The Gata1low murine megakaryocyte–erythroid progenitor cells expand robustly and alter differentiation potential

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Highlights

  • GATA1 low mouse megakaryocyte-erythroid progenitors expand excessively in vitro.

  • Long-term cultured GATA1 low MEP impeded megakaryopoiesis but retained erythropoiesis.

  • TPO-dependent PI3K/AKT pathway partially involve in CD41 reduction in the MEPs.

Abstract

GATA1 is a master transcription factor of megakaryopoiesis and erythropoiesis, and loss-of-function mutation can induce accumulation of megakaryocyte–erythroid progenitors (MEPs) in mice and humans. Accordingly, the murine MEP cell line (termed G1ME2 cells) encoding doxycycline (dox)-inducible anti-Gata1 shRNA on Hprt locus has been developed. The cells were CD41+CD71+KIT+, expand under dox, stem cell factor, and thrombopoietin (TPO), and terminally differentiate into erythroid cells or megakaryocytes upon removal of dox. Surprisingly, in this study, these Gata1low murine MEPs displayed accelerated growth from around 90–100 days after cell culture, impeded megakaryocytic potential, and maintained erythropoiesis. We specified them as late G1ME2 cells and discovered that increased CD41KIT+ population during long-term culture was the main reason for the delayed megakaryopoiesis. The CD41 expression level was partially de-repressed by PI3K/AKT inhibitors, suggesting that TPO-mediated cell survival signaling pathway might have impacted on CD41 in the late G1ME2 cells. Nevertheless, among the late cells, the CD41+KIT+ cells could still generate megakaryocytes on dox withdrawal. Taken together, G1ME2 cells could provide a good model to study molecular mechanism of hematopoiesis because of their ability to expand excessively without artificial immortalization.

Introduction

Proper cell lines are necessary to study the molecular biology of hematopoiesis. Among them, the use of the megakaryocyte–erythroid progenitors (MEPs)/megakaryocyte precursors (MkPs) has been limited. Gata1-deficient murine embryonic stem cell (mESC)-derived MEP-like cell lines, namely G1ME [1] and G1ME2 [2] were established based on the findings that loss-of-GATA1-function mutation could expand MkPs [3,4]. G1ME cells involve GATA1 knockout and have been widely used to understand GATA1 mutation-related disorders or protein-protein interaction during hematopoietic differentiation [5]. Conversely, G1ME2 cells, which express doxycycline (dox)-inducible anti-Gata1 shRNA, expand under dox treatment and show terminal maturation into megakaryocytes or erythroid cells with thrombopoietin (TPO) or erythropoietin (EPO), respectively, presumably because of physiological restoration of GATA1 after dox withdrawal [2]. Thus, G1ME2 cells represent more physiological relevance than G1ME cells, as they are differentiated without retroviral Gata1 gene transduction [6].

Besides its role in megakaryocyte–erythroid differentiation of GATA1, inherited or acquired GATA1 mutations are found in blood cell disorders or in acute megakaryoblastic leukemia associated with Down syndrome [7,8]. In addition, down-regulation of GATA1 is suggested to contribute to the development of myeloproliferative neoplasms (MPNs), as shown in both Gata1low mice and patients [[9], [10], [11]]. Interestingly, the G1ME2 cells thrived without artificial immortalization. Similarly, Gata1low mice (approximately 5% of GATA1 expression) have a high incidence of leukemia [12,13]. Not only GATA1, but other transcription factors related to GATA1 are spatiotemporally regulated during megakaryocyte–erythroid development. Hematopoietic progenitor cells (HPCs) express high levels of GATA2, which is repressed by GATA1 during differentiation [14]. PU.1 (SPI1), a transcription factor required for lymphoid and myeloid differentiation, is inversely regulated by both GATA1 and GATA2 [15]. Intriguingly, reduction of PU.1 in mice and humans is suggested to trigger acute myeloid leukemia [16]. Furthermore, PU.1 expression was reduced in human hematopoietic stem cell (HSCs) from healthy donors aged > 65 years when compared with PU.1 levels in HSCs from individuals aged 25–30 years [16], suggesting that aging-associated factors could contribute to PU.1 suppression.

Despite evidence of the association between the down-regulation of GATA1 and myeloproliferative symptoms, the exact mechanism of action remains to be elucidated. The cytokine TPO plays an important role in the proliferation of HPCs and the differentiation of megakaryocytes through its receptor, MPL [17,18]. Likewise, CD41 is expressed in both HPCs and megakaryocytes and can be induced upon TPO treatment [19]. Downstream signaling of TPO is mainly mediated through phosphorylation of STAT3, ERK, and AKT [[20], [21], [22]]. Therefore, it could be interesting to study the relationship between TPO-dependent expansion of HPCs and CD41 expression. Importantly, gain-of-function mutations of MPL, JAK2, and CALR could evoke MPNs, like primary myelofibrosis, which increases the accumulation of immature MkPs [23,24]. Here, we incidentally found that the Gata1low MEPs/MkPs cell line, G1ME2 cells started to display accelerated replication after about 90–100 days of culture, and this was accompanied by exclusive disappearance of CD41. We hypothesized that this phenomenon could recapitulate myeloproliferative hematopoietic disorders shown in the Gata1low mice, and used the G1ME2 cells to understand the underlying mechanisms.

Section snippets

Cell culture and differentiation

G1ME2, G1ME, Y10 cells were kindly provided by the Children’s Hospital of Philadelphia (Philadelphia, PA). G1ME2 cells were initially obtained and maintained, as previously described [2]. Briefly, mESCs expressing corresponding anti-Gata1 shRNA were used for embryoid body (EB) formation. At day 6 of EB differentiation, the EBs were disaggregated and plated in serum-free differentiation medium (SFD) supplemented with 100 ng/mL murine (m) SCF, 20 ng/mL mTPO and 500 ng/mL dox (Clontech

Results

The G1ME2 cells were classified into two groups according to the culture days or passages, considering the distinctive pattern of cell growth, given that they were morphologically indistinguishable (Fig. 1A). To improve clarity, we considered the early passage of G1ME2 cell culture to be < 50 days (or <25 passages, i.e., early G1ME2) and the late passage ones to be > 90 days (or >50 passages, i.e., late G1ME2), respectively, since development from day 6 EB with dox, SCF, and TPO [2]. Consistent

Discussion

In this study, we employed the previously-developed murine megakaryocyte–erythroid progenitor cell line (G1ME2 cells) to determine the features after long-term expansion. We discovered that the long-term TPO-dependent maintenance affects cell-cycle acceleration and CD41 reduction in G1ME2 cells. Nevertheless, the late G1ME2 cells could generate both megakaryocytes (CD41+CD42b+) and erythroid cells (Ter119+) eventually but were relatively less efficient for megakaryopoiesis. Notably, the

Declaration of competing interest

The authors report no conflict of interests.

Acknowledgements

Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (grant numbers NRF-2016R1C1B3009116, NRF-2019R1A2C3002034); and the KRIBB Research Initiative Program funded by the Korea government.

We are thankful to Prof. Mitchell J. Weiss for sharing cell lines and intellectual support.

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