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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

Forced expression of SPI1 in H1-GATA2−/− restores the generation of granulocytes upon OP9 co-culture. a, b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34+ (left) and CD34+CD43+ (right) HPCs in H1-GATA2−/−. Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34+ cells generation (left) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: *p < 0.05, **p < 0.01 and ***p < 0.001. d Enforced expression of SPI1 in H1-GATA2−/− regenerate G-CFUs. The error bars indicate mean + SEM of three independent experiments. e Morphology of CFU-G regenerated by SPI1 expression; bottom: cytospin of CFU-G. f, g FACS analysis of indicated markers in SPI1 regenerated CFU-M and CFU-G from H1-GATA2−/−
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Fig5: Forced expression of SPI1 in H1-GATA2−/− restores the generation of granulocytes upon OP9 co-culture. a, b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34+ (left) and CD34+CD43+ (right) HPCs in H1-GATA2−/−. Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34+ cells generation (left) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: *p < 0.05, **p < 0.01 and ***p < 0.001. d Enforced expression of SPI1 in H1-GATA2−/− regenerate G-CFUs. The error bars indicate mean + SEM of three independent experiments. e Morphology of CFU-G regenerated by SPI1 expression; bottom: cytospin of CFU-G. f, g FACS analysis of indicated markers in SPI1 regenerated CFU-M and CFU-G from H1-GATA2−/−

Mentions: A mouse embryo with homozygous mutation of SPI1 died at later gestation stage with a complete loss of B cells, T cells, and macrophages, demonstrating the essential role of SPI1 in the proper development of such lineages [41], and SPI1 is one of the four genes used by Sandler et al. to induce hematopoietic MPPs from HUVECs [42]. However, the role of SPI1 in granulocyte development has been conflicting and not yet well documented. Therefore, we sought to examine whether activation of SPI1 could rescue the granulocyte defect of GATA2−/− hESCs. To this end, we constructed an inducible vector expressing SPI1 in a lentiviral backbone [43] and introduced it into GATA2−/− hESCs (Fig. 5a), which could be expressed with Dox addition (Additional file 1: Figure S5). Because SPI1 only started to express at day 6 of differentiation in WT hESCs/OP9 co-culturing (Fig. 4d), we just induced the expression of SPI1 starting at day 6 of differentiation and withdrew the induction during subsequent CFC assay (Fig. 5b). Upon forced induction of SPI1, we showed that CD34+CD43+ HPCs were significantly restored in GATA2−/− hESCs/OP9 co-culture (Fig. 5c). The granulocyte potential cells (G-CFC) did re-appear in GATA2−/− hESCs/OP9 with forced expression of SPI1 (Fig. 5d). However, consistent with the previous finding that SPI1 could antagonize with GATA1 to suppress erythropoiesis [44], we observed a lower level of E-CFCs in GATA2−/− hESCs/OP9 with forced expression of SPI1 (Fig. 5d). Nonetheless, the granulocytes re-established by SPI1 exhibited typical morphology and surface marker expression compared with those from WT hESCs (Fig. 5e–g). In summary, we demonstrated that SPI1 was the target of GATA2 to ensure normal development of HPCs and granulocytes in human ESC-modeled hematopoiesis, thus highlighting a precise and specific role of GATA2 and SPI1 in the regulation of EHT and granulocyte generation.Fig. 5


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)

Forced expression of SPI1 in H1-GATA2−/− restores the generation of granulocytes upon OP9 co-culture. a, b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34+ (left) and CD34+CD43+ (right) HPCs in H1-GATA2−/−. Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34+ cells generation (left) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: *p < 0.05, **p < 0.01 and ***p < 0.001. d Enforced expression of SPI1 in H1-GATA2−/− regenerate G-CFUs. The error bars indicate mean + SEM of three independent experiments. e Morphology of CFU-G regenerated by SPI1 expression; bottom: cytospin of CFU-G. f, g FACS analysis of indicated markers in SPI1 regenerated CFU-M and CFU-G from H1-GATA2−/−
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526303&req=5

Fig5: Forced expression of SPI1 in H1-GATA2−/− restores the generation of granulocytes upon OP9 co-culture. a, b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34+ (left) and CD34+CD43+ (right) HPCs in H1-GATA2−/−. Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34+ cells generation (left) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: *p < 0.05, **p < 0.01 and ***p < 0.001. d Enforced expression of SPI1 in H1-GATA2−/− regenerate G-CFUs. The error bars indicate mean + SEM of three independent experiments. e Morphology of CFU-G regenerated by SPI1 expression; bottom: cytospin of CFU-G. f, g FACS analysis of indicated markers in SPI1 regenerated CFU-M and CFU-G from H1-GATA2−/−
Mentions: A mouse embryo with homozygous mutation of SPI1 died at later gestation stage with a complete loss of B cells, T cells, and macrophages, demonstrating the essential role of SPI1 in the proper development of such lineages [41], and SPI1 is one of the four genes used by Sandler et al. to induce hematopoietic MPPs from HUVECs [42]. However, the role of SPI1 in granulocyte development has been conflicting and not yet well documented. Therefore, we sought to examine whether activation of SPI1 could rescue the granulocyte defect of GATA2−/− hESCs. To this end, we constructed an inducible vector expressing SPI1 in a lentiviral backbone [43] and introduced it into GATA2−/− hESCs (Fig. 5a), which could be expressed with Dox addition (Additional file 1: Figure S5). Because SPI1 only started to express at day 6 of differentiation in WT hESCs/OP9 co-culturing (Fig. 4d), we just induced the expression of SPI1 starting at day 6 of differentiation and withdrew the induction during subsequent CFC assay (Fig. 5b). Upon forced induction of SPI1, we showed that CD34+CD43+ HPCs were significantly restored in GATA2−/− hESCs/OP9 co-culture (Fig. 5c). The granulocyte potential cells (G-CFC) did re-appear in GATA2−/− hESCs/OP9 with forced expression of SPI1 (Fig. 5d). However, consistent with the previous finding that SPI1 could antagonize with GATA1 to suppress erythropoiesis [44], we observed a lower level of E-CFCs in GATA2−/− hESCs/OP9 with forced expression of SPI1 (Fig. 5d). Nonetheless, the granulocytes re-established by SPI1 exhibited typical morphology and surface marker expression compared with those from WT hESCs (Fig. 5e–g). In summary, we demonstrated that SPI1 was the target of GATA2 to ensure normal development of HPCs and granulocytes in human ESC-modeled hematopoiesis, thus highlighting a precise and specific role of GATA2 and SPI1 in the regulation of EHT and granulocyte generation.Fig. 5

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