Embryonic Stem Cell-Derived Platelets as Cellular Therapeutics
Principal Investigator: Mortimer Poncz
Abstract: Present day delivery of platelets for various clinically relevant settings has a number of significant limitations, from issues of storage, contamination, inhibitors in multiply transfused individuals, variable quality and dependency on human donors. We propose a novel strategy for production of clinically relevant numbers of platelets from human embryonic stem cells (hESCs), supported by preliminary data. Such platelets may allow us to avoid many of the present limitations and allow us to develop platelets as a mechanism for the targeted delivery of therapeutic agents to sites of vascular injury. The approach will involve the following steps which will be pursued simultaneously building on our preliminary data: 1) Enhance directed differentiation of hESCs into megakaryocytes (Megs) by improving the efficiency of hESC to hematopoietic mesoderm development and directing hematopoietic progenitors into the Meg lineage. 2) Arrest cells at the Meg-erythroid progenitor (MEP) stage to form self-replicating cells by knockdown of the transcription factor GATA1. Upon re-induction of GATA1, these MEP cells will complete differentiation into Megs. Strategies to enhance MEP to mature Megs will also be pursued. 3) Infuse either ex vivo-generated platelets or mature Megs to generate a vigorous wave of new, functional platelets. 4) Either hESCs or the self-replicating MEP cells will also be modified so that the Megs express an ectopic protein of interest stored in their a-granules for release upon platelet activation. Based on preliminary data, we chose as proof-of-principle to express urinary plasminogen activator in the developing Megs. We will demonstrate that the resulting human platelets allow targeted delivery of this agent to growing thrombi without causing systemic fibrinolysis. These studies will be done in close collaboration with the Seattle Cluster and will involve 3 species: mice, dogs and humans. Mice studies will allow rapid advances that will then be tested in dogs as a large animal model as well as apply our advances to human studies. We believe that over the seven years of support that we will have achieved significant advances in the production of clinically relevant numbers of functional platelets beginning with hESCs and be ready for large-scale confirmatory studies and clinical application.
Funding Period: 2009-09-30 - 2016-04-30
more information: NIH RePORT
- Infusion of mature megakaryocytes into mice yields functional plateletsRudy Fuentes
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
J Clin Invest 120:3917-22. 2010..These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers...
- Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cellsStella T Chou
Division of Hematology, The Children s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Proc Natl Acad Sci U S A 109:17573-8. 2012..Our studies show that T21 confers distinct developmental stage- and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development...
- Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/MafYukako Ono
Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan
Blood 120:3812-21. 2012..These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis...
- SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome modelsRonghua Meng
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, 513 Stellar Chance Laboratories, 422 Curie Blvd, Philadelphia, PA 19104 6100, USA
Blood 120:404-14. 2012....
- Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemiaShilpa Gandre-Babbe
Division of Hematology, Children s Hospital of Philadelphia, Philadelphia, PA, USA
Blood 121:4925-9. 2013..Our studies offer renewable sources of biologically relevant human cells in which to explore the pathophysiology and treatment of JMML. More generally, we illustrate the utility of iPSCs for in vitro modeling of a human malignancy...
- Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell linesJason A Mills
Department of Pathology and Laboratory Medicine, The Children s Hospital of Philadelphia, Philadelphia, PA
Blood 122:2047-51. 2013..Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential...
- The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cellsPrasuna Paluru
Department of Pathology and Laboratory Medicine, Children s Hospital of Philadelphia, Philadelphia, PA, USA Center for Cellular and Molecular Therapeutics, Children s Hospital of Philadelphia, Philadelphia, PA, USA
Stem Cell Res 12:441-51. 2014..These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development. ..
- Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cellsAmita Tiyaboonchai
School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA Center for Cellular and Molecular Therapeutics, The Children s Hospital of Philadelphia, Philadelphia, PA, USA
Stem Cell Res 12:630-7. 2014..Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown. ..
- High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombastheniaSpencer K Sullivan
Division of Hematology
Blood 123:753-7. 2014..Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function. ..