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

Generation of an hESC Line with Inducible GATA2 Overexpression

To explore the function of GATA2 in hematopoietic fate determination, we generated H1-GFP-GATA2 hESCs (G2/hESCs) in which exogenous expression of GATA2 was induced on treatment with Dox using a previously described PB-based Tet-on system (Figure 1A) (Chen et al., 2017). In the absence of Dox, G2/hESCs expressed key stemness-specific markers, such as SOX2, OCT4, and SSEA4 (Figure 1B). PCR analysis verified that the coding sequence (CDS) of GATA2 was integrated into the genome of G2/hESCs (Figure 1C). G2/hESCs could be repeatedly passaged and retained a hESC phenotype without Dox (Figure 1D). Western blotting and qRT-PCR showed increased expression of GATA2 in G2/hESCs and AGM-S3 cocultures (G2/hESC cocultures) with increased concentration of Dox from 0 to 4 μg/mL (Figures 1E and 1F). Treatment with Dox (1μg/mL) increased the percentage of GFP⁺ cells and the mRNA expression of GATA2 in G2/hESCs from 0 to 96 h (Figures 1D, 1G, and 1H). There were three hESC clones used for the experiments with 1, 2, and 4 copies of transgene detected by qPCR, respectively (Figure 1I) (Christodoulou et al., 2016); The three hESC clones showed increased expression of GATA2 mRNA and protein with increased copies of transgene detected by qRT-PCR and western blotting (Figures 1I and 1J). These results demonstrated that G2/hESCs were pluripotent and had a normal phenotype, that the CDS of GATA2 was integrated into the genome of these cells, and that treatment with Dox-induced GATA2-OE from 0 to 4 μg/mL.

GATA2 Overexpression in hESCs Promotes Definitive Hematopoiesis

We firstly elucidated the hematopoietic cells in the H1 hESCs and AGM-S3 cocultures (Figure 2A). KDR⁺ cells, which are mesodermal precursors of hematopoietic and endothelial lineages, gradually express CD34 during human embryonic development (Cortés et al., 1999). In H1 hESCs and AGM-S3 coculture, the KDR⁺CD34⁻CD43^– (K⁺34^–43^–) cells mainly expressed mesodermal proteins and genes compared with the KDR⁺CD34⁺CD43^– (K⁺34⁺43^–) cells (Figures S1A and S1B). CD43 was previously used as a marker of hematopoietic progenitors in cocultures of hPSCs and OP9 cells (Vodyanik et al., 2006). CD34⁺CD43⁺ (34⁺43⁺) cells expressed markers of hematopoietic progenitors and upregulated hematopoietic genes compared with CD34⁺CD43^– (34⁺43^–) cells (Figures S1C and S1D). In cocultures of H1 hESCs and AGM-S3 cells, the former cells differentiate into (APLNR⁺KDR^−/+CD34^–, A⁺K^−/+34^–) mesodermal progenitors (CD34⁺CD43^–, 34⁺43^–) endothelial cells, and (CD34⁺CD43⁺CD45^−/+, 34⁺43⁺45^−/+) hematopoietic progenitors in a stepwise manner (Figures S1E and S1F), as occurs in other hematopoietic systems (Slukvin, 2013). GATA2 expression was upregulated during hematopoiesis (Figure S1G). GATA2 expression in 34⁺43⁺ and 34⁺45⁺ hematopoietic cells derived from H1 hESCs was comparable with that in human CB-derived CD34⁺ cells (Figure S1H). These data demonstrate that the AGM-S3 stromal cell line efficiently induced hematopoietic differentiation of hESCs and simulated the early stages of hematopoiesis (Figure S1I) (Mao et al., 2016, Wang et al., 2018). Expression of GATA2 increased during the development of 34⁺43⁺45^−/+ hematopoietic cells from hESCs.

We further evaluated the role of GATA2 in hematopoiesis by three different culture systems. The hematopoietic efficiency in the promotion of 34⁺45⁺90⁺38^– cells was improved in AGM-S3 coculture than in OP9 and stromal cell-free culture (Figure S1J). Transition of 34⁺K⁺43^– cells into 34⁺43⁺ cells was predominantly enhanced in AGM-S3 cocultures (Figure S1K). A comparable increase of GATA2 expression was paralleled to the increase of Dox dose from 0 to 4 μg/mL, accompanying by an enhanced promotion of 34⁺43⁺ cells in G2/hESC cocultures (Figure S2A). We found 1 μg/mL Dox was enough to trigger GATA2 overexpression, and this dose was used in further experiments. We found that all three hESC lines with different transgene copy numbers demonstrated similar efficacy in promoting hematopoiesis; however, there was a slight difference in enhancement (Figures S2B and S2C). On the other hand, the vector control hESCs showed no difference in differentiation with and without Dox (Figure S2C). To identify barriers to the generation of hematopoietic cells from GATA2-overexpressing hESCs, we analyzed the proportion of hematopoietic cells among G2/hESC cocultures at days 6, 10, and 14, when Dox treatment was initiated at different time points (Figure S2D). Addition of Dox from day 2 until the day of collection promoted the development of K⁺34^–43^–, 34⁺K⁺43^–, 34⁺43⁺, and 34⁺43⁺45⁺ cells (Figure S2D).

We next evaluated the effect GATA2-OE on hematopoiesis from day 2 to 22. The increased level of GATA2 expression mainly relied on exogenous GATA2 expression both at the various times of differentiation and in the 34⁺43⁺-sorted cells with Dox (Figures S2E and S2F). The total cell number was not markedly affected (data not shown). GATA2-OE prolonged the growth of 34⁺43⁺45^−/+ and 34⁺45⁺90⁺38^– cells in cocultures over 22 days (Figures 2B and S2G). Moreover, GATA2-OE increased the generation of 34⁺45⁺90⁺38^– HS-like cells by more than 4.8-fold in cocultures. Total cells collected from Dox-treated cocultures on days 14, 20, and 22 formed an increased number of colonies, which coincided with the increase in hematopoietic cells; however, more colonies were observed when Dox was removed during the colony-formation assay (Figure 2C). BFU-E and Mix colonies recovered when Dox was removed in cloned cultures (Figure 2D).

Fluorescence-activated cell sorting (FACS) analysis demonstrated that the levels of 34⁺43⁺45^– cells (pre-HSPCs), 34⁺43⁺45⁺ cells (HSPCs), and 34⁺45⁺90⁺38^– cells (HS-like cells) were increased in Dox-treated cocultures at day 14 (Figures 2E and 2F). Immunofluorescence staining of CD34 and CD43 showed that dense networks of blood vessels and many hematopoietic cells formed in Dox-treated cocultures on day 14 (Figure 2G).These data demonstrate that GATA2-OE promotes the generation of hematopoietic cells and prolongs the colony-forming unit (CFU) potential of cocultured cells.

GATA2 Overexpression Promotes Definitive Hematopoiesis via Differentiation of hESC-Derived Mesodermal KDR⁺CD34^– (K⁺34^–) Cells and the EHT

The percentage and number of early mesodermal cells with an A⁺K^–34^– phenotype did not markedly differ. However, the number of K⁺34^– cells, including definitive mesodermal cells with an A⁺K⁺34^– phenotype, was higher in Dox-treated cocultures than in untreated cocultures (Figures 3A and S3A). Expression of primitive streak (PS) and LPM genes increased on Dox treatment (Figure 3B). Thus, GATA2-OE promoted the generation of hESC-derived mesodermal K⁺34^– cells, including A⁺K⁺34^– and A⁻K⁺34^– cells.

The increase in 34⁺43⁺ cells in cocultures on GATA2-OE may be because of the promotion of EHT and after EHT. To determine whether GATA2-OE promotes EHT, 34⁺K⁺43^– cells were sorted from cocultures on day 6 and recultured on AGM-S3 cells in the presence or absence of Dox (Figures 3C and 3D). 34⁺K⁺43^– cells gave rise to more 34⁺43⁺ cells, including 34⁺43⁺K^+/– and 34⁺43⁺45^−/+ cells, in the presence of Dox (Figures 3E and S3B). Immunofluorescence staining revealed that 34⁺K⁺43^– cells gave rise to more CD43⁺ cells on GATA2-OE for 7 days (Figure 3F). To investigate the effect of GATA2-OE on expansion of 34⁺43⁺45^– and 34⁺43⁺45⁺ cells after the EHT, both cell populations were sorted from cocultures on day 14 and recultured on AGM-S3 cells (Figure 3C). GATA2-OE did not markedly affect the expansion of 34⁺43⁺45⁺ cells (Figure 3G).

To further elucidate the effect of GATA2-OE on EHT, cocultures on day 4, which lacked emerging CD43⁺ cells, were transfected with GATA2-targeting (si-GATA2: GUCCAAGAAGAGCAAGAAATT) or control siRNA (si-N:UUCUCCGAACGUGUCACGUTT) (Figure S3C). Transfection of 20 nM si-GATA2 significantly decreased endogenous GATA2 expression by more than 42.3% (Figure 3H). Downregulation of GATA2 decreased the emergence of 34⁺43⁺ cells (Figures 3I and S3C).

Relative gene expression profiles differed between 34⁺K⁺43^– cells sorted from untreated and Dox-treated cocultures on day 6. Hematopoietic genes were upregulated in 34⁺K⁺43^– cells on GATA2-OE (Figure 3J).

These data suggest that GATA2-OE promotes the generation of 34⁺43⁺ cells through the EHT in cocultures of hESCs and AGM-S3 cells, and that GATA2 is essential for the EHT during hematopoiesis.

GATA2 Overexpression Inhibits the Differentiation of 34⁺45⁺ HSPCs

GATA2-OE increased production of CD34^highCD45⁺ (34^high45⁺) cells and inhibited production of 34^low45⁺ and 34^–45⁺ cells until day 16 of coculture (Figures 4A and S4A). Flow cytometric analysis demonstrated that the percentages of cells positive for CD144, CD201, KDR, CD90, and CD105 were higher among 34^high45⁺ cells than among 34^low45⁺ and 34^–45⁺ cells, whereas the percentages of cells positive for lineage-specific markers such as CD38, CD13, CD16, and CD14 were lower (Figures 4B and 4C). GATA2-OE increased production of 34^high45⁺ cells and inhibited their differentiation into 34^low45⁺ and 34^–45⁺ cells (Figures 4A, 4B, and S4A). GATA2-OE did not obviously change the immunophenotype of 34^high45⁺ cells (Figure 4D). When Dox was removed from day 10, differentiation of 34^low45⁺ and 34^–45⁺ cells was restored on day 14 (Figure 4E). Thus, GATA2-OE promoted the generation of 34^high45⁺ cells and inhibited their differentiation.

To further elucidate the effect of GATA2-OE on differentiation of 34⁺45⁺ cells, these cells were sorted from cocultures on day 14 (Figure 4F). Western blotting and qRT-PCR analysis demonstrated that GATA2 expression was upregulated in 34⁺45⁺ cells sorted from Dox-treated cocultures (Figure 4G). Removal of Dox increased the numbers of BFU-E, Mix, GM, and M colonies, while GATA2-OE increased the number of G colonies (Figures 4H and 4I). More CD71⁺GPA⁺ erythroid cells were detected by FACS analysis when Dox was removed during the colony-formation assay (Figure S4B). BFU-E formed from GATA2-overexpressing cocultured cells generated erythroid cells that predominantly expressed adult β-globin (Figure 4J). These data demonstrate that GATA2-OE inhibits the differentiation of hematopoietic cells.

To further investigate the effect of GATA2-OE on the differentiation of 34⁺45⁺ cells, we recultured 34⁺45⁺ cells on day 14 in IMDM containing 10% fetal bovine serum (FBS) and seven cytokines. More cells with a 34^–45⁺ phenotype were produced in the presence of Dox (Figures S4C and S4D). Upon suspension of cultures to induce differentiation, Dox treatment decreased the percentage of CD163⁺CD14⁺ cells (macrophages), but increased the percentages of c-KIT⁺CD45⁺ (mast cells) and Siglec-8⁺CD88⁺ cells (eosinophils) (Figures S4C–S4E).

Thus, GATA2-OE increases production of 34⁺43⁺45⁺ cells, but prevents their differentiation in cocultures and the colony-formation assay. By contrast, GATA2-OE increases the granulocytic and mast cell potential of 34⁺45⁺ cells in suspension culture and the colony-formation assay.

GATA2 Overexpression Maintains HSPCs by Inducing Cell-Cycle Arrest

5′-Bromo-2′-deoxyuridine (BrdU) incorporation analysis suggestd that 34⁺45^– cells differentiated into 34^high45⁺ cells, which was accompanied by a decreased percentage of cells in G0/G1 phase and an increased percentage of cells in S stage (Figure 5A). The cell cycle of 34⁺45^– cells did not obviously differ between untreated and Dox-treated cocultures at day 14 (Figure 5A). However, the cell cycle of 34⁺45⁺ cells differed between untreated and Dox-treated samples at day 14 of coculture and day 3 of suspension culture (Figures 5A–5D). The decreased generation of hematopoietic cells from 34⁺45⁺ cells on Dox treatment was due to induction of quiescence, while removal of Dox promoted progression of 34⁺45⁺ cells into S and G2/M phase (Figures 5B and 5D).

Despite confirming GATA2-OE in 34⁺K⁺43^– and 34⁺45⁺ cells treated with Dox, qRT-PCR analysis demonstrated that the expression of cell-cycle-related genes was markedly altered in the latter cells (Figures 5E–5G). Expression of genes encoding cell-cycle activators, such as CCND3, MCM5, and CEBPA, was decreased in GATA2-OE 34⁺45⁺ cells. In addition, AHRR and CYB1P1 expression were reduced in GATA2-OE 34⁺45⁺ cells (Figures 5F and 5G). On removal of Dox from suspension cultures, expression of GATA2 decreased in 34⁺45⁺ cells, while that of CD38 and CYB1P1 increased (Figure 5G). Enforced GATA-2 expression showed no increase in apoptosis of coculture cells. GATA2-OE inhibited apoptosis in CD34^highCD45⁺ cells on coculture day14 (Figure 5H). These data suggest that GATA2-OE prevents the expansion and differentiation of 34⁺45⁺ cells isolated from cocultures on day 14 by inducing quiescence and inhibiting apoptosis.

GATA2 Overexpression Selectively Promotes the Maintenance of 34⁺45⁺90⁺38^– HS-like Cells

CD90⁺ cells were enriched among 34^high45⁺ cells (Figure 4C). The percentages of cells positive for endothelial markers, such as CD201, KDR, CD144, and CD61, were higher among 34⁺45⁺90⁺38^– HS-like cells than among 34⁺45⁺90^–38^– cells (Figure 6A). A two-step differentiation protocol was applied to develop HS-like cells from H1 hESC cocultures on day14 (Figure 6B). Medium was replaced by α-minimum essential medium containing 20% FBS, thrombopoietin, stem cell factor, and FMS-like tyrosine kinase 3 ligand (FL), which supports the maintenance of multipotent human HS-like cells (Figure 6B). To investigate the functional properties of GATA2-OE HS-like cells, 34⁺45⁺90⁺38^– HS-like cells were sorted and replated onto AGM-S3 cells (Figure 6C). Although 34⁺45⁺90⁺38^– cells were detected in the presence of Dox, the percentage of these cells rapidly decreased when Dox was removed (Figures 6D, 6E, and S5A). To further elucidate the effect of GATA2-OE on differentiation of hematopoietic cells, cocultured cells on day 14 were cultured in differentiation medium and their ability to differentiate into CD34⁺CD45⁺CD38⁺ (34⁺45⁺38⁺) progenitors was investigated (Figures 6F and S5B). The percentage of 34⁺45⁺38⁺ cells by FACS and relative expression of CD38 in 34⁺45⁺ cells by qRT-PCR increased when Dox was removed (Figures 5G, 6F, and S5B).

GATA2-OE promoted the development and maintenance of 34⁺45⁺90⁺38^– HS-like cells and inhibited their differentiation into 34⁺45⁺38⁺ hematopoietic cells. These data show that GATA2 selectively promotes the development and maintenance of HS-like cells when expressed in the correct cellular context.

GATA2 Overexpression Maintains 34⁺45⁺90⁺38^– HS-like Cells by Regulating Expression of Cell-Cycle-Related Genes

To dissect the transcriptional features of 34⁺45⁺90⁺38^– HS-like cells, we isolated 34⁺45⁺90⁺38^– and 34⁺45⁺38⁺ cells from Dox-treated and -untreated G2/hESC cocultures and performed RNA-seq. Thereafter, we compared the transcriptional profiles of these cells with those of HSCs (CD34⁺CD38⁻CD90⁺CD45RA⁻) and progenitors (CD34⁺CD38⁺) derived from FL, CB, and bone marrow (BM) (Cesana et al., 2018).

Unsupervised hierarchical clustering of GATA2-OE 34 ⁺ 45⁺90 ⁺ 38⁻ cells (G2-OE 90⁺), 34 ⁺ 45⁺90 ⁺ 38⁻ cells (90⁺) and CD34⁺CD45⁺CD38⁺ cells (38⁺) showed that G2-OE 90⁺ and 90⁺ were positioned next to each other (Figure 7A).

Gene expression patterns of hematopoietic markers, cell surface markers and HSC regulators were very similar in 34 ⁺ 45⁺90 ⁺ 38⁻ cells, 34 ⁺ 45⁺38⁺ cells and that cells derived from FL, CB, and BM (Figures 7B and S6A). Expression of mesodermal and endothelial genes was similar in 90⁺ cells and FL-derived HSCs (Figure 7B). Taken together, the profiling data suggest that 90⁺ cells generated in vitro are transcriptionally very similar to HSCs and are most similar to human FL-derived HSCs.

HSC-specific gene expression was similar in untreated and Dox-treated hESC-derived HS-like cells, suggesting that GATA2-OE did not affect the HSC properties of these cells (Figure 7B). We next assessed how GATA2-OE affects the proliferation and differentiation of 90⁺ cells. In total, 5,058 genes (3,744 downregulated and 1,314 upregulated) were significantly differentially expressed between G2-OE 90⁺ and 90⁺ cells (2.85-fold; Figures 7C and 7D). Repressed genes in G2-OE 90⁺ cells were mostly enriched in gene ontology categories related to cell adhesion, motility, migration, proliferation, and differentiation (Figure 7E). Kyoto Encyclopedia of Genes and Genome (KEGG) analysis suggestd that several pathways important for regulation of HSCs were downregulated in G2-OE 90⁺ cells (Figure 7F). Gene set enrichment analysis (GSEA) showed that the stem cell proliferation and differentiation genes were repressed in G2-OE 90⁺ cells (Figures 7G and S6B). GSEA showed that the positive regulation of mitotic cell-cycle and apoptosis genes were repressed in G2-OE 90⁺ cells (Figures 7H and S6C). Expression of genes related to the AHR signaling pathway, which was previously reported to be inhibited by SR1 (Rentas et al., 2016), was also inhibited in G2-OE 90⁺ cells (Figure S6D).

Maintenance of hESCs

H1 hESCs were provided by Professor Tao Cheng and maintained on Matrigel-coated plates in mTeSR1 medium (STEMCELL Technologies). The cells were dissociated into clumps using ReLeSR (STEMCELL Technologies) every 4–5 days.

Establishment of G2/hESCs

H1 hESCs were seeded into a 24-well plate with 70%–90% confluent at the time of transfection. Plasmid DNA-lipid complexes (containing 0.5 μg of the PB-Tet-on-GFP-T2A-hGATA2 vector, and 0.5 μg of PB200PA vector containing catalytic component) were added to the cells per well and incubated for 2–4 days according to the manufacturer’s description of lipofectamine 3000 (Invitrogen). Transfected cells were selected by culture in medium containing 1 μg/mL puromycin (Sigma). For single-cell culture, cells were dissociated into single cells with Accutase (Gibco) and maintained in mTeSR1 medium supplemented with Y27632 (Cayman). Puromycin-resistant clones were picked, and successful targeting was confirmed by western blotting, qRT-PCR (F1:5′-CAGCAAGGCTCGTTCCTGTTCA-3′, R1:5′-ATGAGTGGTCGGTTCTGCCCAT-3′) and PCR (F2: 5′GAGGAAGTCTTCTAACATGCGGT-3′, R2: 5′CCCTTCGTCTGACGTGGCAG-3′). To minimize variation owing to long-term selection, multiple stable cell lines were used and all experiments were repeated at least three times. The study was approved by the Ethics Committee of the Institute of Blood Transfusion, CAMS & PUMC.

Coculture of hESCs and AGM-S3 Cells

Approximately 50–60 clumps of hESCs, each of which contained 3–4 × 10² cells, were seeded onto 2–3 ×10⁵ irradiated AGM-S3 cells in each well of a 6-well plate and cultured in hPSC maintenance medium. hESCs were differentiated into HSPCs as described previously (Mao et al., 2016). The medium was changed every day.

Colony Assay

Hematopoietic potential of cells was assessed by culture on methylcellulose (H4320, STEMCELL Technologies) supplemented with 1% penicillin/streptomycin (Invitrogen) and cytokines, which were described previously (Ma et al., 2008). BFU-E, CFU-Mix, CFU-GM, CFU-G, and CFU-M colonies were assessed after 10–16 days.

Flow Cytometry and Cell Sorting

Cocultured cells were dissociated with 0.25% trypsin-EDTA solution (Invitrogen) and filtered through a 70-μm nylon mesh to obtain a single-cell suspension. Flow cytometry was performed using a FACSCanto II system (BD Biosciences), and data were analyzed using FlowJo software (v.10.0.8.). Cell sorting was performed using a MoFlo Astrios High Speed Cell Sorter (Beckman Coulter). The antibodies used are presented in Table S1.

Cell-Cycle Analysis

Cells were treated with BrdU for 6 h, stained for surface antigens, and processed using an APC-BrdU Flow Kit (BD) according to the manufacturer's instructions.

Apoptosis Assays

Cells were detected by flow cytometry of cells stained according to the manufacturer's instructions with annexin V–APC and 7-AAD (BioLegend).

Statistical Analysis

All data are presented as the mean ± SD. Statistical analyses were performed using the Student's t test. p < 0.05 was considered significant.