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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01HL099656-06
Application #
8657090
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Mondoro, Traci
Project Start
2009-09-30
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Maguire, Jean Ann; Cardenas-Diaz, Fabian L; Gadue, Paul et al. (2018) Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells. Curr Protoc Stem Cell Biol :e64
Jarocha, Danuta; Vo, Karen K; Lyde, Randolph B et al. (2018) Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors. Blood Adv 2:597-606
Vo, Karen K; Jarocha, Danuta J; Lyde, Randolph B et al. (2017) FLI1 level during megakaryopoiesis affects thrombopoiesis and platelet biology. Blood 129:3486-3494
Gollomp, Kandace; Lambert, Michele P; Poncz, Mortimer (2017) Current status of blood 'pharming': megakaryoctye transfusions as a source of platelets. Curr Opin Hematol 24:565-571
Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R et al. (2017) Intracellular origin and ultrastructure of platelet-derived microparticles. J Thromb Haemost 15:1655-1667
Sim, Xiuli; Jarocha, Danuta; Hayes, Vincent et al. (2017) Identifying and enriching platelet-producing human stem cell-derived megakaryocytes using factor V uptake. Blood 130:192-204
Sim, Xiuli; Poncz, Mortimer; Gadue, Paul et al. (2016) Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets. Blood 127:1227-33
Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R et al. (2016) CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes. Blood 127:675-80
Sim, Xiuli; Cardenas-Diaz, Fabian L; French, Deborah L et al. (2016) A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models. Methods Mol Biol 1353:13-23
Wang, Yuhuan; Hayes, Vincent; Jarocha, Danuta et al. (2015) Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale. Blood 125:3627-36

Showing the most recent 10 out of 51 publications