Mds1CreERT2, an inducible Cre allele specific to adult-repopulating hematopoietic stem cells.
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC8428393 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC8473784 | biostudies-literature
Project description:Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTP?, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTP?-deficient mice revealed that the loss of PTP? substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTP?-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTP? also increased activated RAC1 levels in wild-type HSCs. Functionally, PTP?-deficient BM cells displayed increased cobblestone area-forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34?CD38?CD45RA?lin? PTP?? cells substantially increased the repopulating capacity of human HSCs compared with CD34?CD38?CD45RA?lin? cells and CD34?CD38?CD45RA?lin?PTP?? cells. Our results demonstrate that PTP? regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTP?-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.
| S-EPMC4382260 | biostudies-literature
Project description:BackgroundKupffer cells (KCs) originate from yolk-sac progenitors before birth. Throughout adulthood, they self-maintain independently from the input of circulating monocytes (MOs) at a steady state and are replenished within 2 weeks after having been depleted, but the origin of repopulating KCs in adults remains unclear. The current paradigm dictates that repopulating KCs originate from preexisting KCs or monocytes, but there remains a lack of fate-mapping evidence.MethodsWe first 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. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC-depletion in mice. Finally, we traced the fate of hematopoietic stem cells (HSCs) in an HSC-specific fate-mapping mouse model, in the context of chronic liver inflammation induced by repeated carbon tetrachloride treatment.ResultsBy using fate-mapping mouse models, we found no evidence that repopulating KCs originate from preexisting KCs or MOs and found that in response to KC-depletion, HSCs proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Then, in the chronic liver inflammation context, we confirmed that repopulating KCs originated directly from HSCs.ConclusionTaken together, these findings provided in vivo fate-mapping evidence that repopulating KCs originate directly from HSCs, which presents a completely novel understanding of the cellular origin of repopulating KCs and shedding light on the divergent roles of KCs in liver homeostasis and diseases.
| S-EPMC10712046 | biostudies-literature
Project description:The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). β-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.
| S-EPMC3193263 | biostudies-literature
Project description:Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.
| S-EPMC2977897 | biostudies-literature
Project description:Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage-restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage. This Matters Arising paper is in response to Guo et al. (2013), published in Neuron. See also the Matters Arising Response paper by Eckler et al. (2015), published concurrently with this Matters Arising in Neuron.
| S-EPMC4455040 | biostudies-literature
Project description:The discovery of novel hematopoietic stem cell (HSC) surface markers can enhance understanding of HSC identity and function. We have discovered a population of primitive bone marrow (BM) HSCs distinguished by their expression of the heparan sulfate proteoglycan Syndecan-2, which serves as both a marker and a regulator of HSC function. Syndecan-2 expression was increased 10-fold in CD150+CD48-CD34-c-Kit+Sca-1+Lineage- cells (long-term HSCs [LT-HSCs]) compared with differentiated hematopoietic cells. Isolation of BM cells based solely on syndecan-2 surface expression produced a 24-fold enrichment for LT-HSCs and sixfold enrichment for α-catulin+c-kit+ HSCs, and yielded HSCs with superior in vivo repopulating capacity compared with CD150+ cells. Competitive repopulation assays revealed the HSC frequency to be 17-fold higher in syndecan-2+CD34-KSL cells compared with syndecan-2-CD34-KSL cells and indistinguishable from CD150+CD34-KSL cells. Syndecan-2 expression also identified nearly all repopulating HSCs within the CD150+CD34-KSL population. Mechanistically, syndecan-2 regulates HSC repopulating capacity through control of expression of Cdkn1c (p57) and HSC quiescence. Loss of syndecan-2 expression caused increased HSC cell cycle entry, downregulation of Cdkn1c, and loss of HSC long-term repopulating capacity. Syndecan-2 is a novel marker of HSCs that regulates HSC repopulating capacity via control of HSC quiescence.
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Project description:Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.
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Project description:Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.
| S-EPMC4426797 | biostudies-other
Project description:E-proteins are widely expressed basic helix-loop-helix (HLH) transcription factors that regulate differentiation in many cell lineages, including lymphoid, muscle, and neuronal cells. E-protein function is controlled by HLH inhibitors such as Id and SCL/TAL1 proteins, which recently have been suggested to play a role in hematopoietic stem cell (HSC) differentiation. However, the precise stages when these proteins are expressed and their specific functions are not entirely clear. Using a knock-in mouse model where the sequence for the enhanced green fluorescent protein (GFP) was inserted downstream of the Id1 promoter, we were able to track Id1 expression on an individual cell basis and detected Id1 expression in long-term repopulating HSCs (LT-HSCs). Functional assays showed that the Id1/GFP(+)Lin(-)Sca1(+)c-kit(Hi) population was highly enriched for LT-HSCs. Consistent with this expression pattern, Id1 deficiency led to a 2-fold reduction in the number of LT-HSCs defined as Lin(-)Sca1(+)c-kit(Hi)CD48(-)CD150(+). Primary bone marrow transplantation studies revealed that Id1 is dispensable for short-term engraftment. In contrast, both Id1(-/-) whole bone marrow and Lin(-)Sca1(+)c-kit(Hi)Thy1.1(Lo)-enriched HSCs, but not Id3(-/-) marrow, displayed impaired engraftment relative to wild-type controls in secondary transplantation assays. These findings suggest a unique role for Id1 in LT-HSC maintenance and hematopoietic development.
| S-EPMC1988946 | biostudies-literature