Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors

doi:10.1016/j.stemcr.2019.05.007

. 2019 Jul 9;13(1):31-47.

doi: 10.1016/j.stemcr.2019.05.007. Epub 2019 Jun 6.

Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors

Ya Zhou¹, Yonggang Zhang², Bo Chen¹, Yong Dong¹, Yimeng Zhang¹, Bin Mao¹, Xu Pan¹, Mowen Lai¹, Yijin Chen¹, Guohui Bian¹, Qiongxiu Zhou¹, Tatsutoshi Nakahata³, Jiaxi Zhou⁴, Min Wu⁵, Feng Ma⁶

Affiliations

¹ Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China.
² Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China. Electronic address: biozyg@163.com.
³ Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
⁴ State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China.
⁵ Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks ND 58203, USA.
⁶ Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China; State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China. Electronic address: mafeng@hotmail.co.jp.

PMID: 31178416
PMCID: PMC6626852
DOI: 10.1016/j.stemcr.2019.05.007

Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors

Ya Zhou et al. Stem Cell Reports. 2019.

. 2019 Jul 9;13(1):31-47.

doi: 10.1016/j.stemcr.2019.05.007. Epub 2019 Jun 6.

Authors

Affiliations

¹ Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China.
² Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China. Electronic address: biozyg@163.com.
³ Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
⁴ State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China.
⁵ Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks ND 58203, USA.
⁶ Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Chengdu 610052, China; State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China. Electronic address: mafeng@hotmail.co.jp.

PMID: 31178416
PMCID: PMC6626852
DOI: 10.1016/j.stemcr.2019.05.007

Abstract

GATA2 is essential for the endothelial-to-hematopoietic transition (EHT) and generation of hematopoietic stem cells (HSCs). It is poorly understood how GATA2 controls the development of human pluripotent stem cell (hPSC)-derived HS-like cells. Here, using human embryonic stem cells (hESCs) in which GATA2 overexpression was induced by doxycycline (Dox), we elucidated the dual functions of GATA2 in definitive hematopoiesis before and after the emergence of CD34⁺CD45⁺CD90⁺CD38^- HS-like cells. Specifically, GATA2 promoted expansion of hemogenic precursors via the EHT and then helped to maintain HS-like cells in a quiescent state by regulating cell cycle. RNA sequencing showed that hPSC-derived HS-like cells were very similar to human fetal liver-derived HSCs. Our findings will help to elucidate the mechanism that controls the early stages of human definitive hematopoiesis and may help to develop a strategy to generate hPSC-derived HSCs.

Keywords: GATA2; cell cycle; definitive hematopoiesis; hESCs; hematopoietic stem cells.

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Figures

**Figure 1**
Generation and Analysis of G2/hESCs (A) Schematic illustration of the PB Tet-on inducible expression system and the strategy used to establish G2/hESCs. (B) Immunofluorescence staining of pluripotency markers in G2/hESCs. Scale bars, 50 μm. (C) Confirmation of homologous recombination in G2/hESCs by PCR. Genomic DNA was isolated from wild-type hESCs, GFP-expressing hESCs, and G2/hESCs. (D) Fluorescence and phase-contrast images of G2/hESCs cultured in mTeSR1 medium containing and lacking Dox. Scale bars, 50 μm. (E) Western blotting and qRT-PCR analysis of GATA2 expression in various concentrations of Dox (0–8 μg/mL)-treated G2/hESCs and AGM-S3 cocultures (G2/hESC cocultures) on day 7. (F) qRT-PCR analysis of *GATA2* expression in various concentrations of Dox (0–8 μg/mL)-treated G2/hESCs and AGM-S3 cocultures (G2/hESC cocultures) on day 7. (G) Flow cytometric analysis of the percentage of GFP⁺ cells among Dox-treated G2/hESCs. (H) qRT-PCR analysis of *GATA2* expression in G2/hESCs treated with Dox for 0–96 h. (I and J) Western blotting (I) and qRT-PCR (J) analysis of GATA2 expression in G2/hESC cocultures on day7 derived from three different G2/hESC clones with various transgene copy number. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 2**
GATA2-OE in hESCs Promotes Definitive Hematopoiesis (A) Experimental scheme to study the stage-wise effect of GATA2 on hematopoietic development. (B) Absolute numbers of 34⁺43⁺, 34⁺43⁺45⁺, and 34⁺45⁺90⁺38⁻ cells in untreated and Dox-treated G2/hESC cocultures on days 2–22. (C) Colony formation by total cells collected from untreated and Dox-treated G2/hESC cocultures on days 14, 20, and 22. (D) Formation of BFU-E and Mix colonies by total cells collected from untreated and Dox-treated G2/hESC cocultures on day 14. (E) Percentages and absolute numbers of 34⁺43⁺45^–, 34⁺43⁺45⁺, and 34⁺45⁺90⁺38^– cells in untreated and Dox-treated G2/hESC cocultures on day 14. (F) Flow cytometric analysis of 34⁺43⁺45⁺ cells in untreated and Dox-treated G2/hESC cocultures on day 14. (G) Immunofluorescence staining of CD34 and CD43 in untreated and Dox-treated G2/hESC cocultures on day 14. Scale bars, 100 μm. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 3**
GATA2-OE Promotes Development of 34⁺43⁺45⁺ HSPCs via Mesodermal Differentiation and the EHT (A) Absolute numbers of A⁺K^–34^–, A⁺K⁺34^–, and A⁻K⁺34^– cells in untreated and Dox-treated G2/hESC cocultures on days 0–10. (B) Heatmap of expression of genes associated with the development of germ layers in untreated and Dox-treated G2/hESC cocultures on day 4. PL, pluripotency genes; PS, primitive streak genes; LPM, lateral plate mesoderm genes; PXM, paraxial mesoderm genes; IM, intermediate mesoderm genes; EN, endodermal genes; EC, ectodermal genes. (C) Experimental scheme. 34⁺K⁺43⁻ cells and 34⁺43⁺45^–/+cells were isolated from coculture at days 6 and 14, respectively, then recultured for an additional 14 days onto AGM-S3-coated plates with and without Dox. (D) Flow cytometric analysis of K⁺34⁺43^– cells sorted from G2/hESC cocultures on day 6. (E) The K⁺34⁺43^– cells sorted from G2/hESC cocultures on day 6 were recultured on AGM-S3 cells with and without Dox. The numbers of K^+/–34⁺43⁺ and 34⁺43⁺45^−/+ cells were determined by flow cytometry. (F) Microscopic analysis and immunofluorescence staining of CD43 in K⁺34⁺43^– cells cocultured with AGM-S3 cells with and without Dox for 7 days. Scale bars, 50 μm. (G) 34⁺43⁺45^– and 34⁺43⁺45⁺ cells were sorted from G2/hESC cocultures on day 14 and recultured on AGM-S3 cells for 14 days with and without Dox. The numbers of 34⁺43⁺45⁺ cells were determined by flow cytometry. (H) qRT-PCR analysis of relative *GATA2* expression in siRNA-transfected cells from day 4 G2/hESC cocultures. (I) The absolute number of 34⁺43⁺ cells in cocultures treated with si-GATA2 (20 nM) or control siRNA for 2 and 4 days. (J) Heatmap of gene expression in 34⁺K⁺43^– cells derived from untreated and Dox-treated G2/hESC cocultures on day 6. M, mesodermal genes; EG, endothelial genes; Hem, hematopoietic genes. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 4**
GATA2-OE Inhibits the Differentiation of CD34⁺CD45⁺ HSPCs (A) Absolute numbers of 34^high45⁺, 34^low45⁺, and 34^–45⁺ cells in untreated and Dox-treated G2/hESC cocultures on days 2–16. (B) Flow cytometric analysis of 34⁺45⁺ cells in untreated and Dox-treated G2/hESCs co-cultures on day 14. (C) Flow cytometric analysis of the subphenotype of 34^high45⁺, 34^low45⁺, and 34⁻45⁺ cells in Dox-untreated G2/hESC cocultures on day 14. (D) Flow cytometric analysis of the subphenotype of 34^high45⁺ cells in untreated and Dox-treated G2/hESC cocultures on day 14. (E) The percentage of 34^high45⁺, 34^low45⁺, and 34^–45⁺ cells in untreated and Dox-treated G2/hESC cocultures on day 14. (F–J) Properties of 34⁺45⁺ cells sorted from G2/hESC cocultures on day 14. (F) Flow cytometric analysis of 34⁺45⁺ cells sorted from untreated and Dox-treated cocultures on day 14. (G) Western blotting and RT-qPCR analysis of GATA2 in 34⁺45⁺ cells sorted from untreated and Dox-treated cocultures on day 14. (H–I) Colony formation by 34⁺45⁺ cells sorted from untreated and Dox-treated cocultures on day 14. (J) β-Globin expression in BFU-E colonies derived from 34⁺45⁺ cells with and without Dox. Scale bars, 20 μm. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 5**
GATA2-OE Promotes the Maintenance and Inhibits the Differentiation of HSPCs by Inducing Cell-Cycle Arrest (A–D) Flow cytometric analysis and quantification of BrdU incorporation and 7-AAD staining to determine the cell-cycle distribution of (A and C) 34⁺45⁺ HSPCs on day 14 of coculture and (B and D) day 3 of suspension culture. (E–G) Gene expression profile of 34⁺K⁺43^– cells (E) on day 6 of coculture, and (F) 34⁺45⁺ cells on 14 of coculture and (G) day 3 of suspension culture. (H) Apoptosis detection of hematopoietic cells from G2/hESC cocultures on day14 with and without Dox through flow cytometric analysis. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 6**
GATA2-OE Selectively Promotes the Maintenance of 34⁺45⁺90⁺38^– HS-like Cells (A) Flow cytometric analysis of the subphenotype of 34⁺45⁺90⁺38^– and 34⁺45⁺90^–38^– cells from Dox untreated G2/hESC cocultures on day 14. (B) Analysis of the maintenance of HS-like 34⁺45⁺90⁺38^– cells derived from H1 hESC cocultures on day 14. The absolute number of 34⁺45⁺90⁺38^– cells on day 14 + 7 is shown. (C) Flow cytometric analysis of 34⁺45⁺90⁺38^– HS-like cells sorted from untreated and Dox-treated G2/hESC cocultures on day 14. (D and E) Percentages and absolute numbers of (D) 34⁺45⁺90⁺38^– and (E) 34⁺45⁺90^–38^– cells derived from sorted 34⁺45⁺90⁺38^– cells treated with and without Dox for 14 days. (F) Expansion of 34⁺45⁺38⁺ cells derived from untreated and Dox-treated G2/hESC cocultures on day 14 in medium containing FBS and cytokines. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 7**
GATA2-OE Maintains 34⁺45⁺90⁺38^– HS-like Cells by Regulating Expression of Cell-Cycle-Related Genes (A) Unsupervised hierarchical clustering of RNA-seq data of 34⁺45⁺38⁺ (38⁺), 34⁺45⁺90⁺38^– (90⁺) and G2-OE 34 ⁺ 45⁺90 ⁺ 38⁻ (G2-OE 90⁺) cells. RNA-seq of 38⁺, 90⁺, and G2-OE 90⁺ cells was performed on day 14 of G2/hESC cocultures. Data are from three representative experiments (GEO: GSE122207). (B) Heatmaps showing mesodermal, endothelial, HSC, and hematopoietic progenitor cell (HPC) genes that were differentially expressed in G2-OE 90⁺, 90⁺, and 38⁺ cells. The scale is log₂ RPKM (reads per kilobase of transcript per million). The data are from three FACS experiments. The heatmaps showed significant differences in gene expression in 90⁺ and 38⁺ cells, p < 0.05. Expression of sample genes previously reported to be regulators of mesodermal cells, endothelial cells, HSCs, and HPCs is shown. Expression profiles were investigated in HSCs and progenitor cells derived from FL, CB, and BM (Cesana et al., 2018). (C) Numbers of differentially expressed genes in G2-OE 90⁺ cells (fold change >2, p < 0.05). (D) qRT-PCR analysis of *GATA2* expression in 90⁺ cells and G2-OE 90⁺ cells. (E and F) (E) Gene ontology categories of biological processes and (F) KEGG signaling pathways that were significantly downregulated in G2-OE 90⁺ cells. (G and H) Gene set enrichment analysis of (G) the stem cell proliferation and differentiation and (H) positive regulation of mitotic cell-cycle genes were repressed in G2-OE 90⁺ cells. Error bars represent mean ± SD of samples from at least three independent experiments. NS, not significant; ^∗p < 0.05, ^∗∗p < 0.01.

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References

1. Böiers C., Carrelha J., Lutteropp M., Luc S., Green J.C., Azzoni E., Woll P.S., Mead A.J., Hultquist A., Swiers G. Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells. Cell Stem Cell. 2013;13:535–548. - PubMed
1. Boisset J.C., van Cappellen W., Andrieu-Soler C., Galjart N., Dzierzak E., Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116–120. - PubMed
1. Boitano A.E., Wang J., Romeo R., Bouchez L.C., Parker A.E., Sutton S.E., Walker J.R., Flaveny C.A., Perdew G.H., Denison M.S. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010;329:1345–1348. - PMC - PubMed
1. Cesana M., Guo M.H., Cacchiarelli D., Wahlster L., Barragan J., Doulatov S., Vo L.T., Salvatori B., Trapnell C., Clement K. A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development. Cell Stem Cell. 2018;22:575–588. - PMC - PubMed
1. Chen B., Teng J., Liu H., Pan X., Zhou Y., Huang S., Lai M., Bian G., Mao B., Sun W. Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm. J. Mol. Cell Biol. 2017;9:262–273. - PubMed

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[1] Böiers C., Carrelha J., Lutteropp M., Luc S., Green J.C., Azzoni E., Woll P.S., Mead A.J., Hultquist A., Swiers G. Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells. Cell Stem Cell. 2013;13:535–548. - PubMed

[2] Böiers C., Carrelha J., Lutteropp M., Luc S., Green J.C., Azzoni E., Woll P.S., Mead A.J., Hultquist A., Swiers G. Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells. Cell Stem Cell. 2013;13:535–548. - PubMed

[3] Boisset J.C., van Cappellen W., Andrieu-Soler C., Galjart N., Dzierzak E., Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116–120. - PubMed

[4] Boisset J.C., van Cappellen W., Andrieu-Soler C., Galjart N., Dzierzak E., Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116–120. - PubMed

[5] Boitano A.E., Wang J., Romeo R., Bouchez L.C., Parker A.E., Sutton S.E., Walker J.R., Flaveny C.A., Perdew G.H., Denison M.S. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010;329:1345–1348. - PMC - PubMed

[6] Boitano A.E., Wang J., Romeo R., Bouchez L.C., Parker A.E., Sutton S.E., Walker J.R., Flaveny C.A., Perdew G.H., Denison M.S. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010;329:1345–1348. - PMC - PubMed

[7] Cesana M., Guo M.H., Cacchiarelli D., Wahlster L., Barragan J., Doulatov S., Vo L.T., Salvatori B., Trapnell C., Clement K. A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development. Cell Stem Cell. 2018;22:575–588. - PMC - PubMed

[8] Cesana M., Guo M.H., Cacchiarelli D., Wahlster L., Barragan J., Doulatov S., Vo L.T., Salvatori B., Trapnell C., Clement K. A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development. Cell Stem Cell. 2018;22:575–588. - PMC - PubMed

[9] Chen B., Teng J., Liu H., Pan X., Zhou Y., Huang S., Lai M., Bian G., Mao B., Sun W. Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm. J. Mol. Cell Biol. 2017;9:262–273. - PubMed

[10] Chen B., Teng J., Liu H., Pan X., Zhou Y., Huang S., Lai M., Bian G., Mao B., Sun W. Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm. J. Mol. Cell Biol. 2017;9:262–273. - PubMed

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Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors

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Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors

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