Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function

Abstract

Summary. Background: The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits a IIb and b 3 , is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of a IIb b 3 , thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to a IIb b 3 triggers cytoskeletal changes and granule release (outside-in signaling). Aim: Genetic approaches to characterize the molecular pathways involved in a IIb b 3 signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). Results: a IIb b 3 activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, a þ IIb /GPIba + cells, is readily detectable following stimulation with known platelet agonists. Doseresponse curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of a IIb b 3 outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of a IIb b 3 activation. Conclusion: Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and a IIb b 3 signaling in the native cellular environment.

PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids

“…48,49 The mechanisms that lead to ␣ IIb ␤ 3 activation in megakaryocytes are incompletely understood, but platelet agonists enhance ␣ IIb ␤ 3 -dependent responses in megakaryocytes. 48,50 In this regard, we found that thrombin enhanced megakaryocyte adherence and spreading on immobilized fibrinogen. However, unlike cPAF treatment, binding and spreading of megakaryocytes in response to thrombin was not inhibited by a PAFR blocker ( Figure S6).…”

Platelet bioreactor-on-a-chip

“…Two major quantitative roadblocks persist in the development of donorindependent PLTs for therapeutic use: (1) generating sufficient numbers (;3 3 10 8 ) of human MKs to support the production of 1 PLT transfusion unit (;3 3 10 11 PLTs); and (2) generating physiological numbers of functional human PLTs (;10 3 ) per MK. 18 The development of human embryonic stem cell cultures (hESC), 44,46 and more recently, hiPSC 47 (Feng Q et al, manuscript submitted 2014) offer a physiologically relevant and potentially unlimited source of progenitor cells that can be differentiated into human MKs in vitro to address the first quantitative roadblock. 48 Indeed, because PLTs are anucleate, PLT bioreactor-derived units could be irradiated prior to infusion, addressing concerns that cellular products derived from hESC or hiPSCs could be oncogenic or teratogenic.…”

Transcriptional Regulation of Platelet Formation: Harnessing the Complexity for Efficient Platelet Production In Vitro

“…The first relies on external signals provided by cytokines or stromal cells to mimic embryonic development and direct sequential differentiation of hPSCs into MKs, a process designated as "directed differentiation" (DD) [227][228][229][230][231]. These protocols are impaired by a lack of efficiency, low purity of the MKs and often rely on serum and xenogenic feeder cells.…”