Bioenergetic Profiling of the Differentiating Human MDS Myeloid Lineage with Low and High Bone Marrow Blast Counts

Myelodysplastic syndromes (MDS) encompass a very heterogeneous group of clonal hematopoietic stem cell differentiation disorders with malignant potential and an elusive pathobiology. Given the central role of metabolism in effective differentiation, we performed an untargeted metabolomic analysis of differentiating myeloid lineage cells from MDS bone marrow aspirates that exhibited <5% (G1) or ≥5% (G2) blasts, in order to delineate its role in MDS severity and malignant potential. Bone marrow aspirates were collected from 14 previously untreated MDS patients (G1, n = 10 and G2, n = 4) and age matched controls (n = 5). Following myeloid lineage cell isolation, untargeted mass spectrometry-based metabolomics analysis was performed. Data were processed and analyzed using Metabokit. Enrichment analysis was performed using Metaboanalyst v4 employing pathway-associated metabolite sets. We established a bioenergetic profile coordinated by the Warburg phenomenon in both groups, but with a massively different outcome that mainly depended upon its group mitochondrial function and redox state. G1 cells are overwhelmed by glycolytic intermediate accumulation due to failing mitochondria, while the functional electron transport chain and improved redox in G2 compensate for Warburg disruption. Both metabolomes reveal the production and abundance of epigenetic modifiers. G1 and G2 metabolomes differ and eventually determine the MDS clinical phenotype, as well as the potential for malignant transformation.

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Comprehensive network modeling from single cell RNA sequencing of human and mouse reveals well conserved transcription regulation of hematopoiesis

BMC Genomics ◽

10.1186/s12864-020-07241-2 ◽

2020 ◽

Vol 21 (S11) ◽

Author(s):

Shouguo Gao ◽

Zhijie Wu ◽

Xingmin Feng ◽

Sachiko Kajigaya ◽

Xujing Wang ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factors ◽

Single Cell ◽

Rna Sequencing ◽

Transcription Regulation ◽

Regulatory Networks ◽

Stem Cell Differentiation ◽

Hematopoietic Stem ◽

Single Cell Rna Sequencing ◽

Human And Mouse

Abstract Background Presently, there is no comprehensive analysis of the transcription regulation network in hematopoiesis. Comparison of networks arising from gene co-expression across species can facilitate an understanding of the conservation of functional gene modules in hematopoiesis. Results We used single-cell RNA sequencing to profile bone marrow from human and mouse, and inferred transcription regulatory networks in each species in order to characterize transcriptional programs governing hematopoietic stem cell differentiation. We designed an algorithm for network reconstruction to conduct comparative transcriptomic analysis of hematopoietic gene co-expression and transcription regulation in human and mouse bone marrow cells. Co-expression network connectivity of hematopoiesis-related genes was found to be well conserved between mouse and human. The co-expression network showed “small-world” and “scale-free” architecture. The gene regulatory network formed a hierarchical structure, and hematopoiesis transcription factors localized to the hierarchy’s middle level. Conclusions Transcriptional regulatory networks are well conserved between human and mouse. The hierarchical organization of transcription factors may provide insights into hematopoietic cell lineage commitment, and to signal processing, cell survival and disease initiation.

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IMMU-50. GLIOBLASTOMA MANIPULATES HEMATOPOIETIC STEM CELL DIFFERENTIATION OUTCOMES AND DRIVES ALTERED IMMUNE RECONSTITUTION

Neuro-Oncology ◽

10.1093/neuonc/noab196.409 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi104-vi104

Author(s):

Bayli DiVita Dean ◽

Tyler Wildes ◽

Joseph Dean ◽

David Shin ◽

Connor Francis ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Progenitor Cells ◽

Rna Sequencing ◽

Cell Fate ◽

Immune Reconstitution ◽

Stem Cell Differentiation ◽

Cell Fate Determination ◽

Hematopoietic Stem ◽

Fate Determination

Abstract INTRODUCTION Bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) give rise to the cellular components of the immune system. Unfortunately, immune reconstitution from HSPCs are negatively impacted by solid cancers, including high-grade gliomas. For example, an expansion of myeloid progenitor cells has been previously described across several cancers that originate outside the CNS. A similar expansion of MDSCs coupled with diminished T cell function has also been described in the peripheral blood of patients with newly-diagnosed GBM. Alterations in both lymphoid and myeloid compartments due to CNS malignancy led us to determine how intracranial gliomas impact HSPCs in both their capacity to reconstitute the immune compartment and in their cell fate determination. This is important to better understand the impact of gliomas on immunity and how we can leverage these findings to better develop cellular immunotherapeutics. METHODS HSPCs were isolated from bone marrow of C57BL/6 mice with orthotopic KR158B glioma, or age-matched naïve mice. Experiments were conducted to compare relative changes in: gene expression (RNA-sequencing), precursor frequencies, cell fate determination, and cellular function of cells derived from HSPCs of glioma-bearing mice. RESULTS RNA-sequencing revealed 700+ genes whose expression was significantly up- or downregulated in HSPCs from glioma-bearing mice, particularly those involved with stemness and metabolic activity. Importantly, HSPCs from glioma-bearing mice expressed upregulation of genes involved in myelopoiesis relative to naïve mice. This was coupled with an expansion of granulocyte macrophage precursors (GMPs), the progenitors to gMDSCs. Next, differentiation assays revealed that HSPCs from glioma-bearing mice had higher propensity of differentiating into MDSC under homeostatic conditions relative to controls both in vitro and in vivo. Furthermore, mice bearing intracranial gliomas possess an expansion of MDSCs which are more suppressive on T cell proliferation and hinders T cell-mediated tumor cell killing relative to MDSCs derived from naïve control mice.

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Specialized Bone Marrow Endothelium Defines Microdomains for Tumor and Stem Cell Engraftment.

Blood ◽

10.1182/blood.v104.11.663.663 ◽

2004 ◽

Vol 104 (11) ◽

pp. 663-663

Author(s):

Dorothy A. Sipkins ◽

Xunbin Wei ◽

Juwell W. Wu ◽

Terry K. Means ◽

Andrew D. Luster ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Cells ◽

Stem Cell Differentiation ◽

Multiple Time ◽

Normal Stem Cell ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Vascular Beds

Abstract The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation and regeneration, yet relatively little is known about the architecture of microenvironments that support malignant proliferation. Using dynamic in vivo confocal and multi-photon imaging, we show that the bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas that localize the homing and early engraftment of both leukemic and normal primitive hematopoietic cells. Real-time imaging of cell-cell interactions in SCID mice bone marrow was performed after injection of fluorescently-labeled leukemic and other malignant cell lines. Progressive scanning and optical sectioning through the marrow revealed the existence of unique, spatially-restricted vascular domains to which the majority of marrow-homing tumor cells rolled and arrested. Serial imaging of mice on days 3 – 14 demonstrated that leukemic (Nalm-6 pre-B ALL) extravasation and early proliferation were restricted to these vascular beds. To define the molecular basis of these homing interactions, in vivo labeling of key vascular cell adhesion molecules and chemokines using fluorescent antibodies was performed. We observed that while ICAM-1, VCAM-1, PECAM-1 and P-selectin were expressed diffusely throughout the marrow vasculature, the expression of E-selectin and the chemokine receptor CXCR4 ligand SDF-1 was distinctly limited to vessels that supported leukemic cell engraftment. In vivo co-localization experiments confirmed Nalm-6 binding was restricted to vascular beds expressing both E-selectin and SDF-1. In functional studies, disruption of E-selection had a modest effect on leukemic homing (<20% diminution), while pharmacologic blockade of CXCR4 decreased Nalm-6 binding to vessels by approximately 80%. To explore the normal function of this vascular niche, we next examined whether benign primitive hematopoietic cells might preferentially home to these same vascular microdomains. Fluorescently-labeled stem and progenitor cells (HSPC) isolated from donor balb/c mice were injected into recipient mice and imaging was performed at multiple time points. HSPC were found to adhere to the BM microvasculature in the same restricted domains. At 70 days post-injection, HSPC had extravasated, were persistent in these perivascular areas and had undergone cell division as assessed by dye dilution. Our findings show that these microdomains serve as vascular portals around which leukemic and hematopoietic stem cells engraft, suggesting that this molecularly distinct vasculature provides both a cancer and normal stem cell niche. Specialized vascular structures therefore appear to delineate a stem cell microenvironment that is exploited by malignancy.

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Hematopoietic Stem Cell Differentiation Pathway in Humans Deduced From the Lineage Diversity of PNH-Type Cells in Patients with Bone Marrow Failure.

Blood ◽

10.1182/blood.v114.22.1489.1489 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1489-1489

Author(s):

Takamasa Katagiri ◽

Zhirong Qi ◽

Yu Kiyu ◽

Naomi Sugimori ◽

J. Luis Espinoza ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Refractory Anemia ◽

Hematopoietic Stem

Abstract Abstract 1489 Poster Board I-512 The hematopoietic stem cell (HSC) differentiation pathway in humans remains largely unknown due to the lack of an appropriate in vivo assay allowing the growth of HSCs as well as of clonal markers that enable the tracing of their progenies. Small populations of blood cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as CD55 and CD59 are detectable in approximately 50% of patients with aplastic anemia (AA) and 15% of patients with refractory anemia (RA) of myelodysplastic syndrome defined by the FAB classification. Such blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype (PNH-type cells) are derived from single PIGA mutant HSCs and their fate depends on the proliferation and self-maintenance properties of the individual HSCs that undergo PIG-A mutation by chance (Blood 2008;112:2160, Br J Haematol 2009 in press) Analyses of the PNH-type cells from a large number of patients on the diversity of lineage combination may help clarify the HSC differentiation pathway in humans because PIG-A mutant HSCs in patients with bone marrow failure appear to reflect the kinetics of healthy HSCs. Therefore, different lineages of peripheral blood cells were examined including glycophorin A+ erythrocytes (E), CD11b+ granulocytes (G), CD33+ monocytes (M), CD3+ T cells (T), CD19+ B cells (B), and NKp46+ NK cells (Nk) from 527 patients with AA or RA for the presence of CD55−CD59− cells in E and G, and CD55−CD59−CD48− cells in M,T, B, Nk with high sensitivity flow cytometry. Two hundred and twenty-eight patients (43%) displayed 0.003% to 99.1% PNH-type cells in at least one lineage of cells. The lineage combination patterns of PNH-type cells in these patients included EGM in 71 patients (31%), EGMTBNk in 43 (19%), EG in 37 (16%), T alone 14 (6%), EGMBNk in 11 (5%), G alone in 10 (4%), GM in 10 (4%), EGMNk in 7 (3%), EGMT in 7 (3%), EGMB in 6 (3%), EM in 5 (2%), EGMTB in 3 (1%), EGNk in 1 (0.4%), EGMTNk in 1 (0.4%), GMTB in 1 (0.4%), and GT in 1 (0.4%) (Table). All patterns included G or M, except for 14 patients displaying PNH-type T cells alone. No patients showed TB or TBNk patterns suggestive of the presence of common lymphoid progenitor cells. Peripheral blood specimens from 123 patients of the 228 patients possessing PNH-type cells were examined again after 3 to 10 months and all patients showed the same combination patterns as those revealed by the first examination. PIG-A gene analyses using sorted PNH-type cells from 3 patients revealed the same mutation in G and Nk for 1 patient and in G and T for 2 patients. These findings indicate that human HSCs may take a similar differentiation pathway to that of murine HSCs, the ‘myeloid-based model’ that was recently proposed by Kawamoto et al. (Nature 2008; 10:452), though the cases with PNH-type T cells alone remain to be elucidated. Table. Lineages of cells containing PNH-type cells in patients with AA or RA. The number in the parenthesis denotes the proportion of patients showing each combination pattern in the total patients possessing PNH-type cells. (+ ; presence of PNH-type cells) Disclosures No relevant conflicts of interest to declare.

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Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndrome: A Retrospective Validation From a Single Centre.

Blood ◽

10.1182/blood.v114.22.4846.4846 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4846-4846

Author(s):

Pervin Topcuoglu ◽

Klara Dalva ◽

Sule Mine Bakanay ◽

Sinem Civriz Bozdag ◽

Onder Arslan ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndrome ◽

Antigen Expression ◽

Pathological Examination ◽

Light Scatter ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Altered Expression ◽

Flow Cytometric

Abstract Abstract 4846 Myelodysplastic syndrome (MDS) is heterogeneous clonal hematopoietic stem cell disorder characterized by cytopenia(s) and dysplasia in one or more cell lineage. Though flow cytometry (FCM) is an important diagnostic tool in hematopoietic cell disorders, a prominent immunophenotyping feature in MDS may not be determined. In this study, we retrospectively evaluated flow cytometric features of bone marrow samples diagnosed as MDS with clinical and hematological findings. Patients-Method Between Feb 2004 and March 2009, flow cytometric parameters of 73 patients (M/F: 50/23) with MDS were re-analyzed. Median age was 59 years (17-89 ys). Our general principles are to evaluate quality of bone marrow samples, to determine proportion of the cells and features of their light scatter, and to give percentage of the blast. When detected a finding of dysplasia in the first analysis, the second step includes the determination of the maturation of the cells and the presence of the aberrant antigen expression. Results The samples were interpreted as MDS in % 76.7, MDS-RAEB-1 or RAEB-2 in %16.4, myeloproliferative disorder in %1.4 and non-diagnostic in %6.8 of the cases by flow cytometric examination. We detected variable degrees of hypogranulation in myeloid lineage in %82.2 of the samples by the light scatter features of the cells: 85% of severe and 15% of moderate or mild hypogranulation. The ratio of myeloid and lymphoid was changing from 0.3 to 17.5 (median 2). The decreasing of this ratio (<1) was observed in 19.4% of the samples. We detected altered expression of mature granulocyte. These included decreasing or lack of expression in CD15 45/73 (61.4%), CD13 38/70 (54.3%), CD16 53/67 (79.1%), CD11b 51/71 (71.8%), CD24 44/69 (65.2%), CD10 23/72 (31.9%) and MPO 14/72 (19.4%). Besides, bright expression of CD33 in 53.5% of the samples was observed. CD36 and CD56 in myeloid lineage were co-expressed in about 50 % of the samples. In 80.8 % of the samples dysplasia in erythroid compartment could be evaluated: Expression of CD71 according to glycophorin A (ratio <1) was decreased in 23.7 %. When we made similar analysis in the samples without RAEB-1 and -2 as pathological examination of bone marrow, 13.4 % of the samples could not be evaluated in favor of dysplasia. Of the samples with dysplasia hypogranulation, aberrant antigen expression of myeloid lineage and eryhtroid dysplasia were observed in 92.1%, 34.1% and 31.5%, respectively. In conclusion, FCM events may help to the differantial diagnosis of MDS especially when combining with clinical events. Improving of the analysis by focusing on the blast characteristics may be a standard approach to evaluate for low risk MDS. Disclosures No relevant conflicts of interest to declare.

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RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Blood ◽

10.1182/blood.v118.21.1273.1273 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1273-1273

Author(s):

Xuan Zhou ◽

Jaime Meléndez ◽

Yuxin Feng ◽

Richard Lang ◽

Yi Zheng

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Intracellular Signaling ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Conditional Knockout ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Differentiated Cells ◽

Multipotent Progenitors

Abstract Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

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G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood.v122.21.1181.1181 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1181-1181 ◽

Cited By ~ 1

Author(s):

Laura G. Schuettpelz ◽

Joshua N. Borgerding ◽

Priya Gopalan ◽

Matt Christopher ◽

Molly Romine ◽

...

Keyword(s):

Bone Marrow ◽

Cell Function ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Intestinal Flora ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Tlr Signaling ◽

Hematopoietic Stem

Abstract Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.sci-20.sci-20 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-20-SCI-20

Author(s):

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The Hedgehog receptor Patched controls lymphoid lineage commitment

Blood ◽

10.1182/blood-2007-02-075648 ◽

2007 ◽

Vol 110 (6) ◽

pp. 1814-1823 ◽

Cited By ~ 67

Author(s):

Anja Uhmann ◽

Kai Dittmann ◽

Frauke Nitzki ◽

Ralf Dressel ◽

Milena Koleva ◽

...

Keyword(s):

Bone Marrow ◽

Lineage Commitment ◽

Cell Compartment ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Adult Mice ◽

Multipotent Progenitor Cells ◽

Adult Organism ◽

The Common ◽

B Lymphoid

Abstract A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice, we show that this differentiation step requires Patched (Ptch), the cell surface–bound receptor for Hedgehog (Hh). In the absence of Ptch, the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently, the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally, adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism.

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Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02674-2 ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Francesco Da Ros ◽

Luca Persano ◽

Dario Bizzotto ◽

Mariagrazia Michieli ◽

Paola Braghetta ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Extracellular Matrix ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Adipogenic Differentiation ◽

Stem Cell Differentiation ◽

Dependent Manner ◽

Hematopoietic Stem

Abstract Background Dissection of mechanisms involved in the regulation of bone marrow microenvironment through cell–cell and cell–matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma. Methods Immunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry. Results Emilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging. Conclusion Our data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

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