Identifying and enriching platelet-producing human stem cell–derived megakaryocytes using factor V uptake

Abstract

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FVCD42b human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV HG/CD42b MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

“…The input cell suspension contains MK in several stages of maturation and senescence. After the 2 h of perfusion to produce platelets, a double centrifugation is performed to remove remainder MK and concentrate PLP.…”

Endocytosed factor V is trafficked to CD42b+ proplatelet extensions during differentiation of human umbilical cord blood‐derived megakaryocytes

“…As our studies were being prepared for publication, Sim et al () reported that CD42b + megakaryocytes derived ex vivo from CD34 + mobilized human stem cells endocytose a fluorescently‐labeled recombinant factor V derivative. Upon infusion into a mouse thrombosis model, these megakaryocytes release functional factor V‐containing platelets.…”

Tropomyosin 1 genetically constrains in vitro hematopoiesis

“…Given that these high-scoring putatively active SNPs impacted TPM1 expression, we investigated functions for the TPM1 gene in an in vitro human model of primitive hematopoiesis. 37 We anticipated that total gene deletion would show stronger effects than non-coding SNP modification. 38 Using CRISPR/Cas9, we targeted a ~5kb region containing TPM1 exons 4-8 in iPSCs ( Fig.…”

“…iPS cells were differentiated in HPCs and terminal lineages (MKs, erythroid, myeloid) per published protocols. 37,[55][56][57] Validated flow cytometry gating for pluripotency (SSEA3 + /SSEA4 + ), hemogenic endothelium (KDR + /CD31 + ), hematopoietic progenitors (CD43 + /CD34 + and CD41 + /CD235 + ) and terminal lineages can be found in these references.…”