Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models;therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung d...Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models;therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro?wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.展开更多
The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model ...The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study,using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover,loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs,suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.展开更多
Background: The HOX genes are master regulators of embryogenesis that are also involved inhematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles inhematopoiesis and leukemogenesis.M...Background: The HOX genes are master regulators of embryogenesis that are also involved inhematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles inhematopoiesis and leukemogenesis.Methods: We established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normalpluripotency and potential for hematopoiesis, which could be used to analyze gene function with highaccuracy. HOXA9/hESCs co-cultured with aorta–gonad–mesonephros-derived stromal cells (AGM-S3) wereinduced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started andthen subjected to flow cytometry.Results: Induction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increasedthe production of CD34+ cells and derived populations. The potential for myelogenesis was significantlyelevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion ofS phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κBsignaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could becounteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX.Conclusions: Overexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promotedhematopoiesis and the production of myeloid progenitors while reduced the production of erythroidprogenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoieticlineages.展开更多
目前在体外研究中,主要通过分离胚体和骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导这2种途径从人胚胎干细胞(hESC)分化获取滋养层细胞(TB)。胚体途径可基于细胞的黏附性和培养基中β-hCG的含量从胚体中分离获得TB,进而在...目前在体外研究中,主要通过分离胚体和骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导这2种途径从人胚胎干细胞(hESC)分化获取滋养层细胞(TB)。胚体途径可基于细胞的黏附性和培养基中β-hCG的含量从胚体中分离获得TB,进而在三维培养体系中可检测细胞的侵袭性及细胞与基质的相互作用。BMP4途径通过去除外源性成纤维细胞生长因子及提高氧含量均可促进BMP4诱导TB的分化。虽然hESC分化TB模型目前还存在一些争议,但是相关研究可为探讨人类胚胎的植入及胎盘的形成提供重要的理论依据。展开更多
Human embryonic stem cells(hESCs)are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages,includ-ing neural stem(NS)cells.Diverse regulatory signalin...Human embryonic stem cells(hESCs)are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages,includ-ing neural stem(NS)cells.Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered,and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro.However,the limitations of hESCs resource along with the religious and ethical concerns impede the pro-gress of ESCs application.Therefore,the induced pluri-potent stem cells(iPSCs)via somatic cell reprogramming have opened up another new territory for regenerative medicine.iPSCs now can be derived from a number of lin-eages of cells,and are able to differentiate into certain cell types,including neurons.Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening.Furthermore,with the development of somatic direct reprogramming or lineage reprogramming technique,a more effective approach for regenerative medicine could become a complement for iPSCs.展开更多
文摘Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models;therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro?wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.
基金supported by National Basic Research Program of China (2015CB964902 to Jiaxi Zhou and SQ2016ZY05002105 to Hongtao Wang)CAMS Initiative for Innovative Medicine (2016-I2M-1-018, 2016-I2M-3-002)+2 种基金National Natural Science Foundation of China (81530008, 31671541 to Jiaxi Zhou, 31500949 to Hongtao Wang)Tianjin Natural Science Foundation (16JCZDJC33100 to Jiaxi Zhou)3332015128 supported by PUMC Youth Fund and Fundamental Research Funds for the Central Universities to Dr. Hongtao Wang
文摘The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study,using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover,loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs,suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.
基金This work was supported by awards from the CAMS Initiatives for Innovative Medicine(2016-I2M-1-018 to F.Ma and 2017-I2M-3-021 to J.X.Liu)Sichuan Provincial Science and Technology Department Key R&D projects(020YFSY0023 to B.Chen)the Chengdu Science and Technology Project-Technology Innovation R&D(2018-YF05-01341-SN to B.Chen).
文摘Background: The HOX genes are master regulators of embryogenesis that are also involved inhematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles inhematopoiesis and leukemogenesis.Methods: We established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normalpluripotency and potential for hematopoiesis, which could be used to analyze gene function with highaccuracy. HOXA9/hESCs co-cultured with aorta–gonad–mesonephros-derived stromal cells (AGM-S3) wereinduced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started andthen subjected to flow cytometry.Results: Induction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increasedthe production of CD34+ cells and derived populations. The potential for myelogenesis was significantlyelevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion ofS phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κBsignaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could becounteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX.Conclusions: Overexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promotedhematopoiesis and the production of myeloid progenitors while reduced the production of erythroidprogenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoieticlineages.
文摘目前在体外研究中,主要通过分离胚体和骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导这2种途径从人胚胎干细胞(hESC)分化获取滋养层细胞(TB)。胚体途径可基于细胞的黏附性和培养基中β-hCG的含量从胚体中分离获得TB,进而在三维培养体系中可检测细胞的侵袭性及细胞与基质的相互作用。BMP4途径通过去除外源性成纤维细胞生长因子及提高氧含量均可促进BMP4诱导TB的分化。虽然hESC分化TB模型目前还存在一些争议,但是相关研究可为探讨人类胚胎的植入及胎盘的形成提供重要的理论依据。
基金the National Basic Research Program 973 program(No.2012CB966800)the Thousand Youth Talents Program and the Pioneer Programs of Chinese Academy of Sciences。
文摘Human embryonic stem cells(hESCs)are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages,includ-ing neural stem(NS)cells.Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered,and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro.However,the limitations of hESCs resource along with the religious and ethical concerns impede the pro-gress of ESCs application.Therefore,the induced pluri-potent stem cells(iPSCs)via somatic cell reprogramming have opened up another new territory for regenerative medicine.iPSCs now can be derived from a number of lin-eages of cells,and are able to differentiate into certain cell types,including neurons.Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening.Furthermore,with the development of somatic direct reprogramming or lineage reprogramming technique,a more effective approach for regenerative medicine could become a complement for iPSCs.
