Colony Formation Of Hematopoietic Progenitor Cells Research Articles

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice

AbstractWe hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα−/− and pitpα−/−/β−/−) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα−/−/β−/− BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα−/− and pitpα−/−/β−/− BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα−/− and pitpα−/−/β−/− mice had higher concentrations of TGF-β1. CM from pitpα−/− and pitpα−/−/β−/− MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti–TGF-β antibody, and treatment of pitpα−/−/β−/− mice in vivo with anti–TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα−/−/β−/− MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα−/−/β−/− MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1.

Abstract 1117: Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML

Abstract Background: Over 15 genome-wide association studies (GWASs) have identified a narrow region in chromosome 6q23.3 as a major hematopoietic quantitative trait locus (QTL) for many hematologic traits. The associated phenotypes have yet to be fully explained by genes in the region, including MYB. We noted a newly annotated microRNA within the QTL, miR-3662, and sought to characterize its role in the QTL by exploring its function in normal and malignant hematopoiesis, specifically acute myeloid leukemia (AML). Results: To assess the association between the QTL-defining SNPs and miR-3662 abundance, we genotyped 10 GWAS SNPs in 200 blood samples from non-leukemic individuals. The risk alleles of all 10 SNPs associated with higher miR-3662 abundance. Homozygosity for rs66650371, a three base pair (bp) deletion, showed the strongest association (P<.0001). Reinserting the three bp in cells homozygous for rs66650371 with CRISPR/Cas9 technology lowered miR-3662 abundance by 20%, suggesting rs66650371 directly affects miR-3662 transcription. MiR-3662 was nearly absent in primitive hematopoietic progenitor cells (HPCs) cells, whereafter its abundance increased throughout differentiation. MiR-3662 overexpression increased cell growth and colony formation of HPCs, particularly of the erythroid lineage. To explore the role of miR-3662 in malignant hematopoiesis, we overexpressed miR-3662 in AML cell lines (Kasumi1, KG1a, MV4-11) and primary AML patient blasts (n = 12). Overexpression of miR-3662 reduced cell growth and viability in vitro, and significantly reduced tumor size in a murine xenograft model (scramble vs. miR-3662; n = 9 mice/group). We used targeted RNA sequencing to identify differentially expressed genes between forced miR-3662-expresssing- and control AML patient blasts using a panel of 361 candidate genes (TruSeq RNA platform, Illumina). IKBKB was identified as a promising target, and direct regulation of IKBKB's 3′-UTR by miR-3662 was confirmed via luciferase assay. MiR-3662 overexpression lowered IKBKB mRNA and protein expression in both HPCs and AML patient blasts. As IKBKB promotes activation and nuclear localization of NF-κB, overexpression of miR-3662 led to decreased NF-κB transactivation potential via luciferase assay. Confocal microscopy of HPCs and AML cells, as well as subcellular fraction immunoblots, revealed decreased NF-κB nuclear localization upon miR-3662 overexpression. Re-introduction of IKBKB partly rescued miR-3662's phenotype by increasing nuclear localization of NF-κB, decreasing growth of HPCs, and increasing viability of AML cells. Conclusion: The risk alleles of the 6q23.3 region associated with higher miR-3662 abundance, and miR-3662 abundance was functionally linked to rs66650371. We characterized miR-3662 as an agent in normal and malignant hematopoiesis, and demonstrated that miR-3662 reduces NF-κB activity by targeting IKBKB. Citation Format: Sophia E. Maharry, Christopher J. Walker, Sandya Liyanarachchi, Sujay Mehta, James S. Blachly, Denis C. Guttridge, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1117.

Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner

DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC.Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects.To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. DisclosuresNo relevant conflicts of interest to declare.

Recombinant DEK Regulates Hematopoietic Stem and Progenitor Cells in a CXCR2- GαI Protein-Dependent Manner

DEK, a non-histone nuclear phosphoprotein involved in heterochromatin remodeling, is released from cells and can regulate hematopoiesis (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Marrow from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cells (HSC) engrafting capability. Moreover, recombinant mouse (rmu) DEK inhibited HPC colony formation in vitro. We now show that rmuDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being much more sensitive than control mice to this suppression. In addition, rmuDEK treatment of WT mice in vivo reversibly reduces the phenotypic number of ST-HSC, MPP, CMP, GMP, and MEP in the marrow. In vivo administration of rmuDEK to DEK-/- mice greatly enhanced the number of phenotypic LT-HSC and functional HSC in these mice (competitive HSC repopulation in lethally irradiated mice). DEK-/- mice are also more sensitive to 5-FU than control mice resulting in slower hematopoietic cell recovery, possibly due to the enhanced cycling status of HPC in DEK-/- mice. To determine the biological role of extracellular DEK, we utilized a reagent that could inactivate its function. This inactivating agent, but not its control, neutralized the inhibitory effect of rmuDEK. In addition, treating marrow cells in vitro with truncated rmuDEK by pretreating the DEK with the enzyme DPP4 (which DEK has targeted truncation sites for) also blocked the inhibitory effects of DEK suggesting that DEK must be in its full length form in order to perform its functionUpon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. To determine whether rmuDEK’s inhibitory function is mediated through the CXCR2 receptor, a neutralizing monoclonal antibody against CXCR2 was utilized. Marrow treated in vitro with rmuDEK, rhIL-8, or rmuMIP-2 inhibited colony formation; however pretreating marrow cells with the neutralizing CXCR2 antibody blocked the inhibitory effect of these proteins. Marrow treated with rmuMIP-1α (a chemokine that does not bind to CXCR2) also inhibited colony formation; however neutralizing CXCR2 antibody had no effect on the ability of MIP-1α to inhibit colony formation. Neutralizing CXCR4, a chemokine receptor that binds SDF-1 but not IL-8, MIP-2 or MIP-1α, had no effect on the inhibition of colony formation. DEK inhibition of CFU-GM colony formation is dependent on Gαi-protein-coupled receptor signaling as determined through the use of pertussis toxin. This is a unique mechanism since IL-8 and MIP-1α had been previously reported by us to be insensitive to the inhibitory effects of pertussis toxin.As extracellular DEK can remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 25: 673; Saha et al., 2013, PNAS, 110: 6847), we next assessed the effects of DEK on the heterchromatin marker H3K9Me3 in the nucleus of purified mouse Lineage negative, Sca-1 positive, c-kit positive (LSK) marrow cells by imaging flow cytometry. RmuDEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK marrow cells indicating that rmuDEK can be internalized by LSK cells and mediate heterchromatin formation. These results add to emerging evidence that DEK, a unique nuclear protein, plays a role in regulating hematopoiesis through a CXCR2/Gαi protein signaling pathway. Its modulation may be of practical use. DisclosuresNo relevant conflicts of interest to declare.

Pesticide induced marrow toxicity and effects on marrow cell population and on hematopoietic stroma

Long term inhalation of toxic pesticides used for the domestic and industrial purposes have been shown to cause moderate to severe hematotoxicity and increased incidence of several marrow degenerative diseases, specifically hypoplastic bone marrow failure condition in humans. The progression of pesticide induced hematotoxicity and the exact underlying mechanisms of toxicity that play major role in limiting normal hematopoiesis are not quite well explained. In this present study, we have developed an animal model of hypoplastic bone marrow failure following pesticide exposure to show the deleterious effects of toxic pesticides on mouse hematopoietic system. Here we have presented the results of studying long-term marrow explant culture, IL-2, IL-3 and IL-5 receptors expression profile, fibroblast colony forming unit (CFU-F), hematopoietic progenitor cell colony formation and caspase-3 expression by the bone marrow cells. We have also identified the expression levels of several extracellular apoptosis markers (CD95/Fas) and intracellular apoptosis inducer proteins (pASK1, pJNK, caspase-3 and cleaved caspase-3) in the bone marrow cells of pesticide exposed mice. The long-term marrow explant culture demonstrated the impairment in proliferation of the stromal cells/stromal fibroblasts in culture. Decreased IL-2, IL-3 and IL-5 receptors expression profile essentially hinted at the suppressed cytokine activity in the pesticide exposed marrow. CFU-F analysis showed the defect in functional maturation of the stromal fibroblasts. The decreased hematopoietic progenitor cell colony formation indicated the toxicity induced inhibition of cellular proliferation and functional maturation of hematopoietic stem/progenitor cells in pesticide exposed marrow. We have detected a sharp increase in the expression levels of both the extracellular Fas-antigen and intracellular apoptosis inducer proteins in the bone marrow cells of pesticide exposed mice that explained well, the apoptosis pathway involved following marrow toxicity. The decreased proliferation and functional maturation of marrow stromal cells and hematopoietic progenitors with subsequent increase in marrow cellular apoptosis following pesticide toxicity provided the base necessary for explaining the increased incidence of hypoplastic bone marrow failure in humans exposed to moderate to high concentrations of pesticides.

Alteration in marrow stromal microenvironment and apoptosis mechanisms involved in aplastic anemia: an animal model to study the possible disease pathology.

Aplastic anemia (AA) is a heterogeneous disorder of bone marrow failure syndrome. Suggested mechanisms include a primary stem cell deficiency or defect, a secondary stem cell defect due to abnormal regulation between cell death and differentiation, or a deficient microenvironment. In this study, we have tried to investigate the alterations in hematopoietic microenvironment and underlying mechanisms involved in such alterations in an animal model of drug induced AA. We presented the results of studying long term marrow culture, marrow ultra-structure, marrow adherent and hematopoietic progenitor cell colony formation, flowcytometric analysis of marrow stem and stromal progenitor populations and apoptosis mechanism involved in aplastic anemia. The AA marrow showed impairment in cellular proliferation and maturation and failed to generate a functional stromal microenvironment even after 19 days of culture. Ultra-structural analysis showed a degenerated and deformed marrow cellular association in AA. Colony forming units (CFUs) were also severely reduced in AA. Significantly decreased marrow stem and stromal progenitor population with subsequently increased expression levels of both the extracellular and intracellular apoptosis inducer markers in the AA marrow cells essentially pointed towards the defective hematopoiesis; moreover, a deficient and apoptotic microenvironment and the microenvironmental components might have played the important role in the possible pathogenesis of AA.

Benzene Exposure and Hematotoxicity

In their Report “Hematotoxicity in workers exposed to low levels of benzene” (3 Dec. 2004, p. [1774][1]), Q. Lan et al. present data on blood cell counts and hematopoietic progenitor cell colony formation from sera of benzene-exposed workers (and controls) in China, from which they conclude that

Distinct requirements for Stat4 and Stat6 in hematopoietic progenitor cell responses to growth factors and chemokines.

Hematopoietic progenitor cell (HPC) homeostasis is critical in maintaining innate immunity and healing processes. Recently, we demonstrated that Th1 cells regulate HPC homeostasis, partly based on altered homeostasis in Stat4- and Stat6-deficient mice. To explore changes in HPC responsiveness in altered T helper cell environments, we directly examined growth factor-stimulated colony formation and chemokine-induced myelosuppression of HPC in Stat4- and Stat6-deficient bone marrow cells. Stat6-deficient cells have increased responses to the synergy between granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), compared to wild-type and Stat4-deficient cells. Increased responses are eliminated by in vivo depletion of CD4 cells. Whereas Stat6-deficient bone marrow cells respond to chemokine-mediated myelosuppression, Stat4-deficient bone marrow cells are refractory to the suppressive effects of chemokines. Thus, T helper cell development affects HPC homeostasis through several mechanisms, including the sensitivity to growth factor stimulation and chemokine suppression of HPC colony formation. Since Stat4 and Stat6 regulate opposing programs of T helper differentiation, there are distinct requirements for Stat4 and Stat6 in regulation of growth factor and chemokine responses of HPC.

Colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar

In vitro colony forming unit (CFU) assays have been used to measure the effects of compounds that regulate the growth of hematopoietic progenitor cells. These assays are time consuming and subjective and are therefore not amenable to high throughput of large numbers of compounds. Here we have shown that the traditional murine bone marrow CFU assay can be modified into a robust non-subjective colorimetric assay format. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added after colony formation in an agar based 96-well plate culture system. Optical density correlated with increasing cell input concentrations in the presence of growth factor. The linearity of this response was equivalent to the standard CFU assay. Several hematopoietic inhibitors were tested in both assays. Effects on colony number and size were compared to optical density. Compounds that reduced colony numbers with little effect on colony size had identical IC 50 values in both the colorimetric assay and CFU assay. The IC 50 values of compounds that also decreased colony size did not correlate in the two assays. These results demonstrate the utility of the colorimetric assay to rapidly screen for compounds that specifically inhibit hematopoietic progenitor cell colony formation in vitro.

Basic fibroblast growth factor (B‐FGF) induces early‐ (CFU‐s) and late‐stage hematopoietic progenitor cell colony formation (CFU‐gm, CFU‐meg, and BFU‐e) by synergizing with GM‐CSF, Meg‐CSF, and erythropoietin, and is a radioprotective agent in vitro

Basic fibroblastic growth factor (B-FGF) is a hormone-like protein which belongs to a class of heparin-binding growth factors. B-FGF is synthesized and released to circulate in the blood where it can be recognized by target cells through specific high-affinity plasma membrane receptors. B-FGF is known to be a potent mitogen for a number of specific cell types. We report data which demonstrates B-FGF can influence noncommited and specific lineage-derived hematopoietic progenitors when incubated in vitro. When combined with adherent cell-depleted normal murine marrow cells, B-FGF increased the number of both day 9 and day 12 spleen colony-forming units (CFU-s) from lethally irradiated animals. However, day 12-derived CFU-s were more sensitive to B-FGF, since optimal CFU-s production was observed at 10 ng/ml vs. 100 ng/ml for day 9 CFU-s (p less than 0.05). In adherent cell-depleted murine and human marrow cultures, the addition of B-FGF possessed synergistic activity in combination with the optimal concentration of GM-CSF for CFU-gm at a dose of 10 ng/ml which was inhibited in the presence of protamine sulfate (LD50 dose, 100 mu gm/ml), an inhibitor of B-FGF mitogenic activity, or in the presence of heparin (LD50 dose, 100 U/ml), an effective B-FGF binding agent. B-FGF also expressed synergistic activity in the presence of optimal concentrations of erythropoietin and Meg-CSF for murine and human BFU-e, and murine CFU-meg. No in vitro colony formation was observed when cells were cultured in the presence of B-FGF, but in the absence of the specific hematopoietic growth factor. Finally, B-FGF was also shown to be an effective radioprotective agent in vitro. Murine and human CFU-gm exposed to increasing doses of radiation (0.5 to 5 Gy) combined with GM-CSF and increasing doses of B-FGF (0.1 to 100 ng/ml) produced less radiation-induced toxicity compared to cultures containing GM-CSF alone. This data demonstrates B-FGF influences early- and late-stage hematopoietic progenitors, possesses synergistic activity with hematopoietic growth factors, and is a radioprotective agent in vitro. These results suggest B-FGF must be considered as a member of the family of molecules capable of influencing hematopoiesis in vitro.

Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.