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Rabinovitch Lab

Summary:

Our research program seeks to identify the cellular and molecular programs regulating vascular and lung development. We then determine how these programs are perturbed by genetic abnormalities or injurious processes associated with disease. Our studies use high throughput genomic and microfluidic technologies, a variety of cell biology platforms including confocal and videomicroscopy, genetically modified mouse models of human disease, human tissue samples and induced pluripotent stem cells to answer these questions. Our major disease focus is pulmonary arterial hypertension (PAH), a condition that can be a fatal complication in children with heart defects, but also arises as a condition of unknown etiology primarily in young women. The pathological changes in the lung blood vessels that cause right-sided heart failure include loss of the distal microcirculation and obliterative proliferative changes occluding the lumen of larger arteries. Our goal is to learn how we can activate lung vascular developmental programs to regenerate lost microvessels and to reverse the obliterative changes. Over the past decade our research has led to four novel compounds in clinical trial or being positioned for clinical trial.

Affiliations:

People:

Resources:

Human Studies

  • IPSC DERIVED EC AS SURROGATES USING PULMONARY HYPERTENSION AS A PROTOTYPE DISEASE ( Human Study )

    This proposal investigates the use of induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) as surrogates for native ECs to improve our understanding and to better treat a life-threatening vascular disorder, pulmonary hypertension (PAH). To this end we have assembled a multidisciplinary team with expertise in clinical manifestations and pathobiology of PAH, iPSC generation and differentiation, genetics and genomics, bioinformatics and gene therapy and high throughput phosphoflow and microfluidic technologies. Our proposal reflects a unique opportunity to assess three lines of ECs derived from the same patient. This streamlines our ability to test the efficacy of iPSC-ECs as surrogates for native ECs, and to establish the significance of specific genetic alterations. We have a unique opportunity to compare gene variants, epigenetic and gene expression profiles in fibroblast derived iPSC-ECs to those in pulmonary arterial (PA)EC derived iPSC-ECs from the same PAH patients or controls. This should address in an unprecedented manner, how a genetic vulnerability leads to manifest disease. Moreover, we are well placed to investigate the application of gene and pharmaceutical therapy to reverse PAH pathology in iPSC-ECs. Because studies completed to date in the Wu laboratory have established facility with the production of iPSC-ECs, we are positioned to begin with Phase II of this Proposal. To this end, we propose three Specific Aims in Phase II and three in Phase III. Phase II, Aim 1 applies Hi-Seq, Methyl-Seq and RNA-Seq to obtain information on rare gene variants, epigenetic changes and gene expression in PAH and control fibroblast- iPSC-ECs, PAEC-iPSC-ECs and PAECs. Phase II, Aim 2 correlates this genomic information with function, by extensive analysis of the phenotype of these cells using angiogenesis assays and phosphoflow analyses and by assessing homogeneity using a single cell microfluidic approach. Phase II, Aim 3 initiates a partnership with Progenitor Cell Therapy to transfer the technology of producing iPSC-ECs so that it can be ramped up in the future to benefit large populations of patients. In Phase III, Aim 1 utilizes cutting edge gene therapy approaches, such as minicircles and ribosomal DNA vectors, to correct a gene variant in an iPSC-EC. Phase III, Aim 2 investigates how this reverts the PAH-related phenotype of the cell. Phase III, Aim 3 compares the efficacy of fibroblast and PAEC-iPSC-ECs vs. native PAECs to respond to pharmaceuticals to revert the disease phenotype. In summary, our studies should serve as a model to better understand how iPSC-ECs can be used to uncover and treat genetic predisposition to vascular dysfunction in a wide variety of cardiovascular diseases.

Protocols

  • In vitro monolayer endothelial differentiation of murine iPSCs ( Protocol )

    To induce endothelial differentiation, approximately 1x105 undifferentiated iPSCs were seeded in each well of Matrigel-coated 6-well plates and cultured in differentiation medium containing RPMI and B-27 supplement minus insulin (Life Technologies) with 5 μM CHIR-99021 (a glycogen synthase kinase [GSK]-3 inhibitor; Selleck Chemicals, Houston, TX, USA) for 2 days, followed by RPMI and B-27 supplement minus insulin with 2 μM CHIR-99021 for 2 additional days. The medium was then changed to RPMI and B-27 supplement minus insulin with 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 10 ng/mL fibroblast growth factor basic (FGFb; R&D Systems), and 10 μM Y-27632 (a rho-associated protein kinase [ROCK] inhibitor; Sigma-Aldrich, Saint Louis, MO, USA) for 3 days. For the subsequent 7 days, the medium was changed to RPMI and B-27 supplement (with insulin) with 50 ng/mL VEGF, 10 ng/mL FGFb, 10 μM Y-27632, and 1 μM SB 431542 (a transforming growth factor [TGF]-β inhibitor; Sigma-Aldrich). At day 14, iPSC-ECs were sorted for CD31+/CD144+ markers using FACS and expanded on 0.2% gelatin coated plates. After sorting, iPSC-ECs were cultured in EBM-2 Basal Medium supplemented with the EGM-2 BulletKit (Lonza, Basel, Switzerland) at 37°C, 20% O2, and 5% CO2 in a humidified incubator with medium changes every 48 hours, and cells were passaged once they reached 80-90% confluence. Murine iPSCECs used for in vitro and in vivo characterizations were between passages 3 and 5.

Reagents

  • STAN001i-047-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN002i-161-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN003i-161-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN004i-147-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN005i-147-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN006i-148-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN007i-148-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN008i-165-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN009i-165-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN010i-041-2 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN011i-123-1 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN012i-123-2 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN013i-121-1 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN014i-121-2 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN015i-178-1 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN016i-178-2 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN017i-171-1 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN018i-171-2 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN019i-177-1 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN020i-177-2 ( Induced pluripotent stem cell line )

    Idiopathic pulmonary arterial hypertension

  • STAN021i-170-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN022i-170-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN023i-41-1 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN024i-29-1 ( Induced pluripotent stem cell line )

    Familial pulmonary arterial hypertension

  • STAN025i-29-2 ( Induced pluripotent stem cell line )

    Familial pulmonary arterial hypertension

  • STAN026i-30-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with pulmonary veno-occlusive disease

  • STAN027i-30-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with pulmonary veno-occlusive disease

  • STAN028i-42-1 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN029i-42-2 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN030i-46-1 ( Induced pluripotent stem cell line )

    Familial pulmonary arterial hypertension

  • STAN031i-46-2 ( Induced pluripotent stem cell line )

    Familial pulmonary arterial hypertension

  • STAN032i-48-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN034i-48-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN035i-49-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN036i-49-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN037i-118-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with ventricular septal defect and Eisenmenger's Syndrome

  • STAN038i-118-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with ventricular septal defect and Eisenmenger's Syndrome

  • STAN039i-119-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with Lupus and pulmonary fibrosis

  • STAN040i-119-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with Lupus and pulmonary fibrosis

  • STAN041i-122-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with s/pand patent ductus arteriosus

  • STAN042i-122-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with s/pand patent ductus arteriosus

  • STAN043i-124-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with ventricular septal defect and Eisenmenger's Syndrome

  • STAN044i-124-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with ventricular septal defect and Eisenmenger's Syndrome

  • STAN045i-125-1 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with Eisenmenger's Syndrome

  • STAN046i-125-2 ( Induced pluripotent stem cell line )

    Associated Pulmonary Arterial Hypertension with Eisenmenger's Syndrome

  • STAN047i-126-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN048i-126-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN051i-146-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN052i-146-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN053i-149-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN054i-149-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN055i-160-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN056i-160-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN057i-162-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN058i-162-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN059i-163-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN060i-163-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN061i-164-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN062i-164-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN063i-166-1 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN064i-166-2 ( Induced pluripotent stem cell line )

    Non-affected Control

  • STAN065i-167-1 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN066i-167-2 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN067i-168-1 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN068i-168-2 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN069i-169-1 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN070i-169-2 ( Induced pluripotent stem cell line )

    Drugs and toxins-associated pulmonary arterial hypertension

  • STAN071i-179-1 ( Induced pluripotent stem cell line )

    Familial Pulmonary Arterial Hypertension with Drugs and Toxins-Associated history

  • STAN072i-179-2 ( Induced pluripotent stem cell line )

    Familial Pulmonary Arterial Hypertension with Drugs and Toxins-Associated history

  • STAN073i-180-1 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

  • STAN074i-180-2 ( Induced pluripotent stem cell line )

    Idiopathic Pulmonary Arterial Hypertension

Resource Collection

  • (WiCell) Pulmonary Artery Hypertension Collection - Rabinovitch Lab ( Resource Collection )

    This collection, from Dr. Marlene Rabinovitch (Stanford University), will provide important information about pulmonary hypertension (PAH) pathobiology and the use of iPSC as tools for genetic screening, drug testing, and cell therapy.

    This collection contains 71 cell lines. Individuals giving rise to iPSC lines were from the Cardiovascular Medical Research and Education Fund (CMREF)-Pulmonary Hypertension Breakthrough Initiative (PHBI), a consortium of 10 transplant centers across the US. Disease states for the donors include IPH, PAH, and APH. There are individuals with no reported diagnoses as well. Age of donors range from 1-60 and ethnicities include Caucasian, African American, and Latino.


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Last updated: 2016-06-15T15:42:26.782-05:00

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