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1
Camargo, Fernando D. „In vivo Stem Cell Clonal Dynamics“. Blood 126, Nr. 23 (03.12.2015): SCI—40—SCI—40. http://dx.doi.org/10.1182/blood.v126.23.sci-40.sci-40.
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Abstract Tremendous progress has been achieved in the characterization of the hematopoietic system over the past two decades. Historically, the main experimental approach used to elucidate and define these cellular relationships in the bone marrow (BM) has been the transplantation assay. For this reason, most of our knowledge about the in vivo properties of hematopoietic stem cells (HSCs) and progenitor cells has been derived from studies in the transplant context. Because of the lack of tractable systems, the mechanistic nature of non-transplant hematopoiesis has remained largely unexplored. Over the past several years, my laboratory has developed novel genetic tools for the clonal tracing and imaging of hematopoietic populations in the unperturbed niche that aim to bring insight into the biology of stem and progenitor cells in situ. Our work using a transposon-mediated cellular tagging approach indicated that progenitors, and not the classical long-term HSCs, are the cells mainly responsible for the day-to-day production of blood cells in the adult. Our data also suggested that lineage restricted progenitors are the main contributors to hematopoiesis at steady state. These data represent the first systematic analysis of clonal fate in an unperturbed hematopoietic niche and revealed a novel cellular mechanism for homeostatic blood regeneration. We have now utilized this clonal tracing model to bring insight into the dynamics of stem and progenitor biology during embryonic hematopoiesis and in the severely aged hematopoietic system. These data will be discussed at the meeting. Disclosures Camargo: Cell Signaling Technologies: Consultancy; Vital Therapies: Consultancy.
2
Li, June, Daniel P. Sejas und Qishen Pang. „Nucleophosmin Regulates Differentiation, Cell Cycle Progression, and Stress Response in Hematopoietic Progenitor Cells.“ Blood 106, Nr. 11 (16.11.2005): 312. http://dx.doi.org/10.1182/blood.v106.11.312.312.
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Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Strong evidence indicates that NPM is involved in hematopoiesis and leukemic development. Here we report that NPM enhances the proliferative potential of hematopoietic stem/progenitor cells and increases cell survival upon stress challenge. Specifically, lin-Sca1+c-kit+ bone marrow cells transduced with retroviral vector expressing NPM exhibited higher proliferative rates in both short-term liquid culture and clonogenic progenitor cell assays, compared to the cells transduced with empty vector. Interestingly, NPM overexpression appears to inhibit differentiation of myeloid progenitors. Hematopoietic stem/progenitor cells infected with the NPM retrovirus expressed significantly lower levels of mature cell markers Gr-1 and Mac-1 compared to empty vector transduced cells, and majority of the NPM-overexpressing cells remained Sca1+C-Kit+ during the 5-day culture. Bone marrow transplantation experiments demonstrated that NPM overexpression increases long-term multi-lineage repopulating capacity of hematopoietic progenitors. We have not observed any evidence of proliferative disorders or leukemia in recipients transplanted with NPM-expressing progenitors thus far (4 months posttransplantation). Through cell-cycle profile analysis and single-cell division experiments, we showed that NPM overexpression induces rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G0/G1 to S transition. Furthermore, immunocytochemical and Western-blot analyses demonstrated that NPM-transduced cells expressed higher level of cyclin A compared to vector-transduced cells. Finally, overexpression of NPM significantly increased the survival of hematopoietic progenitors exposed to mitomycin C or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of progenitor differentiation, cell cycle progression, and stress response.
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Kristensen, Helene Bjoerg, Thomas Levin Andersen, Andrea Patriarca, Klaus Kallenbach, Birgit MacDonald, Tanja Sikjaer, Charlotte Ejersted und Jean-Marie Delaisse. „Human hematopoietic microenvironments“. PLOS ONE 16, Nr. 4 (20.04.2021): e0250081. http://dx.doi.org/10.1371/journal.pone.0250081.
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Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- “hematopoietic stem cells and multipotent progenitors” and a population of CD34+/CD38+ “progenitors” based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other “hematopoietic stem cells and multipotent progenitors” and other “progenitors”) within 30 μm of “hematopoietic stem cells and multipotent progenitors”, “progenitors”, and “random cell profiles”. We show that the microenvironment of the “hematopoietic stem cells and multipotent progenitors” is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the “progenitors” is significantly enriched in capillaries, other “progenitors”, bone surfaces and arteries.
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Esmaeli-Azad, Babak, Anand S. Srivastava, Cybele Frederico, Geraldo Martinez, Satoshi Yasukawa und Ewa Carrier. „Artificial Hematopoietic Stem Cell Niche Sustains Growth and Differentiation of Human ES-Derived Early Hematopoietic Progenitors.“ Blood 110, Nr. 11 (16.11.2007): 1415. http://dx.doi.org/10.1182/blood.v110.11.1415.1415.
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Abstract Using a novel Microplate Biomaterial Microarray (MBM™) technology, we have created an artificial hematopoietic stem cell niche that can sustain growth and differentiation of human embryonic stem cells-derived (hES) early hematopoietic progenitors. This hydrogel based ex-vivo niche allows uploading of human embryonal stem cells, human mesenchymal stem cells (MSC), genes (bcl-2 preventing apoptosis and HoxB4 enhancing hematopoiesis) and extracellular matrices to support growth and differentiation of human ES cells. These experiments were done using NIH-approved hES cell lines H1 and H9. Serum-free, feeder-free culture conditions were established and early hematopoietic progenitors grown using SCF, TPO, VEGF and IL-3 with high efficiency. At day 3–5 dual CD34+/CD31+ progenitors were identified, while on day 7–8 CD34+ hematopoietic progenitors were isolated, which formed typical hematopoietic colonies. These progenitors expressed genes related to early hematopoiesis, such as TAL1/SCL, FLT1, GATA2, GATA1, EPOR and TPOR. The early dual endothelio-hematopoietic progenitor (hemangioblast) expressed PECAM-1 and CD34 and showed typical blast-like morphology. Based on mathematical simulations, various micro-niches were designed to establish optimal differentiation conditions for this progenitor using IL-3, IL-6, TPO, EPO, VEGF, SFC, Flt-3 ligand and various extracellular matrices. Specific micro-niches were created for generation of CFU-E, BFU-E, CFU-GM, CFU-GEMM, CFU-M, CFU-G, and CFU-MK progenitors from human ES-derived hemangioblast. Kinetic uploading of TPO, EPO, SCF and VEGF created a niche-sustaining growth of ES-derived hemangioblast with high efficiency and low apoptosis rate. These niches used pulse -delivery of anti-apoptotic bcl-2 gene and hematopoiesis-enhancing Hoxb4 gene. The model of artifical niche sustaing growth and differentiation of human ES-derived hemangioblast was established. In the future, this system will allow optimized and upscaled generation of early hematopoietic progenitors from human ES cells, as a first step towards clinical applications of human embryonic stem cells. Figure Figure
5
Cheng, J., S. Baumhueter, G. Cacalano, K. Carver-Moore, H. Thibodeaux, R. Thomas, HE Broxmeyer et al. „Hematopoietic defects in mice lacking the sialomucin CD34“. Blood 87, Nr. 2 (15.01.1996): 479–90. http://dx.doi.org/10.1182/blood.v87.2.479.bloodjournal872479.
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Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.
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Upadhaya, Samik, Catherine M. Sawai, Efthymia Papalexi, Ali Rashidfarrokhi, Geunhyo Jang, Pratip Chattopadhyay, Rahul Satija und Boris Reizis. „Kinetics of adult hematopoietic stem cell differentiation in vivo“. Journal of Experimental Medicine 215, Nr. 11 (05.10.2018): 2815–32. http://dx.doi.org/10.1084/jem.20180136.
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Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.
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Woolthuis, Carolien M., und Christopher Y. Park. „Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage“. Blood 127, Nr. 10 (10.03.2016): 1242–48. http://dx.doi.org/10.1182/blood-2015-07-607945.
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Abstract The classical model of hematopoiesis has long held that hematopoietic stem cells (HSCs) sit at the apex of a developmental hierarchy in which HSCs undergo long-term self-renewal while giving rise to cells of all the blood lineages. In this model, self-renewing HSCs progressively lose the capacity for self-renewal as they transit into short-term self-renewing and multipotent progenitor states, with the first major lineage commitment occurring in multipotent progenitors, thus giving rise to progenitors that initiate the myeloid and lymphoid branches of hematopoiesis. Subsequently, within the myeloid lineage, bipotent megakaryocyte-erythrocyte and granulocyte-macrophage progenitors give rise to unipotent progenitors that ultimately give rise to all mature progeny. However, over the past several years, this developmental scheme has been challenged, with the origin of megakaryocyte precursors being one of the most debated subjects. Recent studies have suggested that megakaryocytes can be generated from multiple pathways and that some differentiation pathways do not require transit through a requisite multipotent or bipotent megakaryocyte-erythrocyte progenitor stage. Indeed, some investigators have argued that HSCs contain a subset of cells with biased megakaryocyte potential, with megakaryocytes directly arising from HSCs under steady-state and stress conditions. In this review, we discuss the evidence supporting these nonclassical megakaryocytic differentiation pathways and consider their relative strengths and weaknesses as well as the technical limitations and potential pitfalls in interpreting these studies. Ultimately, such pitfalls will need to be overcome to provide a comprehensive and definitive understanding of megakaryopoiesis.
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Jing, Lili, Owen J. Tamplin, Michael J. Chen, Qing Deng, Shenia Patterson, Peter G. Kim, Ellen M. Durand et al. „Adenosine signaling promotes hematopoietic stem and progenitor cell emergence“. Journal of Experimental Medicine 212, Nr. 5 (13.04.2015): 649–63. http://dx.doi.org/10.1084/jem.20141528.
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Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.
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Mazo, Irina B., Steffen Massberg und Ulrich H. von Andrian. „Hematopoietic stem and progenitor cell trafficking“. Trends in Immunology 32, Nr. 10 (Oktober 2011): 493–503. http://dx.doi.org/10.1016/j.it.2011.06.011.
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Mitjavila-Garcia, Maria Teresa, Michel Cailleret, Isabelle Godin, Maria Manuela Nogueira, Karine Cohen-Solal, Valérie Schiavon, Yann Lecluse, Françoise Le Pesteur, Anne Hélène Lagrue und William Vainchenker. „Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells“. Development 129, Nr. 8 (15.04.2002): 2003–13. http://dx.doi.org/10.1242/dev.129.8.2003.
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In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.
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Li, Xiaxin, P. Artur Plett, Yanzhu Yang, Ping Hong, Brian Freie, Edward F. Srour, Christie M. Orschell, D. Wade Clapp und Laura S. Haneline. „Fanconi anemia type C–deficient hematopoietic stem/progenitor cells exhibit aberrant cell cycle control“. Blood 102, Nr. 6 (15.09.2003): 2081–84. http://dx.doi.org/10.1182/blood-2003-02-0536.
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Abstract The pathogenesis of bone marrow failure in Fanconi anemia is poorly understood. Suggested mechanisms include enhanced apoptosis secondary to DNA damage and altered inhibitory cytokine signaling. Recent data determined that disrupted cell cycle control of hematopoietic stem and/or progenitor cells disrupts normal hematopoiesis with increased hematopoietic stem cell cycling resulting in diminished function and increased sensitivity to cell cycle–specific apoptotic stimuli. Here, we used Fanconi anemia complementation type C–deficient (Fancc–/–) mice to demonstrate that Fancc–/– phenotypically defined cell populations enriched for hematopoietic stem and progenitor cells exhibit increased cycling. In addition, we established that the defect in cell cycle regulation is not a compensatory mechanism from enhanced apoptosis occurring in vivo. Collectively, these data provide a previously unrecognized phenotype in Fancc–/– hematopoietic stem/progenitor cells, which may contribute to the progressive bone marrow failure in Fanconi anemia.
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Chen, Yuhong, Mei Yu, Xuezhi Dai, Mark Zogg, Renren Wen, Hartmut Weiler und Demin Wang. „Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells“. Journal of Experimental Medicine 208, Nr. 5 (18.04.2011): 923–35. http://dx.doi.org/10.1084/jem.20101192.
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Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1—which is essential for HSC survival—and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.
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Velu, Chinavenmeni S., Michael Berk, Haiming Xu, Tristan Bourdeau, Avedis Kazanjian, David A. Williams, Clemencia Colmenares und H. Leighton Grimes. „The Ski oncoprotein regulates Hematopoietic Stem Cell Fitness and Functions as a Corepressor for Gfi1.“ Blood 112, Nr. 11 (16.11.2008): 1403. http://dx.doi.org/10.1182/blood.v112.11.1403.1403.
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Abstract Ski is a corepressor protein originally identified as a retrovirally transduced oncoprotein. Genetic deletion of Ski has revealed essential roles in multiple developmental processes. Suggestion that Ski may play a role in hematopoiesis first came from expression of v-Ski and c-Kit, which induced the continuous in vitro growth of primary avian multipotent progenitors. However, the hematopoietic phenotype of Ski−/− mice has not been described. Here, we show that Ski loss of function results in loss of hematopoietic stem cell (HSC) fitness and abnormal regulation of myeloid progenitor numbers. Fetal liver Ski−/− HSC engraft well in ablated recipients, but are not competitive in engraftment. Moreover, Ski null embryonic stem cells generate many tissues in chimeras, but infrequently participate in hematopoiesis. Thus, Ski null HSC are not competitive in both transplant and chimera settings, indicating a defect in stem cell fitness. Engrafted Ski−/− fetal liver cells generate fewer myeloid lineage cells than wild type littermates, and accumulate granulocytemonocyte progenitors. Growth factor independent -1 (Gfi1) is a transcriptional repressor that controls HSC maintenance and myeloid progenitor differentiation. Gfi1−/− and Ski−/− hematopoietic stem and myeloid progenitor phenotypes are strikingly similar. We find that Ski functions as a corepressor for Gfi1. Both endogenous and synthetic Gfi1 and Ski physically interact in vitro and upon Gfi1 target genes. Knockdown of Gfi1 or Ski results in derepression of these targets. Thus, our results provide a molecular link between the similar HSC and myeloid progenitor phenotypes engendered by Gfi1 or Ski deletion.
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Cheng, Yuanming, Hanzhi Luo, Franco Izzo, Brian F. Pickering, Ly P. Vu, Alexandra Schurer, Saroj Gourkanti, Samie R. Jaffrey, Dan A. Landau und Michael G. Kharas. „m6A Maintains Hematopoietic Stem and Progenitor Cell Identity“. Blood 132, Supplement 1 (29.11.2018): 327. http://dx.doi.org/10.1182/blood-2018-99-117191.
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Abstract N-6-methyladenosine (m6A) is one of the most abundant posttranscriptional modifications in eukaryotic mRNAs and long noncoding RNAs. We previously found a critical role for m6A in promoting human myeloid leukemia (Vu et al. Nature Medicine 2017). Targeting the RNA methylation program in leukemias has been suggested as a potential novel therapeutic strategy. However, it is unknown whether the m6A modification controls normal adult hematopoiesis and hematopoietic stem cells (HSC) function. To investigate the role of m6A in adult hematopoiesis, we crossed with the Mettl3 conditional knockout (cKO) mouse model with the interferon inducible Mx1-Cre system to abolish m6A in the hematopoietic compartment. Deletion of Mettl3 (3 weeks post pIpC injections) resulted in pancytopenia (white blood count 9.44 k/ul in WT versus 4.35 k/ul cKO) and a 55% reduction in red blood cell counts and nearly 70% loss in platelet counts. Most remarkably, METTL3 depletion resulted in a 5-fold increase in the number of overall hematopoietic stem and progenitors HSPCs (LSKs; Lin-c-kit+Sca1+). Within the HSPC compartment there was a about a 10-fold expansion in immunophenotypic long term hematopoietic stem cells LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-), multipotent progenitors (MPP-2s), (Lin-c-kit+Sca1+CD150+CD48+) and MPP4s (Lin-c-kit+Sca1+CD150-CD48+). In contrast to this general increase in HSPCs, we observed a decrease in common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) populations by 70% and 60% respectively. Altogether, these results suggest that loss of METTL3 results in a partial blockage in hematopoietic stem and progenitor cell differentiation, and an accumulation of LT-HSC and MPPs. Despite this phenotypic expansion of LT-HSCs and MPPs, their function was impaired as demonstrated by a reduction in long-term chimerism in non-competitive transplants into congenic mice compared to the remaining wildtype host cells (66% in WT vs 26% in cKO). Interestingly, the relative differentiation block and accumulation of HSCs remained after transplantation. Furthermore, Mettl3 cKO HSCs are less quiescent (79% WT vs. 44% cKO) and more proliferative based on cell cycle profiling with pyronin Y/Hoechst or Ki67/Hoechst. RNA-seq in sorted LT-HSC, MPP1, MPP2 and MPP4s from WT and Mettl3 cKO mice demonstrated that Mettl3 cKO HSCs lose the HSC signature while MPP1 and MPP2 cells exhibited gene signatures resembling WT HSCs. Interestingly, genes uniquely upregulated in Mettl3 cKO LT-HSC showed a significant enrichment of the Mettl3 KO mouse embryonic stem cells (mESCs) expression signature. To decipher METTL3 regulation of transcriptional states in individual cells along the hematopoietic hierarchy, we performed single-cell RNA-seq using sorted WT and Mettl3 cKO cKit+ cells. Surprisingly, tSNE analysis of scRNA-seq data uncovered the loss of the normal HSC cluster and the emergence of three unique HSC-like populations in Mettl3 cKO mice. Single cell analysis identified a reduction in c-MYC levels in the HSPCs that was then confirmed by immunofluorescence in sorted HSCs. More importantly, when forced into division, Mettl3 cKO HSCs exhibited increased symmetric c-MYC low division pattern compared to control (60% to 48%) based on c-MYC IF staining in paired daughter cells. We further confirmed that sorted HSCs underwent increased symmetric renewal divisions in the Mettl3 cKO compared to controls (65% to 49%) without any effect on asymmetric division based on NUMB IF staining in paired daughter cells. These data suggest that without METTL3, HSCs are partially blocked in a self-renewing state albeit with reduced fitness compared to wildtype HSCs. Overall, our studies uncovered a novel role for METTL3 and RNA methylation to maintain normal HSC identity and progenitor differentiation. Also, these studies suggest that inhibiting METTL3 could result in significant hematopoietic defects. Disclosures No relevant conflicts of interest to declare.
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Schuettpelz, Laura G., Priya K. Gopalan, Felipe O. Giuste, Molly P. Romine, Ronald van Os und Daniel C. Link. „Kruppel-like factor 7 overexpression suppresses hematopoietic stem and progenitor cell function“. Blood 120, Nr. 15 (11.10.2012): 2981–89. http://dx.doi.org/10.1182/blood-2012-02-409839.
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AbstractIncreased expression of Kruppel-like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7−/− cells was comparable with control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, although resulting in multilineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21Cip1/Waf1) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival.
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Kuramoto, Ken, Dean A. Follmann, Peiman Hematti, Stephanie Sellers, Brian A. Agricola, Mark E. Metzger, Robert E. Donahue, Christof von Kalle und Cynthia E. Dunbar. „Effect of chronic cytokine therapy on clonal dynamics in nonhuman primates“. Blood 103, Nr. 11 (01.06.2004): 4070–77. http://dx.doi.org/10.1182/blood-2003-08-2934.
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Abstract Hematopoietic cytokines such as filgrastim are used extensively to stimulate granulocyte production or to mobilize hematopoietic progenitors into the circulation; however, their effect on more primitive hematopoietic progenitor and stem cells in vivo is unknown, particularly in large animals or humans. In particular, there is concern that chronic therapy with cytokines could result in stem cell exhaustion or clonal dominance; however, direct assessment of the dynamics of individual stem and progenitor cell clones in vivo has not been previously reported. A number of models can be proposed regarding the mechanisms by which the marrow responds to cytokine stimulation, including recruitment of previously quiescent clones, stimulation of proliferation of already active clones, or prevention of apoptosis of more mature progenitors from all clones. Using retroviral marking and comprehensive insertion site tracking of individual stem and progenitor cell clones in 2 rhesus macaques, we analyzed the effect of chronic administration of granulocyte colony-stimulating factor (G-CSF), or a combination of G-CSF plus stem cell factor (SCF). The overall number of contributing clones remained constant, and the relative output from each clone did not change significantly during or following cytokine treatments. These results suggest that individual transduced stem or progenitor cells can contribute to hematopoiesis for prolonged periods, with no evidence for an effect of G-CSF or G-CSF/SCF on the number, the lifespan, or the relative activity of individual stem or progenitor cell clones. These relevant large animal studies are reassuring regarding clinical applications of cytokines and provide new insights into their mechanisms of action.
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Keller, U., MJ Aman, G. Derigs, C. Huber und C. Peschel. „Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor“. Blood 84, Nr. 7 (01.10.1994): 2189–96. http://dx.doi.org/10.1182/blood.v84.7.2189.2189.
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Abstract Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.
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Keller, U., MJ Aman, G. Derigs, C. Huber und C. Peschel. „Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor“. Blood 84, Nr. 7 (01.10.1994): 2189–96. http://dx.doi.org/10.1182/blood.v84.7.2189.bloodjournal8472189.
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Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.
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Lin, Fan-ching, Megan Karwan, Bahara Saleh, Deborah L. Hodge, Tim Chan, Kimberly C. Boelte, Jonathan R. Keller und Howard A. Young. „IFN-γ causes aplastic anemia by altering hematopoietic stem/progenitor cell composition and disrupting lineage differentiation“. Blood 124, Nr. 25 (11.12.2014): 3699–708. http://dx.doi.org/10.1182/blood-2014-01-549527.
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Key Points IFN-γ alone leads to aplastic anemia by disrupting the generation of common myeloid progenitors and lineage differentiation. The inhibitory effect of IFN-γ on hematopoiesis is intrinsic to hematopoietic stem/progenitor cells.
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Liu, Yuanyuan, Feifei Xiao, Bijie Yang, Zhiwei Chen, Jieping Chen und Yu Hou. „Wtapblock Cell Differentiation of Hematopoietic Stem and Progenitor Cells“. Blood 136, Supplement 1 (05.11.2020): 30. http://dx.doi.org/10.1182/blood-2020-137027.
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Wilms' tumor 1-associating protein (WTAP) is a ubiquitously expressed nuclear protein has been associated with regulation of cell proliferation, apoptosis, embryonic development, cell cycle, RNA splicing and stabilization, N6-Methyladenosine RNA modification in various physiological processes. Recently,WTAPwas reported to promoter tumorigenicity in Glioblastoma and cholangiocarcinoma. Besides solid tumors,WTAPplays an important role in abnormal proliferation and arrested differentiation in acute myeloid leukemia (AML) cell, suggesting its oncogenic activity. For its promising novel therapeutic target in AML, a systematic investigation of the roles ofWTAPin normal hematopoiesis is warranted.To investigate the function ofWTAPin normal hematopoietic system, we firstly determined the mRNA level ofWTAPin different hematopoietic stem and progenitor cells (HSPCs) and several mature populations in C57/B6 mouse bone marrow (BM).WTAPwas ubiquitously expressed in different cell populations and especially elevated in HSPCs. For WTAP-null and heterozygous caused early embryonic lethality, we generated endothelial system conditional knockout (cKO) mice by crossing WTAP floxed mice with poly (I:C) induced Mx1-Cre transgenic mice. In poly (I:C) inducedWTAPfl/fl-Mx1-Cre, WTAP deficiency lead to approximately 2-fold increase in HSC and LSK pool size, and modest expansion of HPC, CLP and LMPP population. In competitive BM transplantation assay, lossWTAPshowed a significantly decreased repopulation capacity. WhileWTAPknockout did not significantly affect the proliferation, cell cycle and apoptosis of HSPCs tested by Brdu, Ki67 and Annexin-V straining assay. Mechanistically, deletion ofWTAPin HSC resulted in decreased transcription of myeloid cell and erythrocyte differentiation gene (including Jak3, Jun and Junb) and genes regulating pluripotency of stem cells (induding Akt2, Fzd1/9 and Mapk3).Collectively, we speculateWTAPplay important role in blocking cell differentiation of HSPCs. Currently, we are conducting a series of studies to reveal the underlying molecular mechanism(s) ofWTAPregulating normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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Miyoshi, Hirotoshi, Chiaki Sato, Yuichiro Shimizu und Misa Morita. „Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer“. International Journal of Artificial Organs 42, Nr. 7 (12.02.2019): 374–79. http://dx.doi.org/10.1177/0391398819827596.
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With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.
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Gonzalez-Nieto, Daniel, Gabriel Ghiaur, Lina Li, Jorden Arnett, Susan Dunn, Glenn Fishman, David Gutstein, Roberto Civitelli und Jose Cancelas. „Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.“ Blood 114, Nr. 22 (20.11.2009): 1500. http://dx.doi.org/10.1182/blood.v114.22.1500.1500.
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Abstract Abstract 1500 Poster Board I-523 Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Creflox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G0). Interestingly, the expression of cyclin D1 and p21cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G1 restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.
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Salter, Alice B., Sarah K. Meadows, Garrett G. Muramoto, Heather Himburg, Phuong Doan, Pamela Daher, Lauren Russell, Benny Chen, Nelson J. Chao und John P. Chute. „Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo“. Blood 113, Nr. 9 (26.02.2009): 2104–7. http://dx.doi.org/10.1182/blood-2008-06-162941.
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Hematopoietic stem cells (HSCs) reside in association with bone marrow (BM) sinusoidal vessels in vivo, but the function of BM endothelial cells (ECs) in regulating hematopoiesis is unclear. We hypothesized that hematopoietic regeneration following injury is regulated by BM ECs. BALB/c mice were treated with total body irradiation (TBI) and then infused with C57Bl6-derived endothelial progenitor cells (EPCs) to augment endogenous BM EC activity. TBI caused pronounced disruption of the BM vasculature, BM hypocellularity, ablation of HSCs, and pancytopenia in control mice, whereas irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, and platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti–VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non–EPC-treated mice compared with irradiated controls. These data demonstrate that allogeneic EPC infusions can augment hematopoiesis and suggest a relationship between BM microvascular recovery and hematopoietic reconstitution in vivo.
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Chan, Charles, Ching-Cheng Chen, Daniel L. Kraft, Cynthia Luppen, Jae-Beom Kim, Anthony DeBoer, Kevin Wei Wei und Irving L. Weissman. „Identification and Isolation of the Hematopoietic Stem Cell Niche Initiating Cell Population“. Blood 112, Nr. 11 (16.11.2008): 3574. http://dx.doi.org/10.1182/blood.v112.11.3574.3574.
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Abstract Introduction: Identification and understanding of the cells and processes that can generate, sustain and influence the HSC niche and hematopoiesis are critical for the development of a more comprehensive knowledge of normal hematopoiesis, stem cell homing, trafficking, differentiation and hematopoietic pathology. Growth and renewal in many tissues are initiated by stem cells, supported by the microenvironment (niche) in which they reside. While recent work has begun to describe functional interactions between stem cells and their niches, little is known about the formation of stem cell niches. Methods & Results: We established a functional, in vivo assay (via implantation of cells under the renal capsule) to isolate the determinants of hematopoietic stem cell (HSC) niche formation and activity. Using this novel assay, we show that a population of progenitor cells (CD45−Tie2-aV+CD105+Thy1.1−; CD105+Thy1−) sorted from 15.5 dpc fetal limbs and transplanted under the adult mouse renal capsule recruit host-derived vasculatures in a VEGF dependent manner, produce donor-derived ectopic bones through endochondral ossification, and generate a marrow cavity populated by host-derived long term reconstituting HSC (LT-HSC). In contrast, CD45−Tie2-aV+CD105+Thy1a+ (CD105+Thy1+) progenitors form bone that does not contain a marrow cavity. While analyzing these and other sorted populations, we did not observe any instances where niche was present without bone, suggesting that skeletal progenitors are necessary for initiating an HSC niche but osteoblasts alone cannot initiate and support niche activity. Suppression of factors important for HSC maintenance, such as steel factor (SLF), in progenitor populations prior to transplant did not alter their ability to initiate and support an HSC niche. On the other hand, suppression of factors involved in endochondral ossification, such as osterix and VEGF, inhibited niche generation. Furthermore, CD105+Thy1− progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification form only bone without marrow in our assay. Conclusions: In addition to identifying the limb-derived skeletal progenitor capable of endochondral ossification involved and the basic mechanisms of HSC niche initiation, our study provides a functional framework by which future studies on HSC-niche interactions at the cellular level can be carried out.
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., Hirokazu Tanaka, Itaru Matsumura . und Yuzuru Kanakura . „Cell Cycle Regulation in Hematopoietic Stem/progenitor Cells“. Journal of Biological Sciences 5, Nr. 1 (15.12.2004): 50–60. http://dx.doi.org/10.3923/jbs.2005.50.60.
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Ezoe, Sachiko, Itaru Matsumura, Yusuke Satoh, Hirokazu Tanaka und Yuzuru Kanakura. „Cell Cycle Regulation in Hematopoietic Stem/Progenitor Cells“. Cell Cycle 3, Nr. 3 (März 2004): 312–16. http://dx.doi.org/10.4161/cc.3.3.710.
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Park, Christopher Y., Wendy W. Pang, Peter L. Greenberg und Irving L. Weissman. „Myelodysplastic Syndromes Are Characterized by Gene Expression Changes in Hematopoietic Stem Cells and Alterations in Hematopoietic Stem Cell and Myeloid Progenitor Composition.“ Blood 114, Nr. 22 (20.11.2009): 1762. http://dx.doi.org/10.1182/blood.v114.22.1762.1762.
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Abstract Abstract 1762 Poster Board I-788 The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders characterized by peripheral cytopenias due to impaired hematopoietic differentiation. To date, most data characterizing the immature hematopoietic compartment in MDS have relied on evaluation of CD34+ bone marrow cells, which are a heterogeneous population containing a predominance of oligo- and unilineage-potent progenitors and few hematopoietic stem cells (HSC). In this study we show that MDS are disorders of HSC, evidenced by the presence of recurrent cytogenetic alterations, including -5q, -7, and -20q, in highly purified HSC (Lin-CD34+CD38-CD90+CD45RA-) by FISH. Because MDS HSC harbor cytogenetic changes, we sought to better characterize the molecular basis of MDS HSC function by performing whole transcriptome analysis of highly purified HSC and committed myeloid progenitor populations from low-risk (n=8) and high-risk (n=2) MDS patients. When compared to control HSC from healthy patients (n=10), MDS HSC showed broad transcriptional changes. Using the significance analysis of microarrays (SAM) algorithm and Ingenuity Pathways Analysis software, we identified 3,258 differentially expressed genes (FDR < 0.1) with increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). Interestingly, while MDS common myeloid progenitors (CMP, Lin-CD34+CD38+CD123+CD45RA-) showed increased expression of cell death-related genes when compared to normal CMP (p < 0.001), neither MDS HSC nor multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-) showed significant differential expression of these genes when compared to their normal counterparts. To assess the cellular and developmental correlates of HSC/committed progenitor transcriptional changes, we evaluated by flow cytometry the frequency of HSC and committed myeloid progenitors in bone marrow aspirates from 35 low-risk MDS, 6 high-risk MDS and 32 healthy patient samples (range 4-84 yo). Low-risk MDS bone marrow samples showed significantly increased numbers of HSC compared to normal bone marrow samples (+3-fold change, p < 0.03). In addition, myeloid progenitor composition was frequently altered in low-risk MDS patients, with decreased percentages of granulocyte-macrophage progenitors (GMP, Lin-CD34+CD38+CD123+CD45RA+) when expressed as a percentage of total myeloid progenitors [including GMP, CMP and megakaryocyte-erythroid progenitors (MEP, Lin-CD34+CD38+CD123loCD45RA-)] (-2.3-fold change, p < 1e-6). This altered myeloid progenitor profile was highly specific to MDS, even when MDS patient samples were compared to a group of control bone marrow samples from non-MDS patients exhibiting at least one cytopenia (n=34, p < 1e-5), allowing for the distinction of MDS samples from non-MDS cytopenias with 0.89 sensitivity and 0.89 specificity. Together, these data indicate that MDS HSC exhibit significantly altered gene expression profiles and suggest that gene expression changes in MDS HSC induce the altered developmental fate decisions and transcriptional changes observed in MDS committed myeloid progenitors. These data also demonstrate that the changes in MDS myeloid progenitor composition may provide a novel, flow cytometric method for distinguishing MDS from other hematologic conditions that mimic MDS. Finally, these studies indicate that molecular characterization of MDS phenotypes may require evaluation of purified hematopoietic progenitors in order to account for the differential effect of MDS-associated changes on specific hematopoietic progenitor populations. Disclosures Weissman: Amgen: Equity Ownership; Cellerant Inc.: Founder; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.
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McRae, Helen M., Alexandra L. Garnham, Yifang Hu, Matthew T. Witkowski, Mark A. Corbett, Mathew P. Dixon, Rose E. May et al. „PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia“. Blood 133, Nr. 16 (18.04.2019): 1729–41. http://dx.doi.org/10.1182/blood-2018-07-860726.
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Abstract Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and β receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.
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Carlo-Stella, C., und V. Rizzoli. „In Vitro Manipulation of Peripheral Blood Progenitor Cell Collections“. International Journal of Artificial Organs 21, Nr. 6_suppl (Mai 1998): 1–10. http://dx.doi.org/10.1177/039139889802106s01.
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Mobilized peripheral blood progenitor cells (PBPC) are increasingly used to reconstitute hematopoiesis in patients undergoing high-dose chemoradiotherapy. PBPC collections comprise a heterogeneous population containing both committed progenitors and pluripotent stem cells and can be harvested (i) in steady state, (ii) after chemotherapeutic conditioning, (iii) growth factor priming, or (iv) both. The use of PBPC has opened new therapeutic perspectives mainly related to the availability of large amounts of mobilized hematopoietic stem and progenitor cells. Extensive manipulation of the grafts, including the possibility of exploiting these cells as vehicles for gene therapy strategies, are now possible and will be reviewed.
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Law, Ping, Linda Traylor und Diether J. Recktenwald. „Cell analysis for hematopoietic stem/progenitor cell transplantation“. Cytometry 38, Nr. 2 (15.04.1999): 47–52. http://dx.doi.org/10.1002/(sici)1097-0320(19990415)38:2<47::aid-cyto1>3.0.co;2-2.
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Bhatia, Ravi, Khristine Van Heijzen, Ann Palmer, Asako Komiya, Marilyn L. Slovak, Karen L. Chang, Henry Fung et al. „Longitudinal Assessment of Hematopoietic Abnormalities After Autologous Hematopoietic Cell Transplantation for Lymphoma“. Journal of Clinical Oncology 23, Nr. 27 (20.09.2005): 6699–711. http://dx.doi.org/10.1200/jco.2005.10.330.
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Purpose Autologous hematopoietic cell transplantation (HCT) is being increasingly used as an effective treatment strategy for patients with relapsed or refractory Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL) but is associated with therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) as a major cause of nonrelapse mortality. The phenomenon of hematopoietic reconstitution after autologous HCT and the role of proliferative stress in the pathogenesis of t-MDS/AML are poorly understood. Patients and Methods Using a prospective longitudinal study design, we evaluated the nature and timing of alterations in hematopoietic progenitors and telomere length after HCT in patients undergoing autologous HCT at City of Hope Cancer Center (Duarte, CA). Results A significant reduction in primitive and committed progenitors was observed before HCT compared with healthy controls. Further profound and persistent reduction in primitive progenitors but only transient reduction in committed progenitors were seen after HCT. Primitive progenitor frequency in pre-HCT marrow and peripheral-blood stem cells predicted for primitive progenitor recovery after HCT. Shortening of telomere length was observed in marrow cells early after HCT, with subsequent restoration to pre-HCT levels. Patients within this cohort who developed t-MDS/AML had reduced recovery of committed progenitors and poorer telomere recovery, possibly indicating a functional defect in primitive hematopoietic cells. Conclusion Our studies suggest that hematopoietic regeneration after HCT is associated with increased proliferation and differentiation of primitive progenitors. Increased proliferative stress on stem cells bearing genotoxic damage could contribute to the pathogenesis of t-MDS/AML. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor and telomere recovery predict for increased risk of t-MDS/AML.
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Schreiter, Jessica, Kaomei Guan, Karim Nayernia, Bjoern Chapuy, Lorenz Truemper, Gerd Hasenfuss, Wolfgang Engel und Gerald G. Wulf. „Hematopoietic and Endothelial Progenitor Cells from Mouse Adult Testis Derived Stem Cell Lines.“ Blood 108, Nr. 11 (16.11.2006): 1686. http://dx.doi.org/10.1182/blood.v108.11.1686.1686.
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Abstract Adult mouse testis was recently found to harbour multipotent adult germline stem cells (maGSC) with the differentiation capacity along several mesodermal and epithelial lineages. We here addressed the hematopoietic potential from three multipotent germline stem cell lines in vitro. In the undifferentiated state, these cells expressed stem cell markers including Sca1, Thy1 and c-kit, but lacked markers of definitive hematopoiesis such as CD45. On exposure to in vitro culture conditions favouring hematopoietic differentiation, these cells gave rise to both primitive erythropoiesis with nucleated red blood cells expressing ζ-globin, and definitive hematopoiesis, forming β-globin positive erythroid and myeloid colonies in colony assays. On OP9 stroma cells, the maGSC lines formed mesoderm-like colonies, from which progenitor cell populations for either endothelial or hematopoietic differentiation were isolated. The differentiation patterns and efficiencies were similar to the results obtained from the mouse embryonic stem cell line MPI2. In summary, adult germline derived stem cell lines can be induced to recapitulate early embryonic hemangioblastic development and may thus serve as tools for studies on hematopoietic and angioblastic development as well as applications in regenerative medicine.
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Gordon, M. Y., und N. M. Blackett. „Reconstruction of the Hematopoietic System after Stem Cell Transplantation“. Cell Transplantation 7, Nr. 4 (Juli 1998): 339–44. http://dx.doi.org/10.1177/096368979800700401.
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The practice of hematopoietic stem cell transplantation to rescue patients from the myeloablative effects of chemo- or radiotherapy, or to replace defective hematopoiesis, is based on the assumption that hematopoietic stem cells in the graft have sufficient proliferative potential to supply mature blood cells for the remainder of the recipient's lifespan. However, the mechanism(s) whereby this is achieved are not well understood. Here we address the reconstruction of the hematopoietic system by considering the effects of stem cell and progenitor cell renewal and differentiation. We conclude that stem cell self-renewal is necessary for hematological recovery and that infused committed progenitor cells (CFU-GM) may contribute to the neutrophil count in the early posttransplant period.
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Pinto do Ó, Perpétua, Karin Richter und Leif Carlsson. „Hematopoietic progenitor/stem cells immortalized byLhx2 generate functional hematopoietic cells in vivo“. Blood 99, Nr. 11 (01.06.2002): 3939–46. http://dx.doi.org/10.1182/blood.v99.11.3939.
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Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whetherLhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor–dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell–deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell–deficient hosts.
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Li, Cheng, Daniel R. George, Nichole M. Helton, Jeffery M. Klco, Jacqueline L. Mudd und Timothy J. Ley. „Functional Early Hematopoietic Progenitor Cells Derived From Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells“. Blood 122, Nr. 21 (15.11.2013): 2421. http://dx.doi.org/10.1182/blood.v122.21.2421.2421.
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Abstract We have previously reported a method to produce early hematopoietic progenitor cells from C57BL/6J-derived mouse embryonic stem cells (mESCs). After co-culture on OP9 stromal cells for one week, four different C57Bl/6 mESC lines consistently differentiated into hematopoietic progenitors, as determined by immunophenotyping; we detected cells that mark as KLS (Lin- Kit+ Sca+), CMPs, GMPs, and MEPs (but not SLAMs) from all four lines. In addition, functional progenitors for erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) were detected after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). These findings were replicated using four different lots of fetal bovine serum, and with three different lots of OP9 cells from ATCC. We injected 1x106 “OP9-induced” progenitor cells retroorbitally into unconditioned NSG mice, and detected multilineage hematopoietic engraftment (myeloid compartments marked by CD34, CD11b, Kit, and Gr-1, lymphoid compartments marked by CD3 and B220, and erythroid compartments marked by Ter119) in the bone marrow and/or spleens of 10 out of 19 recipients at 3 months. Using the OP9 co-culture system, we have differentiated miPSC clones from three independent iPSC experiments, using an integrating polycistronic lentivirus expressing OCT4, SOX2, and KLF4 as the reprogramming vector. One set of miPSC clones was produced from mouse embryonic fibroblasts (MEFs) from pooled C57BL/6J embryos, and two sets were made from adult mouse fibroblasts derived from a single animal, producing 6, 12, and 12 independent iPS clones for analysis, respectively. All thirty clones had pluripotent features, as determined by alkaline phosphatase staining and immunophenotyping (SSEA1, Oct4, and Nanog). We have injected the OP9-induced progenitors derived from one miPSC clone into NSG mice; thus far, 2 out of 14 recipients have demonstrated engraftment in the peripheral blood. However, the efficiency of hematopoietic progenitor generation with OP9 induction (based on the immunophenotyping and progenitor assays noted above) was highly variable for miPSCs from all three experiments. Among all three sets of miPSC clones, 18/30 exhibited differentiation efficiencies comparable to wild-type B6 ESCs, 5/30 clones exhibited moderately reduced differentiation efficiencies, 5/30 clones exhibited markedly reduced differentiation efficiencies, and 2/30 clones (from two different iPSC experiments) did not produce any detectable hematopoietic progenitors with OP9 induction. These phenotypes were stable and highly reproducible. The 2 clones that did not yield any hematopoietic progenitors had robust pluripotency marks, and one that was injected into the hindflank of NSG mice produced cystic teratomas. We found that 2% DMSO pretreatment of mESCs for 24 hours prior to OP9 co-culture improved the differentiation efficiency of wild-type B6 ESCs by 50% (Chetty et al. Nature Methods 10(6):553-6, 2013), but it did not rescue the phenotype of miPSC clones that did not produce hematopoietic progenitors. We are currently performing exome sequencing on the 24 miPSC clones from the adult fibroblast reprogramming experiments to determine whether phenotypic heterogeneity is due to specific mutations in the iPSC clones (Young et al. Cell Stem Cell 10(5):570-82, 2012). In summary, we have developed a simple system to derive functional early hematopoietic progenitor cells from mouse embryonic stem cells and/or induced pluripotent stem cells. OP9-induced progenitor cells engraft into NSG mice without the need for forced expression of HoxB4 (Wang et al. Proc Natl Acad Sci USA 102(52):19081-6, 2005). We have detected functional heterogeneity in miPSC clones derived from the same parental cells, which could be due to genetic variation in the founding cell from which each clone was derived, to different integration sites of the OSK lentivirus in each clone, or to as yet undefined epigenetic mechanisms. Exome sequencing may help to resolve this issue. Regardless, this approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines and/or miPSC lines derived from genetically altered mice. Disclosures: No relevant conflicts of interest to declare.
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Hirabayashi, Yoko, Byung-Il Yoon, Isao Tsuboi, Yan Huo, Yukio Kodama, Jun Kanno, Thomas Ott, James E. Trosko und Tohru Inoue. „The Cell Cycle in Hematopoietic Stem Cells, Lin−/C-kit+/Sca1+ Fraction, Is Regulated by Connexin 32, Specifically Expressed in the Stem Cell Compartment“. Blood 112, Nr. 11 (16.11.2008): 4775. http://dx.doi.org/10.1182/blood.v112.11.4775.4775.
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Abstract Connexin (Cx) functions in the organization of cell-cell communication via gap junctions in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control, cellular differentiation, apoptosis and the synchronization of electrotonic and metabolic functions. As Cxs are essential molecules for multicellular organisms, Cxs that organize cell-cell communication within the hematopoietic progenitor cell compartment are surmised to be present in bone marrow tissue. Recently, we first found that Cx32 is only Cx molecule expressed in the bone marrow in wild-type mice by means of comparison with Cx32-knockout (KO) mice, studied by a reverse biological approach. Cx32 is specifically expressed in primitive hematopoietic stem/progenitor cells, i.e., the lineage marker-negative (Lin−)/c-kit positive (c-kit+)/stem cell antigen-1-positive (Sca1+) (=LKS) fraction, and likely playing a role of restoration of stem/progenitor cell-quiescence, thereby preventing primitive stem cells from exhaustion. In this study, we present results on cell cycle analyses with respect to the function of Cx32; one for colony-forming progenitors by the method evaluating the cycling progenitor cells using incorporation of bromodeoxyuridine (BrdUrd) followed by ultraviolet-light cytocide and the other for primitive progenitor cells using a cell sorter with bioactive AT-rich DNA-binding dye Hoechst 33342. In the colonization assay on CFU-S-13 (primitive hematopoietic progenitor cells), the incorporation of BrdUrd starts from a higher percentage with rapid increase in Cx32-KO mice, suggesting suppression of cell cycle in these primitive hematopoietic progenitor cells with Cx32-mediated cell-cycle regulation in the wild-type steady state. This suppression may be attenuated in CFU-S-9, a differentiated progenitor cell compartment. The progenitor cells assayed by in vitro colonization on CFU-GM also showed accelerated cell cycle in the Cx32-KO mice. Following the incorporation of Hoechst 33342, the lineagedepleted bone marrow cells were analyzed by flow cytometry. The population sizes of the LKS fraction obtained were 0.052% in the Cx32-KO bone marrow cells and 0.035% in the wild-type bone marrow cells (p=0.0458<0.05). The lineage-depleted bone marrow cells were analyzed their cell-cycle patterns by flow cytometry, and the G0/G1 was calculated for the LKS fractions in both, the Cx32-KO mice and wild-type mice. The percentage of G0/G1 calculated for the LKS fractions were significantly lower in the Cx32-KO mice than those in wild-type mice (60.6% vs. 87.9% for Cx32-KO vs. wild-type; p=0.001). The results suggest that Cx32 may have suppressive functions on the hematopoietic stem cell compartment, the LKS fraction, under the physiological function of Cx32. The Cx32 in the wild-type mice is, thus considered to be expressed in the primitive hematopoietic stem/progenitor cells to prevent from their exhaustion.
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Hewitt, Kyle, Kirby D. Johnson, Duk-Hyoung Kim, Prithvia Devadas, Rajalekshmi Prathibha, Chandler Zuo, Colin Dewey et al. „Cistrome Control of Hematopoieitic Stem/Progenitor Cell Function“. Blood 126, Nr. 23 (03.12.2015): 43. http://dx.doi.org/10.1182/blood.v126.23.43.43.
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Abstract Cis-regulatory mechanisms control chromatin structure and cellular identity. At the GATA2 locus, two cis-elements are linked to human pathologies, including a primary immunodeficiency (MonoMAC syndrome) associated with multiple complex phenotypes, myelodysplastic syndrome, and acute myeloid leukemia (AML). Mutations that disrupt the function of an intronic GATA2 +9.5 element cause MonoMAC syndrome, while an inversion that relocates the distal GATA2 -77 element to the EVI1 locus induces AML. The +9.5 and -77 cis-elements are GATA-2-occupied and confer context-dependent enhancer activities in select hematopoietic cell types in vivo. In knockout mouse models, the Gata2 +9.5 cis-element is required for hematopoietic stem cell (HSC) genesis, whereas the Gata2 -77 cis-element governs a unique sector of the myeloid progenitor cell transcriptome without impacting HSC genesis. Three other GATA-2-occupied cis-elements (-1.8, -2.8 and -3.9) were not individually required for hematopoietic development, and had relatively mild effects on Gata2 expression; the -1.8 site was required to maintain Gata2 repression in late-stage erythroblasts, the -2.8 conferred maximal Gata2 expression, and the -3.9 had no effect on Gata2 expression. We predict that additional cis-elements exist in the genome with functions resembling the +9.5 and -77, and their analysis will provide important mechanistic and biological insights. We utilized the known properties of Gata2 cis-elements as learning tools to identify prospective constituents of a hematopoietic stem/progenitor cell (HSPC) regulatory cistrome genome-wide. Using sequence attributes shared with the critically-important +9.5 element, namely a CATCTG-8bp spacer-AGATAA, we generated a list of 797 candidate cis-elements ("+9.5-like" elements). This list was prioritized using chromatin occupancy by GATA-2 and Scl/TAL-1, among others, chromatin accessibility, evolutionary conservation, and histone modifications in a multitude of biologically-relevant cell types. Gene editing was used to delete three high-ranked elements (Samd14 +2.5, Bcl2l1 +12.2, and Dapp1 +23.5), revealing their importance for transcriptional activation, GATA-2 occupancy and chromatin accessibility, while deletion of two low-ranked elements (Mrps9 +17.6 and Mgmt +182) had no effect on gene transcription. One such cis-element (Samd14 +2.5) resided in Samd14, a gene with undescribed biological function. Samd14 has a conserved sterile α-motif and coiled-coil domain, and is highly expressed in hematopoietic progenitors and differentiated progeny. Mouse knockout of the Samd14 +2.5 element dramatically lowered expression of Samd14 in hematopoietic progenitors. We conducted loss-of-function analysis to elucidate Samd14 function in lineage-depleted (Lin-) E14.5 fetal liver cells infected with control or Samd14 shRNA-expressing retrovirus. In a CFU assay, Samd14 knockdown reduced BFU-E and CFU-GM colonies 3.4-fold. Early erythroid precursor R1 (CD71low, Ter119-) and R2 (CD71high, Ter119-) cell populations decreased ~2-fold, concomitant with increases in more mature R3 and R4/5 populations (Ter119+). In R1/R2 cells, Samd14 knockdown reduced surface c-Kit expression by 1.6-fold and prevented Stem Cell Factor/c-Kit activation of AKT. Cellular deficits resulting from Samd14 knockdown could be rescued by c-Kit. In -77-/- common myeloid progenitors, Samd14 was ~20-fold downregulated. Thus, the importance of Samd14 and the Samd14 +2.5 element on progenitor function and SCF/c-Kit signaling validates our strategy for identifying cis-elements relevant for hematopoiesis. Our findings demonstrate that +9.5-like elements control cell signaling (Samd14 +2.5) and apoptosis (Bcl2l1 +12.2), and we predict that additional cistrome constituents will control these and other important HSPC processes. I will discuss the mechanistic and biological properties of additional cis-elements analyzed from a cohort of 68 GATA-2-occupied elements and general principles arising from the HSPC cistrome analysis, which provide unique insights into the control of hematopoiesis and GATA-2-linked pathologies. Disclosures No relevant conflicts of interest to declare.
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Zeng, Yi, Karl Staser, Keshav Mohan Menon, Su-jung Park, Muithi Mwanthi, Li Jiang und D. Wade Clapp. „Ezrin Regulates Hematopoietic Stem/Progenitor Cell Motility“. Blood 118, Nr. 21 (18.11.2011): 1282. http://dx.doi.org/10.1182/blood.v118.21.1282.1282.
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Abstract Abstract 1282 Ezrin is a member of the ERM (ezrin, moesin and radixin) protein family that links plasma membrane proteins to the actin cytoskeleton. Ezrin in other in vitro cell systems has been hypothesized to participate in cell-cell contact and could have a role in stem/ progenitor cell mobilization and adhesion. To test this hypothesis, we crossed ezrinflox/flox mice with Mx1 cre transgenic mice to generate an inducible ezrin knock out mouse model. Inducible disruption of the ezrin gene in hematopoietic cells was achieved by the administration of polyIC. Ezrin knock out HSPCs exhibited a 30–40% decrease in baseline and chemokine stromal cell-derived factor-1 (SDF-1) stimulated motility in transwell migration assays in vitro. In addition, loss of ezrin led to a 60% decrease in the homing capacity of HSPCs in lethally irradiated recipient mice following transplantation. There was a 40–55% decrease in colony forming cells in peripheral blood and spleen of the mice following ezrin knock out, suggesting that ezrin knock out HSPCs may be deficient in egressing out of the bone marrow. To further understand the cause of the impaired motility of ezrin knock out HSPCs, we examined F-actin level of HSPCs at baseline and in response to SDF-1. Ezrin knock out HSPCs displayed 1.5 to 2 fold higher level of F-actin at baseline when compared with wild type cells. Following stimulation with SDF-1, wild type HSPCs that migrated to the bottom compartment of the transwell demonstrated a 2 time greater decrease in F-actin level when compared with ezrin knock out cells, suggesting that ezrin may participate in the regulation of F-actin depolymerization in HSPCs. In summary, we demonstrate that ezrin modulates HSPC migration and homing likely through its regulation on F-actin organization. Disclosures: No relevant conflicts of interest to declare.
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Colvin, Gerald A., Jean-Fran�ois Lambert, Brian E. Moore, Jane E. Carlson, Mark S. Dooner, Mehrdad Abedi, Jan Cerny und Peter J. Quesenberry. „Intrinsic hematopoietic stem cell/progenitor plasticity: Inversions“. Journal of Cellular Physiology 199, Nr. 1 (2004): 20–31. http://dx.doi.org/10.1002/jcp.10436.
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Li, Yan, Shi Chen, Jin Yuan, Yanzhu Yang, Jingling Li, Jin Ma, Xiaohua Wu et al. „Mesenchymal stem/progenitor cells promote the reconstitution of exogenous hematopoietic stem cells in Fancg−/− mice in vivo“. Blood 113, Nr. 10 (05.03.2009): 2342–51. http://dx.doi.org/10.1182/blood-2008-07-168138.
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AbstractFanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow failure and complex congenital anomalies. Although mutations in FA genes result in a characteristic phenotype in the hematopoietic stem/progenitor cells (HSPCs), little is known about the consequences of a nonfunctional FA pathway in other stem/progenitor cell compartments. Given the intense functional interactions between HSPCs and the mesenchymalmicroenvironment, we investigated the FA pathway on the cellular functions of murine mesenchymal stem/progenitor cells (MSPCs) and their interactions with HSPCs in vitro and in vivo. Here, we show that loss of the murine homologue of FANCG (Fancg) results in a defect in MSPC proliferation and in their ability to support the adhesion and engraftment of murine syngeneic HSPCs in vitro or in vivo. Transplantation of wild-type (WT) but not Fancg−/− MSPCs into the tibiae of Fancg−/− recipient mice enhances the HSPC engraftment kinetics, the BM cellularity, and the number of progenitors per tibia of WT HSPCs injected into lethally irradiated Fancg−/− recipients. Collectively, these data show that FA proteins are required in the BM microenvironment to maintain normal hematopoiesis and provide genetic and quantitative evidence that adoptive transfer of WT MSPCs enhances hematopoietic stem cell engraftment.
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Zhang, Yiyue, Hao Jin, Li Li, F. Xiao-Feng Qin und Zilong Wen. „cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish hematopoiesis“. Blood 118, Nr. 15 (13.10.2011): 4093–101. http://dx.doi.org/10.1182/blood-2011-03-342501.
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Abstract The establishment of the HSC pool in vertebrates depends not only on the formation and the propagation of these stem cells but also on their proper trafficking among the defined hematopoietic organs. However, the physiologic mechanisms that regulate HSC mobilization remain elusive. Through analysis of the zebrafish cmyb mutant cmybhkz3, we show that the suppression of cMyb function abrogates larval and adult hematopoiesis, with concomitant accumulation of hematopoietic stem/progenitor cells (HSPCs) in their birthplace, the ventral wall of the dorsal aorta (VDA). Cell tracking and time-lapse recording reveal that the accumulation of HSPCs in cmybhkz3 mutants is caused by the impairment of HSPC egression from the VDA. Further analysis demonstrates that the HSPC migratory defects in cmybhkz3 mutants are at least partly because of adversely elevated levels of chemokine stromal cell-derived factor 1a (Sdf1a). Our study reveals that cMyb plays a hitherto unidentified role in dictating physiologic HSPC migration by modulating Sdf1a signaling.
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Hsu, Jingmei, Hsuan-Ting Huang, Chung-Tsai Lee, Shuqian Yu, Leonard I. Zon und Nancy A. Speck. „Chromatin Remodeling Enzyme CHD7 Negatively Regulate Hematopoietic Stem Cell Function“. Blood 122, Nr. 21 (15.11.2013): 2413. http://dx.doi.org/10.1182/blood.v122.21.2413.2413.
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Abstract ATP-dependent chromatin remodeling enzymes alter histone/DNA interactions, and are involved in the regulation of transcription, chromosome segregation, DNA replication and repair. We identified Chromodomain Helicase DNA binding protein 7 (CHD7) as a negative regulator of hematopoietic stem cell function. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Although several cases of T and B cell immunodeficiency in a subset of CHARGE syndrome patients have been reported, no previous role for CHD7 in hematopoiesis had been proposed. We show that morpholino knockdown of Chd7 in zebrafish embryos results in an increased number of runx1 and c-myb expressing hematopoietic stem and progenitor cells in the aorta/gonad/mesonephros (AGM) region, and this effect is cell autonomous as determined by blastula transplantation. Heterozygous germline Chd7 deletion in mice also results in an increased number of phenotypic hematopoietic stem and progenitor cells (Runx1+c-kit+CD31+) in the AGM region. Downstream lineages such as myeloid and erythroid cells are expanded in zebrafish, and similarly conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre results in myeloid lineage expansion and increased granulocyte/monocyte progenitors. Consistent with these results, microarray analysis of murine CD48- CD150+ Lin- Sca-1+ c-kit+ (SLAM LSK) phenotypic long term repopulating HSCs shows up-regulation of genes in several subclasses of the myeloid lineages. Interestingly, although CHD7 deficient mouse bone marrow had a normal frequency of SLAM LSK cells, it had a two-fold higher frequency of functional LT-HSCs as determined by whole bone marrow, purified LSK and SLAM LSK limiting dilution transplants. ChIP-seq performed in human CD34+ hematopoietic stem and progenitor cells show CHD7 localizes to genes encoding many key hematopoietic transcription factors including MYB and RUNX1. The most abundant transcription factor motif under CHD7 genomic binding sites is a RUNX motif, indicating that CHD7 and Runx1 function together. We show CHD7, Runx1 and c-Myb interact both physically and genetically. CHD7 function in hematopoietic stem cells is dependent on both Runx1 and c-Myb since the increase in hematopoiesis in fish upon morpholino knockdown of Chd7 is abolished when either Runx1 or c-Myb is mutated. In summary, our study identifies CHD7 as a novel evolutionarily conserved negative epigenetic regulator of HSCs and progenitors through its interaction with Runx1 and c-Myb. Disclosures: No relevant conflicts of interest to declare.
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Ribeiro-Filho, Antonio Carlos, Débora Levy, Jorge Luis Maria Ruiz, Marluce da Cunha Mantovani und Sérgio Paulo Bydlowski. „Traditional and Advanced Cell Cultures in Hematopoietic Stem Cell Studies“. Cells 8, Nr. 12 (12.12.2019): 1628. http://dx.doi.org/10.3390/cells8121628.
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Hematopoiesis is the main function of bone marrow. Human hematopoietic stem and progenitor cells reside in the bone marrow microenvironment, making it a hotspot for the development of hematopoietic diseases. Numerous alterations that correspond to disease progression have been identified in the bone marrow stem cell niche. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells determine the balance between the proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation. However, our understanding of human hematopoiesis and the associated niche biology remains limited due to accessibility to human material and the limits of in vitro culture models. Traditional culture systems for human hematopoietic studies lack microenvironment niches, spatial marrow gradients, and dense cellularity, rendering them incapable of effectively translating marrow physiology ex vivo. This review will discuss the importance of 2D and 3D culture as a physiologically relevant system for understanding normal and abnormal hematopoiesis.
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Paz, Helicia, Maureen R. Lynch, Clifford W. Bogue und Judith C. Gasson. „The homeobox gene Hhex regulates the earliest stages of definitive hematopoiesis“. Blood 116, Nr. 8 (26.08.2010): 1254–62. http://dx.doi.org/10.1182/blood-2009-11-254383.
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Abstract The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41+ and CD45+ hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex−/− ES-derived progeny accumulate as CD41+ and CD41+c-kit+ cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex−/− ES-derived progeny display a significantly reduced ability to develop into mature CD45+ hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex−/− CD41+CD45−c-kit+ hematopoietic progenitors accumulated in the G2 phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.
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Kundu, Mondira, Amy Chen, Stacie Anderson, Martha Kirby, LiPing Xu, Lucio H. Castilla, David Bodine und Pu Paul Liu. „Role of Cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb-MYH11“. Blood 100, Nr. 7 (01.10.2002): 2449–56. http://dx.doi.org/10.1182/blood-2002-04-1064.
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Core-binding factor β (CBFβ) and CBFα2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFβ or CBFα2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFβ and CBFα2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion ofCBFB with MYH11, the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for aCbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb−/−embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfβ in hematopoietic cells. In midgestation embryos, Cbfβ was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and thatCbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfβ was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfβ was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.
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Suh, Hyung Chan, Ming Ji, John Gooya, Michael Lee, Kimberly Klarmann und Jonathan R. Keller. „Id1 Provides a Proper Hematopoietic Progenitor Niche Function“. Blood 112, Nr. 11 (16.11.2008): 2427. http://dx.doi.org/10.1182/blood.v112.11.2427.2427.
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Abstract Development of hematopoietic stem cells (HSC) and their progeny is maintained by the interaction with cells in the microenvironment. In addition to hematopoietic cells, Id1 is expressed in stromal cells known to support hematopoiesis, and is involved in cell proliferation, differentiation and senescence. Therefore, to investigate the role of Id1 in hematopoiesis, we examined hematologic phenotypes of Id1−/− mice. In this study, we found increased neutrophils and macrophages, and decreased B cells and platelets in peripheral blood, and decreased BM cellularity. While the percentages of hematopoietic stem cells (HSC) in Id1−/− mice were increased relative to the Id1+/+ mice, their total numbers and function appeared normal. For example, Id1 was not required for self-renewal or repopulation of HSC. In contrast, we found that there were increased numbers of hematopoietic progenitor cells (HPC) in S phase of cell cycle in Id1−/− mice BM, suggesting that the loss of Id1 within HPC promotes proliferation. However, purified Id1−/− HPC had the same proliferation potential as Id1+/+ HPC when cultured in vitro. In transplantation experiments, we proved that BM microenvironment in Id1−/− mice is defective by showing that the Id1+/+ HSC showed impaired hematopoietic development in Id1−/− mice, while the Id1−/− HSC had normal repopulation potential in an Id1+/+ microenvironment. In agreement with these findings, Id1−/− BM stromal cell cultures supported enhanced proliferation of hematopoietic progenitors. Furthermore, quantitative PCR showed that SCF, M-CSF, OPN, SDF-1 and TGF-α mRNA expression was decreased in Id1−/− stromal cells relative to Id1+/+ stromal cells, while G-CSF, GM-CSF, and VEGF mRNA expression was significantly increased. Id1−/− BM showed decreased number of mesenchymal stem/progenitor cells. Thus, Id1 does not play a role in maintaining HSC, but is involved in regulating hematopoietic progenitor niche. Funded by NCI contract No. N01-CO-12400.
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Manz, Markus G. „Microbial Impact on Hematologic Homeostasis“. Blood 120, Nr. 21 (16.11.2012): SCI—40—SCI—40. http://dx.doi.org/10.1182/blood.v120.21.sci-40.sci-40.
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Abstract Abstract SCI-40 During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation. Disclosures: No relevant conflicts of interest to declare.
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Qian, Hong, Karl Tryggvason, Sten Eirik Jacobsen und Marja Ekblom. „Contribution of α6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with α4 integrins“. Blood 107, Nr. 9 (01.05.2006): 3503–10. http://dx.doi.org/10.1182/blood-2005-10-3932.
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The laminin receptor integrin α6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti–integrin α6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice, with a corresponding retention of progenitors in blood. Remarkably, the anti–integrin α6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells, studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against α4 integrin, studied in parallel. Furthermore, the anti–integrin α6 and α4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti–integrin α6 antibodies, in contrast to antibodies against α4 integrin, did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin α6 receptor during hematopoietic stem and progenitor cell homing and collaboration of α6 integrin with α4 integrin receptors during homing of short-term stem cells.
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Li, Hojun, Vincent Butty, Guinevere Connelly, Vivian Morris, George Q. Daley, Jennifer Whangbo, Edroaldo Lummertz da rocha und Robert Grant Rowe. „Single Cell Resolution Mapping of Hematopoietic Stem and Progenitor Cell States throughout Human Life“. Blood 136, Supplement 1 (05.11.2020): 31. http://dx.doi.org/10.1182/blood-2020-136380.
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The hematopoietic system continuously generates and replenishes the supply of circulating blood cells from embryonic life throughout the entirety of human lifespan. Studies in mouse development have shown that the repertoire of mature blood cell types produced changes dramatically during development and aging, with hematopoietic stem and progenitor cells (HSPCs) adapting their output to meet age-specific physiologic needs. In humans, it is presumed that age-dependent changes in the production of mature blood lineages underlie the tendency of blood disorders to skew toward certain ages of onset. The observations that mature cell output changes throughout life but mechanisms of terminal hematopoietic differentiation within each lineage remain consistent suggest that age-specific hematopoietic states are programmed at the level of HSPCs. Although the developmental changes occurring in mouse hematopoiesis are well documented, the specific changes in human HSPC ontogeny occurring during prenatal development and postnatal aging from newborn, through childhood, and into adulthood are completely unknown. We hypothesized that temporal changes in human hematopoiesis are mediated by age-specific, occasionally transient, HSPC states and that mechanisms of HSPC lineage commitment change over time in order to meet the changing physiologic demands of the developing and aging human. To test this hypothesis, we comprehensively profiled human HSPC cell states from human fetal hematopoiesis through adulthood using single cell RNA sequencing (scRNAseq). We obtained CD34+ HSPCs from 14 different human donors covering a range of ages from first and second trimester fetal liver, umbilical cord blood, and pediatric and adult bone marrow. We obtained high quality sequencing data on a total of 38,873 individual HSPCs after filtering out apoptotic cells, hepatocytes, stromal cells, and endothelial cells. We then identified differentially expressed genes and performed dimensionality reduction and uniform manifold approximation and projection followed by Louvain clustering to identify 32 distinct cell types encompassing primitive stem and multipotent progenitor cells as well as committed progenitors in the myeloid, erythroid and lymphoid lineages. We found age-specific alterations in hematopoietic differentiation trajectories, particularly in the myeloid lineages. Additionally, we discovered an HSC, emerging in mid-gestation and diminishing at birth, with a characteristic immunophenotype and megakaryocyte (Meg) differentiation bias. Differential gene expression analysis of the Meg-biased fetal HSC identified increased expression of the MYB transcription factor relative to other HSCs, potentially illuminating a mechanistic role for MYB in driving megakaryocyte-erythroid progenitor (MEP) differentiation of fetal HSCs, that is distinct from the role of MYB in conferring erythroid differentiation bias at the MEP stage itself. Finally, we used this atlas of human developmental hematopoiesis to map lineage commitment and progenitor states in leukemia, highlighting the translational applicability of this resource. In summary, we have compiled the first comprehensive atlas of HSPCs across human development and aging. This resource allowed us to identify age-specific differentiation trajectories in human hematopoiesis and enabled identification of a Meg-biased fetal-specific HSC. Our research reveals novel mechanisms of maturation and aging of the human hematopoietic system, uncovers transient HSPC states and differentiation trajectories, and establishes a framework for interrogating the differentiation and maturation states of human leukemias that can likely be applied to other blood diseases. As a resource, we expect that this atlas will broadly impact the study of human hematopoietic development and aging, developmental immunology, and the pathophysiology of age-biased blood diseases. Disclosures No relevant conflicts of interest to declare.
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van Dijken, PJ, J. Wimperis, JM Crawford und JL Ferrara. „Effect of graft-versus-host disease on hematopoiesis after bone marrow transplantation in mice“. Blood 78, Nr. 10 (15.11.1991): 2773–79. http://dx.doi.org/10.1182/blood.v78.10.2773.bloodjournal78102773.
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We have examined the effect of graft-versus-host disease (GVHD) on the reconstitution of donor hematopoiesis in a murine bone marrow transplant (BMT) model of GVHD to minor histocompatibility antigens. GVHD had no effect on peripheral blood counts, which normalized by 1 month after BMT, and did not affect numbers of hematopoietic progenitors in the BM, which remained decreased in all transplant recipients. Donor stem cells (colony-forming unit-spleen day 8) and stem cell self-renewal remained low in all mice for 5 months after transplant, but GVHD further damaged the stem cell compartment. Peripheral counts 1 month after transplant were supported by increased numbers of stem cells in cycle and increased splenic hematopoiesis. However, GVHD altered the pattern of extramedullary hematopoiesis, causing dramatically decreased activity in the spleen and increased activity in the liver. We conclude that GVHD further decreases hematopoietic reserve and causes damage to the donor stem cell compartment during hematopoietic reconstitution after transplant, despite unaffected progenitor frequencies and peripheral blood counts.