An Early Myelosuppression in the Acute Mouse Sepsis Is Partly Outcome-Dependent

Adult hematopoietic stem and progenitor cells (HSPCs) respond to bacterial infections by expansion to myeloid cells. Sepsis impairs this process by suppressing differentiation of stem cells subsequently contributing to an ineffective immune response. Whether the magnitude of HSPCs impairment in sepsis is severity-dependent remains unknown. This study investigated dynamics of the HSPC immune-inflammatory response in the bone marrow, splenic, and blood compartments in moribund and surviving septic mice. The 12-week-old outbred CD-1 female mice (n=65) were subjected to a cecal ligation and puncture (CLP) sepsis, treated with antibiotics and fluid resuscitation, and stratified into predicted-to-die (P-DIE) and predicted-to-survive (P-SUR) cohorts for analysis. CLP strongly reduced the common myeloid and multipotent progenitors, short- and long-term hematopoietic stem cell (HSC) counts in the bone marrow; lineage−ckit+Sca-1+ and short-term HSC suppression was greater in P-DIE versus P-SUR mice. A profound depletion of the common myeloid progenitors occurred in the blood (by 75%) and spleen (by 77%) of P-DIE. In P-SUR, most common circulating HSPCs subpopulations recovered to baseline by 72 h post-CLP. Analysis of activated caspase-1/-3/-7 revealed an increased apoptotic (by 30%) but not pyroptotic signaling in the bone marrow HSCs of P-DIE mice. The bone marrow from P-DIE mice revealed spikes of IL-6 (by 5-fold), CXCL1/KC (15-fold), CCL3/MIP-1α (1.7-fold), and CCL2/MCP-1 (2.8-fold) versus P-SUR and control (TNF, IFN-γ, IL-1β, -5, -10 remained unaltered). Summarizing, our findings demonstrate that an early sepsis-induced impairment of myelopoiesis is strongly outcome-dependent but varies among compartments. It is suggestive that the HSCPC loss is at least partly due to an increased apoptosis but not pyroptosis.

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Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

Blood ◽

10.1182/blood.v124.21.5117.5117 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5117-5117

Author(s):

Valentina Giai ◽

Elona Saraci ◽

Eleonora Marzanati ◽

Christian Scharenberg ◽

Monica De Stefanis ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Healthy Volunteers ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Progenitor Compartment

Abstract BACKGROUND: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01). No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation. A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

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Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽

10.1182/blood-2020-137426 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Maria Rosa Lidonnici ◽

Giulia Chianella ◽

Francesca Tiboni ◽

Matteo Barcella ◽

Ivan Merelli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Gene Set Enrichment Analysis ◽

Lineage Commitment ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Hematopoietic Stem ◽

Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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The Disease-Related Bone Marrow Microenvironment Alters Hematopoietic Stem and Progenitor Function in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v118.21.2898.2898 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2898-2898

Author(s):

Ingmar Bruns ◽

Ron-Patrick Cadeddu ◽

Ines Brückmann ◽

Sebastian Buest ◽

Julia Fröbel ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Microenvironment ◽

Tgf Beta ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Stem And Progenitor Cells ◽

Related Bone Marrow

Abstract Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

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Abstract 32: Hypercholesterolemia-induced Priming of Hematopoietic Stem and Progenitor Cells Aggravates Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a32 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Tom Seijkens ◽

Marten A. Hoeksema ◽

Linda Beckers ◽

Svenja Meiler ◽

Esther Smeets ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Myeloid Cells ◽

Blood Monocytes ◽

Blood Leukocytes ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

Stem And Progenitor Cells ◽

Inflammatory Phenotype

During homeostasis hematopoietic stem and progenitor stem cells (HSPCs) give rise to lymphoid and myeloid cells as well as platelets and erythrocytes. However, during chronic inflammatory conditions hematopoiesis is often skewed towards the myeloid lineage, thereby potentially aggravating the ongoing inflammation. Here we investigated the effects of hypercholesterolemia on HSPCs during atherogenesis. Hypercholesterolemia increased HSPCs, defined as Lin - Sca1 + cKit - , in the bone marrow (BM) of LDLr -/- mice by 253.1%. The number of granulocyte-monocyte progenitors, BM granulocytes and BM monocytes was increased by 18.1%, 34.8% and 13.2%, respectively. In accordance, the myeloid colony forming potential of hypercholesterolemic BM was increased by 25.8%. Peripheral blood monocytes and granulocytes were increased by 203.0% and 161.1%, respectively. Competitive bone marrow transplantations (cBMT) in which we compared the effects of normo- vs. hypercholesterolemia primed HSPCs confirmed that the hypercholesterolemic microenvironment activates HSPCs, as reflected by a 26.5% increased reconstitution of peripheral blood leukocytes 10 weeks after the cBMT. Moreover, hypercholesterolemia-primed, and not normocholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C high monocytes, even under long-term normocholesterolemic conditions in the recipient animals. cBMT demonstrated that hypercholesterolemia-induced activation of HSPCs increased atherosclerosis in LDLr -/- mice by 122.1% and increased CD45.1 + plaque leukocytes by 76.1%. Macrophages differentiated from hypercholesterolemia-primed BM produced increased levels of TNFα (+21.3%), IL6 (+17.4%) and MCP1 (+10.5%) compared to their normocholesterolemic counterparts, demonstrating that hypercholesterolemia-induced priming of HSPCs increased the inflammatory phenotype of their mature offspring. These results demonstrate that hypercholesterolemia-induced priming of HSPCs aggravates atherosclerosis by skewing hematopoiesis towards the pro-inflammatory myeloid lineages. Inhibition of this pro-inflammatory differentiation pathway on HSPC level has the potential to reduce atherosclerosis.

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Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽

10.1182/blood.v110.11.2237.2237 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2237-2237

Author(s):

Ravindra Majeti ◽

Christopher Y. Park ◽

Irving L. Weissman

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Human Cord Blood ◽

Self Renewal ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

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DNA Methyltransferase 1 Is Essential for and Uniquely Regulates Hematopoietic Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.392.392 ◽

2009 ◽

Vol 114 (22) ◽

pp. 392-392 ◽

Cited By ~ 1

Author(s):

Jennifer J. Trowbridge ◽

Jonathan W. Snow ◽

Jonghwan Kim ◽

Stuart H. Orkin

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Progenitor Cells ◽

Dna Methyltransferase ◽

Adult Stem Cells ◽

Global Gene Expression ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Stem And Progenitor Cells

Abstract Abstract 392 DNA methylation is essential for development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we crossed Dnmt1fl/fl mice with Mx1-Cre transgenic mice, and by injection of poly(I)-poly(C) we selectively deleted Dnmt1 in the hematopoietic system (Dnmt1Δ/Δ). In Dnmt1Δ/Δ mice, peripheral blood counts and mature multilineage composition of the bone marrow was found to be normal. Interestingly, specific defects were observed in Dnmt1Δ/Δ HSC self-renewal as assessed by long-term and secondary competitive transplantation, in retention of Dnmt1Δ/Δ HSCs within the bone marrow niche, and in the ability of Dnmt1Δ/Δ HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had unique impact on myeloid progenitor cells (including common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythrocyte progenitors), regulating their cycling and transcriptional lineage fidelity. To determine the molecular mechanisms underlying these defects, we performed global gene expression microarray analysis and bisulfite sequencing of select loci (IAP, Car1, and Gata1) in purified populations of control and Dnmt1Δ/Δ long-term HSCs, short-term HSCs/multipotent progenitor cells, and myeloid restricted progenitor cells. Through this approach, we demonstrate that loss of Dnmt1 has cell type-specific molecular consequences. For example, demethylation of the Car1 and Gata1 loci in Dnmt1Δ/Δ long-term HSCs is not sufficient to activate gene transcription, whereas demethylation of these genes in Dnmt1Δ/Δ short-term HSCs is associated with activation of transcription. In Dnmt1Δ/Δ myeloid restricted progenitor cells, we observed increases in DNA methylation at specific gene loci such as Car1, indicating that methylation can be established by other methyltransferases in the absence of Dnmt1. Our global gene expression microarray analysis clearly demonstrates that Dnmt1 regulates expression of distinct gene families in these closely related, primitive hematopoietic populations. We were unable to attribute specific functional defects in Dnmt1Δ/Δ hematopoietic stem and progenitor cells to alterations in expression of previously characterized genes, supporting the existence of novel, uncharacterized regulators of HSC and progenitor cell function to be explored from candidates in our data set. We conclude that maintenance methylation induced by Dnmt1 appears to be especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of long-term HSCs to multipotent progenitors, multipotent progenitors to myeloid restricted progenitors, stem cell mobilization, and regulating cell cycle entry. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment. Furthermore, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. Disclosures: No relevant conflicts of interest to declare.

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AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v90.12.5002 ◽

1997 ◽

Vol 90 (12) ◽

pp. 5002-5012 ◽

Cited By ~ 1209

Author(s):

Amy H. Yin ◽

Sheri Miraglia ◽

Esmail D. Zanjani ◽

Graca Almeida-Porada ◽

Makio Ogawa ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Umbilical Vein ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

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Geographic clonal tracking in macaques provides insights into HSPC migration and differentiation

Journal of Experimental Medicine ◽

10.1084/jem.20171341 ◽

2017 ◽

Vol 215 (1) ◽

pp. 217-232 ◽

Cited By ~ 20

Author(s):

Chuanfeng Wu ◽

Diego A. Espinoza ◽

Samson J. Koelle ◽

E. Lake Potter ◽

Rong Lu ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Nk Cells ◽

Local Production ◽

Developmental Pathway ◽

Hematopoietic Stem ◽

Cell Staining ◽

Multipotent Progenitors ◽

Stem And Progenitor Cells ◽

Over Time

The geographic distribution of hematopoiesis at a clonal level is of interest in understanding how hematopoietic stem and progenitor cells (HSPCs) and their progeny interact with bone marrow (BM) niches during regeneration. We tagged rhesus macaque autologous HSPCs with genetic barcodes, allowing clonal tracking over time and space after transplantation. We found marked geographic segregation of CD34+ HSPCs for at least 6 mo posttransplantation, followed by very gradual clonal mixing at different BM sites over subsequent months to years. Clonal mapping was used to document local production of granulocytes, monocytes, B cells, and CD56+ natural killer (NK) cells. In contrast, CD16+CD56− NK cells were not produced in the BM, and in fact were clonally distinct from multipotent progenitors producing all other lineages. Most surprisingly, we documented local BM production of CD3+ T cells early after transplantation, using both clonal mapping and intravascular versus tissue-resident T cell staining, suggesting a thymus-independent T cell developmental pathway operating during BM regeneration, perhaps before thymic recovery.

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Identification of Lin–Sca1+kit+CD34+Flt3– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients

Blood ◽

10.1182/blood-2004-06-2159 ◽

2005 ◽

Vol 105 (7) ◽

pp. 2717-2723 ◽

Cited By ~ 271

Author(s):

Liping Yang ◽

David Bryder ◽

Jörgen Adolfsson ◽

Jens Nygren ◽

Robert Månsson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Transplant Recipients ◽

Short Term ◽

Hematopoietic Stem ◽

Regulatory Pathways ◽

Stem And Progenitor Cells ◽

Bone Marrow Ablation

AbstractIn clinical bone marrow transplantation, the severe cytopenias induced by bone marrow ablation translate into high risks of developing fatal infections and bleedings, until transplanted hematopoietic stem and progenitor cells have replaced sufficient myeloerythroid offspring. Although adult long-term hematopoietic stem cells (LT-HSCs) are absolutely required and at the single-cell level sufficient for sustained reconstitution of all blood cell lineages, they have been suggested to be less efficient at rapidly reconstituting the hematopoietic system and rescuing myeloablated recipients. Such a function has been proposed to rather be mediated by less well-defined short-term hematopoietic stem cells (ST-HSCs). Herein, we demonstrate that Lin–Sca1+kithiCD34+ short-term reconstituting cells contain 2 phenotypically and functionally distinct subpopulations: Lin–Sca1+kithiCD34+flt3– cells fulfilling all criteria of ST-HSCs, capable of rapidly reconstituting myelopoiesis, rescuing myeloablated mice, and generating Lin–Sca1+kithiCD34+flt3+ cells, responsible primarily for rapid lymphoid reconstitution. Representing the first commitment steps from Lin–Sca1+kithi CD34–flt3– LT-HSCs, their identification will greatly facilitate delineation of regulatory pathways controlling HSC fate decisions and identification of human ST-HSCs responsible for rapid reconstitution following HSC transplantations.

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The Trithorax-Group Protein ASH1L Regulates Hematopoietic Stem Cell Homeostasis Independently of Its Histone Methyltransferase Activity

Blood ◽

10.1182/blood-2018-99-117367 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1270-1270 ◽

Cited By ~ 1

Author(s):

Gloria Jih ◽

Jennifer Chase ◽

Yile Zhou ◽

Ann Friedman ◽

Xiaomin Feng ◽

...

Keyword(s):

Bone Marrow ◽

Wild Type ◽

Set Domain ◽

Hematopoietic Stem ◽

Trithorax Group ◽

Multipotent Progenitors ◽

Donor Bone Marrow ◽

A Cell ◽

Group Protein

Abstract Absent, small or homeotic discs 1-like (Ash1l) encodes the mammalian homolog of a Trithorax-group protein with a conserved SET domain that harbors histone H3 lysine 36 dimethyltransferase activity. Using mice with constitutive knockdown of Ash1l due to the insertion of a "gene trap" cassette in the first intron (Ash1lGT/GT mice), we previously reported that Ash1l is a critical regulator of quiescence and self-renewal in adult hematopoietic stem cells (HSCs). While wild-type HSCs could readily establish long-term bone marrow reconstitution after transplantation into irradiated recipients, Ash1l-deficient HSCs had markedly decreased quiescence and failed to establish long-term bone marrow reconstitution (Jones, Chase et al., JCI 2015). Here, we addressed three important questions to better understand the role of Ash1l in hematopoiesis: 1) What is the impact of complete, as opposed to partial, Ash1l loss on adult hematopoiesis? 2) Does Ash1l regulate HSC homeostasis in a cell-autonomous manner? 3) Is the catalytic activity of ASH1L required for its function? To move beyond the limitations of the constitutive knockdown Ash1lGT allele and address the first two questions, we studied newly generated mice carrying conditional knockout (cKO) Ash1l alleles (exon 13 flanked by loxP sites) along with the Mx1-Cre transgene. Induction of Mx1-Cre expression with poly(I:C) in Mx1-Cre+Ash1lf/f mice resulted in nonsense-mediated decay of Ash1l mRNA in hematopoietic cells, thereby completely knocking out Ash1l in adult hematopoietic stem and progenitor cells. Four weeks after inducing Ash1l inactivation in the hematopoietic compartment, we observed a profound depletion of HSCs and multipotent progenitors in Ash1l cKO mice similar to the phenotype of Ash1lGT/GTmice, indicating that conditional Ash1l knockout has a comparable impact on adult hematopoiesis to that of constitutive Ash1l knockdown. Of note, overt hematopoietic failure in steady-state conditions was not observed in either model. To address the second question, we transplanted a mixture of wild-type and Mx1-Cre+Ash1lf/f bone marrow into irradiated wild-type hosts, allowed donor bone marrow to occupy the wild-type niche and establish hematopoiesis, then induced Cre-mediated excision to inactivate Ash1l in Mx1-Cre+Ash1lf/f hematopoietic cells. Upon Cre induction, donor-derived Ash1lΔ/Δ HSCs and myeloid progeny were depleted or outcompeted by wild-type cells, consistent with the model that Ash1l regulates HSC homeostasis in a cell-autonomous manner. Given that Ash1l encodes a SET domain, we next sought to directly examine whether its catalytic activity is required for its role in regulating HSCs. We studied an Ash1l allele with an in-frame deletion of exon 11 and 12, resulting in preserved expression of ASH1L with internally deleted SET domain (ΔSET) (Miyazaki et al., PLOS Genetics 2013). Homozygous ΔSET mice were viable, and phenotypic analysis of adult ΔSET mice revealed normal frequencies of HSCs and multipotent progenitors, in contrast to our observations in Ash1lGT/GTand Ash1l cKO mice. Furthermore, transplanting ΔSET donor bone marrow into irradiated wild-type hosts resulted in sustained long-term reconstitution throughout primary, secondary and tertiary competitive transplantation assays, consistent with preserved HSC function and no alterations in cell cycle regulation. These findings establish that Ash1l regulates HSC homeostasis independently of its SET domain and histone methyltransferase activity. As ASH1L is a very large protein encoding multiple chromatin binding domains, we speculate that ASH1L may serve as a platform to recruit other partners to form novel protein complex(es) that regulate genes critical for HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

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