Lineage-committed Hematopoietic Progenitors Research Articles

EPH/Ephrin Signaling in Normal Hematopoiesis and Hematologic Malignancies: Deciphering Their Intricate Role and Unraveling Possible New Therapeutic Targets.

Erythropoietin-producing hepatocellular carcinoma receptors (EPHs) represent the largest family of receptor tyrosine kinases (RTKs). EPH interaction with ephrins, their membrane-bound ligands, holds a pivotal role in embryonic development, while, though less active, it is also implicated in various physiological functions during adult life. In normal hematopoiesis, different patterns of EPH/ephrin expression have been correlated with hematopoietic stem cell (HSC) maintenance and lineage-committed hematopoietic progenitor cell (HPC) differentiation, as well as with the functional properties of their mature offspring. Research in the field of hematologic malignancies has unveiled a rather complex involvement of the EPH/ephrinsignaling pathway in the pathophysiology of these neoplasms. Aberrations in genetic, epigenetic, and protein levels have been identified as possible players implicated both in tumor progression and suppression, while correlations have also been highlighted regarding prognosis and response to treatment. Initial efforts to therapeutically target the EPH/ephrin axis have been undertaken in the setting of hematologic neoplasia but are mainly confined to the preclinical level. To this end, deciphering the complexity of this signaling pathway both in normal and malignant hematopoiesis is necessary.

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Multipotent Progenitor Niche Factors

Signal transducer and activator of transcription 5 (STAT5a and STAT5b isoforms) are latent transcription factors that are intrinsically critical for normal hematopoiesis but are also expressed in bone marrow stromal cells. However, despite known STAT5a (prolactin) and STAT5b (growth hormone) functions in breast and liver tissue respectively, hematopoiesis-supporting stromal functions have not been explored. Here, floxed STAT5ab double knockout (KO) was generated with a variety of hematopoietic and stromal Cre-expressing transgenic mouse strains to test whether STAT5a or STAT5b play a role in extrinsic regulation of hematopoiesis. Pan-hematopoietic deletion with Vav1-Cre was the positive control for loss of multipotent hematopoietic function but surprisingly Vav1-Cre also deleted STAT5ab in CD45neg cells, dysregulated stromal niche factor mRNA expression, and altered MSC osteo/chondro-lineage differentiation priming. Single cell transcriptome analysis of bone marrow from wild-type or Vav1-Cre KO mice long-term repopulating hematopoietic stem cells (LT-HSC) showed upregulated oxidative phosphorylation (P<10-10) and protein translation (P<10-39) genes in LT-HSCs and throughout myeloid-primed clusters, while in contrast common lymphoid progenitor-like cells were completely eliminated. Similarly, like Vav1+ cells and based on scRNAseq analysis, Nes+ cells were also detected in both CD45neg and CD45+ cell clusters. Deletion of STAT5ab with Nes-Cre reduced the number of LT-HSCs (30% reduction compared to Nes-Cre control mice, P=0.02, N=4) and caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45+ and CD45neg bone marrow cell populations, additional stroma-specific Cre strains (LepR, Osx, Ocn, Prrx1, Nes-CreERT2) as well as endothelial-specific (Cdh5) strains were generated lacking STAT5ab. Viable mice were assessed for peripheral blood hematology, competitive repopulation, as well as hematopoietic stem/progenitor subsets (LT-HSC, MPP1, MPP2, MPP3, MPP4, KLS) using well-accepted flow cytometry antibody panels. Deletion of STAT5 in stromal cells with LepR-Cre significantly reduced MPP4 (35% reduction, P=0.025, N=4). Overall, extrinsic stromal (but not endothelial) cells were deficient in supporting multipotent and lineage-committed hematopoietic progenitors. However, peripheral blood hematology was rescued through homeostatic production. Gene regulation profiles resembled that of cytokine-starved MPPs. Functional support for niche-supporting activity was also assessed using STAT5ab KO MSCs in vitro. With Lepr-Cre mediated STAT5ab deletion, bone marrow niche factor mRNAs were downregulated (Cxcl12, Angpt1, Igfbp4, Igf1, Il34) and support of multipotent hematopoietic cells was significantly impaired. ELISA assay showed that SDF-1a was reduced 3-fold (P<0.001, N=5) and IGF-1 was reduced 2-fold (P<0.01, N=5) compared to wild type MSCs. Inversely, mRNAs for Kitl and Grem1 were increased in the same stromal cells, consistent with loss of multipotency and mixed HSC vs. MPP stromal support activities. Furthermore, novel scRNAseq analysis on simultaneous bone marrow hematopoietic and stromal cells compared wild-type and KO mice. Differentially expressed gene sets with Maximum Mean Discrepancy (MMD) co-expression revealed a key central role for coordinated STAT5ab/Cish balance as a biomarker of the cellular progenitor activity state (metabolic, protein translation, survival) associated with multipotency, comparing KO to wild-type cells. Importantly, Cish was strongly co-expressed in cells primed for lineage-specific differentiation (MSC - osteo/chondrogenic-primed; HSC - myeloid-primed) with STAT5ab co-expressed genes being associated with multipotent potential. Therefore, novel STAT5ab/Cish-mediated gene regulation programs operate within the bone marrow microenvironment to mediate cytokine production and responsiveness. This work highlights the importance of examining heterotypic cell types within bone marrow to elucidate functional cross-talk underlying hematopoietic homeostasis and defines conserved STAT5ab-mediated gene signatures as potential biomarkers to test in human hematopoietic and immune cell disorders.

Direct pharmacologic regulation of the ETS transcription factor PU.1

The ETS‐family transcription factor PU.1 is essential for the self‐renewal of hematopoietic stem cells (HSC) as well as their differentiation to various lineage‐committed hematopoietic progenitors. Specific to the bone marrow niche, PU.1 regulates the expression of several key cytokines (e.g. CXCR4) and their receptors (e.g. GM‐CSF receptor, FLT3) involved in HSC mobilization. Extensive studies have shown that incomplete loss of PU.1 activity, to ~20% wildtype levels, leads to lineage arrest and aggressive leukemic disease. Conversely, lentiviral overexpression of PU.1 resolves the differentiation block in leukemic blasts, and suggests the activation of PU.1 as a potential therapeutic approach. To this end, we have identified by phage display technology a panel of selective PU.1‐binding oligopeptides. Several of these peptides promote PU.1 binding to cognate DNA sites in a dose‐dependent manner and confer resistance to PU.1 against inhibition by netropsin. To determine the essential residues responsible for their activity, we screened a targeted library of truncated hit peptides and found that as few as four residues were sufficient to enhance site‐specific DNA binding by PU.1. These peptide sequences represent leads for developing direct‐acting PU.1‐specific modulators with respect to target DNA binding and interactions with major binding partners. The resulting low‐MW reagents could significantly expand the pharmacology of transcription factors and help overcome barriers in hematopoietic research and therapy that are currently imposed by genetic approaches.Support or Funding InformationSupported by NIH HL129063.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dasatinib promotes the activation of quiescent hematopoietic stem cells in mice.

Dasatinib is an orally available broad-spectrum tyrosine kinase inhibitor that is widely used to treat chronic myeloid leukemia. It is also in clinical trials for the treatment of other malignancies, including solid tumors. Despite its wide use, little is known of its effects on normal hematopoietic stem and progenitor cells. Here, we study wild-type mice dosed with dasatinib and find that it causes the transient induction of proliferation of quiescent hematopoietic stem cells (HSCs). This finding was unexpected given the ability of dasatinib to inhibit c-Kit signaling and promote cell cycle arrest in many cell types. The transient induction of HSC proliferation in dasatinib-dosed mice coincided with a marked induction in the expression of Sca-1 and phospho-S6. Also evident at this time was a rapid but transient loss of lineage-committed hematopoietic progenitors that express high levels of c-Kit and the induction of stem cell factor in the serum. These findings suggest that activation of quiescent HSCs is part of a rapid rescue response that restores hematopoietic progenitors to pretreatment levels. This restoration coincides with HSCs returning to quiescence, and the expression of Sca-1 and phospho-S6 reverting to pre-treatment levels, even though dasatinib dosing is maintained. These data suggest that equilibrium is reached between the opposing forces of dasatinib and hematopoietic growth factors. The transient induction of HSC proliferation provided a window of opportunity whereby these cells became sensitive to killing by the cytotoxic drug 5-fluorouracil.

The hematopoietic stem cell niche in homeostasis and disease

AbstractThe bone marrow microenvironment contains a heterogeneous population of stromal cells organized into niches that support hematopoietic stem cells (HSCs) and other lineage-committed hematopoietic progenitors. The stem cell niche generates signals that regulate HSC self-renewal, quiescence, and differentiation. Here, we review recent studies that highlight the heterogeneity of the stromal cells that comprise stem cell niches and the complexity of the signals that they generate. We highlight emerging data that stem cell niches in the bone marrow are not static but instead are responsive to environmental stimuli. Finally, we review recent data showing that hematopoietic niches are altered in certain hematopoietic malignancies, and we discuss how these alterations might contribute to disease pathogenesis.

Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells

Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1-which is essential for HSC survival-and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.

Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis

AbstractMembers of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3′-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

Chromatin Modifications in Hematopoietic Multipotent and Committed Progenitors Are Independent of Gene Subnuclear Positioning Relative to Repressive Compartments

To further clarify the contribution of nuclear architecture in the regulation of gene expression patterns during differentiation of human multipotent cells, we analyzed expression status, histone modifications, and subnuclear positioning relative to repressive compartments, of hematopoietic loci in multipotent and lineage-committed primary human hematopoietic progenitors. We report here that positioning of lineage-affiliated loci relative to pericentromeric heterochromatin compartments (PCH) is identical in multipotent cells from various origins and is unchanged between multipotent and lineage-committed hematopoietic progenitors. However, during differentiation of multipotent hematopoietic progenitors, changes in gene expression and histone modifications at these loci occur in committed progenitors, prior to changes in gene positioning relative to pericentromeric heterochromatin compartments, detected at later stages in precursor and mature cells. Therefore, during normal human hematopoietic differentiation, changes in gene subnuclear location relative to pericentromeric heterochromatin appear to be dictated by whether the gene will be permanently silenced or activated, rather than being predictive of commitment toward a given lineage.

Targeted Disruption of Zfp36l2, Encoding a CCCH Tandem Zinc Finger RNA-Binding Protein, Results in Defective Hematopoiesis.

Members of the tristetraprolin (TTP) family of tandem CCCH finger proteins can bind to AU-rich elements in the 3′-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. In previous work, we disrupted the first exon of one of the four mouse TTP family members, Zfp36l2 (Tis11d, Brf2, Erf2), resulting in the production of decreased levels of a truncated protein lacking the first 29 amino acids. These mice exhibited complete female infertility, with embryos not progressing past the two-cell stage. In order to establish a true null phenotype for this gene, we have generated mice completely lacking the second exon, encoding the RNA-binding tandem zinc finger domain, resulting in a true knockout (KO) with complete lack of mRNA and protein. Surprisingly, these mice exhibited a completely unexpected phenotype involving the development of the hematopoietic system; they appeared otherwise anatomically normal. Homozygous Zfp36l2 KO mice on a mixed C57Bl/6 – 129SvEv background were born with normal Mendelian frequency but generally died about two weeks after birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice at two weeks of age showed significant decreases in red cells (2.3 fold), white cells (2.1 fold), and platelets (11 fold). Flow cytometric analysis of spleen cells demonstrated significant decreases in myeloid cells (Gr-1+; Gr-1+/Mac-1+; 4 fold) and megakaryocytes (CD41+; 14 fold), as well as in c-Kit+ hematopoietic progenitors, without changes in lymphoid cell populations. Bone sections from rare surviving adult mice exhibited hematopoietic cell depletion. In addition, analysis of bone marrow revealed nine-fold decreases in lin-/Sca-1+/c-Kit+ cells; colony forming assays revealed that no hematopoietic progenitors grew from the KO bone marrow, compared to an average of 54 colonies per mouse from the wild-type (WT) mice. We therefore analyzed the development of the hematopoietic system. To do this, we cultured fetal liver cells in semisolid media that supported the proliferation and differentiation of multipotential and lineage-committed hematopoietic progenitors. There were significant decreases in the numbers of erythroid (BFU-E, 10-fold), granulocyte- macrophage (CFU-GM, 32-fold), granulocyte macrophage/macrophage (CFU-GM/M, 14-fold), and multipotential (CFU-GEMM, 14-fold) progenitor cells obtained from Zfp36l2 KO fetal liver cells at embryonic day (E) 14.5 as compared to WT cells from littermates. There were also statistically significant decreases in these progenitors in heterozygous mice compared to WT, suggesting a gene dosage effect. Similar studies of yolk sacs from E11.5 mice revealed significant decreases in myeloid (CFU-GM, 2-fold) and multipotential (CFU-GEMM, 1.7-fold) progenitors in the KO yolk sacs. Primitive hematopoiesis was unaffected, as assessed by in vitro colony forming assays with E8-8.25 yolk sac cells. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal cells from E14.5 mice were markedly defective in reconstituting the hematopoietic system of lethally irradiated recipients after 1, 2 and 6 months of follow-up; engraftment rates for these dates were, respectively, 45% (WT) vs. 8% (KO); 65% vs. 3%; and 86% vs. 1%. These studies demonstrated that the development of the definitive hematopoietic system was severely adversely affected in the Zfp36l2 KO mice. This led to the hypothesis that elimination of the RNA destabilizing protein ZFP36L2 leads to the accumulation of one or more transcripts that are toxic to hematopoiesis. To explore this hypothesis, microarray analyses were performed on RNA samples from fetal liver at E14.5. We identified 239 significantly elevated transcripts in the KO samples, including several possible candidates for direct binding targets for ZFP36L2. In addition, 175 transcripts were significantly down-regulated in the KO fetal livers, in many cases in pathways of hematopoiesis or platelet development and function. These results are currently being validated by a variety of functional biochemical approaches, and are being supplemented by analogous microarray assays using transcripts from E11.5 yolk sacs. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis, and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

Pum2 Protein Supports Maintenance and Suppresses Differentiation of Multipotent Hematopoietic Progenitors by Regulating the Function and Activation of c-kit Receptor.

Intrinsic mechanisms that regulate self-renewal of mammalian stem cells remain largely unknown. Stem cell maintenance and self-renewal in Drosophila and C. elegans are regulated by members of the conserved Pumilio family of RNA-binding proteins. We have previously described cloning and characterization of two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the role of mammalian Pum proteins in HSC, Pum2 was over-expressed in a SCF-dependent multipotent progenitor cell line EML, which has the capacity for multilineage (erythroid, myeloid, B and T lymphoid) differentiation in vitro. In the presence of SCF EML cells undergo SCF-dependent self-renewal, thus remaining undifferentiated and retaining an immature phenotype. When cultured with hematopoietic cytokines (IL-3, GM-CSF, Epo, Tpo) EML cells differentiate into lineage-committed hematopoietic progenitors (e.g. granulocyte/macrophage (CFU-GM), burst-forming unit erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors). Pum2 over-expression leads to uncoupling of the survival and differentiation signals in EML cells, and their SCF-independent maintenance. EML cells over-expressing Pum2 (Pum2-EML cells) also exhibit almost complete block of differentiation into multiple lineages in the absence of SCF. Moreover, although the culture with cytokine cocktail (IL-3, Epo, Tpo and GM-CSF) and retinoic acid enhances differentiation capacity of wild type EML cells, it was not sufficient to overcome the differentiation block in Pum2-EML cells. However, the repression of Pum2-EML cell differentiation is a reversible phenomenon, since the addition of SCF to Pum2-EML cell cultures, for at least 48 hours, restores their capacity to undergo multilineage differentiation and generate hematopoietic colonies. The SCF-independent maintenance of Pum2-EML cells seems to be caused by upregulated expression and constitutive activation of the SCF receptor c-kit, and is accompanied by constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. More importantly, Pum2-EML cells also exhibit upregulated expression and constitutive activation of a novel truncated form of c-kit receptor called tr-kit, which was found previously to be expressed preferentially in HSC. Tr-kit could play a critical role in the SCF-independent activation of the full-length c-kit receptor, leading to SCF-independent maintenance of Pum2-EML cells, and inhibition of their multilineage differentiation. The observation that Pum2-EML cells, maintained with or without SCF, are resistant to treatment with blocking anti-c-kit antibody (ACK2) and c-kit inhibitor STI-571, supports the notion that maintenance and survival of Pum2-EML cells in the presence of SCF is not due to an external activation of c-kit receptor through ligand binding. Taken together, these findings suggest a model in which survival and maintenance of multipotent hematopoietic progenitors are mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. In summary, mouse Pum2 protein could play an important role in supporting maintenance of HSC and multipotent progenitors through regulation of the SCF/c-kit signaling pathway, and could represent a part of the mechanism through which HSCs balance their self-renewal and commitment to differentiation.

Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor.

Hematopoietic progenitor cells can be mobilized from the bone marrow to the blood by a wide variety of stimuli, including hematopoietic growth factors, chemotherapy, and chemokines. Increasingly, mobilized peripheral blood hematopoietic progenitor cells instead of bone marrow hematopoietic progenitor cells have been used to reconstitute hematopoiesis after myeloablative therapy because of their reduced engraftment times and relative ease of collection. A striking feature of hematopoietic progenitor cell mobilization is the ability of hematopoietic growth factors with distinct cellular targets and biologic activities to mobilize a similar spectrum of pluripotent and lineage-committed hematopoietic progenitor cells into the blood. Recent studies have identified some of the key adhesive interactions that regulate hematopoietic progenitor cell trafficking in the bone marrow. In addition, pathways linking mobilizing agents to hematopoietic progenitor cell mobilization have begun to be elucidated. This review summarizes these advances, emphasizing the mechanisms regulating granulocyte colony-stimulating factor-induced mobilization.

Differential expression of lymphoid and myeloid markers on differentiating hematopoietic stem cells in normal and tumor-bearing adult human liver.

The presence and phenotype of lineage-committed hematopoietic progenitors in the normal adult human liver (AHL) were investigated and compared with the profiles of differentiating hematopoietic precursor populations detected in liver bearing metastases of colonic origin. Levels of hematopoietic stem cells (HSCs) (CD34(+)CD45(+)) detected in hepatic mononuclear cell (HMNC) populations were increased 6-fold when compared with matched peripheral blood samples. In normal liver, less than 5% of HSCs expressed the myeloid-associated antigen, CD33, whereas considerable proportions expressed lymphoid-associated markers (T cell, 33.39%; B cell, 17.39%; and natural killer [NK] cell, 37.17%). Significant increases were observed in the relative proportions of hepatic HSCs coexpressing CD33 (20.53%; P =.001), and the T-cell marker (CD7, 58. 13%; P =.02) in tumor-bearing liver compared with normal liver. HSCs with B-cell progenitor phenotype (CD19(+)) were significantly decreased in tumor-bearing liver (0.06%; P =.02). Despite these differences, the activation status of hematopoiesis, as measured by the coexpression of the differentiation and activation markers, CD38 and CD45RA, did not differ significantly between normal and tumor-bearing liver. These results indicate that the normal AHL harbors lineage-committed hematopoietic progenitors, and the vast majority of these progenitors express lymphoid-associated antigens with changes occurring in both the myeloid and lymphoid compartments of the hepatic hematopoietic pathway on tumor challenge. While tumor-bearing livers are enriched for intrahepatic myeloid precursors and T-cell progenitor cells, further studies are required to establish the origin and in situ development potential of hepatic HSCs in the adult human and their role in tumor immunity.

Effect of flt3 Ligand on the Ex Vivo Expansion of Human CD34+ Hematopoietic Progenitor Cells

A ligand for the tyrosine kinase receptor flt3/flk-2, referred to here as flt3 ligand (flt3L), was recently cloned. The effect of flt3L on purified human CD34+ progenitor cells was examined. flt3 receptor (flt3R) was detected on the surface of human bone marrow cells that were enriched for CD34 expression. The effects of flt3L and the c-kit ligand Steel factor (SLF) on hematopoietic progenitors were compared in clonal colony assays. Both factors synergized with Pixy321 (interleukin-3 [IL-3]-granulocyte-macrophage colony-stimulating factor fusion protein) to induce granulocytic-monocytic (GM) and high proliferative potential (HPP) colonies and synergized with Pixy321 + erythropoietin (EPO) to induce multipotent granulocytic-erythroid-monocytic-megakaryocytic colonies. Although SLF had a potent effect on colony formation of erythroid restricted progenitor cells (burst-forming unit-erythroid), no effect by flt3L was observed. The addition of flt3L to irradiated long-term marrow cultures seeded with CD34+ cells augmented both total and progenitor cell production. Ex vivo expansion studies with isolated CD34+ bone marrow cells from normal donors showed that flt3L alone supported maintenance of both GM and HPP progenitors for 3 to 4 weeks in vitro. The addition of flt3L to a growth factor combination of IL-1 alpha + IL-3 + IL-6 + EPO resulted in a synergistic effect on progenitor cell expansion comparable to that observed with the addition of SLF to IL-1 alpha + IL-3 + IL-6 + EPO. These data show a function for flt3L in the regulation of both primitive multipotent and lineage-committed hematopoietic progenitor cells.

CD34-Expressing Human Thymocyte Precursors Proliferate in Response to Interleukin-7 But Have Lost Myeloid Differentiation Potential

AbstractCD34 is a marker for pluripotent stem cells also present on lineage-committed hematopoietic progenitors from bone marrow and a subpopulation of immature thymocytes. To characterize these early immature thymocytes, we have studied 24 pediatric thymus samples for CD34/7 expression. Three subpopulations could be defined from these T-cell receptor (TcR-) immature thymocytes: CD34+7++ (12.0 +/- 5.8), CD34- 7++ (12.6 +/- 8.6), and CD34-7+ (71.5 +/- 17.0%). CD7++ represents upregulation of this antigen and is expressed by cells of a blast-like morphology. Three-color flow cytometric analysis of these three subsets suggests the following ordered differentiation sequence: CD34+7++1-4-8- 45RA+–>CD34+7++1+ 4+8-45RA+/- –>CD34-7++1+4+8-+45RO+–>CD34- 7+1++4+8+45RO+. Early immature thymocyte cell division is essential in the thymus to generate a large number of precursors before the initiation of the selection process. We observed that both CD2 as well CD28 activation pathways were inefficient to serve as costimulant with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate or interleukin-2 (IL-2) to induce the proliferation of the three CD34/7 subsets isolated by cell sorting. However, whereas IL-1, IL-2, IL-3, IL-4, granulocyte colony-stimulating factor, and granulocyte-macrophage colony- stimulating factor were ineffective, IL-7 was a potent cytokine, alone or in synergy with stem cell factor (SCF) to induce immature thymocyte proliferation. The proliferation induced by IL-7 or IL-7 + SCF is restricted to the CD34+ cells and, after 4 or 8 days of culture with IL- 7, some CD34+7++ acquire the expression of CD4 and/or CD8, but remain CD3/TcR-. We also tested the myeloid differentiation capacity of these CD34 immature thymocytes. Using two different approaches, myeloid colony formation in methylcellulose and limiting dilution analysis in the presence of myeloid growth factors, we were unable to detect myeloid differentiation capacity from CD34+ early thymocytes, whereas CD34+7+ from bone marrow contained about 10% of the clonogenic cells present in the CD34+7- fraction. Together, these data support the concept that thymic CD34+7++ represents the earliest thymic subset of fully committed T-lineage cells, capable of proliferating specifically to IL-7.

Changing patterns of radiosensitivity of hematopoietic progenitors from chronically irradiated dogs prone either to aplastic anemia or to myeloproliferative disease

Hematopoietic patterns have been assessed in chronic 60Co gamma irradiated dogs during preclinical phases of evolving aplastic anemia (AA) or myeloproliferative disease (MPD), principally myeloid leukemia. Within the AA-prone dog, a singular phase of progressive decline in blood levels of granulocytes and monocytes was noted along with a similar reduction in marrow progenitors committed to granulocyte/monocyte differentiation (CFU-GM). Measured radioresistance of the preAA CFU-GM in vitro, relative to control CFU-GM from nonirradiated animals, revealed only slightly increased resistance to gamma rays, but significantly increased resistance to fission neutrons. Within the MPD-prone dogs, four preclinical phases (i.e. suppression, partial recovery, accommodation, and preleukemic transition) preceding development of overt MPD were evidenced by the monitored change in blood granulocyte/monocyte counts and marrow progenitor levels. Analysis of radioresistance of preMPD CFU-GM revealed marked changes with time of exposure and, in turn, with preclinical phase transitions. Gamma ray resistance increased in the initial phases of exposure, with maximal levels occurring during the middle phase of exposure (accommodation, phase III) followed by a tailing off of resistance at later times. Resistance to fission neutrons by preMPD CFU-GM was observed as well, but somewhat later in the exposure course and at a much lower, more consistent level. These differential patterns of radioresistance expressed by marrow CFU-GM of chronically irradiated MPD-prone dogs to gamma rays and fission neutrons gave rise to preclinical phase-specific ‘relative biological effectiveness’ (RBE) values. From these observations, we conclude that: (i) CFU-GM of MPD-prone dogs acquire and maintain marked radioresistance to low linear energy transfer (LET) gamma rays, but only marginally elevated radioresistance to high-LET fission neutrons during the course of chronic gamma ray exposure; and (ii) CFU-GM of the AA-prone dog, in contrast, acquire little change in resistance to gamma rays, but, surprisingly, marked resistance to neutrons relative to progenitors from nonirradiated controls. These results support the concept that acquired radioresistance of vital granulocyte/monocyte lineage-committed hematopoietic progenitors is temporally, perhaps causally, linked to the processes mediating hematopoietic recovery and accommodation under chronic irradiation, and in turn to preclinical events of evolving MPD. In addition, the marked differential responses of progenitors to gamma and neutron irradiation in vitro might suggest differences in the nature of cellular lesions elicited by chronic gamma irradiation, in vivo.