RUNXI is absolutely required for definitive hematopoiesis, but the function of RUNXlb/c, two isoforms of human RUNX1, is unclear. We established inducible RUNXlb/c-overexpressing human embryonic stem cell （hESC） lin...RUNXI is absolutely required for definitive hematopoiesis, but the function of RUNXlb/c, two isoforms of human RUNX1, is unclear. We established inducible RUNXlb/c-overexpressing human embryonic stem cell （hESC） lines, in which RUNXlb/c overexpression prevented the emergence of CD34＋ cells from early stage, thereby drastically reducing the production of hematopoi- etic stem/prognnitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 ceU co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor （TGF）-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models.展开更多
The hematopoietic function of HOXC4 has not been extensively investigated.Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenito...The hematopoietic function of HOXC4 has not been extensively investigated.Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells.CD34−CD43+cells could be clearly classified into CD34−CD43^(low) and CD34−CD43^(high) sub-populations at D14.The former cells had greater myelogenic potential,and their production was not significantly influenced by induction of HOXC4.By contrast,the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells,and thus had properties of erythroid–megakaryocyte common progenitors,which abundance was increased by∼2-fold when HOXC4 was induced from D10.For CD34−CD43^(low),CD34+CD43+,and CD34−CD43^(high) sub-populations,CD43 level served as a natural index for the tendency to undergo hematopoiesis.Induction of HOXC4 from D10 caused more CD43+cells sustain in S-phase with up-regulation of NF-κB signaling,which could be counteracted by inhibition of NF-κB signaling.These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status,which containing potential of clinical applications.展开更多
The aryl hydrocarbon receptor(AHR)plays an important role during mammalian embryo development.Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells.AHR also regulates the function...The aryl hydrocarbon receptor(AHR)plays an important role during mammalian embryo development.Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells.AHR also regulates the functional maturation of blood cells,such as T cells and megakaryocytes.However,little is known about the role of AHR modulation during the development of erythroid cells.In this study,we used the AHR antagonist StemRegenin 1(SR1)and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR.We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation.RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes.We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes,favoring their maturation of the cytoskeleton and enucleation.We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling.Our findings help uncover the mechanism for AHRmediated human erythroid cell differentiation.We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.展开更多
Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other c...Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other connective tissues and expresses both tryptase and chymase, while the second, termed MCT, which only expresses tryptase, is found primarily in the mucosa. MCs induced from human adult-type CD34+ cells are reported to be of the MCT type, but the development of MCs during embryonic/fetal stages is largely unknown. Using an efficient coculture system, we identified that a CD34+c-kit+ cell population, which appeared prior to the emergence of CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), stimulated robust production of pure Tryptase+Chymase+ MCs (MCTCs). Single-cell analysis revealed dual development directions of CD34+c-kit+ progenitors, with one lineage developing into erythro-myeloid progenitors (EMP) and the other lineage developing into HSPC. Interestingly, MCTCs derived from early CD34+c-kit+ cells exhibited strong histamine release and immune response functions. Particularly, robust release of IL-17 suggested that these early developing tissue-type MCTCs could play a central role in tumor immunity. These findings could help elucidate the mechanisms controlling early development of MCTCs and have significant therapeutic implications.展开更多
Runt-related transcription factor 1(RUNX1)is required for definitive hematopoiesis;however,the functions of most human RUNX1 isoforms are unclear.In particular,the effects of RUNX1-205(a novel splice variant that lack...Runt-related transcription factor 1(RUNX1)is required for definitive hematopoiesis;however,the functions of most human RUNX1 isoforms are unclear.In particular,the effects of RUNX1-205(a novel splice variant that lacks exon 6 in comparison with RUNX1b)on human hematopoiesis are not clear.In this study,a human embryonic stem cell(hESC)line with inducible RUNX1-205 overexpression was established.Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+cells,and the production of hematopoietic stem/progenitor cells was significantly reduced.In addition,the expression of hematopoiesis-related factors was downregulated.However,these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells,indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells,while the promotive effect could be observed during the late stage of hematopoiesis.This is very similar to that of RUNX1b.Interestingly,the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b,and the protein stability of RUNX1-205 was much higher than that of RUNX1b.Thus,the function of RUNX1-205 in normal and diseased models should be further explored.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
Before committing to an erythroid cell lineage,hematopoietic stem cells differentiate along a myeloid cell pathway to generate megakar yocyte-er ythroid biopotential progenitor cells in bone marrow.Recent studies sugg...Before committing to an erythroid cell lineage,hematopoietic stem cells differentiate along a myeloid cell pathway to generate megakar yocyte-er ythroid biopotential progenitor cells in bone marrow.Recent studies suggest that erythroid progenitors(EryPs)could be generated at the level of common myeloid progenitors(CMPs).However,due to a lack of suitable markers,little is known about the early differentiation of these committed EryP cells during CMP development.Herein,using miR-144/451-eGFP knock-in mice,we found that early differentiation of committed erythroid cells could be defined by miR-144/451 expression within CMPs.Single-cell RNA sequencing showed that miR-144/451+progenitors show obvious differentiation characteristics of erythroid lineage cells and diverge from megakaryocyte and other myeloid cell lineages.These progenitors exclusively give rise to erythroid cells,both in vitro and in vivo,and the commitment to an erythroid cell lineage is accompanied by loss of CD53 expression.Our findings will facilitate further understanding of the molecular mechanisms governing erythroid development and support the identification of therapeutic targets for diseases related to erythrocyte development.展开更多
基金This work was supported by the National Program on Key Basic Research Project of China （973 Program 2015CB964902）, the National Natural Science Foundation of China （NSFC H81170466 and H81370597）, and the CAMS Initiatives for Innovative Medicine （2016-12M-1-018） awarded to F.M.
文摘RUNXI is absolutely required for definitive hematopoiesis, but the function of RUNXlb/c, two isoforms of human RUNX1, is unclear. We established inducible RUNXlb/c-overexpressing human embryonic stem cell （hESC） lines, in which RUNXlb/c overexpression prevented the emergence of CD34＋ cells from early stage, thereby drastically reducing the production of hematopoi- etic stem/prognnitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 ceU co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor （TGF）-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models.
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The hematopoietic function of HOXC4 has not been extensively investigated.Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells.CD34−CD43+cells could be clearly classified into CD34−CD43^(low) and CD34−CD43^(high) sub-populations at D14.The former cells had greater myelogenic potential,and their production was not significantly influenced by induction of HOXC4.By contrast,the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells,and thus had properties of erythroid–megakaryocyte common progenitors,which abundance was increased by∼2-fold when HOXC4 was induced from D10.For CD34−CD43^(low),CD34+CD43+,and CD34−CD43^(high) sub-populations,CD43 level served as a natural index for the tendency to undergo hematopoiesis.Induction of HOXC4 from D10 caused more CD43+cells sustain in S-phase with up-regulation of NF-κB signaling,which could be counteracted by inhibition of NF-κB signaling.These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status,which containing potential of clinical applications.
基金supported by the National Basic Research Program(973 Program,2015CB964902)the National Natural Science Foundation of China(H81170466 and H81370597)the CAMS Initiatives for Innovative Medicine(2016-I2M-1-018,2019-I2M-1-006,and 2017-I2M-2005)to F.M.,the National Natural Science Foundation of China Youth Fund(82000119)to Yonggang Zhang.
文摘The aryl hydrocarbon receptor(AHR)plays an important role during mammalian embryo development.Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells.AHR also regulates the functional maturation of blood cells,such as T cells and megakaryocytes.However,little is known about the role of AHR modulation during the development of erythroid cells.In this study,we used the AHR antagonist StemRegenin 1(SR1)and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR.We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation.RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes.We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes,favoring their maturation of the cytoskeleton and enucleation.We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling.Our findings help uncover the mechanism for AHRmediated human erythroid cell differentiation.We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.
基金This work was supported by the National Basic Research Program(973 Program2015CB96A902)+4 种基金the National Natural Science Foundation of China(H81170466 and H81370597)and the CAMS Initiatives for Innovative Medicine(2016-I2M-1-018)awarded to F.M.the CAMS Initiatives for Innovative Medicine(2017-12M-2005)the Union Youth Fund of Chinese Academy of Medical Sciences(81572089)to G.B.and the National Nature Science Foundation of China Youth Fund(81700107)to B.M.
文摘Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other connective tissues and expresses both tryptase and chymase, while the second, termed MCT, which only expresses tryptase, is found primarily in the mucosa. MCs induced from human adult-type CD34+ cells are reported to be of the MCT type, but the development of MCs during embryonic/fetal stages is largely unknown. Using an efficient coculture system, we identified that a CD34+c-kit+ cell population, which appeared prior to the emergence of CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), stimulated robust production of pure Tryptase+Chymase+ MCs (MCTCs). Single-cell analysis revealed dual development directions of CD34+c-kit+ progenitors, with one lineage developing into erythro-myeloid progenitors (EMP) and the other lineage developing into HSPC. Interestingly, MCTCs derived from early CD34+c-kit+ cells exhibited strong histamine release and immune response functions. Particularly, robust release of IL-17 suggested that these early developing tissue-type MCTCs could play a central role in tumor immunity. These findings could help elucidate the mechanisms controlling early development of MCTCs and have significant therapeutic implications.
基金supported by grants from the CAMS Initiatives for Innovative Medicine(2016-I2M-1-018 to F.M.and 2017-I2M-3-021 to J.L.)the Sichuan Provincial Health and Family Planning Commissi on research project(17PJ489 to B.C.)Chengdu Science and Technology Project-Technology Innovation R&D(2018-YF05-01341-SN to B.C.).
文摘Runt-related transcription factor 1(RUNX1)is required for definitive hematopoiesis;however,the functions of most human RUNX1 isoforms are unclear.In particular,the effects of RUNX1-205(a novel splice variant that lacks exon 6 in comparison with RUNX1b)on human hematopoiesis are not clear.In this study,a human embryonic stem cell(hESC)line with inducible RUNX1-205 overexpression was established.Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+cells,and the production of hematopoietic stem/progenitor cells was significantly reduced.In addition,the expression of hematopoiesis-related factors was downregulated.However,these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells,indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells,while the promotive effect could be observed during the late stage of hematopoiesis.This is very similar to that of RUNX1b.Interestingly,the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b,and the protein stability of RUNX1-205 was much higher than that of RUNX1b.Thus,the function of RUNX1-205 in normal and diseased models should be further explored.
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.
基金supported in part by grants from the National Natural Science Foundation of China(H82170121)Sichuan Natural Science Foundation(2023NSFSC1628 and 2024ZYD0185).
文摘Before committing to an erythroid cell lineage,hematopoietic stem cells differentiate along a myeloid cell pathway to generate megakar yocyte-er ythroid biopotential progenitor cells in bone marrow.Recent studies suggest that erythroid progenitors(EryPs)could be generated at the level of common myeloid progenitors(CMPs).However,due to a lack of suitable markers,little is known about the early differentiation of these committed EryP cells during CMP development.Herein,using miR-144/451-eGFP knock-in mice,we found that early differentiation of committed erythroid cells could be defined by miR-144/451 expression within CMPs.Single-cell RNA sequencing showed that miR-144/451+progenitors show obvious differentiation characteristics of erythroid lineage cells and diverge from megakaryocyte and other myeloid cell lineages.These progenitors exclusively give rise to erythroid cells,both in vitro and in vivo,and the commitment to an erythroid cell lineage is accompanied by loss of CD53 expression.Our findings will facilitate further understanding of the molecular mechanisms governing erythroid development and support the identification of therapeutic targets for diseases related to erythrocyte development.
