University of Minnesota Projects
The projects undertaken at our institution will define the key regulatory nodes in the networks that promote stem cells and progenitor cells to adopt a hematopoietic and cardiovascular cell fate – with an aim toward improving clinical options. We will decipher the intracellular, extracellular and paracrine factors that promote stem/progenitor cell specification and differentiation to cardiac, vascular and hematopoietic restricted lineages. In these projects, we will utilize an array of tools and emerging technologies including transgenic mouse models, genetically engineered embryonic and induced pluripotent stem cells, clonal cardiac progenitor cell populations, the decellularized heart, and state-of-the-art high resolution imaging/spectroscopy technologies, which will facilitate our studies and those of our partners at the University of Wisconsin and those associated with the consortium. The overall goal of this proposal is to mechanistically decipher the intracellular and extracellular networks that govern specification and differentiation of stem cells to the cardiovascular and hematopoietic lineages. To address this overall goal, we will pursue the following projects:
Project 1: To define the transcriptional mechanisms that specify the hematopoietic program in hES- and hiPS-derived mesoderm.
Project 2: To define the transcriptional and signaling networks that specify the cardiovascular program in mES/EBs, hESC, hiPSC, and cardiac progenitors.
Project 3: To define the extracellular cues including cell-matrix interactions that direct cardiovascular differentiation and function.
Project 4: To define the optimal cell populations derived from hESCs and hiPSCs for myocardial regeneration.
To successfully address these projects and the associated specific aims, we have assembled an outstanding, collaborative research team that has established expertise in stem cell biology and regenerative medicine. The members of the team include:
Daniel J. Garry, M.D., Ph.D., Professor, Medicine and Integrative Biology and Physiology Departments, St. Jude Medical Endowed Chair in Cardiology, Director of the Lillehei Heart Institute and Chief of the Cardiovascular Division will serve as the lead Principle Investigator for the hub and this U01 grant and will direct Project 2.
Michael Kyba, Ph.D., Assistant Professor, Pediatrics Department, Lillehei Endowed Scholar has expertise in hematopoiesis, transcriptional biology and gene targeting in stem cell populations will direct Project 1.
Dan Kaufman, M.D., Ph.D., Associate Professor of Medicine and Co-Director of the Stem Cell Institute has expertise in hESCs and hematopoiesis, completed his training in the Thomson laboratory at UW and will serve as a Co-Investigator on Projects 1, 2 and 4.
University of Wisconsin Projects
Project 1, “The De Novo Generation of Hematopoietic Stem Cells (Hscs) from Human ESC/iPCs and Somatic Cells.”
Aim 1. Identify the hierarchy of mesodermal progenitors of vascular endothelial and hematopoietic lineages using human ES cell lines with targeted FOXF1, GATA-2, GATA-3, RUNX1, and SCL genes.
Aim 2. Determine the most critical molecular events leading to the formation of hematopoietic cells and HSCs during embryogenesis in mouse and human, and following in vitro differentiation of human pluripotent stem cells.
Aim 3. Develop technologies for de novo generation of HSCs from human ESC/iPSCs and somatic cells.
Project 2, “Cardiovascular Progenitors and Cardiomyocytes from Human Pluripotent Stem Cells and Somatic Cells.”
Aim 1. Isolate and define human embryonic cardiovascular progenitors derived from pluripotent stem cells.
Aim 2. Reprogram human pluripotent stem cells and somatic cells using transcription factors to generate cardiovascular progenitors and ventricular myocytes.
Project 3, “Artificial Transcription Factors for Reprogramming/Transdifferentation.”
Aim 1. Design ATFs that activate the expression of Oct4, Sox2 or Nanog in primary fibroblasts.
Aim 2. Perform genome-wide location analysis (ChIP-chip or ChIP-seq), CSI and transcriptome analysis to determine the gene targets and specificity of the designed ATFs.
Aim 3. Combine ATFs, alone or in combination with natural transcription factors, to induce pluripotency in primary fibroblasts.
Project 4, “Rapid In Vitro Generation of Affinity Reagents for Hematopoietic and Cardiovascular Precursors.”
Aim 1. Develop methods for rapidly generating aptamers that bind to transmembrane protein targets with high affinity and specificity.
Aim 2. Develop methods to accelerate the discovery of aptamers with sub nanomolar affinities using high-throughput DNA sequencing technologies.
Aim 3. Generate high affinity aptamers for the surface markers of cardiovascular and hematopoietic precursors.
Genomics and Bioinformatics Core that will support each of the four proposed UO1 projects.
Core aims include:
Aim 1. Isolate rare hematopoietic and cardiac precursor cell populations for genomic analysis.
Aim 2. Provide high throughput sequencing for RNA-Seq and ChiP-Seq.
Aim 3. Provide Bioinformatics support for RNA-seq and ChIP-Seq.
Aim 4. Provide pools of heart and blood precursor cell cDNAs in lentiviral vectors.
Aim 5. Provide genetically modified human ES and iPS cells.