Generation of megakaryocytes and functional platelets from human embryonic stem cells
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PROVIDER: PRJNA894477 | ENA |
REPOSITORIES: ENA
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Project description:We developed a novel differentiation system that directionally induces hESCs into megakaryocytes and functional platelet in vitro by highly mimicking the in vivo developmental process of megakaryocytes and platelets. We then performed gene expression profiling analysis using data obtained by RNA-seq at different stages of differentiation during the differentiation of hESCs into megakaryocyte lineages.
2024-08-14 | GSE216593 | GEO
Project description:Thrombopoietin (TPO) acting via its receptor Mpl is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO over-stimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.
2014-06-10 | E-MTAB-2389 | biostudies-arrayexpress
Project description:The shortage of platelets is becoming increasingly prominent owing to their short shelf life, limited supply, and increasing demand in response to public health incidents. It is an attractive idea to obtain large numbers of transfusible megakaryocytes (MKs) and platelets from somatic cells via cell lineage reprogramming. However, generating human MKs from somatic cells using a pharmacological reprogramming approach has not been widely explored. Here, we report the successful generation of human induced MKs (iMKs) from cord blood erythroblasts (EBs) using a chemical reprogramming strategy with a combination of four small molecules (4M): Bix01294, RG108, VPA, and PD0325901. The iMKs exhibited the ability to produce proplatelets and release vital functional platelets in vitro, demonstrating their similarity to natural MKs. Importantly, after injection into mice, iMKs were able to mature and give rise to functional platelets that were incorporated into newly formed thrombi. The reprogramming process was carefully examined using single-cell RNA sequencing, which revealed an efficient, rapid, and successful cell fate conversion of EBs to iMKs by 4M via the intermediate state of bipotent precursors. Assay for transposase-accessible chromatin sequencing results indicated that 4M induced genome-wide chromatin remodeling during MK reprogramming from EBs. 4M drove the transition of the transcription factor gene network by downregulating the key erythroid transcription factor genes KLF1 and MYB and subsequently upregulating MK development-associated transcription factor genes, including FLI1 and MEIS1. This process eventually led human cord blood EBs to acquire the MK fate. Thus, our chemical reprogramming of cord blood EBs to iMKs provides a simple and efficient approach to generating clinically transfusible MKs and platelets.
2022-08-28 | GSE207654 | GEO
Project description:Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel therapies. Thus, we evaluated the effect of iMPs on wound healing in vitro. iMPs specifically released FGF2 and exhibited superior enhancement on HUVEC proliferation compared to PRP. RNA-seq revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. These mechanisms were suggested to provide the superior effects of iMPs.
2024-10-23 | GSE260954 | GEO
Project description:Dominant-negative mutations in transcription factor Growth Factor Independence-1B (GFI1B) cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that normal and mutant GFI1B interacted most strongly with the LSD1-RCOR-HDAC corepressor complex in megakaryoblasts. Sequestration of this complex by mutant GFI1B and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1B-mutant induced pluripotent stem cells (iPSC) also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant iPSC-derived megakaryocytes identified a multitude of deregulated pathways downstream of mutant GFI1B. Proteome studies on primary normal and GFI1B-mutant platelets showed reduced expression of proteins implicated in platelet function, and sustained expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B regulates a broad developmental program during megakaryopoiesis. Mutant GFI1B deregulates this program through LSD1-RCOR-HDAC sequestering.
2019-01-25 | PXD009020 | Pride
Project description:Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. To exploit this clinical observation, we engineered wildtype (WT) murine ES cells to express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs. In vitro differentiation with dox and thrombopoietin (Tpo) resulted in approximately 1013-fold expansion of immature hematopoietic progenitors. Upon dox withdrawal with multilineage cytokines, GATA1 expression was restored and the cells differentiated into erythroblasts and megakaryocytes. With Tpo alone, dox-deprived progenitors formed mainly mature megakaryocytes that generated functional platelets in vivo. Our findings provide a novel, readily reproducible strategy to expand ES-cell derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes producing functional platelets in clinically relevant numbers. 3 classes of samples were compared 1) fetal liver derived megkaryocytes 2) G1ME (Gata1â megakaryocyte-erythroid) 3) G1ME2 (engineered wildtype (WT) murine ES cells to express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs)
2015-12-09 | E-GEOD-66479 | biostudies-arrayexpress
Project description:Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. To exploit this clinical observation, we engineered wildtype (WT) murine ES cells to express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs. In vitro differentiation with dox and thrombopoietin (Tpo) resulted in approximately 1013-fold expansion of immature hematopoietic progenitors. Upon dox withdrawal with multilineage cytokines, GATA1 expression was restored and the cells differentiated into erythroblasts and megakaryocytes. With Tpo alone, dox-deprived progenitors formed mainly mature megakaryocytes that generated functional platelets in vivo. Our findings provide a novel, readily reproducible strategy to expand ES-cell derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes producing functional platelets in clinically relevant numbers.
2015-12-09 | GSE66479 | GEO
Project description:Effect of iPSC-derived megakaryocytes and platelets on gene expression of HUVEC
| PRJNA1084727 | ENA
Project description:Megakaryocytes are bone marrow (BM) resident cells that derive from hematopoietic stem cells. A pivotal function of megakaryocytes is the generation of platelets through the release of long protrusions called proplatelets into sinusoidal vessels. single-cell RNA-sequencing on murine BM megakaryocytes has previously revealed transcriptional heterogeneity with segmentation into four distinct categories. These studies postulated functions beyond platelet production with evidence for immunoregulatory and stem cell niche supporting subtypes, as well as a cycling population. The spatial context and transcriptional heterogeneity of megakaryocytes is of great interest as localization of for instance the vasculature is a necessity for platelet production. For single-cell RNA sequencing this spatial orientation is however lost due to the dissociation of tissues. Recent technological advances have enabled the interrogation of gene expression profiles of tissues in situ. This enables the integration of morphological, situational and transcriptional information to classify cells in the context of their microenvironment. In the following we present, for the first time, the application of this technology to BM megakaryocytes at a single cell level.
2023-04-27 | GSE224838 | GEO
Project description:Post-transcriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biological processes and disease. We systematically dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and healthy controls, and integrated these data with transcriptomic and proteomic platforms. We identified and validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a discrete 3-member subset effectively defines ET phenotypes. The genetic signature includes functional guide and passenger strands of the previously-uncharacterized miR 490 (5p and 3p), both of which displayed restricted, low-level expression in megakaryocytes/platelets (compared to leukocytes), and aberrant expression during thrombocytosis, most profound in essential thrombocythemia (ET). Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic stem cell colony differentiation and/or lineage specification. Systematic integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a unique miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively modulated by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis, and collectively support a developmentally-restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation. Human platelets from a total of 79 samples in 3 phenotypic groups: essential thrombocythemia (ET, N =27), reactive thrombocytosis (RT, N=22) and healthy controls (NO, N=30) were measured on array platform
2012-11-06 | E-GEOD-39046 | biostudies-arrayexpress