Transcription Factor Levels after Forward Programming of Human Pluripotent Stem Cells with GATA1, FLI1, and TAL1 Determine Megakaryocyte versus Erythroid Cell Fate Decision

Abstract

SummaryThe production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or erythroblasts. Using “rainbow” lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.

AHR:IKAROS Interaction Promotes Platelet Biogenesis in Response to SR1

“…A number of hematopoietic TFs have been identified to promote or favor MK progenitor specification, MK maturation and platelet formation, including GATA-1, Friend of GATA-1 (FOG-1, ZFPM1), Fli-1, RUNX-1 and NF-E2 [7]. These TFs have also been described to promote the expression of megakaryocytic receptors [3][4][5]. In contrast, IKAROS (IKZF1),a member of the zing-finger protein family important for early lymphocyte development, has been shown to exert a repressor effect on MK differentiation and to restrain terminal MK maturation [8,9].…”

“…Megakaryopoiesis (MKP) is a hierarchized and complex process where hematopoietic stem cells (HSCs) differentiate to give rise to megakaryocytic progenitors, which will differentiate into mature MKs capable of extending cytoplasmic extensions into the sinusoid vessel to release platelets under flow [2]. These successive cell fate decisions are under the coordination of an array of extrinsic differentiation or growth factor, small metabolites, stromal mesenchymal cells (MSCs), and intrinsic transcription factors (TFs) [3][4][5]. In a previous study, we reported that the Aryl-Hydrocarbon Receptor (AHR) antagonist, StemRegenin 1 (SR1), favors the expansion in culture at day 10 of a specific population of progenitors, phenotypically defined as CD34 + CD41 low cells, empowered with an enhanced potential to generate mature MKs allowing us to obtain a yield of 50-100 platelets/CD34 progenitor [6].…”

Forming megakaryocytes from murine induced pluripotent stem cells by the inducible overexpression of supporting factors

“…This can be achieved by the use of cytokines and specified culture conditions; however, supporting factors may also be expressed in the cells. In this respect, the combined overexpression of the transcription factors GATA‐binding factor 1 (GATA1), stem cell leukemia 1/T‐cell acute lymphocytic leukemia protein 1 (SCL/TAL1), and Friend leukemia integration 1 (FLI1) 13,15 in human iPSCs has been shown to enhance MK differentiation. In addition, expression of MYC proto‐oncogene, bHLH transcription factor in combination with BMI1 proto‐oncogene, polycomb ring finger and B‐cell lymphoma‐extra large in a different approach generated expandable and cryopreservable MKs 16 .…”

“…This can be achieved by the use of cytokines and specified culture conditions; however, supporting factors may also be expressed in the cells. In this respect, the combined overexpression of the transcription factors GATA-binding factor 1 (GATA1), stem cell leukemia 1/T-cell acute lymphocytic leukemia protein 1 (SCL/TAL1), and Friend leukemia integration 1 (FLI1) 13,15 in human iPSCs has been shown to enhance MK differentiation.…”

Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

“…These MKs produce functional platelets which can contribute to thrombus formation (Shepherd et al, 2018). Interestingly, early timepoints of the culture produce both erythroid cells and MKs, tuned by the rheostat of FLI1 expression (Dalby et al, 2018), mirroring in vivo megakaryopoiesis (Chen et al, 2014). One of the key characteristics of the cultures is their ability, beyond reaching maturity at day 20 (D20), to survive for a further 100 days whilst maintaining cell proliferation and purity by CD41 and CD42 expression (Moreau et al, 2016), indicating the presence of cells capable of reconstituting the culture when mature MKs fragment to generate platelets.…”