Early Stages Of Hematopoietic Differentiation Research Articles

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

AbstractHematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity.

Arsenic impairs the lineage commitment of hematopoietic progenitor cells through the attenuation of GATA-2 DNA binding activity

Arsenic exposure produces significant hematotoxicity in vitro and in vivo. Our previous work shows that arsenic (in the form of arsenite, AsIII) interacts with the zinc finger domains of GATA-1, inhibiting the function of this critical transcription factor, and resulting in the suppression of erythropoiesis. In addition to GATA-1, GATA-2 also plays a key role in the regulation of hematopoiesis. GATA-1 and GATA-2 have similar zinc finger domains (C4-type) that are structurally favorable for AsIII interactions. Taking this into consideration, we hypothesized that early stages of hematopoietic differentiation that are dependent on the function of GATA-2 may also be disrupted by AsIII exposure. We found that in vitro AsIII exposures disrupt the erythromegakaryocytic lineage commitment and differentiation of erythropoietin-stimulated primary mouse bone marrow hematopoietic progenitor cells (HPCs), producing an aberrant accumulation of cells in early stages of hematopoiesis and subsequent reduction of committed erythro-megakaryocyte progenitor cells. Arsenic significantly accumulated in the GATA-2 protein, causing the loss of zinc, and disruption of GATA-2 function, as measured by chromatin immunoprecipitation and the expression of GATA-2 responsive genes. Our results show that the attenuation of GATA-2 function is an important mechanism contributing to the aberrant lineage commitment and differentiation of early HPCs. Collectively, findings from the present study suggest that the AsIII-induced disruption of erythro-megakaryopoiesis may contribute to the onset and/or exacerbation of hematological disorders, such as anemia.

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation.

Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells

Reactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activities. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+.

SIRT2 Plays Essential Role in Early Hematopoiesis through Deacetylation of LMO2 Protein

Nicotinamide phosphoribosyltransferase (NAMPT) is known as a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) and supplies NAD+ to sirtuins (SIRTs) that require NAD+ to activate their protein deacetylase functions. SIRTs are members of the NAD+-dependent class III histone deacetylase family. We already reported that NAMPT-NAD+-SIRT1 pathway is essential for granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis (Skokowa J, et al. Nat Med. 2009).In the present study, we analyzed the mechanism of NAMPT-NAD+ -SIRTs-triggered hematopoiesis and myelopoiesis using human induced pluripotent stem (iPS) cells model. We applied a serum- and feeder-free monolayer hematopoietic differentiation system of iPS cells by sequential incubation of iPS cells with combinations of mesoderm- and hematopoiesis-inducing cytokines. We found that addition of NAMPT to granulopoiesis-inducing iPS cells culture medium resulted in a marked increase of CD15+ and CD16+ myeloid and granulocytic cell numbers. These results are compatible with our published data in healthy donor derived CD34+ cells. To evaluate at which stage of hematopoietic differentiation NAMPT plays a role, we cultured iPS cells in the presence or absence of the specific inhibitor of NAMPT, FK866. Interestingly, we found marked reduction of VEGFR2+ CD34+ early hematopoietic progenitor cells at day 6 of differentiation after inhibition of NAMPT. These data argue for the requirement of NAMPT and its downstream effectors at early stages of hematopoietic differentiation. To evaluate which of the SIRTs is essential in this process, we tested early hematopoietic differentiation of iPS cells in the presence of specific inhibitors of several SIRTs. We found that inhibition of SIRT2 using its specific inhibitor AC93253 significantly suppressed differentiation into VEGFR2+ CD34+ early hematopoietic progenitor fraction. Furthermore, the emergence of CD43+ hematopoietic progenitor cells at day 13 was completely suppressed after inhibition of SIRT2. These results indicate that NAMPT-NAD+-SIRT2 signaling is needed for early hematopoiesis. We further evaluated downstream targets of NAMPT-NAD+-SIRT2. qRT-PCR analysis of well-known early hematopoietic genes (GATA2, RUNX1, LMO2, TAL1) revealed that mRNA expression of these factors was not down-regulated after NAMPT or SIRT2 inhibition. We therefore tested whether hematopoietic functions of these proteins might be affected by post-transcriptional modifications via NAMPT-NAD+-SIRT2-triggered deacetylation. We tested protein-protein interaction between SIRT2 and these candidate proteins in differentiated iPS cells on day 6 of culture using Duolink technique, which allows identification of protein complexes in single cells. We found that RUNX1, LMO2 and TAL1 proteins interact with SIRT2 protein. To identify if these proteins could be deacetylated by SIRT2, we compared the signals from total- and de-/acetylated- candidate proteins in differentiated iPS cells on day6 treated or not with SIRT2 inhibitor using Duolink-FACS, Duolink technique in combination with flow cytometry, as a readout method. This analysis revealed that only LMO2 is deacetylated by SIRT2. LMO2 is known as an essential transcriptional regulator in early hematopoiesis. It is a component of TAL1 complex, which consist of LMO2, TAL1, LDB1, E47 and GATA1/2 proteins and has transcriptional activity as a complex on hematopoiesis-specific target genes as KIT, GATA1, GYPA, KLF1 and KDR. Interestingly, mRNA expression levels of all these genes were markedly downregulated after inhibition of NAMPT or SIRT2, which was in line with severely reduced numbers of early hematopoietic progenitors.Overall, these results indicate that NAMPT, known to be essential for myeloid differentiation, induces early stages of hematopoietic differentiation by SIRT2-triggered deacetylation of LMO2. DisclosuresNo relevant conflicts of interest to declare.

MicroRNAs expressed in hematopoietic stem/progenitor cells are deregulated in acute myeloid leukemias

MicroRNAs are key regulators of hematopoiesis, specifically involved in regulating the maintenance of stemness of primitive hematopoietic progenitor cells (HPCs) and the early and late stages of hematopoietic differentiation. Some microRNAs have been found to be expressed in hematopoietic stem cells (HSCs) and primitive HPCs, and play a relevant role in regulation of the early steps of hematopoietic cell differentiation. Notable examples of these microRNAs are given by miR-22, miR-29, miR-125 and miR-126. These HSC/HPC-regulating microRNAs are often deregulated in some subsets of acute myeloid leukemia (AML), with pathogenic, diagnostic and prognostic implications. Therefore, elucidation of the pattern of microRNA expression at the level of the early stages of hematopoietic cell differentiation has essential implications, not only for elucidation of the molecular bases of the early stages of hematopoietic differentiation, but also for a better understanding of the pathogenic mechanisms underlying AML.

Mutational Status of TET2, JAK2, and MPL in Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis.

Abstract 418According to the WHO classification, myelodysplastic/myeloproliferative neoplasms include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). The best characterized of these latter conditions is the provisional entity defined as refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T); up to 60% of RARS-T patients harbor the JAK2 (V617F) mutation. Somatic mutations of TET2 have been recently described in myeloid neoplasms, where they appear to be associated with the amplification of the mutated clone at the early stages of hematopoietic differentiation [N Engl J Med. 2009 May 28;360(22):2355-7]. In order to gain a deeper insight into the pathophysiology of RARS-T, we studied a cohort of 187 patients with myeloid neoplasms and investigated the relationship between ringed sideroblasts, thrombocytosis, and mutational status of TET2, JAK2 and MPL. RARS-T was defined according to the following WHO criteria: i) refractory anemia associated with erythroid dysplasia and ringed sideroblasts ≥ 15%; ii) < 5% blasts in the bone marrow; iii) platelet count ≥ 450 × 109/L; iv) presence of large atypical megakaryocytes similar to those observed in BCR/ABL1-negative myeloproliferative neoplasms; v) absence of del(5q), t(3;3)(q21;q26) or inv(3)(q21q26). The combination of ringed sideroblasts ≥ 15% and platelet count ≥ 450 × 109/L was found in 19 subjects fulfilling the diagnostic criteria for RARS-T, while 24 patients had RARS without thrombocytosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells - but not in T-lymphocytes - from 11 out of 19 (58%) RARS-T patients. Three RARS patients, who initially had low to normal platelet counts, progressed to RARS-T, and two of them acquired JAK2 (V617F) at this time. Somatic mutations of TET2 were found in three of the 15 RARS-T patients studied, and the presence of multiple mutant genes allowed analysis of subclones in two of them. One of these patients carried the following three somatic mutations: TET2 (C1271Y), JAK2 (V617F) and MPL (W515L). Analysis of genomic DNA from circulating granulocytes showed 50% TET2 (C1271Y) mutant alleles but smaller proportions of JAK2 (V617F) and MPL (W515L) mutant alleles (5.8% and 20% respectively). We then analyzed five BFU-E grown from peripheral blood mononuclear cells obtained from this patient. All these five colonies were heterozygous for TET2 (C1271Y), while three of them were heterozygous also for MPL (W515L) and the remaining two were heterozygous also for JAK2 (V617F), clearly indicating that erythroid progenitors carrying JAK2 or MPL mutants belonged to subclones of the dominant TET2 (C1271Y) clone. A woman with the TET2 (S1612LfsX4) mutation (50% granulocyte mutant alleles) and fully clonal hematopoiesis as indicated by X-chromosome inactivation patterns, carried 28% JAK2 (V617F) mutant alleles in circulating granulocytes, indicating that granulocytes harboring JAK2 mutant alleles belonged to a subclone of the initial TET2 (S1612LfsX4) mutant clone. Over a 5-year period, in fact, the initial TET2 mutant clone was completely replaced by the TET2/JAK2 mutant subclone. In other two female patients with RARS-T and no somatic mutation of TET2, granulocytes carrying JAK2 (V617F) represented only a fraction (11 to 22%) of clonal granulocytes as determined by X-chromosome inactivation patterns (96 to 100%). Somatic mutations of TET2 were detected also in a significant proportion of patients with RARS without thrombocytosis, while no JAK2 or MPL mutation was identified in these individuals. These observations suggest that the occurrence of a TET2 mutation may represent the initial event determining clonal dominance of hematopoietic cells both in RARS and RARS-T patients, while the subsequent occurrence of JAK2 and/or MPL mutations likely generates myelodysplastic/myeloproliferative subclones in RARS-T patients. In conclusion, RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical level, and it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL or other as-yet-unknown genes on the background of clonal hematopoiesis caused by somatic mutations of TET2 or other similar (as-yet-unknown) mutant genes. Disclosures:No relevant conflicts of interest to declare.

Jedi—a novel transmembrane protein expressed in early hematopoietic cells

Self-renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short- and long-term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF-like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non-adherent co-cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway.

Two types of precursor cells in a multipotential hematopoietic cell line.

The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.

Early stages of hematopoietic differentiation.

Mouse bone marrow contains hematopoietic stem cells as well as progenitor cells, which are partially differentiated offspring of stem cells. We have utilized several approaches to separate progenitors from stem cells in order to characterize essential differences between these two stages of development. As a first approach, we utilized the supravital fluorescent dye rhodamine-123 (Rh-123) to distinguish quiescent stem cells (Rh-123(low)) from metabolically active progenitor cells (Rh-123(hi)). Analysis of megakaryocyte potential in a tissue culture assay demonstrated that Rh-123(hi) progenitor cells were capable of robust megakaryocyte differentiation, while Rh-123(low) stem cells produced fewer colonies containing megakaryocytes. Transplantation of the two cell populations into irradiated recipients revealed the opposite outcome, suggesting that the tissue culture assay failed to predict behavior in a transplant setting. We also evaluated functional potential of lymphoid progenitors isolated by selecting for differential expression of Thy-1.1 and c-kit. The potential of defined cell populations to differentiate as T or B lymphocytes in vivo was dependent upon the time post transplant at which animals were evaluated. These studies underscore the need for caution in the interpretation of lineage potentials evaluated by both in vitro and in vivo assays.

Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells

An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase- positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C- 3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.

Hematopoietic Factor Production by a Cell Line (TC-1) Derived From Adherent Murine Marrow Cells

An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase-positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C-3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.