Decrease In Hematopoietic Stem Cells Research Articles

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

Role of Mir-29b in T-Cell Development and in Cutaneous T-Cell Lymphoma Pathogenesis

The role of microRNA (miR) is rapidly advancing with numerous functions discovered in cancer pathogenesis. We had previously shown a significant decrease in miR-29b levels in malignant cells isolated from peripheral blood of cutaneous T-cell lymphoma (CTCL) (Kohnken et al. Blood, 2018). Replenishing miR-29b levels in vitro induced apoptosis in leukemic cells suggesting its role as tumor suppressor miR in CTCL patients. In this study, we focused on the role of mir-29b on early T-cell development and homeostasis using the miR-29b-/- mouse model. Using knockout mouse model, we show that the homozygous deletion of miR-29b locus results in an overall decrease in T-cell numbers and density in primary and secondary lymphoid organs. We observed an early thymic involution in miR-29b-/- mice, with a 5-fold decrease in the total number of thymocytes and altered T-cell development. Using the surface expression of CD25 and CD44 on double-negative (DN) cells, we observed significant decrease of the absolute counts in the four early differentiation stages (DN1-4) in miR-29b-/- mice compared to age-matched wild-type (WT) mice: DN1 (miR-29b-/- vs. WT: 4.5 x 105 ± 1.04 vs. 1.12 x 105 ± 0.201, N=4 and 6 respectively, p < 0.05); DN2 (miR-29b-/- vs. WT: 0.83 x 105 ± 0.07 vs. 0.051 x 105 ± 0.021, N=4 and 6 respectively, p < 0.001); DN3 (miR-29b-/- vs. WT: 4.05 x 105 ± 0.38 vs. 0.54 x 105 ± 0.16, N=4 and 6 respectively, p < 0.001) and DN4 (miR-29b-/- vs. WT: 2.871 x 105 ± 0.578 x 105 vs. 0.569 ± 0.14, N=4 and 6 respectively, p < 0.01). Furthermore, miR-29b-/- mice show a significant increase in regulatory T cells in comparison to WT mice in the spleen (miR-29b-/- vs. WT: 18.48 ± 0.89 vs. 10.89 ± 0.41, N=4 and 6 respectively, p < 0.001) thymus (miR-29b-/- vs. WT: 5.80 ± 0.44 vs. 3.65 ± 0.004, N=4 and 6 respectively, p < 0.01) and bone marrow (miR-29b-/- vs. WT: 50.04 ± 3.14 vs. 37.52 ± 3.29, N=4 and 6 respectively, p < 0.05). Using single-cell RNA sequencing (scRNA-seq), we found a framework of putative genes in miR-29b deficient T-cells that overlap with CTCL pathogenesis: Ccr7, Cd69, Cd74, and Lef-1. Among thymic T cells, Ccr7 affects physiologic homing of T-cells to lymph nodes and facilitate nodal metastasis in CTCL patients. As T-cells progenitors originate from the bone marrow, we observed a severe impairment in the progenitor cell population in miR-29b-/- mice and over 4-fold reduction in the absolute number of Lin-Sca1+ckit+ cell (miR-29b-/- vs. WT: 28.42 x 103 ± 4.13 vs. 5.98 x 103 ± 1.01, N=4 and 3 respectively, p < 0.01). Using the surface expression of CD48 and CD150 on Lin-Sca1+ckit+, we observed a significant decrease in hematopoietic stem cells and multipotent progenitor cells in miR-29b-/- mice. Since impairment in T-cells development can be linked to hematopoietic stem cell defects, we interrogated the engraftment capacity of Lin-Sca1+ckit+ cells into committed cell lineages in vivo. To evaluate whether the defect in T-cells development is due to reduced ability of precursor cells, we performed transplantation of Lin-Sca1+ckit+ cells from the bone marrow of miR-29b and WT (CD45.1) mice in lethally irradiated WT (CD45.2) mice. Our results show a significant reduction in T-cells reconstitution in mice transplanted with miR-29b-/- Lin-Sca1+ckit+ cells versus the bone marrow counterpart. These deficits in T-cells populations were observed in peripheral blood, thymus, spleen, and bone marrow in miR-29b-/- transplanted mice. ScRNA-seq profiling of Lin- bone marrow cells show significant changes in miR-29b-/- and WT mice. Among the upregulated genes in the mir-29b-/- mice, several genes showed greater than a 10-fold increase in the thymus (Ifna2, Zmynd10, Plekha4, Etl4, and Gm38004). In conclusion, our results highlight the importance of mir-29b in early defects in T-cells development and help us understand the complex miR-29b regulated cellular transformation machinery in CTCL pathogenesis, thus paving path to develop novel therapeutic approaches targeting miR-29b in CTCL therapy. Disclosures No relevant conflicts of interest to declare.

Site-1 protease ablation in the osterix-lineage in mice results in bone marrow neutrophilia and hematopoietic stem cell alterations.

ABSTRACTSite-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro, recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche.

[Reference guide for the treatment of aplastic anemia].

Aplastic anemia is a syndrome characterized by the decrease in hematopoietic stem cells along with bone marrow hypoplasia and pancytopenia, which is likely to be a T cell-mediated autoimmune disease. Since the response rate to immunosuppressive therapy is higher if started ahead of time, early initiation of treatment is recommended even in non-severe cases. Among treatment approaches in severe cases, immunosuppressive therapy with anti-thymocyte globulin (ATG) plus cyclosporin is the basic approach. However, the effectiveness of thrombopoietin receptor agonists has also been reported, with recovery of hematopoiesis in three blood lineages observed in some patients. Despite no evidence of increased incidence of genetic mutations with thrombopoietin receptor agonist treatment, bone marrow testing is recommended after three to six months of long-term treatment to detect the presence of chromosomal abnormalities. With regard to hematopoietic stem cell transplantation for aplastic anemia, cyclophosphamide is reduced as a pretreatment therapy, and instead, fludarabine is used in combination in order to reduce cardiotoxicity. Since HLA haploidentical hematopoietic stem cell transplantation has been developed and is being reportedly used in patients with no suitable donors, this transplantation approach might also be extended to aplastic anemia patients who were not considered eligible for transplantation in the past.

The VWRPY Domain Is Essential for RUNX1 Function in Hematopoietic Progenitor Cell Maturation and Megakaryocyte Differentiation

RUNX1 is a transcription factor essential during definitive hematopoiesis. Germline mutations in RUNX1 results in a disorder called Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients have abnormal bleeding due to reduced platelet count and/or function. Importantly, 20-60% of the FPDMM patients develop hematological malignancies, which are mainly myeloid. Reported RUNX1 mutations in FPDMM families are mostly clustered in the N-terminal runt domain and the C-terminal transactivation domain. Recently, three mutations have been reported at or near the end of the C-terminal repression domain, the VWRPY motif. However, the mechanism behind the VWRPY motif involvement in the FPDMM pathogenesis has not been studied. Interestingly, these VWRPY-mutated RUNX1 proteins still have intact runt and transactivation domains, but patients still show FPDMM phenotype. Here, we evaluate the functional defects of a RUNX1 mutation, L472fsX, which was reported in a FPDMM family, using three different experimental models. Our study is aimed to unravel the significance of the VWRPY motif in FPDMM pathogenesis.The RUNX1 L472fsX mutation is caused by a GC insertion upstream of the VWRPY motif. The mutation results in a frameshift and a run on protein for an additional 123 amino acids. The frameshift abolishes the VWRPY motif, which is responsible for the binding between RUNX1 and a co-repressor protein, TLE1. As expected, from both FRET and co-IP assays, the mutated RUNX1 lost binding with TLE1. Interestingly, we observed increased binding between the mutated RUNX1 and its co-factor CBFβ in the FRET assay, as compared to the wildtype RUNX1. Furthermore, in reporter assays we found that TLE1 failed to repress the expression of a RUNX1 target, M-CSFR promoter, when co-transfected with the mutated RUNX1, which is contrary to what has been seen with wildtype RUNX1. Consistent with increased binding between the mutated RUNX1 and CBFβ in the FRET assay, co-transfecting mutant RUNX1 and CBFβ resulted in a significant increase of M-CSFR promoter expression as compared to wildtype RUNX1 with CBFβ. For another RUNX1 target, Hmga2, wildtype RUNX1 and CBFβ decreased Hmga2 expression, which could be restored by adding TLE1. Transfected mutant RUNX1 and CBFβ also decreased Hmga2 expression, but TLE1 could not restore Hmga2 expression when co-transfected with the mutant RUNX1. These findings suggest that the VWRPY-disrupting L472fsX mutation leads to the loss of binding between mutant RUNX1 and TLE1, which in turn resulted in defective repression of the RUNX1 activity by TLE1.To assess for hematopoietic defects in the FPDMM patients with the L472fsX mutation, blood cells from two family members were reprogrammed to induced pluripotent stem cells (iPSCs). Similar to previous studies, iPSCs from these patients gave rise to fewer megakaryocyte progenitors and mature megakaryocytes during in vitro differentiation. In addition, these FPDMM iPSCs showed decrease in hematopoietic stem cell (HSCs) maturation and differentiation to progenitors. The L472fsX mutation in the iPSCs was then corrected by genome editing using zinc finger nuclease. Importantly, the hematopoietic defects of the FPDMM iPSCs mentioned above were rescued after mutation correction. Overall, the findings in these iPSCs differentiation assays showed that the VWRPY motif is essential for RUNX1 activity in megakaryocytes differentiation. In addition, the VWRPY motif is important for HSCs maturation and differentiation to progenitors.To evaluate the impact of this VWRPY-deletion mutation on hematopoiesis in an in vivo model, CRISPR-mediated genome editing was used to generate mice with frameshift mutations that remove the VWRPY domain. Preliminary observations showed that the mutant mice have minor defects in the peripheral blood. More data on this mouse model will be presented at the meeting.In conclusion, we present a novel RUNX1 mutation (L472fsX) with unique hematopoietic defect that has not been reported previously in FPDMM. Our findings imply the significance of the VWRPY motif in megakaryopoiesis, as well as HSCs maturation and differentiation. DisclosuresNo relevant conflicts of interest to declare.

舌の血腫を初発症状とした再生不良性貧血の１例

Aplastic anemia is a blood disease characterized by pancytopenia due to a decrease in hematopoieticstem cells. We describe a patient who presented with submucosal hematoma of the tongue and was given a diagnosisof aplastic anemia. The patient was a 24-year-old man who visited our department because of a hematoma ofthe tongue. Two days before presentation, a dark reddish tumor with a diameter of 1-2mm was noticed at the leftmargin of the tongue. At presentation, a 20×11mm dark reddish elevation was seen at the left lateral margin ofthe tongue. On the basis of the above course and findings, a hematoma was diagnosed, and blood tests were conducted.A blood disease was suspected. The white blood cell count was 500/μl, the red blood cell count was320×104/μl, the Hb was 11.2g/dl, the Ht was 31.1%, the MCV was 96.9 fl, the MCH was 35.0 pg, the MCHCwas 36.1%, and the platelet count was 0.3×104/μl, indicating pancytopenia. A submucosal hematoma of thetongue accompanied by pancytopenia was diagnosed, and detailed examinations were performed at the hematologydepartment. A bone marrow puncture test showed that the bone marrow cell count was 4, 000/μl and themegakaryocyte count was 0/μl; aplastic anemia was thus diagnosed, and the patient received immunosuppressivetherapy. Even when the onset is gradual, aplastic anemia can be lethal if detection is late and appropriate therapyis not administered. Therefore, if the regional cause of an oral hematoma is not clear, close follow-up is necessary.If systemic factors are suspected, blood tests should be promptly performed.

LIM Domain Binding Protein 1 (Ldb1) is Required for the Maintenance of Hematopoietic Stem Cells (81.4)

Abstract Ldb1 is a ubiquitous nuclear protein that functions by interacting with and/or recruiting specific partners (including LMO2, SCL and GATA-1) to form multi-molecular DNA-binding transcription complexes. In the hematopoietic lineage, expression of Ldb1 is highest in lineage−Sca1+c-kit+ (LSK) multipotent progenitors. Ldb1 gradually decreases as cells mature within lymphoid or granulocyte/macrophage lineages, but remains high in erythroid lineage cells. We investigated the role of Ldb1 in hematopoiesis by conditional deletion of Ldb1 using a cre transgene driven by the Tie2 promotor; or by following the fate of Ldb1-null embryonic stem (ES) cells in mouse blastocyst chimeras. The number of Ter119+ (erythroid lineage) cells was decreased dramatically in Tie2Cre+Ldb1flox/flox mice, revealing a critical role for Ldb1 in erythropoiesis. Ldb1-null ES cells were capable of generating LSKs in the fetal liver and these cells could give rise to both myeloid and lymophoid lineage cells; however, the number of Ldb1−/− LSKs was markedly decreased at later stages of development. Following adoptive transfer, Ldb1-null fetal liver LSKs were rapidly lost indicating a failure of self-renewal or survival. Loss of Ldb1 was associated with an increase in multipotent progenitor (MPP) cells and a decrease in hematopoietic stem cells (HSCs), suggesting that Ldb1 participates in stem cell maintenance. These data provide the first evidence that LIM domain binding proteins function in hematopoiesis, and identify Ldb1 as a critical factor in regulating HSC maintenance/self-renewal.

Granulocyte-colony stimulating factor impedes recovery from damage caused by cytotoxic agents through increased differentiation at the expense of self-renewal.

G-CSF is routinely used to hasten recovery from chemotherapy-induced neutropenia. We have recently shown that G-CSF, when combined with stem cell-damaging cytotoxic agents, results in enhanced stem cell damage and loss of marrow reserve. To investigate the mechanisms of stem cell damage caused by G-CSF, we gave C57BL/6 (B6) mice repeated doses of cyclophosphamide ([CY] 84 mg/kg) or carmustine ([BCNU] 13.2 mg/kg) and G-CSF (250 microg/kg/day) for either four days or eight days. Two different regimens of G-CSF were chosen to study the influence of increased proliferation on hematopoiesis which was measured at the end of the first, third and sixth 14-day cycle of each cytotoxic agent and 7 and 20 weeks after completion of all cycles. A spectrum of hematopoietic indices was measured including WBC, bone marrow cellularity, granulocyte/macrophage-colony-forming cells (GM-CFC), colony-forming cells with high proliferative-potential (HPP-CFC), cobblestone area-forming cells ([CAFC]-day 7 and CAFC-day 28), and long-term marrow repopulating ability in vivo. Despite the absence of differences in peripheral blood cell counts or bone marrow cellularity 14 days after each dose, progenitor cell levels (HPP-CFC, GM-CFC, and CAFC-7) were increased up to 2.5-fold with cytotoxic agent and G-CSF administration compared with cytotoxic agent administration alone. Mice given G-CSF for eight days had the greatest number of progenitors suggesting a dose-response relationship for G-CSF administration. G-CSF resulted in a decrease in hematopoietic stem cell (CAFC-28) content when measured two weeks after each cycle of saline, CY, and BCNU. Twenty weeks after six cycles of BCNU, the reduction in stem cell levels persisted and was further decreased when G-CSF was added to BCNU for four or eight days. Data from this study suggest that the most likely explanation for the damaging effects of G-CSF is that G-CSF directly or indirectly induces stem cells to differentiate into more committed hematopoietic cells resulting in a loss of marrow reserve. This effect is enhanced in animals with an already compromised hematopoietic stem cell compartment as seen with repeated doses of BCNU.