Generation of Megakaryocytic Progenitors from Human Embryonic Stem Cells in a Feeder- and Serum-Free Medium

Pick, Marjorie; Azzola, Lisa; Osborne, E; Stanley, Edouard G.

doi:10.1371/journal.pone.0055530

Cited by 50 publications

(44 citation statements)

References 34 publications

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“…A recent study demonstrated that the generation of MKs from HPCs is achievable under feeder‐free conditions [18]. However, that study only examined hESCs and used animal‐derived products such as bovine serum albumin (BSA) [18, 21].…”

Section: Introductionmentioning

confidence: 99%

Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents

Liu

Wang

Gao

et al. 2015

Stem Cells Translational Medicine

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Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animalderived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administrationapproved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34 + CD45 + hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41 CD42a+ MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy ( ‡16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:309-319

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Section: Introductionmentioning

confidence: 99%

Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents

Liu

Wang

Gao

et al. 2015

Stem Cells Translational Medicine

View full text Add to dashboard Cite

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“…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.…”

Section: Mks and Platelets From Human Pluripotent Stem Cellsmentioning

confidence: 99%

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

Tijssen

Moreau

Ghevaert

2016

Molecular and Cellular Biology of Platelet Formation

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It is now common knowledge that specific repertoires of transcription factors (TFs) determine a cell's protein content and thereby its phenotype. The expression of a given TF is not necessarily cell specific, and many TFs play a pivotal role in several different cell types. For example, TAL1, FLI1, RUNX1, ERG and GATA2 are important regulators of stem cells, but also play a vital role in megakaryopoiesis. Although the megakaryocyte (MK) and its closest relative, the red blood cell, share key TFs like GATA1 and NFE2, the bifurcation between the two lineages has been associated with pairs of TFs that act as a toggle switch (such as FLI1 and KLF1). This chapter will summarise the current knowledge of key transcriptional regulators of MK differentiation and how some of these TFs, despite being expressed in several cell types, can impose MK cell identity. Since the discovery of TPO in 1994, our knowledge of MK biology and differentiation has increased exponentially, but we still lack a deep understanding of what triggers the transition from MK growth and maturation to proplatelet formation. We describe how some well-known TFs control the expression of proteins that play a pivotal role in the dramatic cytoplasmic and cytoskeletal events that accompany proplatelet formation. Finally, we show how TFs can be harnessed in a powerful way to produce MKs and, potentially, platelets in vitro for future clinical applications.

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“…Recent studies have shown that functional megakaryocytes and platelets can be generated from hESCs and hiPSCs by optimizing culture conditions [40,41,42]. Currently, yields of ~ 60 megakaryocytes per input hESC have been reported [40].…”

Section: Discussionmentioning

confidence: 99%

MicroRNA Screen of Human Embryonic Stem Cell Differentiation Reveals miR‐105 as an Enhancer of Megakaryopoiesis from Adult CD34+ Cells

et al. 2014

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MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by 4-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235+CD41−), megakaryocyte (CD41+CD42+) and myeloid (CD18+CD235−) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a 6-fold enhancement in megakaryocyte production. MiR-513a, miR-571 and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34+ cells derived from peripheral blood. In addition, adult CD34+ cells express endogenous miR-105 during megakaryocyte differentiation. SiRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3’UTR construct and western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis.

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Generation of Megakaryocytic Progenitors from Human Embryonic Stem Cells in a Feeder- and Serum-Free Medium

Cited by 50 publications

References 34 publications

Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents

Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents

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

MicroRNA Screen of Human Embryonic Stem Cell Differentiation Reveals miR‐105 as an Enhancer of Megakaryopoiesis from Adult CD34+ Cells

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