Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

Abstract Definitive hematopoietic stem cells (HSCs) emerge in the embryo and sustain major adult hematopoietic lineages. Although their functional potential is detected by transplant, nascent HSC contribution during development is both unknown and difficult to address due to the overlapping emergence of HSC-independent progenitors-cells that lack the multipotency and/or longevity of HSCs but express many of the same markers. Using sorted hematopoietic stem and progenitor cells from zebrafish embryos, we performed single cell RNA sequencing to decipher HSC and HSC-independent progenitor heterogeneity during the time frame of their emergence and initial maturation. After batch correction and dimensional reduction, we identified seven distinct populations that are inferred from RNA velocity analysis to originate from pre-hemogenic endothelium and develop into three main differentiation trajectories. We also determined that HSCs can be distinguished from HSC-independent progenitors based on the temporal regulation and differential activity of the draculin (drl) promoter that was previously shown to mark adult-contributing HSCs. From these studies, we found that the drl promoter is active in HSCs and HSC-independent progenitors at 1-day post-fertilization (dpf) but becomes highly expressed primarily in HSCs by 2 dpf. We applied a drl:cre-ER T2 tamoxifen-inducible Cre-loxP lineage-tracing approach to selectively lineage trace HSCs starting at 2 dpf and track their myeloid and lymphoid contribution during larval development and adulthood. We determined that HSC-independent progenitors primarily contribute to developmental lymphomyelopoiesis with minimal HSC contribution until after 7 dpf. Consistent with this result, we demonstrated that although HSCs robustly regenerated after hematopoietic injury using a novel inducible larval HSC injury model, their depletion had almost no impact on lymphoid and myeloid cell numbers up to 7 dpf. These findings suggest that HSCs are not entirely dormant during development and that there exists an uncoupling of HSC self-renewal and differentiation in development. In conclusion, we determine that it is the HSC-independent progenitors, and not HSCs, that sustain embryonic and early larval lymphomyelopoiesis. Acquiring a greater understanding regarding developmental differences in progenitor and HSC specification and maturation will inform and improve the generation of functional HSCs from renewable pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

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Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.728057 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wanbo Tang ◽

Jian He ◽

Tao Huang ◽

Zhijie Bai ◽

Chaojie Wang ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Lineage Tracing ◽

Genetic Lineage ◽

Hematopoietic Stem ◽

Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

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Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

10.1101/2021.05.02.442359 ◽

2021 ◽

Author(s):

Bianca A Ulloa ◽

Samima S Habbsa ◽

Kathryn S Potts ◽

Alana Lewis ◽

Mia McKinstry ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

De Novo ◽

Marker Gene ◽

Lineage Tracing ◽

Hematopoietic Stem ◽

Functional Potential ◽

Injury Model ◽

Rare Cells

Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single cell transcriptomics, we defined gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a new lineage tracing approach, we selectively tracked HSC output in situ and discovered significantly delayed lymphomyeloid contribution. Using a novel inducible HSC injury model, we demonstrated a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding the differences among embryonic HSC and progenitor populations will guide improved de novo generation and expansion of functional HSCs.

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Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

10.21203/rs.3.rs-1034533/v1 ◽

2021 ◽

Author(s):

Xu Fan ◽

Pei Lu ◽

Xianghua Cui ◽

Peng Wu ◽

Weiran Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Kupffer Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Fate Mapping ◽

Monocytic Cell ◽

Hematopoietic Stem ◽

Cellular Origin

Abstract Kupffer cells (KCs) originate from yolk sac progenitors before birth, but the origin of repopulating KCs in adult remains unclear. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells, which present a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

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Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis

Blood Advances ◽

10.1182/bloodadvances.2018016295 ◽

2018 ◽

Vol 2 (11) ◽

pp. 1220-1228 ◽

Cited By ~ 24

Author(s):

Richard H. Chapple ◽

Yu-Jung Tseng ◽

Tianyuan Hu ◽

Ayumi Kitano ◽

Makiko Takeichi ◽

...

Keyword(s):

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Lineage Tracing ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Key Points ◽

Active Contribution

Key Points HSCs contribute robustly to steady-state hematopoiesis. Platelets receive extensive influx from HSCs compared with other myeloid or lymphoid cells.

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Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Archivum Immunologiae et Therapiae Experimentalis ◽

10.1007/s00005-016-0411-0 ◽

2016 ◽

Vol 65 (1) ◽

pp. 69-81 ◽

Cited By ~ 7

Author(s):

Geoffrey Brown ◽

Aleksandra Marchwicka ◽

Alan Cunningham ◽

Kai-Michael Toellner ◽

Ewa Marcinkowska

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Hematopoietic Stem Cells ◽

Myeloid Cell ◽

Retinoic Acid Receptors ◽

Hematopoietic Stem ◽

Cell Production

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Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.

Blood ◽

10.1182/blood.v114.22.1478.1478 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1478-1478

Author(s):

Kathryn M. Shinnick ◽

Kelly A. Barry ◽

Elizabeth A. Eklund ◽

Thomas J. McGarry

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Transcription Factors ◽

Cell Division ◽

Dna Replication ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.

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Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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Characterization of Histone Modifications on Lineage-Affiliated Genes During Hematopoietic Stem Cell Differentiation

Blood ◽

10.1182/blood.v116.21.4784.4784 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4784-4784

Author(s):

Chen Fangping ◽

Huarong Tang

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Histone Modifications ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Low Level ◽

Lineage Specific Genes

Abstract Abstract 4784 Hematopoietic stem cells (HSCs) are multipotent stem cells capable of self-renewal and multi-lineage differentiation. Though it has been shown that multiple factors take part in the maintenance of HSCs’ multipotency and differentiation potential, the mechanisms are unclear. Recent studies showed that histone modifications play an important role in maintenance of embryonic stem cells pluripotency and differentiation. To characterize the histone modification patterns of different lineages, HSCs were collected from umbilical cord blood and induced to differentiate to granulocytic, erythroid, and megakarytic in vitro. genes during HSC differentiation. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) technology was adopted to investigate the dynamic changes of histone modifications on lineage specific transcription factors and lineage–affiliated genes. Our results showed a certain level of H4 acetylation and H3 acetylation together with high level of H3K4me2 and low level of H3K4me3, H3K9me3 and H3K27me3 were present in lineage specific genes in CD34+CD38- HSCs. As CD34+CD38- cells differentiated, the modification level of acH3, acH4, H3K4me2, H3K9me3 and H3K27me3 on lineage specific genes remained the same, while H3K4me3 level increased greatly. In non-lineage specific genes, the acH3 and acH4 levels decreased, and H3K4me3 level remain at low level, while H3K9me3 and H3K27me3 levels increased. Thus, our data suggested that histone modifications played an important role in maintenaning the multipotency and differentiation capability of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Stability of Self-Renewal in Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v116.21.sci-42.sci-42 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Norman N. Iscove

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Single Cells ◽

Gene Expression Profiles ◽

Short Term ◽

Blood Leukocytes ◽

Hematopoietic Stem

Abstract Abstract SCI-42 For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58 Disclosures: No relevant conflicts of interest to declare.

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Marking of Embryonic Precursors of Adult Hematopoietic Stem Cells At Gestational Day E7.5-E8.5 Using An Abcg2-CreERT2 Lineage Tracing Mouse Model

Blood ◽

10.1182/blood.v118.21.1287.1287 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1287-1287

Author(s):

Sheng Zhou ◽

Soghra Fatima ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Lineage Tracing ◽

Tamoxifen Treatment ◽

Specific Expression ◽

Hematopoietic Stem ◽

Tissue Specific ◽

Tissue Specific Expression

Abstract Abstract 1287 Abcg2 is a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs), and is required for the side population phenotype of adult bone marrow HSCs as well as other adult tissue-specific stem cells. We have used these properties of Abcg2 expression for lineage tracing of stem cell development in mice, particularly for HSCs. An ires-CreERT2 cassette was inserted into the Abcg2 locus, down-stream of its stop codon but upstream of its endogenous polyadenylation site, so that both Abcg2 and CreER are co-expressed from a single bicistronic transcript. This design allows for minimum disruption of Abcg2 expression and tissue specific expression of CreERT2 under the control of endogenous Abcg2 transcription elements. The Abcg2CreER/CreER mouse was crossed with flox-STOP-flox-YFP (Rosa26RYFP/YFP) mouse to generate compound heterozygous Abcg2CreER/+ Rosa26RYFP/+ mice. Treatment of adult Abcg2CreER/+ Rosa26RYFP/+ mice with tamoxifen resulted in robust YFP expression in kidney proximal tubule cells and hepatocytes demonstrating the expected tissue-specific expression of the Abcg2CreER allele. We also observed tamoxifen-dependent appearance of YFP+ cells in all hematopoietic lineages in the peripheral blood and bone marrow, confirming our prior observations that Abcg2 is expressed in adult stem cells. Unexpectedly, we observed long term marking in intestinal epithelial cells and in seminiferous tubules 9 to 20 months after tamoxifen treatment, recapitulating classic progeny tracking patterns, proving that intestinal stem cells and spermatogonial stem cells express the Abcg2 marker. Pregnant females were treated with a single dose of 4-hydroxytamoxifen (4-HT) at gestational days E7.5 and E8.5 using overnight timed breeding pairs. We chose 4-HT rather than tamoxifen because 4-HT has been shown to decay relatively quickly in the fetus so that no recombination can occur 24 hours after the pulse. After maternal treatment, mice were born, grew to adulthood, and were analyzed 14–17 weeks after birth for expression of YFP in distinct peripheral blood lineages. In the majority of the 18 mice born from mothers treated with 4-HT at day E7.5 and from 17 mice born of mothers treated at day E8.5, a small but distinct YFP+ subpopulation could be clearly detected in all hematopoietic lineages (Figure A and B). The numbers of marked cells have been stable for approximately 4 months and are strictly dependent on 4-HT treatment of the mother. These results demonstrate that a precursor to adult hematopoietic stem cells exists at gestational day E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood. The low level of marking could reflect inefficient recombination due to either relatively low levels of expression of the recombinant allele in these embryonic HSC precursors or due to inefficient nuclear localization with the single 4-HT pulse. Alternatively, these marked embryonic HSC precursors may be generating only a minor population of adult HSCs that are competing against a larger fraction of HSCs that arise from precursors that originate later in gestation after the 24 hour 4-HT washout. We are in the process of determining the embryonic source of the E7.5 – E8.5 adult HSC precursor and have not yet determined whether it originates in the yolk sac, in another extra-embryonic source, or within the embryo proper. We are following these mice for longer periods of time to determine the stability of marking in primary and serial transplant experiments. Altogether, we expect that studies with this novel lineage tracing model will provide a better understanding of steady-state, uninterrupted embryonic hematopoietic development that does not require transplant assays to detect HSC activity. Disclosures: No relevant conflicts of interest to declare.

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