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1.
Nat Cell Biol ; 26(5): 719-730, 2024 May.
Article in English | MEDLINE | ID: mdl-38594587

ABSTRACT

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.


Subject(s)
Embryonic Development , Receptors, IgG , Humans , Embryonic Development/genetics , Receptors, IgG/metabolism , Receptors, IgG/genetics , Hemangioblasts/metabolism , Hemangioblasts/cytology , Cell Differentiation , Endothelial Cells/metabolism , Endothelial Cells/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Lineage , Cells, Cultured , Gene Expression Regulation, Developmental , Hematopoiesis , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Transcriptome , Gene Expression Profiling , Embryo, Mammalian/metabolism , Embryo, Mammalian/cytology
2.
Nat Cell Biol ; 25(8): 1135-1145, 2023 08.
Article in English | MEDLINE | ID: mdl-37460694

ABSTRACT

Definitive haematopoietic stem and progenitor cells (HSPCs) generate erythroid, lymphoid and myeloid lineages. HSPCs are produced in the embryo via transdifferentiation of haemogenic endothelial cells in the aorta-gonad-mesonephros (AGM). HSPCs in the AGM are heterogeneous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. Here we discovered that loss of microRNA (miR)-128 in zebrafish leads to an expansion of HSPCs in the AGM with different cell cycle states and a skew towards erythroid and lymphoid progenitors. Manipulating miR-128 in differentiating haemogenic endothelial cells, before their transition to HSPCs, recapitulated the lineage skewing in both zebrafish and human pluripotent stem cells. miR-128 promotes Wnt and Notch signalling in the AGM via post-transcriptional repression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b. De-repression of cskn1a1 resulted in replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in G2/M and lymphoid-biased HSPCs with long-term consequence on the respective blood lineages. We propose that HSPC heterogeneity arises in the AGM endothelium and is programmed in part by Wnt and Notch signalling.


Subject(s)
Hemangioblasts , MicroRNAs , Animals , Humans , Zebrafish/genetics , Hematopoietic Stem Cells/metabolism , Cell Differentiation/genetics , Endothelium , MicroRNAs/metabolism , Hematopoiesis/genetics
3.
Stem Cell Res ; 62: 102808, 2022 07.
Article in English | MEDLINE | ID: mdl-35569347

ABSTRACT

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.


Subject(s)
Hemangioblasts , Pluripotent Stem Cells , Antigens, CD1d/metabolism , Antigens, CD34/metabolism , Cell Differentiation/physiology , Glycophorins/metabolism , Hemangioblasts/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Mesoderm/metabolism , Pluripotent Stem Cells/metabolism
4.
Nat Cell Biol ; 24(5): 616-624, 2022 05.
Article in English | MEDLINE | ID: mdl-35484246

ABSTRACT

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. During embryonic development, HSCs derive from haemogenic endothelium (HE) in a NOTCH- and retinoic acid (RA)-dependent manner. Although a WNT-dependent (WNTd) patterning of nascent hPSC mesoderm specifies clonally multipotent intra-embryonic-like HOXA+ definitive HE, this HE is functionally unresponsive to RA. Here we show that WNTd mesoderm, before HE specification, is actually composed of two distinct KDR+ CD34neg populations. CXCR4negCYP26A1+ mesoderm gives rise to HOXA+ multilineage definitive HE in an RA-independent manner, whereas CXCR4+ ALDH1A2+ mesoderm gives rise to HOXA+ multilineage definitive HE in a stage-specific, RA-dependent manner. Furthermore, both RA-independent (RAi) and RA-dependent (RAd) HE harbour transcriptional similarity to distinct populations found in the early human embryo, including HSC-competent HE. This revised model of human haematopoietic development provides essential resolution to the regulation and origins of the multiple waves of haematopoiesis. These insights provide the basis for the generation of specific haematopoietic populations, including the de novo specification of HSCs.


Subject(s)
Hemangioblasts , Pluripotent Stem Cells , Cell Differentiation/physiology , Cell Lineage , Female , Hematopoiesis , Humans , Pregnancy , Tretinoin/pharmacology
5.
Development ; 149(8)2022 04 15.
Article in English | MEDLINE | ID: mdl-35178561

ABSTRACT

Tissue-resident macrophages are increasingly recognized as important determinants of organ homeostasis, tissue repair, remodeling and regeneration. Although the ontogeny and function of tissue-resident macrophages has been identified as distinct from postnatal hematopoiesis, the inability to specify, in vitro, similar populations that recapitulate these developmental waves has limited our ability to study their function and potential for regenerative applications. We took advantage of the concept that tissue-resident macrophages and monocyte-derived macrophages originate from distinct extra-embryonic and definitive hematopoietic lineages to devise a system to generate pure cultures of macrophages that resemble tissue-resident or monocyte-derived subsets. We demonstrate that human pluripotent stem cell-derived extra-embryonic-like and intra-embryonic-like hematopoietic progenitors differentiate into morphologically, transcriptionally and functionally distinct macrophage populations. Single-cell RNA sequencing of developing and mature cultures uncovered distinct developmental trajectories and gene expression programs of macrophages derived from extra-embryonic-like and intra-embryonic-like hematopoietic progenitors. These findings establish a resource for the generation of human tissue resident-like macrophages to study their specification and function under defined conditions and to explore their potential use in tissue engineering and regenerative medicine applications.


Subject(s)
Macrophages , Pluripotent Stem Cells , Cell Differentiation/genetics , Hematopoiesis , Homeostasis , Humans , Macrophages/metabolism
6.
Dev Cell ; 53(2): 229-239.e7, 2020 04 20.
Article in English | MEDLINE | ID: mdl-32197069

ABSTRACT

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.


Subject(s)
Cell Differentiation , Cell Lineage , Embryonic Stem Cells/cytology , Erythroid Precursor Cells/cytology , Hematopoietic Stem Cells/cytology , Killer Cells, Natural/pathology , Myeloid Progenitor Cells/cytology , Animals , Embryonic Stem Cells/metabolism , Erythroid Precursor Cells/metabolism , Female , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Humans , Killer Cells, Natural/metabolism , Male , Mice , Myeloid Progenitor Cells/metabolism , Yolk Sac
7.
PLoS One ; 13(9): e0203991, 2018.
Article in English | MEDLINE | ID: mdl-30231080

ABSTRACT

Megakaryocytes (Mks) derive from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and develop into large, polyploid cells that eventually give rise to platelets. As Mks mature, they migrate from the bone marrow niche into the vasculature, where they are exposed to shear forces from blood flow, releasing Mk particles (platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs)) into circulation. We have previously shown that transcription factor p53 is important in Mk maturation, and that physiological levels of shear promote Mk particle generation and platelet biogenesis. Here we examine the role of p53 in the Mk shear-stress response. We show that p53 is acetylated in response to shear in both immature and mature Mks, and that decreased expression of deacetylase HDAC1, and increased expression of the acetyltransferases p300 and PCAF might be responsible for these changes. We also examined the hypothesis that p53 might be involved in the shear-induced Caspase 3 activation, phosphatidylserine (PS) externalization, and increased biogenesis of PLPs, PPTs, and MkMPs. We show that p53 is involved in all these shear-induced processes. We show that in response to shear, acetyl-p53 binds Bax, cytochrome c is released from mitochondria, and Caspase 9 is activated. We also show that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not. This is the first report to show that shear flow stimulates TIPA and that Caspase 9 activation and Mk-particle biogenesis are directly modulated by TIPA.


Subject(s)
Apoptosis/physiology , Megakaryocytes/cytology , Megakaryocytes/metabolism , Thrombopoiesis/physiology , Tumor Suppressor Protein p53/metabolism , Acetylation , Antigens, CD34/metabolism , Apoptosis/genetics , Blood Platelets/cytology , Blood Platelets/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , Cell Differentiation , Cell Line , Cell-Derived Microparticles/physiology , Cytochromes c/metabolism , Enzyme Activation , Gene Knockdown Techniques , Humans , Mitochondria/metabolism , Models, Biological , Phosphatidylserines/metabolism , Stress, Mechanical , Thrombopoiesis/genetics , Transcription, Genetic , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics
8.
J Biol Chem ; 291(15): 7831-43, 2016 Apr 08.
Article in English | MEDLINE | ID: mdl-26814129

ABSTRACT

Megakaryocytes (MKs) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulating blood. Shear forces promote DNA synthesis, polyploidization, and maturation in MKs, and platelet biogenesis. To investigate mechanisms underlying these MK responses to shear, we carried out transcriptional analysis on immature and mature stem cell-derived MKs exposed to physiological shear. In immature (day (d)9) MKs, shear exposure up-regulated genes related to growth and MK maturation, whereas in mature (d12) MKs, it up-regulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, six activator protein 1 (AP-1) transcripts (ATF4,JUNB,JUN,FOSB,FOS, andJUND) were up-regulated at d9 and two AP-1 proteins (JunD and c-Fos) were up-regulated both at d9 and d12. We show that mitogen-activated protein kinase (MAPK) signaling is linked to both the shear stress response and AP-1 up-regulation. c-Jun N-terminal kinase (JNK) phosphorylation increased significantly following shear stimulation, whereas JNK inhibition reduced shear-induced JunD expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos expression. JNK inhibition reduced fibrinogen binding and P-selectin expression of d12 platelet-like particles (PLPs), whereas p38 inhibition reduced fibrinogen binding of d12 PLPs. AP-1 expression correlated with increased MK DNA synthesis and polyploidization, which might explain the observed impact of shear on MKs. To summarize, we show that MK exposure to shear forces results in JNK activation, AP-1 up-regulation, and downstream transcriptional changes that promote maturation of immature MKs and platelet biogenesis in mature MKs.


Subject(s)
MAP Kinase Signaling System , Mechanotransduction, Cellular , Megakaryocytes/cytology , Mitogen-Activated Protein Kinases/metabolism , Transcription Factor AP-1/genetics , Up-Regulation , Cells, Cultured , Gene Expression Regulation, Developmental , Humans , Megakaryocytes/metabolism , Phosphorylation , Polyploidy , Rheology , Stress, Mechanical , Transcription Factor AP-1/metabolism , Transcriptional Activation
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