Platelet Production System Using An Immortalized Megakaryocyte Cell Line Derived From Human Pluripotent Stem Cells

Abstract MRG-1

Human induced pluripotent stem cells (hiPSCs) are a promising source of blood cells, including platelets, for transfusion. However, there remains a need for: 1) a method to obtain large numbers of cells, 2) a system to provide cells of a predefined quality, and 3) a method to overcome the storage limitations of platelets caused by their short shelf life.

To address these issues, we attempted to establish an immortalized megakaryocyte progenitor cell line derived from hiPSCs. We recently showed that the temporal profile of c-MYC activation during megakaryopoiesis is critical for normal platelet production from hiPSCs; that is, peak activation of c-MYC in megakaryocyte progenitors must be followed by a reduction of c-MYC expression for further maturation (Takayama et al. J Exp Med, 2010). Mechanistic analysis revealed that overexpression (O/E) of c-MYC increased megakaryocyte numbers but also induced apoptosis and senescence. Here we demonstrate that this phenomenon is primarily regulated by induction of the INK4A and ARF genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/E and BCL-XL (negative regulator of caspase family) O/E, and c) c-MYC O/E and BMI1 (negative regulator for both INK4A and ARF genes) O/E in hematopoietic progenitors derived from human embryonic stem cells (hESCs), we found that only c-MYC and BMI1 O/E (protocol b) increased numbers of CD41a+/CD42b+ non-polyploid megakaryocytes in an exponential manner for over 3 months. Neither c-MYC O/E and p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficient to maintain an increase in the megakaryocyte population, which suggests that down-regulation of INK4A and ARF contributes mainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thus appears that we were able to establish an immortalized megakaryocyte cell line (MKCL).

As mentioned, a decline in c-MYC activation during hiPSC-derived megakaryocyte maturation is required for generation of functional CD41a+/CD42b+ platelets in vitro. Excessively sustained c-MYC expression in megakaryocytes was accompanied by increased ARF and INK4A expression and decreased GATA1 and NF-E2 expression, eventually leading to megakaryocyte senescence and apoptosis, and CD41a+/CD42b^low/− platelet generation. By using an inducible expression vector system with c-MYC and BMI1 in this context, the MKCL was capable of generating polyploid megakaryocytes (>8N; 40%). The MKCL also subsequently showed proplatelet formation leading to the release of “functional” CD41a+/CD42b+ platelets. Furthermore, following transfusion of 6×10⁸ platelets originally derived from the our immortalized MKCL into immunodeficient NOG mice, the platelets appeared to exhibit normal circulation a high degree of chimerism (human CD41a/ human CD41a + mouse CD41 was ∼67% at 2 hrs and 26% at 24 hrs post transfusion). We therefore propose that establishment of immortalized MKPCs through gene manipulation could potentially provide a stable supply of platelets at a predefined quality and quantity for transfusion therapy.