Using human induced pluripotent stem cells (iPSCs) as a source of platelets (Plt)s for transfusion medicine is being pursued by a number of groups with several proposing that such a product will soon be available. To achieve this goal, it would be important to generate a Plt product that overcomes challenges associated with donors Plts, including the issue that iPSC-derived hematopoietic progenitors tend to be primitive in nature and the subsequent megakaryocytes, termed Prim-Megs, and the platelets they produce are primitive as well with many quantitative and qualitative limitations due to this immature developmental stage. In our parent R01 application, we follow a number of strategies to define the proper hematopoietic progenitor to generate the highest quality of megakaryocyte with the potential to generate the highest yield and function of derivative Plts. We have focused in the parent grant on obtaining hematopoietic lineages of later developmental stages collectively called definitive hematopoiesis that can generate definitive megakaryocytes, termed Def-Megs. These cells are distinct and have differing abilities to generate Plts with appropriate biologic activity than Prim- Megs. While our parent R01 aims to compare these two populations generated from pluripotent stem cells and optimize the generation of the highest quality megakaryocyte possible, we did not address a final maturation event that occurs after birth: While both neonatal and adult hematopoietic progenitors are definitive and are derived from the same developmental program, the megakaryocytes derived from these populations are distinct. Neonate-Megs are low ploidy and generate Plts with poor activity in clotting assays compared to adult- Megs. Recent work suggests that the RNA binding protein IGF2BP3 may regulate this maturation process. Its expression is lost in adult-Megs and suppression of IGF2BP3 in cord blood neonate-Megs results in maturation more similar to adult-Megs. We show preliminary data that iPSC-derived megakaryocytes (iMegs) also express high levels of IGF2BP3 and its suppression results in higher ploidy megakaryocytes. We now wish to apply these findings to enhance maturation of iMegs in the following 2 Specific Aims (SAs): SA#1: To determine the effect of IGF2BP3 inhibition on maturation of iMegs in vitro. We will utilize various methods to inhibit IGF2BP3 in iMegs and examine the impact on megakaryocyte maturation in vitro. SA#2: To determine the effect of IGF2BP3 inhibition on release of platelets from iMegs in vivo. Using the same methodologies to inhibit IGF2BP3 as in SA#1, we will examine the ability of the megakaryocytes to function in a mouse xeno- infusion model, examining Plt yield, half-life and ability to contribute to clot formation. We believe that these studies will not only be important in bringing such Plts closer to clinical application, but serve as a role model of how one can improve regenerative medicine in general by obtaining adult rather than less functional embryonic tissues.

Public Health Relevance

The transfusion of platelets is used to treat patients with bleeding problems caused by a number of reasons including having low platelet counts in cancer patients receiving chemotherapy. Donor platelets can only be stored for less than one week requiring a consistent donation to prevent supply issues, and in this revised application, we use a new approach in regenerative medicine to generate platelets from stem cells grown in culture for such platelet transfusions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL130698-04
Application #
9572994
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Welniak, Lisbeth A
Project Start
2015-09-10
Project End
2019-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Gollomp, Kandace; Friedman, David F; Poncz, Mortimer (2018) Platelets Can Soak It Up and Then Spit It Out. Arterioscler Thromb Vasc Biol 38:2544-2545
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
Hanby, Hayley A; Bao, Jialing; Noh, Ji-Yoon et al. (2017) Platelet dense granules begin to selectively accumulate mepacrine during proplatelet formation. Blood Adv 1:1478-1490
Johnston, Ian; Hayes, Vincent; Poncz, Mortimer (2017) Threading an elephant through the eye of a needle: Where are platelets made? Cell Res 27:1079-1080
Borst, Sara; Sim, Xiuli; Poncz, Mortimer et al. (2017) Induced Pluripotent Stem Cell-Derived Megakaryocytes and Platelets for Disease Modeling and Future Clinical Applications. Arterioscler Thromb Vasc Biol 37:2007-2013
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
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
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

Showing the most recent 10 out of 11 publications