During embryogenesis,hematopoietic stem progenitor cells(HSPCs)are believed to be derived from hemogenic endothelial cells(HECs).Moreover,arterial feature is proposed to be a prerequisite for HECs to generate HSPCs wi...During embryogenesis,hematopoietic stem progenitor cells(HSPCs)are believed to be derived from hemogenic endothelial cells(HECs).Moreover,arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential.Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper,the functional and molecular characteristics of HECs in the extraembryonic yolk sac(YS)remain largely unresolved.In this study,we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing(scRNA-seq)datasets and validated the arterial vasculature distribution of Gja5+ECs using a Gja5-EGFP reporter mouse model.Further,we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels.The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days(E)8.5–E9.5 and gradually concentrated in CD44-positive ECs from E10.0.Unexpectedly,B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features.Furthermore,the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0–E10.5.Importantly,the distinct identities of E10.0–E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis.Cumulatively,these findings extend our knowledge regarding the hemogenic potential of ECs from anatomically and molecularly different vascular beds,providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.展开更多
Prior to the generation of hematopoietic stem cells(HSCs)from the hemogenic endothelial cells(HECs)mainly in the dorsal aorta in midgestational mouse embryos,multiple hematopoietic progenitors including erythro-myeloi...Prior to the generation of hematopoietic stem cells(HSCs)from the hemogenic endothelial cells(HECs)mainly in the dorsal aorta in midgestational mouse embryos,multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs.These HSCindependent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth.However,little is known about yolk sac HECs.Here,combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays,we reveal that Neurl3-EGFP,in addition to marking the continuum throughout the ontogeny of HSCs from HECs,can also serve as a single enrichment marker for yolk sac HECs.Moreover,while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper,the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression.Interestingly,the B lymphoid potential of hematopoietic progenitors,but not for myeloid potentials,is exclusively detected in Neurl3-negative subpopulations in midgestational embryos.Taken together,these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.展开更多
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
Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clar...Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells(PSCs-embryonic stem cells(ESCs) and induced pluripotent stem cells(i PSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.展开更多
目的:比较二维平铺法和三维悬滴法两种培养体系对小鼠胚胎期生血内皮细胞体外诱导生成造血产物的支持作用。方法:分别利用二维平铺法和三维悬滴法将流式细胞术分选的造血干细胞潜能的生血内皮细胞PK44(CD41-CD43-CD45-CD31^(+)CD201^(+)...目的:比较二维平铺法和三维悬滴法两种培养体系对小鼠胚胎期生血内皮细胞体外诱导生成造血产物的支持作用。方法:分别利用二维平铺法和三维悬滴法将流式细胞术分选的造血干细胞潜能的生血内皮细胞PK44(CD41-CD43-CD45-CD31^(+)CD201^(+)Kit^(+)CD44^(+))群体与OP9-DL1-GFP基质细胞共孵育6 d后,在显微镜下观察两种培养体系产生造血产物的形态特点;利用流式细胞术分析两种培养体系孵育后的造血产物,并统计分析造血产物的比例和绝对数量;分别对两种培养体系共孵育后的造血产物进行造血细胞集落形成单位培养(colony-forming unit culture,CFU-C)实验,并对不同形态的细胞集落进行计数。结果:(1)利用二维平铺法形成的造血细胞平铺或悬浮于基质细胞上,而利用三维悬滴法共孵育后的产物形成了以基质细胞为中心,造血细胞黏附在周围的细胞聚集体。(2)二维平铺法产生更多CD45^(+)造血细胞;三维悬滴法对造血产物的分化具有谱系偏向性,更有利于肥大细胞的分化。(3)二维平铺法诱导生血内皮的分化产物在体外CFU-C实验中可以产生更多的造血集落。结论:二维平铺法更支持小鼠胚内生血内皮细胞体外诱导产生CD45^(+)的造血细胞以及有集落形成能力的造血干祖细胞。展开更多
基金supported by the National Key Research and Development Program of China(2020YFA0112402,2017YFA0103401,and 2016YFA0100601)the National Natural Science Foundation of China(81890991,31930054,31871173,82000111,and 81900115)+1 种基金the Program for Guangdong Introducing Innovative and Entrepreneurial Teams(2017ZT07S347)the Key Research and Development Program of Guangdong Province(2019B020234002)。
文摘During embryogenesis,hematopoietic stem progenitor cells(HSPCs)are believed to be derived from hemogenic endothelial cells(HECs).Moreover,arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential.Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper,the functional and molecular characteristics of HECs in the extraembryonic yolk sac(YS)remain largely unresolved.In this study,we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing(scRNA-seq)datasets and validated the arterial vasculature distribution of Gja5+ECs using a Gja5-EGFP reporter mouse model.Further,we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels.The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days(E)8.5–E9.5 and gradually concentrated in CD44-positive ECs from E10.0.Unexpectedly,B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features.Furthermore,the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0–E10.5.Importantly,the distinct identities of E10.0–E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis.Cumulatively,these findings extend our knowledge regarding the hemogenic potential of ECs from anatomically and molecularly different vascular beds,providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.
基金supported by the National Key R&D Program of China(2022YFA1103501,2020YFA0112400,2021YFA1100102)the National Natural Science Foundation of China(82000111,81890991,31930054,82200121,82122004,82270118)the Program for Guangdong Introducing Innovative and Entrepreneurial Teams(2017ZT07S347).
文摘Prior to the generation of hematopoietic stem cells(HSCs)from the hemogenic endothelial cells(HECs)mainly in the dorsal aorta in midgestational mouse embryos,multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs.These HSCindependent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth.However,little is known about yolk sac HECs.Here,combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays,we reveal that Neurl3-EGFP,in addition to marking the continuum throughout the ontogeny of HSCs from HECs,can also serve as a single enrichment marker for yolk sac HECs.Moreover,while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper,the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression.Interestingly,the B lymphoid potential of hematopoietic progenitors,but not for myeloid potentials,is exclusively detected in Neurl3-negative subpopulations in midgestational embryos.Taken together,these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.
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supported by the National Key Basic Research Program of China(2015CB964903)the National Natural Science Foundation of China(81270640)
文摘Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells(PSCs-embryonic stem cells(ESCs) and induced pluripotent stem cells(i PSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.
文摘目的:比较二维平铺法和三维悬滴法两种培养体系对小鼠胚胎期生血内皮细胞体外诱导生成造血产物的支持作用。方法:分别利用二维平铺法和三维悬滴法将流式细胞术分选的造血干细胞潜能的生血内皮细胞PK44(CD41-CD43-CD45-CD31^(+)CD201^(+)Kit^(+)CD44^(+))群体与OP9-DL1-GFP基质细胞共孵育6 d后,在显微镜下观察两种培养体系产生造血产物的形态特点;利用流式细胞术分析两种培养体系孵育后的造血产物,并统计分析造血产物的比例和绝对数量;分别对两种培养体系共孵育后的造血产物进行造血细胞集落形成单位培养(colony-forming unit culture,CFU-C)实验,并对不同形态的细胞集落进行计数。结果:(1)利用二维平铺法形成的造血细胞平铺或悬浮于基质细胞上,而利用三维悬滴法共孵育后的产物形成了以基质细胞为中心,造血细胞黏附在周围的细胞聚集体。(2)二维平铺法产生更多CD45^(+)造血细胞;三维悬滴法对造血产物的分化具有谱系偏向性,更有利于肥大细胞的分化。(3)二维平铺法诱导生血内皮的分化产物在体外CFU-C实验中可以产生更多的造血集落。结论:二维平铺法更支持小鼠胚内生血内皮细胞体外诱导产生CD45^(+)的造血细胞以及有集落形成能力的造血干祖细胞。
