Limits...
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

H1-GATA2−/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1-GATA2−/− ES cells were co-cultured with OP9 for 9 days, then the CD34+ HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b+ myeloid cells or CD11b+CD14− granulocytes were shown at the left. The right bar charts represent the statistic results of relative generation of CD11b+ cells of indicated test (up) and the generation of CD11b+CD14− cells (down). The data of H1 and H1-GATA2−/− from the OP9 co-culture for CD11b+ cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: *p < 0.05 and **p < 0.01. c FACS analysis of CD86 and CD14 expression in day 12 of OP9/OP9-DL1-mediated myeloid differentiation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4526303&req=5

Fig6: H1-GATA2−/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1-GATA2−/− ES cells were co-cultured with OP9 for 9 days, then the CD34+ HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b+ myeloid cells or CD11b+CD14− granulocytes were shown at the left. The right bar charts represent the statistic results of relative generation of CD11b+ cells of indicated test (up) and the generation of CD11b+CD14− cells (down). The data of H1 and H1-GATA2−/− from the OP9 co-culture for CD11b+ cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: *p < 0.05 and **p < 0.01. c FACS analysis of CD86 and CD14 expression in day 12 of OP9/OP9-DL1-mediated myeloid differentiation

Mentions: It has been shown that Notch signaling could inhibit myelopoiesis on normal stem cells and uncommitted hematopoietic progenitors. This inhibition largely depended on the normal function of GATA2 and could be rescued after GATA2 knockout [45]. However, it remained elusive whether granulocyte commitment could be restored after GATA2 knockout. Therefore, we seeded WT or GATA2−/− hESC-derived CD34+ HPCs directly onto wild-type OP9 or OP9 expressing DL1 (OP9-DL1), the Notch signaling ligand for further myeloid differentiation with addition of cytokines [46] (Fig. 6a). As shown in Fig. 6b, the total number of CD11b+ myeloid cells generated from WT hESCs was significantly reduced upon co-culturing with OP9-DL1 compared with OP9, indicating that Notch signaling indeed inhibits myelopoiesis in human cells. However, the Notch-mediated inhibition of myelopoiesis was not obvious on GATA2−/ hESC-derived CD34+ HPCs (Fig. 6b, upper panel), demonstrating that GATA2 was the critical downstream factor for Notch signaling to inhibit myelopoiesis. Furthermore, we showed that the CD11b+CD14− granulocytes from GATA2−/− hESCs were significantly restored in the presence of Notch signaling (Fig. 6b, lower panel). Additional markers, such as CD86, also confirmed the generation of granulocytes of GATA2−/− hESCs on OP9-DL1 (Fig. 6c). These findings demonstrate that the granulocyte potential of GATA2−/− hESCs could be restored in the presence of Notch signaling.Fig. 6


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)

H1-GATA2−/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1-GATA2−/− ES cells were co-cultured with OP9 for 9 days, then the CD34+ HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b+ myeloid cells or CD11b+CD14− granulocytes were shown at the left. The right bar charts represent the statistic results of relative generation of CD11b+ cells of indicated test (up) and the generation of CD11b+CD14− cells (down). The data of H1 and H1-GATA2−/− from the OP9 co-culture for CD11b+ cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: *p < 0.05 and **p < 0.01. c FACS analysis of CD86 and CD14 expression in day 12 of OP9/OP9-DL1-mediated myeloid differentiation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526303&req=5

Fig6: H1-GATA2−/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1-GATA2−/− ES cells were co-cultured with OP9 for 9 days, then the CD34+ HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b+ myeloid cells or CD11b+CD14− granulocytes were shown at the left. The right bar charts represent the statistic results of relative generation of CD11b+ cells of indicated test (up) and the generation of CD11b+CD14− cells (down). The data of H1 and H1-GATA2−/− from the OP9 co-culture for CD11b+ cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: *p < 0.05 and **p < 0.01. c FACS analysis of CD86 and CD14 expression in day 12 of OP9/OP9-DL1-mediated myeloid differentiation
Mentions: It has been shown that Notch signaling could inhibit myelopoiesis on normal stem cells and uncommitted hematopoietic progenitors. This inhibition largely depended on the normal function of GATA2 and could be rescued after GATA2 knockout [45]. However, it remained elusive whether granulocyte commitment could be restored after GATA2 knockout. Therefore, we seeded WT or GATA2−/− hESC-derived CD34+ HPCs directly onto wild-type OP9 or OP9 expressing DL1 (OP9-DL1), the Notch signaling ligand for further myeloid differentiation with addition of cytokines [46] (Fig. 6a). As shown in Fig. 6b, the total number of CD11b+ myeloid cells generated from WT hESCs was significantly reduced upon co-culturing with OP9-DL1 compared with OP9, indicating that Notch signaling indeed inhibits myelopoiesis in human cells. However, the Notch-mediated inhibition of myelopoiesis was not obvious on GATA2−/ hESC-derived CD34+ HPCs (Fig. 6b, upper panel), demonstrating that GATA2 was the critical downstream factor for Notch signaling to inhibit myelopoiesis. Furthermore, we showed that the CD11b+CD14− granulocytes from GATA2−/− hESCs were significantly restored in the presence of Notch signaling (Fig. 6b, lower panel). Additional markers, such as CD86, also confirmed the generation of granulocytes of GATA2−/− hESCs on OP9-DL1 (Fig. 6c). These findings demonstrate that the granulocyte potential of GATA2−/− hESCs could be restored in the presence of Notch signaling.Fig. 6

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