transition (EHT). During EHT, endothelial features are lost while switching on hematopoietic transcriptional programs leading to drastic functional and morphological changes that generate free-floating, round hematopoietic cells from large, flat, anchorage-dependent ECs. Key steps regulating EHT are difficult to elucidate using in vivo models because the hemogenic capacity of HECs occurs within a 2-day developmental window …

Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle. Here, we report an in vitro method to derive HIOs with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, these populations expand and organize to support organoid maturation and function. Functional experiments demonstrate enteric nervous system function, with HIOs undergoing peristaltic-like contractions, suggesting the development of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices to introduce vascular-like flow, and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. Collectively, we report an in vitro model of the human gut that simultaneously co-differentiates epithelial, stromal, endothelial, neural, and organized muscle populations.

Vascular patterning is dictated by transcription factors, such as the Notch pathway. Molecular profiling has uncovered snapshots of the transcriptional specification of endothelial cell tubulogenesis. Here, the authors leverage the output of Notch signaling, demonstrating that vascular remodeling is poorly predicted by transcriptional profiling.

Methods: We developed strategies to obtain large numbers of viable ISECs, acinar specific ECs (ASECs), and stromal cells from three human pancreas donors. Single cell RNA-sequencing and comparison with the vascular signatures of other organs enabled us to uncover the unique transcriptomic signatures of ISECs, ASECs, and corresponding perivascular cells. Candidate ISEC-transcription factors were subsequently transduced into generic endothelium, studied in microfluidic devices, and transplanted into diabetic mice.Results: ISECs are characterized by expression of distinct combinations of structural genes COL13A1, PLVAP, UNC5B, ESM1, and LAMA4, instructive angiocrine factors THBS1, BMP2, and Dll4, metabolic modulators PASK, CD36, and FABP5, and immune trafficking gatekeepers CXCR4, CXCR7, CD320, and SELE. ISECs manifest signaling pathways VEGF-A: VEGFR2, NOTCH1, 4: DLL4 …

The present disclosure is directed to method of generating human glomeruli endothelial cells (HGECs) from human endothelial cells (ECs), comprising expressing in human ECs an exogenous nucleic acid encoding a T-box transcrip tion factor 3 (Tbx3), alone or in combination with one or more of PR domain zinc finger protein 1 (Prdml), GATA Binding Protein 5 (Gata5) and Pre-B-Cell Leukemia Tran scription Factor 1 (Pbx1). Disclosed also are HGECs pro duced by the methods of the instant disclosure, as well as methods for using the same.

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)–induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS …

Little is known about the maintenance of endothelial cell fate in adults. We show that deletion of Erg and Fli1 in endothelial cells of adult mice causes loss of endothelial cell fate and vascular collapse. Overexpression of ERG and FLI1 in non-vascular cells activates an endothelial cell program, confirming their regulation of endothelial cell identity.

The production of blood cells, including some immune cells, relies heavily on the bone-marrow microenvironment. Cardiovascular diseases are now found to corrupt this niche, leading to imbalances in blood-cell production.

Endothelial cells in vivo are subjected to a wide array of mechanical stimuli, such as cyclic stretch. Notably, a 10% stretch is associated with an atheroprotective endothelial phenotype, while a 20% stretch is associated with an atheroprone endothelial phenotype. Here, a systems biology-based approach is used to present a comprehensive overview of the functional responses and molecular regulatory networks that characterize the transition from an atheroprotective to an atheroprone phenotype in response to cyclic stretch. Using primary human umbilical vein endothelial cells (HUVECs), we determined the role of the equibiaxial cyclic stretch in vitro, with changes to the radius of the magnitudes of 10% and 20%, which are representative of physiological and pathological strain, respectively. Following the transcriptome analysis of next-generation sequencing data, we identified four key endothelial responses to pathological cyclic stretch: cell cycle regulation, inflammatory response, fatty acid metabolism, and mTOR signaling, driven by a regulatory network of eight transcription factors. Our study highlights the dynamic regulation of several key stretch-sensitive endothelial functions relevant to the induction of an atheroprone versus an atheroprotective phenotype and lays the foundation for further investigation into the mechanisms governing vascular pathology. This study has significant implications for the development of treatment modalities for vascular disease.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on …

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 stem cells maintain regenerative tissue structure and function by producing tissue-specific progeny, but the factors that preserve their tissue identities are not well understood. The small and large intestines differ markedly in cell composition and function, reflecting their distinct stem cell populations. Here we show that SATB2, a colon-restricted chromatin factor, singularly preserves LGR5+ adult colonic stem cell and epithelial identity in mice and humans. Satb2 loss in adult mice leads to stable conversion of colonic stem cells into small intestine ileal-like stem cells and replacement of the colonic mucosa with one that resembles the ileum. Conversely, SATB2 confers colonic properties on the mouse ileum. Human colonic organoids also adopt ileal characteristics upon SATB2 loss. SATB2 regulates colonic identity in part by modulating enhancer binding of the intestinal transcription factors CDX2 and HNF4A. Our …

Hematopoietic stem cells (HSCs) develop from hemogenic endothelium within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, here we use single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from hemogenic endothelium to HSCs, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering reveals dynamics of gene expression during the hemogenic endothelium to HSC transition, identifying surface receptors specifically expressed on developing HSCs. Transcriptional profiling of niche endothelial cells identifies corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, are sufficient to …

Ovarian cancer (OC) is a heterogeneous disease characterized by its late diagnosis (FIGO stages III and IV) and the importance of abdominal metastases often observed at diagnosis. Detached ovarian cancer cells (OCCs) float in ascites and form multicellular spheroids. Here, we developed endothelial cell (EC)-based 3D spheroids to better represent in vivo conditions. When co-cultured in 3D conditions, ECs and OCCs formed organized tumor angiospheres with a core of ECs surrounded by proliferating OCCs. We established that Akt and Notch3/Jagged1 pathways played a role in angiosphere formation and peritoneum invasion. In patients’ ascites we found angiosphere-like structures and demonstrated in patients’ specimens that tumoral EC displayed Akt activation, which supports the importance of Akt activation in ECs in OC. Additionally, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCC–EC crosstalk relating to OCC proliferation and during peritoneal invasion. Our results support the use of multicellular spheroids to better model tumoral and stromal interaction. Such models could help decipher the complex pathways playing critical roles in metastasis spread and predict tumor response to chemotherapy or anti-angiogenic treatment.

Method: Given the limitations of adult ECs, we have engineered adaptable human endothelium by transient transduction of ETS variant transcription factor 2, Reprogramming adult Vascular ECs (R-VECs) to an adaptable tubulogenic state. The ability of R-VECs to sustain islet function and survival was tested via in vitro and in vivo approaches.Results: Within microfluidic devices, R-VECs assemble into perfused plexi that can carry blood and avidly vascularize islets. Islets arborized by R-VECs in microfluidic devices demonstrate preserved function by glucose stimulated insulin secretion. To test if this rich, expedited vascularization from R-VECs can enable islets to survive in the subcutaneous space, streptozotocin-induced diabetic SCID-Beige mice were transplanted; human islets achieved euglycemia for 6 months, while transplantation with islets alone or islets/generic ECs did not. Furthermore, mice transplanted …

The fetal liver (FL) serves as the primary site for hematopoietic stem cell (HSC) expansion during development. Understanding the mechanisms regulating proliferation and self-renewal of FL-HSC is of importance to engineering HSC expansion in vitro, with translational applications in transplantation, gene and cellular therapies, and disease modeling. Moreover, while heterogeneity in the self-renewal and engraftment properties of adult HSC has been extensively studied, heterogeneity of FL-HSC in this regard remains poorly characterized. To address these issues at the single cell level, we engineered an in vitro culture platform designed to recapitulate the murine FL endothelial niche and used it to expand colonies from single FL-HSC. This approach yielded multiple colony types as determined by flow cytometric analysis. FL-HSC-derived colonies providing multilineage engraftment, as measured by …

Niche cells within the bone marrow (BM) microenvironment support the maintenance and reconstitution of hematopoiesis. In this issue of Blood, Yu et al demonstrate that angiopoietin-like 2 (ANGPTL2) secreted by the vascular niche endothelial cells serves as the seminal paracrine angiocrine factor, orchestrating hematopoietic stem cell (HSC) homeostasis and regeneration after myelosuppression. 1Tissue-specific endothelium, such as BM endothelial cells (BMECs), is endowed with unique organotypic specialized functions, including modulation of metabolism, immune cell trafficking, inflammatory response, vascular tone, regeneration, and repair. 2, 3 It is well documented that blood vessels in the BM are not just passive conduits delivering nutrients and oxygen to hematopoietic cells. BMECs execute specialized hematopoietic-supportive functions that promote hematopoietic stem and progenitor cell (HSPC …

Paper 2 of the paediatric regenerative medicine Series focuses on recent advances in postnatal approaches. New gene, cell, and niche-based technologies and their combinations allow structural and functional reconstitution and simulation of complex postnatal cell, tissue, and organ hierarchies. Organoid and tissue engineering advances provide human disease models and novel treatments for both rare paediatric diseases and common diseases affecting all ages, such as COVID-19. Preclinical studies for gastrointestinal disorders are directed towards oesophageal replacement, short bowel syndrome, enteric neuropathy, biliary atresia, and chronic end-stage liver failure. For respiratory diseases, beside the first human tracheal replacement, more complex tissue engineering represents a promising solution to generate transplantable lungs. Genitourinary tissue replacement and expansion usually involve …

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors …

BACKGROUND Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS We analyzed colony-forming, self-renewal, and …

The instant disclosure is directed to a method for vascularizing a pancreatic islet comprising culturing the pancreatic islet or β-cells with an endothelial cell comprising an exogenous nucleic acid encoding an ETV2 transcription factor under conditions wherein the endothelial cell expresses the ETV2 transcription factor. The instant disclosure is further directed to a method for making a vascularized β-cell organoid comprising culturing the pancreatic islet or β-cells with an endothelial cell comprising an exogenous nucleic acid encoding an ETV2 transcription factor under conditions wherein the endothelial cell expresses the ETV2 transcription factor. Disclosed also are vascularized islets and vascularized β-cell organoids produced by the methods of the instant disclosure, as well as methods for using the same.

Endothelial cells are critical mediators of health and disease. Regenerative medicine techniques that target the endothelium hold vast promise for improving lifespan and quality of life worldwide. Regenerative therapies via induced pluripotent stem cells (IPSCs) have helped demonstrate disease mechanisms, but so far, concerns regarding their function, malignant potential, and expense have limited therapeutic potential. One alternative approach is direct reprogramming of somatic cells, which avoids the pluripotent state and allows for in vivo reprogramming. Transcription factors from endothelial development have yielded essential transcription factors and small molecules that induce endothelial cell fate. Most direct cell reprogramming strategies targeting endothelial cells use ETV2, a pioneer transcription factor to specify endothelial lineage via histone-modifying enzymes. Many different types of starting cells and …

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 …

A method of enhancing or initiating regeneration of an organ in a subject in need thereof comprising the administration of endothelial cells specific to said organ, or inductive endothelial cells specific to said organ, into the area of the body in which organ regeneration is desired in said subject.

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFβ-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFβ signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFβ signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing …

Disclosures: DJ Nolan reported personal fees from Angiocrine Bioscience outside the submitted work; in addition, DJ Nolan had a patent number 8,465,732 issued (Angiocrine Bioscience) and a patent number 9,944,897 issued; and is an employee and equity holder of Angiocrine Bioscience. M. Ginsberg reported personal fees from Angiocrine Bioscience outside the submitted work; in addition, M. Ginsberg had a patent to 8,465,732 issued and a patent to 9,944,897 issued. In addition, M. Ginsberg is a current employee and equity holder of Angiocrine Bioscience. FJ Martinez reported non-financial support from ProterrixBio, Nitto, Zambon;" other" from Afferent/Merck, Biogen, Veracyte, Prometic, Bridge Biotherapeutics, and Abbvie; grants from Gilead; and personal fees from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Chiesi, Sunovion, Patara/Respivant, Bayer, Promedior/Roche, Teva, Col Behring …

The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2+ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity …

Hematopoietic stem cells (HSCs) possess the unique ability to engraft a suitably conditioned host, generate all cell types of the adult hematopoietic system, and self-renew for life, making them clinically valuable for the treatment of a variety of blood disorders in the setting of hematopoietic cell transplantation. Efforts to expand the therapeutic potential of HSCs, such as by generating them de novo from pluripotent stem cells, have been hampered by an incomplete understanding of their embryonic origin. Complicating the study of HSC development, embryonic hematopoiesis is characterized by the initial generation of abundant HSC-independent hematopoietic progenitors with restricted lineage potentials that emerge prior to rare HSCs. How hemogenic endothelial cells (HE), which serve as the embryonic precursors to most blood cells, are uniquely endowed with the potential to generate functional HSCs remains …

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes—Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)—together with three reprogramming-helper genes—dominant-negative (DN)-TGFβ, DN-Wnt8a, and acid ceramidase (AC)—to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G …

Brain microvascular endothelial cells (BMECs) possess unique properties that are crucial for many functions of the blood-brain-barrier (BBB) including maintenance of brain homeostasis and regulation of interactions between the brain and immune system. The generation of a pure population of putative brain microvascular endothelial cells from human pluripotent stem cell sources (iBMECs) has been described to meet the need for reliable and reproducible brain endothelial cells in vitro. Human pluripotent stem cells (hPSCs), embryonic or induced, can be differentiated into large quantities of specialized cells in order to study development and model disease. These hPSC-derived iBMECs display endothelial-like properties, such as tube formation and low-density lipoprotein uptake, high transendothelial electrical resistance (TEER), and barrier-like efflux transporter activities. Over time, the de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.

Vascular injury is a menacing element of acute respiratory distress syndrome (ARDS) pathogenesis. To better understand the role of vascular injury in COVID-19 ARDS, we used lung autopsy immunohistochemistry and blood proteomics from COVID-19 subjects at distinct timepoints in disease pathogenesis, including a hospitalized cohort at risk of ARDS development (“at risk”, N=59), an intensive care unit cohort with ARDS (“ARDS”, N=31), and a cohort recovering from ARDS (“recovery”, N=12). COVID-19 ARDS lung autopsy tissue revealed an association between vascular injury and platelet-rich microthrombi. This link guided the derivation of a protein signature in the at risk cohort characterized by lower expression of vascular proteins in subjects who died, an early signal of vascular limitation termed the maladaptive vascular response. These findings were replicated in COVID-19 ARDS subjects, as well as when bacterial and influenza ARDS patients (N=29) were considered, hinting at a common final pathway of vascular injury that is more disease (ARDS) then cause (COVID-19) specific, and may be related to vascular cell death. Among recovery subjects, our vascular signature identified patients with good functional recovery one year later. This vascular injury signature could be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic …

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment.Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements.Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing.Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR …

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 …

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together …

Adult organs are vascularized by specialized blood vessels. In addition to inter-organ vascular heterogeneity, each organ is arborized by structurally and functionally diversified populations of endothelial cells (ECs). The molecular pathways that are induced to orchestrate inter- and intra- organ vascular heterogeneity and zonation are shaped during development and fully specified postnatally. Notably, intra-organ specialization of ECs is associated with induction of angiocrine factors that guide cross-talk between ECs and parenchymal cells, establishing co-zonated vascular regions within each organ. In this review, we describe how microenvironmental tissue-specific biophysical, biochemical, immune, and inflammatory cues dictate the specialization of ECs with zonated functions. We delineate how physiological and biophysical stressors in the developing liver, lung, and kidney vasculature induce specialization of …

Signals related to liver regeneration (LR) have been identified by triggering proliferation in hepatocyte cultures, delay of regeneration associated with signal ablation in genomically modified mice, or response of intact liver in vivo by injecting specific signaling substances. This chapter discusses these signals with emphasis on their effects on LR. Autophagy regulation is an important part of LR. Even though LR after partial hepatectomy is not representative of types of liver injury most commonly seen in liver disease, it has been a very useful model for understanding the fundamental signaling mechanisms controlling LR. The chapter infers that nuclear Yap plays a role in regulating liver size and that combined signaling of hepatocyte growth factor/hepatocyte growth factor receptor and epidermal growth factor/ epidermal growth factor receptor are essential for maintaining the hepatostat; combined elimination …

The molecular triggers of organotypic tissue repair are unknown. The thymus, which is the primary site of T cell development, is a model of tissue damage and regeneration as it is particularly sensitive to insult, but also has a remarkable capacity for repair. However, acute and profound damage, such as that caused by common cytoreductive therapies or age-related decline, lead to involution of the thymus and prolonged T cell deficiency, precipitating life-threatening infections and malignant relapse. Consequently, there is an unmet need to boost thymic function and enhance T cell immunity. Here, we demonstrate an innate trigger of the reparative response in the thymus, centered on the attenuation of signaling directly downstream of apoptotic cell detection as thymocytes are depleted after acute damage. We found that the intracellular pattern recognition receptor NOD2, via induction of microRNA-29c, suppressed the induction of the regenerative factors IL-23 and BMP4, from thymic dendritic cells (DCs) and endothelial cells (ECs), respectively. During steady-state, when a high proportion of thymocytes are undergoing apoptosis (as a consequence of selection events during T cell development), this suppressive pathway is constitutively activated by the detection of exposed phosphatidylserine on apoptotic thymocytes by cell surface TAM receptors on DCs and ECs, with subsequent downstream activation of the Rho GTPase Rac1. However, after damage, when profound cell depletion occurs across the thymus, the TAM-Rac1-NOD2-miR29c pathway is abrogated, therefore triggering the increase in IL-23 and BMP4 levels. Importantly, this pathway …

Introduction: Despite various clinical modalities, ischemic heart disease remains among the leading causes of mortality and morbidity worldwide. The elemental problem is the immense loss of cardiomyocytes (CMs) post-myocardial infarction (MI). Reprogramming non- cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells in vivo is a promising strategy for cardiac regeneration: however, the traditional viral delivery method hampered its application into clinical settings due to low and erratic transduction efficiency. Methods: We used a modified mRNA (modRNA) gene delivery platform to deliver different stoichiometry of cardiac-reprogramming genes (Gata4, Mef2c, Tbx5 and Hand2) together with reprogramming helper genes (Dominant Negative (DN)-TGFβ, DN- Wnt8a and Acid ceramidase (AC)), named 7G, to induce direct cardiac reprogramming post myocardial infarction (MI). Results: Here, we identified …

The concept of cancer as a cell-autonomous disease has been challenged by the wealth of knowledge gathered in the past decades on the importance of tumor microenvironment (TM) in cancer progression and metastasis. The significance of endothelial cells (ECs) in this scenario was initially attributed to their role in vasculogenesis and angiogenesis that is critical for tumor initiation and growth. Nevertheless, the identification of endothelial-derived angiocrine factors illustrated an alternative non-angiogenic function of ECs contributing to both physiological and pathological tissue development. Gene expression profiling studies have demonstrated distinctive expression patterns in tumor-associated endothelial cells that imply a bilateral crosstalk between tumor and its endothelium. Recently, some of the molecular determinants of this reciprocal interaction have been identified which are considered as …

Erythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor β1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFβ signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with …

The limited impact of treatments for COVID-19 has stimulated several phase 1 clinical trials of whole-lung low-dose radiation therapy (LDRT; 0.3–1.5 Gy) that are now progressing to phase 2 randomized trials worldwide. This novel but unconventional use of radiation to treat COVID-19 prompted the National Cancer Institute, National Council on Radiation Protection and Measurements and National Institute of Allergy and Infectious Diseases to convene a workshop involving a diverse group of experts in radiation oncology, radiobiology, virology, immunology, radiation protection and public health policy. The workshop was held to discuss the mechanistic underpinnings, rationale, and preclinical and emerging clinical studies, and to develop a general framework for use in clinical studies. Without refuting or endorsing LDRT as a treatment for COVID-19, the purpose of the workshop and this review is to provide …

Main Text:(Cancer Cell 19, 740–753; June 14, 2011) In the originally published version of this article, during final figure preparation the authors inadvertently selected an incorrect western blot image for Figure 3C (lower right; p65 blot for BMSC+ CML). The blot image for phospho-p65 was mistakenly copied into the p65 panel. The densitometric quantification of band intensities was based on the correct blot images. The correction to the image in Figure 3C does not affect the conclusions of the paper. The authors apologize for any confusion or inconvenience that this oversight may have caused.

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSC), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. Prior studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSC originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent and multipotent progenitors originate from the same population of HE that gives rise to HSC. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We found that …

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and …

Disclosures: Dr. Nolan reported personal fees from Angiocrine Bioscience during the conduct of the study; in addition, Dr. Nolan had a patent number 9,944,897 issued" Angiocrine Bioscience." Dr. Panagis reported personal fees from Regeneron outside the submitted work. Dr. Zippin reported grants from Pfizer and personal fees from Hoth outside the submitted work. Dr. Romano reported," I am an employee of Regeneron Pharmaceuticals, however there are no products or services of this company related to the work presented in this manuscript." Dr. Elemento reported" other" from Volastra Therapeutics," other" from One Three Biotech," other" from Freenome, and" other" from Owkin outside the submitted work. Dr. Rafii reported non-financial support from Angiocrine BioScience during the conduct of the study. No other disclosures were reported.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration,. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens,. In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable …

Rationale Chronic obstructive pulmonary disease (COPD) is a disorder marked by small airway occlusion and distal airspace enlargement (emphysema) leading to progressive airflow obstruction and clinical symptoms. Microvasculature abnormalities are key features of COPD lungs and correlate with severity of disease. However, it is not known if abnormal lung endothelium drives COPD and/or if correcting endothelial dysfunction has therapeutic potential. Investigations have uncovered the instructive role for endothelial cells (ECs) in organ regeneration and repair. Lung-specific endothelium coordinate propagation and behavior of adjacent parenchymal and mesenchymal cells through the release of growth factors, known as angiocrine factors. We hypothesized that maladaptive ECs are critical to the pathogenesis of COPD and that targeting lung EC biology has great therapeutic potential. Methods We performed …

To understand Zena’s beginnings is to understand the essence of who Zena was as a person, mentor, matriarch, and as a world-class scientist. We are fortunate to have crossed paths with her. Zena’s achievements are remarkable, considering the hardships she faced as she navigated through the adventurous journey of life. She endured a difficult childhood, some life-threatening illnesses, and challenging transitions from Europe to Canada and then to the United States to evolve into one of the most decorated renowned scientists of our time. Zena was an imposing figure of a woman that demanded respect, with a wicked sense of humor and brilliance that rubbed off on anyone that was near her. She was a powerhouse of principles to reckon with, and she was a woman of authority to learn from.Zena’s very start was harrowing and heart-wrenching—she was born at the Bergen–Belsen concentration camp in …