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GATA2(-/-) human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment.

Huang K, Du J, Ma N, Liu J, Wu P, Dong X, Meng M, Wang W, Chen X, Shi X, Chen Q, Yang Z, Chen S, Zhang J, Li Y, Li W, Zheng Y, Cai J, Li P, Sun X, Wang J, Pei D, Pan G - Cell Regen (Lond) (2015)

Bottom Line: Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT).Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes.Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China ; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.

Results: To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

Conclusion: Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.

No MeSH data available.


Related in: MedlinePlus

Hematopoietic differentiation of the H1-GATA2−/− ES cell line. a CFUs of H1 or H1-GATA2−/− derived CD34+ cells. H1 or H1-GATA2−/− cells were co-cultured with OP9 cells for 9 days. The CD34+ HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1-GATA2−/− cells co-cultured with OP9 for the indicated time was analyzed by FACS (left and middle panels). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: ***p < 0.001. e Time course analysis of the expression of indicated genes of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The gene expression was analyzed by qRT-PCR by using GAPDH as an internal reference. f HPCs with CD34+CD31+CD43+ were developed from CD34+CD31+CD43− HEs. CD34+CD31+CD43− populations were sorted out at day 8 of differentiation and replated on OP9 for one additional day and analyzed by FACS. g FACS analysis of CD114 and KDR on HEs from H1 or H1-GATA2−/−. h H1 or H1-GATA2−/− derived HEs produced endothelial cells. Left: Morphology of endothelial cells; middle: immunostaining of CD31 on endothelial cells; right: capillary structure formation by endothelial cells
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Fig2: Hematopoietic differentiation of the H1-GATA2−/− ES cell line. a CFUs of H1 or H1-GATA2−/− derived CD34+ cells. H1 or H1-GATA2−/− cells were co-cultured with OP9 cells for 9 days. The CD34+ HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1-GATA2−/− cells co-cultured with OP9 for the indicated time was analyzed by FACS (left and middle panels). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: ***p < 0.001. e Time course analysis of the expression of indicated genes of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The gene expression was analyzed by qRT-PCR by using GAPDH as an internal reference. f HPCs with CD34+CD31+CD43+ were developed from CD34+CD31+CD43− HEs. CD34+CD31+CD43− populations were sorted out at day 8 of differentiation and replated on OP9 for one additional day and analyzed by FACS. g FACS analysis of CD114 and KDR on HEs from H1 or H1-GATA2−/−. h H1 or H1-GATA2−/− derived HEs produced endothelial cells. Left: Morphology of endothelial cells; middle: immunostaining of CD31 on endothelial cells; right: capillary structure formation by endothelial cells

Mentions: Since GATA2 has been known to be a master regulator for hematopoiesis, we sought to analyze the hematopoietic potential of GATA2−/− hESCs. In a stromal-free defined condition that could drive blood differentiation, we showed that in contrast to WT hESCs, GATA2−/− hESCs generated a few HPCs (CD34+CD43+) and blood colony-forming units (CFUs) (Additional file 1: Figure S3). However, when co-culturing with OP9 stromal cells, GATA2−/− hESCs exhibited CFC potential (Fig. 2a). This data is consistent with previous findings in vivo in the mouse model that mouse ES cells lacking Gata2 could generate certain blood lineages, such as erythrocytes [10]. We confirmed that the GATA2−/− hESCs failed to express full-length GATA2 mRNAs during the whole process of blood differentiation driven by OP9 co-culture (Fig. 2e and Additional file 1: Figure S1D). Then, we attempted to analyze this process in detail. Upon OP9 co-culture, the HPCs with CFU potential were believed to develop through the EHT process from HEs, the endothelial cells with hematopoietic potential [21, 22, 29–35]. Through further analyzing the surface markers at different differentiation stages during OP9 co-culture, we showed that GATA2−/− hESCs exhibited little difference in the generation of CD34+CD31+ HEs compared with WT hESCs, but significant reduction in the production of CD34+CD43+ HPCs (Fig. 2b–d). These data indicated that GATA2 was critical for EHT to generate HPCs but not essential for HE determination. Consistently, the transcription factors (TFs) critical for HE determination such as RUNX1 and SCL/TAL1 [30] were successfully activated in GATA2−/− hESCs, albeit a little lower than in WT hESCs (Fig. 2e, Additional file 1: Figure S4).Fig. 2


GATA2(-/-) human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment.

Huang K, Du J, Ma N, Liu J, Wu P, Dong X, Meng M, Wang W, Chen X, Shi X, Chen Q, Yang Z, Chen S, Zhang J, Li Y, Li W, Zheng Y, Cai J, Li P, Sun X, Wang J, Pei D, Pan G - Cell Regen (Lond) (2015)

Hematopoietic differentiation of the H1-GATA2−/− ES cell line. a CFUs of H1 or H1-GATA2−/− derived CD34+ cells. H1 or H1-GATA2−/− cells were co-cultured with OP9 cells for 9 days. The CD34+ HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1-GATA2−/− cells co-cultured with OP9 for the indicated time was analyzed by FACS (left and middle panels). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: ***p < 0.001. e Time course analysis of the expression of indicated genes of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The gene expression was analyzed by qRT-PCR by using GAPDH as an internal reference. f HPCs with CD34+CD31+CD43+ were developed from CD34+CD31+CD43− HEs. CD34+CD31+CD43− populations were sorted out at day 8 of differentiation and replated on OP9 for one additional day and analyzed by FACS. g FACS analysis of CD114 and KDR on HEs from H1 or H1-GATA2−/−. h H1 or H1-GATA2−/− derived HEs produced endothelial cells. Left: Morphology of endothelial cells; middle: immunostaining of CD31 on endothelial cells; right: capillary structure formation by endothelial cells
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Related In: Results  -  Collection

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Fig2: Hematopoietic differentiation of the H1-GATA2−/− ES cell line. a CFUs of H1 or H1-GATA2−/− derived CD34+ cells. H1 or H1-GATA2−/− cells were co-cultured with OP9 cells for 9 days. The CD34+ HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1-GATA2−/− cells co-cultured with OP9 for the indicated time was analyzed by FACS (left and middle panels). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: ***p < 0.001. e Time course analysis of the expression of indicated genes of H1 and H1-GATA2−/− cells upon co-culturing with OP9. The gene expression was analyzed by qRT-PCR by using GAPDH as an internal reference. f HPCs with CD34+CD31+CD43+ were developed from CD34+CD31+CD43− HEs. CD34+CD31+CD43− populations were sorted out at day 8 of differentiation and replated on OP9 for one additional day and analyzed by FACS. g FACS analysis of CD114 and KDR on HEs from H1 or H1-GATA2−/−. h H1 or H1-GATA2−/− derived HEs produced endothelial cells. Left: Morphology of endothelial cells; middle: immunostaining of CD31 on endothelial cells; right: capillary structure formation by endothelial cells
Mentions: Since GATA2 has been known to be a master regulator for hematopoiesis, we sought to analyze the hematopoietic potential of GATA2−/− hESCs. In a stromal-free defined condition that could drive blood differentiation, we showed that in contrast to WT hESCs, GATA2−/− hESCs generated a few HPCs (CD34+CD43+) and blood colony-forming units (CFUs) (Additional file 1: Figure S3). However, when co-culturing with OP9 stromal cells, GATA2−/− hESCs exhibited CFC potential (Fig. 2a). This data is consistent with previous findings in vivo in the mouse model that mouse ES cells lacking Gata2 could generate certain blood lineages, such as erythrocytes [10]. We confirmed that the GATA2−/− hESCs failed to express full-length GATA2 mRNAs during the whole process of blood differentiation driven by OP9 co-culture (Fig. 2e and Additional file 1: Figure S1D). Then, we attempted to analyze this process in detail. Upon OP9 co-culture, the HPCs with CFU potential were believed to develop through the EHT process from HEs, the endothelial cells with hematopoietic potential [21, 22, 29–35]. Through further analyzing the surface markers at different differentiation stages during OP9 co-culture, we showed that GATA2−/− hESCs exhibited little difference in the generation of CD34+CD31+ HEs compared with WT hESCs, but significant reduction in the production of CD34+CD43+ HPCs (Fig. 2b–d). These data indicated that GATA2 was critical for EHT to generate HPCs but not essential for HE determination. Consistently, the transcription factors (TFs) critical for HE determination such as RUNX1 and SCL/TAL1 [30] were successfully activated in GATA2−/− hESCs, albeit a little lower than in WT hESCs (Fig. 2e, Additional file 1: Figure S4).Fig. 2

Bottom Line: Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT).Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes.Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China ; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.

Results: To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

Conclusion: Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.

No MeSH data available.


Related in: MedlinePlus