Large-scale production of megakaryocytes from human pluripotent stem cells

Large-scale production of megakaryocytes from human pluripotent stem cells

Megakaryocytes (MKs) generate blood platelets whose primary role is to stop haemorrhages via localized clot formation at the site of vessel injury. MKs are polyploid cells derived from haematopoietic stem cells residing in the bone marrow where they represent only 0.01% of the total nucleated blood cells. By extension of cytoplasmic protrusions through bone marrow sinusoids, they release daily ~1 × 1011 platelets into the blood stream to sustain the count of short-lived (7–10 days) circulating platelets between 150–450 × 109 per litre of blood.

A decrease in platelet number, or thrombocytopenia, may occur following bone marrow failure (inherited or acquired, such as post-cancer treatment) or severe peripheral bleeding after trauma or surgery, and potentially leads to life-threatening haemorrhages. Currently, prophylactic and therapeutic treatment essentially relies on transfusion of ABO and Rhesus-D-matched platelet concentrates—at >2.4 × 1011 platelets per unit—from voluntary donations.

Recently, the increase in high-dose cancer therapy, advanced surgical procedures and the ageing population has led to a rising demand for platelets with over 4.5 million platelet units transfused per year in Europe and the United States. In addition, platelet transfusion refractoriness in HLA class I alloimmunized chronically transfused patients and multiparous women necessitates the special provision of matched platelet units sourced from a small pool of genotyped recallable donors. Altogether, the dependence on donations combined with the limited shelf life of platelet concentrates (5–7 days) represents a logistical, financial and biosafety challenge for health organizations worldwide.

Human pluripotent stem cells (hPSCs)—including embryonic stem cells (hESCs) derived from embryos and induced PSCs (hiPSCs) generated from post-natal somatic cells—can be maintained in vitro for prolonged periods while retaining the capacity to differentiate towards virtually any cell type upon adequate stimulation. Therefore, they offer huge opportunities for basic research and clinical applications.

The production of platelets in vitro from genetically defined hPSC lines could revolutionize transfusion medicine by providing a controllable source of platelets. Moreover, platelets are anucleate and do not proliferate which means they can be irradiated before transfusion. This provides a marked safety advantage over other hPSC-derived therapeutic cells which can potentially retain oncogenic cell fractions. However, in vitro systems for the production of large amounts of MKs and subsequent platelet release to match the needs for making transfusion units still require considerable optimization.

Researchers describes a novel approach for generating large quantities of functional MKs from hPSCs with unique advantages for clinical development. Existing protocols have so far relied on external signals provided by cytokines or stromal cells to mimic embryonic development in vitro and thus direct sequential differentiation of hPSCs into MKs, a process designated as ‘directed differentiation.

While mature MKs showing functional platelet release are produced, this strategy has been limited by the relatively low number of MKs generated or by the complex genetic modifications and clonal selection required to immortalize MKs post differentiation.

Urged by the recent discoveries on the plasticity of cell identities controlled by limited sets of transcription factors (TFs), authors adopted a radically different approach for the generation of MKs by exploring the potential of exogenous TFs to drive the differentiation process from hPSCs, a strategy called ‘forward programming’ (FOP).

 Proceeding from a methodically curated list of candidate genes, authors discovered that the combination of GATA1–FLI1–TAL1 uniquely promoted highly efficient MK-FOP from an array of hPSC lines in chemically defined conditions.

Critically, the forward programmed MKs (fopMKs) matured into platelet-producing cells that could be cryopreserved, maintained and amplified in vitro for over 90 days showing an average yield of 200,000 MKs per input hPSC with 90% purity. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs.

This unprecedented efficiency combined with minimal cell manipulation and low cytokine requirements makes MK-FOP a promising platform for basic research as well as future clinical applications in the field of transfusion medicine.