Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

Abstract

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro–derived transfusion products toward the clinic.

The Analysis of the Human Megakaryocyte and Platelet Coding Transcriptome in Healthy and Diseased Subjects

“…In this case, plate-based or combinatorial indexing methods form a good alternative. Other studies [ 92 , 93 , 94 , 95 ] have, however, successfully constructed high-quality MK libraries using 10X Chromium. Table 1 compares the advantages of the two most frequently used scRNA-seq methods, plate-based and droplet-based.…”

“…In Sun et al [ 91 ], three MK subpopulations were reported with distinct transcriptomes linked to the inflammatory response, HSC niche interactions and platelet generation. scRNA-seq has also driven transcriptomic research into primary MK cells [ 89 , 94 ], for which extensive sample purification is needed, most often through flow cytometry [ 90 , 99 ]. The study of Lu et al [ 90 ] identified key genes involved in MEP fate determination and demonstrated that differential modulation of cell cycle speed dictates MEP differentiation into either erythrocytes or megakaryocytes using scRNA-seq.…”

“…Although scRNA-seq analysis has been carried out on MK differentiation in vivo [ 89 , 90 , 91 ] and in vitro from iPSCs or CD34+ HSCs [ 94 ], the transcriptomic difference between different types of in vitro and in vivo MKs is largely unknown. Recently, the first single-cell study on this subject [ 94 ] assessed the transcriptomic differences between megakaryopoiesis in vivo and in vitro through iPSC forward programming.…”

“…Although scRNA-seq analysis has been carried out on MK differentiation in vivo [ 89 , 90 , 91 ] and in vitro from iPSCs or CD34+ HSCs [ 94 ], the transcriptomic difference between different types of in vitro and in vivo MKs is largely unknown. Recently, the first single-cell study on this subject [ 94 ] assessed the transcriptomic differences between megakaryopoiesis in vivo and in vitro through iPSC forward programming. Cells from the latter type were compared to primary hematopoietic stem and progenitor cells from the bone marrow, and they found that the in vitro cells did not pass through states resembling primary HSCs or MPP cells.…”

“…These studies are summarized in Table 2 and are briefly discussed below. In MKs and their precursors, scRNA-seq has been proven useful for studying their dynamic transcriptome throughout their various developmental trajectories [89][90][91]94,96]. Polyploidization during maturation, for example, is associated with a shift from transcripts associated with platelet degranulation, coagulation, hemostasis, wound healing and vesiclemediated transport towards transcripts associated with translational initiation, elongation and termination, protein localization and cellular protein complex disassembly [89].…”