Pluripotent Stem Cell Modeling of Normal and Abnormal Megakaryocyte Development and Platelet Production

Abstract 1276

Thrombocytopenia is a multi-factorial blood disorder characterized by an abnormally low number of circulating platelets that can have devastating effects upon a wide swath of patients independent of age, race, or socioeconomic group. The two major reasons for thrombocytopenia are increased turnover in immune thrombocytopenia purpura (ITP) and decreased production due to bone marrow failure as a result of chemotherapy, aging, or drugs. Even in ITP, there is some evidence that decreased production may play a role in the etiology of the disease. Thus, patients not making enough platelets are usually treated with platelet transfusions, which carry risks of allergic reactions, infections, and eventually sensitization to allo-antigens making patients refractory to transfusions. With these facts in mind, there is a clear need for the development of novel, autologous sources of mature platelets, and the ability to produce patient-specific megakaryocytes from pluripotent stem cells would have a potential therapeutic role.

We have developed a novel, excisable reprogramming vector (STEMCCA) capable of generating ‘clinical grade’ induced Pluripotent Stem Cells (iPSC) free of any residual reprogramming transgenes, and have employed this vector in the derivation of both normal and megakaryocyte disease-specific cell lines. To develop a novel source of platelet precursors for hematopoietic and cell-based therapy studies, we have established conditions for the efficient directed differentiation of these lines into a virtually unlimited supply of functional megakaryocyte-lineage cells that express a constellation of accepted megakaryocyte markers, appropriate Wright-Giemsa stained morphology, expected polyploidy via endoreduplication, and both normal and aberrant platelet production.

iPSC-derived megakaryocytes were subsequently tagged with viral vectors expressing fluorescent proteins (for quantification of platelet contribution in peripheral blood) and/or luciferase (for in vivo imaging studies) and administered to mouse models via the retro orbital sinus. Transplanted mice were monitored for the presence of the transferred megakaryocytes and resulting platelets via Ly 5.1/5.2 chimerism as well as for the presence of GFP positive cells using FACS analysis. Peripheral blood from these mice was screened at 1 day post transplantation for chimerism and expression of GFP, and at subsequent 2 day time periods when GFP positive cells were noted in order to track the continued viability or death of the megakaryocyte-lineage cells and resulting platelets. Following these cell transfer experiments, the presence of green platelets in the peripheral blood of these mice indicated that the megakaryocyte-lineage cells produced from the directed differentiation of iPSC are indeed viable in vivo and are capable of the production of platelets. The duration of reconstitution and the functionality of the platelets derived from the iPSC generated megakaryocytes as well as those generated from embryonic stem cell (ESC) controls are currently being assessed by quantifying the labeled platelets over time, and carrying out tests of platelet function in vivo (bleeding time) and in vitro (platelet aggregation studies).

Our current work focuses on the hypothesis that an iPSC-based system is capable of producing sufficient numbers of fully functional megakaryocytes to ameliorate thrombocytopenia in vivo. The implications of successfully testing this hypothesis are profound, for they suggest that early megakaryocyte and platelet development can be directly evaluated in vitro and, moreover, that megakaryocyte-lineage cells produced from patient-specific, directly differentiated iPSC lines can become a potent source for transfusion studies and regenerative medicine.