The role of PDK1 in the transition of endothelial to hematopoietic cells / 中华血液学杂志

Objective: To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods: PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(fl/fl) AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31(+)c-Kit(high) cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results: In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31(+)c-Kit(high) in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion: PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.

The role of PDK1 in the transition of endothelial to hematopoietic cells / 中华血液学杂志

Objective@#To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC.@*Methods@#PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1fl/fl, PDK1fl/+ and Vec-Cre; PDK1fl/fl AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31+c-Kithigh cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells.@*Results@#In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31+c-Kithigh in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster.@*Conclusion@#PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro / 中国病理生理杂志

AIM:To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells(HSC) in vitro.METHODS:The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum(dpc) mouse embryo and 6 week mouse.After identification by Wright's staining and flow cytometry,the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived,cytokine-induced HSCs,and the maintenance and expansion of HSCs were evaluated by detecting CD34+,CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell(BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS:AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype,and had common character of stromal cells.Supported by AGM stromal cells or by BM stromal cells,more primitive progenitor cells HPP-CFC were expanded,but BL-CFC expansion was only detected in AGM-derived stromal cells.In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times,but no significant expansion in CD34+Sca-1+ cells was observed.While in the supporting of AGM stromal cells,both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times,respectively(P