Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression.

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its 'undruggable' nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be 'undruggable' as previously thought.

Experimental basis of cord blood transplantation.

Efforts are needed to enhance the efficacy of cord blood (CB) transplantation. Laboratory information set the stage for the first and subsequent CB transplants, and will be instrumental in continuing to advance the field. This paper offers a brief understanding of the current state of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) biology, a look back at laboratory studies leading to the first CB transplants, and a discussion of the possible means to enhance CB transplantation. Results show that physical recovery of greater numbers of HPCs is possible after CB is collected by perfusing the placenta, but how realistic this procedure is for collection of CB to be banked is open to question. We also show that the chemokine stromal cell-derived factor-1/CXCL12 can enhance the ex vivo expansion of CB HPCs beyond that of the combination of SCF, Flt3-ligand and TPO. Advances in cytokine and stromal cell biology, and in intracellular signals mediating the effects of cytokines/stromal cells should be considered in the context of future efforts to enhance functional activities of donor CB HSCs and HPCs and the microenvironmental niche of the recipient, which is required for acceptance and nurturing these HSCs/HPCs.

SDF-1alpha/CXCL12 enhances retroviral-mediated gene transfer into immature subsets of human and murine hematopoietic progenitor cells.

Genetic modification of hematopoietic stem and progenitor cells has the potential to treat diseases affecting blood cells. Oncoretroviral vectors have been used for gene therapy; however, clinical success has been limited in part by low gene transfer efficiencies. We found that the presence of stromal-derived factor 1 (SDF-1alpha)/CXCL12 during retroviral transduction significantly enhanced, in a dose-dependent fashion, gene transfer into immature subsets of high proliferative human and murine hematopoietic progenitor cells. Murine mononuclear bone marrow cells and purified c-Kit(+)Lin(-) bone marrow cells were prestimulated and transduced with the bicistronic retroviral vector MIEG3 on Retronectin-coated surfaces in the presence and absence of SDF-1. SDF-1 enhanced gene transduction of murine bone marrow and c-Kit(+)Lin(-) cells by 35 and 29%, respectively. Moreover, SDF-1 enhanced transduction of progenitors in these populations by 121 and 107%, respectively. SDF-1 also enhanced transduction of human immature subsets of high proliferative progenitors present in either nonadherent mononuclear or CD34(+) umbilical cord blood cells. Transduction of hematopoietic progenitors was further increased by preloading Retronectin-coated plates with retrovirus using low-speed centrifugation followed by increasing cell-virus interactions through brief centrifugation during the transduction procedure. These results may be of clinical relevance.

Chemokine regulation of hematopoiesis and the involvement of pertussis toxin-sensitive G alpha i proteins.

Chemokines have been implicated in regulation of various aspects of hematopoiesis, including negative regulation of the proliferation of immature subsets of myeloid progenitor cells (MPCs), chemotaxis of MPCs, and survival enhancement of MPCs after delayed growth factor addition. Since chemokine receptors are seven-transmembrane-spanning G-protein-linked receptors and the chemotactic effect in vitro of the CXC chemokine SDF-1 is pertussis toxin (PT)-sensitive, implying the involvement of G alpha i proteins as mediators of SDF-1-induced chemotaxis, we evaluated the effects of PT on other chemokine actions influencing MPCs. While the in vitro survival-enhancing effects of SDF-1 on GM-CSF and steel factor-dependent mouse bone marrow granulocyte macrophage progenitors (CFU-GM) were pertussis toxin-sensitive, the suppressive effects of the CC chemokine MIP-1 alpha and the CXC chemokine IL-8 on colony formation by GM-CSF and steel factor-sensitive CFU-GM were insensitive to pertussis toxin. These results suggest that not all chemokine-mediated effects on MPCs are necessarily mediated through pertussis toxin-sensitive G alpha i proteins.

Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3 have been characterized, the involvement of the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably expressing human Flt3 receptor. Stat5a, but not Stats 1-4, 5b, or 6, was potently activated by Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a required the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of primary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors from marrows of Stat5a(-/-) mice, but did stimulate/costimulate proliferation of these cells from Stat5a(+/+), Stat5b(-/-), and Stat5b(+/+) mice. Thus, Stat5a is essential for at least certain effects of FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

The exodus subfamily of CC chemokines inhibits the proliferation of chronic myelogenous leukemia progenitors.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3alpha, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKbeta11/MIP-3beta, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines. (Blood. 2000;95:1506-1508)

Altered responsiveness to chemokines due to targeted disruption of SHIP.

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.

Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.

Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors.

Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.

Dominant myelopoietic effector functions mediated by chemokine receptor CCR1.

Macrophage inflammatory protein (MIP)-1alpha, a CC chemokine, enhances proliferation of mature subsets of myeloid progenitor cells (MPCs), suppresses proliferation of immature MPCs, and mobilizes mature and immature MPCs to the blood. MIP-1alpha binds at least three chemokine receptors. To determine if CCR1 was dominantly mediating the above activities of MIP-1alpha, CCR1-deficient (-/-) mice, produced by targeted gene disruption, were used. MIP-1alpha enhanced colony formation of marrow granulocyte/macrophage colony-forming units (CFU-GM), responsive to stimulation by granulocyte/macrophage colony-stimulating factor (GM-CSF), and CFU-M, responsive to stimulation by M-CSF, from littermate control CCR1(+/+) but not CCR1(-/-) mice. Moreover, MIP-1alpha did not mobilize MPCs to the blood or synergize with G-CSF in this effect in CCR1(-/-) mice. However, CCR1(-/-) mice were increased in sensitivity to MPC mobilizing effects of G-CSF. Multi-growth factor-stimulated MPCs in CCR1(-/-) and CCR1(+/+) marrow were equally sensitive to inhibition by MIP-1alpha. These results implicate CCR1 as a dominant receptor for MIP-1alpha enhancement of proliferation of lineage-committed MPCs and for mobilization of MPCs to the blood. CCR1 is not a dominant receptor for MIP-1alpha suppression of MPC proliferation, but it does negatively impact G-CSF-induced MPC mobilization.

p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells.

A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21(cip-1/waf-1). Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21(cip-1/waf-1) deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21(cip-1/waf-1) expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21(cip-1/waf-1) in p21(cip-1/waf-1) antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.

Enhanced myeloid progenitor cell cycling and apoptosis in mice lacking the chemokine receptor, CCR2.

Chemokines regulate hematopoiesis in part by influencing the proliferative status of myeloid progenitor cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin- populations of BM from CCR2-deficient (-/-) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with progenitors from spleens of CCR2 (-/-) mice. Increased cycling status of CCR2 (-/-) BM MPC did not result in increased numbers of nucleated cells or MPC in BM or spleens of CCR2 (-/-) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin- BM cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin- population of BM cells from CCR2 (-/-) mice had a significantly higher percentage of apoptotic cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin- cells. The increased percentage of apoptotic c-kit+lin- cells was due to elevated apoptosis within the c-kitdimlin-, but not the c-kitbrightlin-, subpopulations of cells. Consistent with enhanced apoptosis of phenotypically defined cells, MPC from CCR2 (-/-) BM and purified c-kit+lin- cells demonstrated decreased cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of progenitor cells in the BM, but also enhance survival of these cells.

Isolation and characterization of LMC, a novel lymphocyte and monocyte chemoattractant human CC chemokine, with myelosuppressive activity.

By searching the Expressed Sequence Tag (EST) data base, we identified a partial cDNA sequence encoding a novel human CC chemokine. The entire cDNA sequence was determined and revealed a CC chemokine whose mature protein consisted of 100 amino acids with predicted molecular weight of 11 kd. The chemokine preferantially chemoattracted lymphocytes and monocytes but not neutrophils. It was, therefore, named LMC (Lymphocyte and Monocyte Chemoattractant). LMC exhibited potent myelosuppressive activity, which was comparable to that of MIP-1alpha. We identified several bacterial artificial clones (BAC) containing the LMC gene along with two human CC chemokine subfamily members; leukotactin-1 (Lkn-1) and CKbeta8-1/CKbeta8. This data suggests that the LMC gene is located at human chromosome 17q which encompasses a human CC chemokine gene cluster.

Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic cells from Fac-/- mice.

We used a murine model containing a disruption of the murine homologue (Fac) of Fanconi Anemia group C (FAC) to evaluate the role of Fac in the pathogenesis of bone marrow (BM) failure. Methylcellulose cultures of BM cells from Fac-/- and Fac+/+ mice were established to examine the growth of multipotent and lineage-restricted progenitors containing inhibitory cytokines, including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-1alpha (MIP-1alpha). Clonogenic growth of Fac-/- progenitors was reduced by 50% at 50- to 100-fold lower concentrations of all inhibitory cytokines evaluated. We hypothesized that the aberrant responsiveness to inhibitory cytokines in clonogenic cells may be a result of deregulated apoptosis. To test this hypothesis, we performed the TUNEL assay on purified populations of primary BM cells enriched for hematopoietic progenitors or differentiated myeloid cells. After stimulation with TNF-alpha, accentuated apoptosis was observed in both populations of Fac-/- cells. In addition, deregulated apoptosis was also noted in the most immature phenotypic population of hematopoietic cells after stimulation with MIP-1alpha. Together these data suggest a role of Fac in affecting the signaling of multiple cytokine pathways and support cytokine-mediated apoptosis as a major mechanism responsible for BM failure observed in FA patients.

The contribution of animal models to the development of treatments for hematologic recovery following myeloablative therapy: a review.

This review describes the role that animal models have played in the development of clinical procedures for growth factor and hematopoietic cell therapies following high-dose cancer chemotherapy, radiotherapy or both. Data are discussed describing animal models that add to the understanding of human hematopoiesis, including myeloid and lymphoid lineage localization and in vivo maturation. Finally, current animal models of cytokine and cell therapies are presented in the context of their contributions to early clinical trials and future therapies. These studies underscore the past and current contributions animal investigations have made to improving clinical therapies.

Regulation of myelopoiesis by murine fibroblastic and adipogenic cell lines.

The regulation of hematopoiesis has been suggested to take place in close association with various cell types found in the bone marrow (BM) microenvironment. In the present study the role of fibroblasts, adipocytes and cell surface heparan sulfate in regulating hematopoiesis in an in vitro mouse system was examined. Mouse BM cells were allowed to adhere to a mouse embryo fibroblast cell line (C3H 10T1/2) or a clonally derived adipogenically determined derivative (Clone D) of the 10T1/2 cell line. Nonadherent cells were removed, cultures were overlaid with semisolid media supplemented with growth factors and colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells was quantitated. Adherence and co-culture of BM cells with the fibroblast cell line resulted in increased numbers of total CFU-GM and CFU-GEMM colonies. In contrast, adherence and co-culture of BM cells with the adipocytic cell line resulted in an increase only in CFU-GEMM colonies. Morphological analysis revealed a preferential adherence/growth of granulocyte and macrophage progenitors at the expense of bipotent granulocyte-macrophage progenitors to the fibroblastic cell line and an increase in the adherence/growth of granulocyte progenitors to the adipogenic cell line. Progenitor cell adherence was abolished when the fibroblastic or adipocytic cell lines were pretreated with heparitinase. These results demonstrate enhanced proliferation/differentiation of hematopoietic progenitor cells when there is direct contact between hematopoietic progenitors and cell types characteristic of those found in the microenvironment and that heparan sulfate and different types of stromal cells appear to play different roles in this interaction.

In vivo effects of recombinant interleukin-11 on myelopoiesis in mice.

Purified recombinant human interleukin-11 (rhuIL-11) was assessed for its in vivo effects on the proliferation and differentiation of hematopoietic progenitors as well as its capacity to accelerate the recovery of a drug-suppressed hematopoietic system. Dosage and time sequence studies demonstrated that administration of IL-11 to normal mice resulted in increases in absolute numbers of femoral marrow and splenic myeloid (granulocyte-macrophage colony-forming unit [CFU-GM], burst-forming unit-erythroid [BFU-E], CFU-granulocyte, erythroid, macrophage, megakaryocyte) progenitor cells and in stimulation of these progenitors to a higher cell cycling rate. This was associated with increased numbers of circulating neutrophils. Administration of IL-11 to mice pretreated with cyclophosphamide decreased the time required to regain normal levels of neutrophil and platelet counts in peripheral blood. In addition, IL-11 accelerated reconstitution to normal range of myeloid progenitors from bone marrow and spleen of myelosuppressed mice. These data suggest that IL-11 may play an important role in the regulation of hematopoiesis, and the application of this novel cytokine may have clinical therapeutic benefits.