Publications by authors named "Shahin Rafii"

231 Publications

Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFβ target genes.

Genes Dev 2021 Oct 21. Epub 2021 Oct 21.

Abramson Family Cancer Research Institute, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia Pennsylvania 19104, USA.

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 chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications.
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http://dx.doi.org/10.1101/gad.348738.121DOI Listing
October 2021

Attenuation of apoptotic cell detection triggers thymic regeneration after damage.

Cell Rep 2021 Oct;37(1):109789

Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Department of Immunology, University of Washington, Seattle, WA 98109, USA. Electronic address:

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 function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised.
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http://dx.doi.org/10.1016/j.celrep.2021.109789DOI Listing
October 2021

SATB2 preserves colon stem cell identity and mediates ileum-colon conversion via enhancer remodeling.

Cell Stem Cell 2021 Sep 22. Epub 2021 Sep 22.

Division of Regenerative Medicine & Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA. Electronic address:

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 study uncovers a conserved core regulator of colonic stem cells able to mediate cross-tissue plasticity in mature intestines.
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http://dx.doi.org/10.1016/j.stem.2021.09.004DOI Listing
September 2021

Endothelial reprogramming for vascular regeneration: Past milestones and future directions.

Semin Cell Dev Biol 2021 Sep 18. Epub 2021 Sep 18.

Division of Regenerative Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, NY 10065, USA. Electronic address:

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 strategies, including lentiviral transduction, inducing innate immunity, and small molecule signaling have been leveraged for reprogramming. However, so far therapeutic benefit of these strategies remains unproven. Future research will have to solve scalability, safety, and efficacy hurdles before being ready for the clinic. However, researchers have already discovered meaningful insights into disease mechanisms and development through direct reprogramming.
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http://dx.doi.org/10.1016/j.semcdb.2021.09.003DOI Listing
September 2021

Multipotent progenitors and hematopoietic stem cells arise independently from hemogenic endothelium in the mouse embryo.

Cell Rep 2021 Sep;36(11):109675

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98105, USA. Electronic address:

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, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties.
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http://dx.doi.org/10.1016/j.celrep.2021.109675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478150PMC
September 2021

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Mol Ther 2021 10 29;29(10):3042-3058. Epub 2021 Jul 29.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

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-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.
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http://dx.doi.org/10.1016/j.ymthe.2021.07.014DOI Listing
October 2021

Reversal of emphysema by restoration of pulmonary endothelial cells.

J Exp Med 2021 Aug 21;218(8). Epub 2021 Jul 21.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.
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http://dx.doi.org/10.1084/jem.20200938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298104PMC
August 2021

Morphological characterization of Etv2 vascular explants using fractal analysis and atomic force microscopy.

Microvasc Res 2021 Nov 17;138:104205. Epub 2021 Jun 17.

Bioengineering Program, DeMatteis School of Engineering and Applied Science, Hofstra University, Hempstead, NY, USA; Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA. Electronic address:

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, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2 HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2 HUVECs had decreased fractal dimension and lacunarity compared to Etv2 HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.
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http://dx.doi.org/10.1016/j.mvr.2021.104205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446305PMC
November 2021

Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells: Current Controversies.

Front Physiol 2021 31;12:642812. Epub 2021 Mar 31.

Division of Regenerative Medicine, Department of Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, NY, United States.

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 . 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 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.
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http://dx.doi.org/10.3389/fphys.2021.642812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044318PMC
March 2021

Endothelial Jak3 expression enhances pro-hematopoietic angiocrine function in mice.

Commun Biol 2021 03 25;4(1):406. Epub 2021 Mar 25.

Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY, 10065, USA.

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.
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http://dx.doi.org/10.1038/s42003-021-01846-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994450PMC
March 2021

Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.

Proc Natl Acad Sci U S A 2021 02;118(8)

Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY 10065;

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 (, , and ) 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.
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http://dx.doi.org/10.1073/pnas.2016950118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923590PMC
February 2021

Low-Dose Radiation Therapy (LDRT) for COVID-19: Benefits or Risks?

Radiat Res 2020 11;194(5):452-464

National Council on Radiation Protection and Measurements, Bethesda, Maryland.

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 guidance to clinicians and researchers who plan to conduct preclinical and clinical studies, given the limited available evidence on its safety and efficacy.
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http://dx.doi.org/10.1667/RADE-20-00211.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009137PMC
November 2020

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Nature 2020 09 9;585(7825):426-432. Epub 2020 Sep 9.

Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA.

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, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.
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http://dx.doi.org/10.1038/s41586-020-2712-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480005PMC
September 2020

Megakaryocyte TGFβ1 partitions erythropoiesis into immature progenitor/stem cells and maturing precursors.

Blood 2020 08;136(9):1044-1054

Laboratory of Molecular Hematopoiesis, Hematology and Oncology.

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 low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.
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http://dx.doi.org/10.1182/blood.2019003276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453150PMC
August 2020

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

J Exp Med 2020 06;217(6)

Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.
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http://dx.doi.org/10.1084/jem.20190730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971135PMC
June 2020

Angiocrine endothelium: from physiology to cancer.

J Transl Med 2020 02 3;18(1):52. Epub 2020 Feb 3.

Nice Breast Institute, 57 bld de la Californie, 06000, Nice, France.

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 potential targets for developing novel anti-angiocrine therapeutic strategies.
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http://dx.doi.org/10.1186/s12967-020-02244-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998193PMC
February 2020

Molecular determinants of nephron vascular specialization in the kidney.

Nat Commun 2019 12 13;10(1):5705. Epub 2019 Dec 13.

Division of Regenerative Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, NY, 10065, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-12872-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910926PMC
December 2019

Haematopoietic stem cell reprogramming and the hope for a universal blood product.

FEBS Lett 2019 12 2;593(23):3253-3265. Epub 2019 Dec 2.

Division of Regenerative Medicine, Department of Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, NY, USA.

Haematopoietic stem cells (HSCs) are the only adult stem cells with a demonstrated clinical use, even though a tractable method to maintain and expand human HSCs in vitro has not yet been found. Owing to the introduction of transplantation strategies for the treatment of haematological malignancies and, more recently, the promise of gene therapy, the need to improve the generation, manipulation and scalability of autologous or allogeneic HSCs has risen steeply over the past decade. In that context, reprogramming strategies based on the expression of exogenous transcription factors have emerged as a means to produce functional HSCs in vitro. These approaches largely stem from the assumption that key master transcription factors direct the expression of downstream target genes thereby triggering haematopoiesis. Both somatic and pluripotent cells have been used to this end, yielding variable results in terms of haematopoietic phenotype and functionality. Here, we present an overview of the haematopoietic reprogramming methods reported to date, provide the appropriate historical context and offer some critical insight about where the field stands at present.
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http://dx.doi.org/10.1002/1873-3468.13681DOI Listing
December 2019

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

Nat Cell Biol 2019 11 4;21(11):1403-1412. Epub 2019 Nov 4.

Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41556-019-0404-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354005PMC
November 2019

Akt-activated endothelium promotes ovarian cancer proliferation through notch activation.

J Transl Med 2019 06 10;17(1):194. Epub 2019 Jun 10.

Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Education City, Qatar Foundation, Doha, Qatar.

Background: One main challenge in ovarian cancer rests on the presence of a relapse and an important metastatic disease, despite extensive surgical debulking and chemotherapy. The difficulty in containing metastatic cancer is partly due to the heterotypic interaction of tumor and its microenvironment. In this context, evidence suggests that endothelial cells (EC) play an important role in ovarian tumor growth and chemoresistance. Here, we studied the role of tumor endothelium on ovarian cancer cells (OCCs).

Methods: We evaluated the effect of activated endothelial cells on ovarian cancer cell proliferation and resistance to chemotherapy and investigated the survival pathways activated by endothelial co-culture.

Results: The co-culture between OCCs and E4ECs, induced an increase of OCCs proliferation both in vitro and in vivo. This co-culture induced an increase of Notch receptors expression on OCC surface and an increase of Jagged 1 expression on E4ECs surface and activation of survival pathways leading to chemoresistance by E4ECs.

Conclusion: The targeting of aberrant NOTCH signaling could constitute a strategy to disrupt the pro-tumoral endothelial niche.
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http://dx.doi.org/10.1186/s12967-019-1942-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558713PMC
June 2019

Endothelial cell adaptation in regeneration.

Science 2018 Dec;362(6419):1116-1117

Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA.

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http://dx.doi.org/10.1126/science.aar4800DOI Listing
December 2018

In vitro conversion of adult murine endothelial cells to hematopoietic stem cells.

Nat Protoc 2018 12;13(12):2758-2780

Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine (WCM), New York, NY, USA.

The ability to generate hematopoietic stem cells (HSCs) in vitro would have an immeasurable impact on many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach that converts adult murine endothelial cells (ECs) to HSCs, termed 'reprogrammed ECs into hematopoietic stem and progenitor cells' (rEC-HSPCs). The conversion, which is achieved without cells transitioning through a pluripotent state, 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 (TFs)-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 hematopoietic 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 for interrogating the generation of engraftable hematopoietic cells, advancing the mechanistic understanding of hematopoietic development and HSC self-renewal.
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http://dx.doi.org/10.1038/s41596-018-0060-3DOI Listing
December 2018

Generation of BMEC Lines and BMEC-HSPC Co-culture Assays.

Bio Protoc 2018 Nov 5;8(21):e3079. Epub 2018 Nov 5.

Department of Medicine, Division of Regenerative Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, NY, USA.

Endothelial cells (ECs) sustain the self-renewal and regeneration of adult hematopoietic stem and progenitor cells (HSPCs) via deployment of EC-derived paracrine factors, termed as angiocrine factors. Generation of durable vascular niche that maintains EC identity and preserves the angiocrine profile of organ of origin offers platforms for dissection of the mechanism by which angiocrine factors execute their instructive function for stem cell maintenance and tissue regeneration. This protocol describes detailed methods to isolate primary bone marrow ECs (BMECs), to subsequently transduce lentiviral vector carrying myristoylated-Akt1 into primary BMECs, and to use the Akt1-BMECs to expand engraftable murine HSPCs. The BMEC-HSPC co-culture system serves as bioreactor prototype to generate scalable populations of the blood and immune systems.
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http://dx.doi.org/10.21769/BioProtoc.3079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342070PMC
November 2018

Testicular endothelial cells are a critical population in the germline stem cell niche.

Nat Commun 2018 10 22;9(1):4379. Epub 2018 Oct 22.

Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA, 19104, USA.

Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer.
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http://dx.doi.org/10.1038/s41467-018-06881-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197186PMC
October 2018

Blood flow forces liver growth.

Nature 2018 10;562(7725):42-43

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http://dx.doi.org/10.1038/d41586-018-06741-2DOI Listing
October 2018

Isolation and Characterization of Mouse Organ-Specific Endothelial Transcriptomes.

Methods Mol Biol 2018 ;1846:301-308

Department of Regenerative Medicine, Weill Cornell Medical College, New York, NY, USA.

Endothelial cells play an essential role in homeostasis and regeneration and their transcriptional profile has been found to be tissue specific. Here we describe the isolation of organ specific mouse endothelial cells using either magnetic beads or fluorescence-activated cell sorting. This efficient protocol should allow for the transcriptional characterization of organotypic endothelial cells.
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http://dx.doi.org/10.1007/978-1-4939-8712-2_20DOI Listing
May 2019

A computational approach to identify cellular heterogeneity and tissue-specific gene regulatory networks.

BMC Bioinformatics 2018 06 7;19(1):217. Epub 2018 Jun 7.

Department of Pharmacology, The University of Illinois College of Medicine, 835 S. Wolcott Ave. Rm. E403, Chicago, IL, 60612, USA.

Background: The heterogeneity of cells across tissue types represents a major challenge for studying biological mechanisms as well as for therapeutic targeting of distinct tissues. Computational prediction of tissue-specific gene regulatory networks may provide important insights into the mechanisms underlying the cellular heterogeneity of cells in distinct organs and tissues.

Results: Using three pathway analysis techniques, gene set enrichment analysis (GSEA), parametric analysis of gene set enrichment (PGSEA), alongside our novel model (HeteroPath), which assesses heterogeneously upregulated and downregulated genes within the context of pathways, we generated distinct tissue-specific gene regulatory networks. We analyzed gene expression data derived from freshly isolated heart, brain, and lung endothelial cells and populations of neurons in the hippocampus, cingulate cortex, and amygdala. In both datasets, we found that HeteroPath segregated the distinct cellular populations by identifying regulatory pathways that were not identified by GSEA or PGSEA. Using simulated datasets, HeteroPath demonstrated robustness that was comparable to what was seen using existing gene set enrichment methods. Furthermore, we generated tissue-specific gene regulatory networks involved in vascular heterogeneity and neuronal heterogeneity by performing motif enrichment of the heterogeneous genes identified by HeteroPath and linking the enriched motifs to regulatory transcription factors in the ENCODE database.

Conclusions: HeteroPath assesses contextual bidirectional gene expression within pathways and thus allows for transcriptomic assessment of cellular heterogeneity. Unraveling tissue-specific heterogeneity of gene expression can lead to a better understanding of the molecular underpinnings of tissue-specific phenotypes.
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http://dx.doi.org/10.1186/s12859-018-2190-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019795PMC
June 2018

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

Microcirculation 2018 07 30;25(5):e12455. Epub 2018 May 30.

Bioengineering Program, Fred DeMatteis School of Engineering and Applied Science, Hofstra University, Hempstead, NY, USA.

Objective: Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response.

Methods: Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness.

Results: Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness.

Conclusions: Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.
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http://dx.doi.org/10.1111/micc.12455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407863PMC
July 2018

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

Nat Cell Biol 2018 05 16;20(5):565-574. Epub 2018 Apr 16.

Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.
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http://dx.doi.org/10.1038/s41556-018-0086-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937285PMC
May 2018
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