Human Hemopoietic Progenitor Cells Research Articles

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The human hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but remain incompletely characterized. Here, we demonstrated cord blood lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP), granulocyte-macrophage progenitor (GMP) and multi-lymphoid progenitor (MLP) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Though most progenitors had uni-lineage myeloid or lymphoid potential, bi- and rarer multi-lineage progenitors occurred in LMPP, GMP and MLP. This, coupled with single cell expression analyses, suggested a continuum of progenitors execute lymphoid and myeloid differentiation rather than only uni-lineage progenitors being present downstream of stem cells.

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.

Regulation of cellular therapy in Australia

Use of cellular products for therapeutic purposes has predominantly been unregulated in Australia until recently. Transplant of haemopoietic progenitor cells (HPC) for bone marrow regeneration is now a routine treatment for many disorders with an established mechanism of facility accreditation. However, other cellular therapies do not have any form of accreditation, are not well evaluated and may be associated with significant risks. On 31 May 2011 the Therapeutic Goods Administration (TGA) implemented a long heralded regulatory biologicals framework for cell and tissue based therapies. The framework currently excludes human HPC, organs for direct transplantation and reproductive materials which are already covered by various forms of existing peer review and accreditation. This new framework is a practical approach for applying regulation based on the risk of the product to the recipient with four classes of product. Class 1 is reserved for the least regulated products and currently does not contain any proposed products. Class 2 will be for minimally manipulated products which will only require manufacturing compliance and evaluation against product and other mandatory standards before entry onto the Australian Register of Therapeutic Goods (ARTG). Class 3 and 4 products will be more than minimally manipulated and these cells and tissues may be used in a non-homologous manner. Class 3 and 4 products will represent a spectrum of risk where Class 4 therapies will represent the highest potential risk to the recipient, with the same requirements for Class 2 approvals but with additional requirements for comprehensive evaluation of a dossier for quality, safety and efficacy of the product. The extent of this quality, safety and efficacy data will depend upon the nature of the product and its associated risks, but will be more comprehensive for Class 4 as opposed to Class 3 products. The only truly contentious feature of this framework is the extremely high cost for dossier evaluation and the puzzling absence of an orphan drug scheme for biologicals.

Vascular endothelial growth factor (VEGF) and ovarian carcinoma cell supernatant activate signal transducers and activators of transcription (STATs) via VEGF receptor-2 (KDR) in human hemopoietic progenitor cells

Objective. To investigate the STATs signaling pathway activated by VEGF in human hemopoietic progenitor cells. Methods. CD34 + hemopoietic progenitor cells, which isolated from umbilical cord blood, were treated with VEGF or culture supernatant of ovarian carcinoma cell line which could secrete large amount of VEGF, phosphorylation and nuclear translocation of STAT3 and STAT5 were then detected by Western Blot and immunocytochemistry. Expression of VEGFR2/KDR on CD34 + cells was studied by immunocytochemistry. The specific VEGFR2/KDR heptapeptide antagonist ATWLPPR was used to identify whether the activation of STATs signaling pathway was specifically mediated by VEGFR2/KDR. Results. The concentration of VEGF in SKOV3-supernatant was 4024.84± 505.59 pg/ml. CD34 + progenitor cells could express VEGFR2/KDR. When CD34 + cells were stimulated by VEGF and SKOV3-supernatant, STAT3 appeared tyrosine-phosphorylation and nuclear translocation, but STAT5 was only phosphorylated, and not translocated. When ATWLPPR was used to block the binding of VEGF to KDR, VEGF and the SKOV3-supernatant failed to activate the phosphorylation of STAT3 and STAT5. Conclusions. STAT3 may participate in the signal transduction pathways activated by VEGF specifically mediated by VEGFR2/KDR in human hemopoietic progenitor cells, and the aforementioned signaling pathway participated in the interaction of ovarian carcinoma cells and progenitor cells.

The human granulocyte/macrophage colony-stimulating factor receptor alpha2 isoform influences haemopoietic lineage commitment and divergence.

A number of alternatively spliced isoforms of haemopoietic growth factor receptors (HGFRs) have been described, but their role in human haemopoiesis remains undetermined. We have investigated the relative expression of the alpha1 and alpha2 isoforms of human granulocyte/macrophage colony-stimulating factor receptor (hGM-CSFR) during haemopoietic cell differentiation, and have shown that both subunits are independently regulated during differentiation of CD34+ human haemopoietic progenitor cells. To further investigate these ex-vivo observations, we established a series of murine FDCP mix cell lines, which, as a consequence of the ectopic expression of alpha1 or alpha2 hGM-CSFR, demonstrated differential differentiation responses to hGM-CSF. In this model system, hGM-CSFR-alpha2-expressing cells showed increased hGM-CSF-mediated erythroid/megakaryocytic differentiation compared with hGM-CSFR-alpha1-expressing cells.

A novel monoclonal antibody recognizing a cation-dependent epitope within the regulatory loop of human beta(1) integrin (CD29).

Cell adhesion receptors of the integrin superfamily can be expressed in different affinity states towards their ligands. It has been previously demonstrated that beta(1) integrins alpha4beta(1) and alpha5beta(1) are expressed in a nonligand binding form by human hemopoietic progenitor cells but can be activated into a ligand binding form by a variety of stimuli including intracellular stimuli generated by cytokine receptors and extracellular stimuli generated by function-activating anti-beta(1) integrin monoclonal antibodies (MAbs). In both instances, the activation of beta(1) integrins is believed to be the result of conformational changes propagating along the beta(1) integrin chain which in turn increase accessibility to the ligand. A cluster of either function-activating or function-inhibiting anti-beta(1) integrin MAbs have been shown to bind within a 12 amino acid long regulatory loop between residues 207 and 218 of the human beta(1) integrin chain. We describe in this report the first MAb (96.9H9) specific for this regulatory loop whose binding is cation-dependent and requires either Ca(2+) or Mn(2+) but not Mg(2+). In addition, the activation of alpha4beta(1) and alpha5beta(1) integrins by 96.9H9 is a two-step process with distinct cation requirements. Whereas Ca(2+) is sufficient to promote binding of the antibody to the beta(1) integrin chain, Mg(2+) is necessary for activating function following 96.9H9 binding. Our data therefore suggest that the regulatory epitope of the human beta(1) integrin chain is flexible with multiple conformations according to the cationic environment.

Transient Disruption of Autocrine TGF-β Signaling Leads to Enhanced Survival and Proliferation Potential in Single Primitive Human Hemopoietic Progenitor Cells

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-beta negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-beta type II receptor (TbetaRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-beta signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TbetaRIIDN and green fluorescence protein in the same CD34(+)CD38(-)Lin(-) cells were constructed. Overexpression of TbetaRIIDN specifically disrupted TGF-beta-mediated signaling. Autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34(+)CD38(-)Lin(-) cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-beta-negative regulatory loop of primitive hemopoietic progenitor cells.

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

Naturally-occurring anti-G-CSF antibodies produced by human cord blood B-cell lines infected with Epstein-Barr virus

Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. Mononuclear cells from cord blood samples of different newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. Six different, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing lambda clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. The secreted Abs did not affect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing.

SDF-1 suppresses cytokine-induced adhesion of human haemopoietic progenitor cells to immobilized fibronectin.

The process of haemopoietic cell homing to the microenvironment includes migration and adhesion. SDF-1 is a C-X-C chemokine that acts as a chemoattractant for haemopoietic progenitors. Adhesion of haemopoietic progenitors to immobilized fibronectin is up-regulated by stimulation with cytokines. We assessed the effects of SDF-1 on cytokine-induced adhesion of progenitor cells to fibronectin. In factor-dependent human MO7e cells and primary CD34+ cord blood cells, treatment with SDF-1 dose-dependently suppressed cytokine-induced adhesion. Pretreatment with pertussis toxin reversed adhesion-inhibition, suggesting that activation of G-coupled proteins are involved in the intracellular signalling of this process. These data suggest that SDF-1 not only acts as a chemoattractant but also participates in modulating adhesiveness of haemopoietic progenitors to extracellular matrix components.

Hepatocyte growth factor/scatter factor (HGF/SF) affects proliferation and migration of myeloid leukemic cells.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by mesenchymal cells, including bone marrow (BM) stromal cells, and has mitogenic and motogenic effects on a variety of cell types. Recently, a role has been assigned to HGF/SF and its receptor, c-MET, in both normal and malignant hemopoiesis. We investigated the function of HGF/SF on hemopoietic mononuclear cells (MNC) from patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with circulating blasts. In contrast to results with normal MNC, HGF/SF alone stimulated the proliferation and colony formation of MNC from these patients. MNC from some (4/13) of the AML patients also produced HGF/SF (0.1-0.2 ng/ml/day), while we could not detect HGF/SF in cultures from normal MNC. Furthermore, it appeared that HGF/SF induced migration of leukemic cells in Boyden using KG1a cells as a model for leukemic blasts. The membranes dividing the two compartments of the Boyden chambers were coated with fibronectin. HGF/SF significantly promoted migration in 3/5 samples of MDS patients and in 5/7 samples of AML patients. Supernatant of human BM stromal cells, which is chemoattractive for normal human hemopoietic progenitor cells, also promoted migration of MNC from 4/5 MDS patients and 6/7 AML patients. Since HGF/SF is one of the growth factors produced by BM stromal cells, a neutralizing antibody directed against HGF/SF was added to the BM stroma supernatant, which reduced migration significantly in 2/3 MDS and in 3/6 AML responders to BM stroma supernatant. In conclusion, HGF/SF promotes proliferation and migration of hemopoietic cells from AML and MDS patients in vitro and may therefore contribute to the malignant potential of these cells.

Activation of phospholipase A2 during differentiation of human haemopoietic progenitor cells.

Activation of phospholipase A2 during differentiation of human haemopoietic progenitor cells.

The Phenomenon of Blood Stem Cell Mobilisation

AbstractThe use of mobilised blood to effect haematological rescue now exceeds that of bone marrow. Despite the now broad application of blood stem cells (BSC) in a variety of clinical settings little is known of the precise mechanisms responsible for their release into the peripheral circulation. Under steady state conditions, primitive haemopoietic progenitor cells (HPC) are restricted to the bone marrow and circulate at low to undetectable levels. Although the basis for this specific retention of HPC within the BM remains to be defined, adhesive interactions between HPC and their surrounding stromal cell and ECM environment are hypothesised to play a major role.Mobilisation occurs in response to a variety of perturbations including physical exercise, following myelosupressive chemotherapy and following administration of a number of different cytokines. The kinetics of release may vary from as little as a few minutes to several weeks. Such observations suggest that a single unifying mechanism is therefore unlikely to be responsible for mobilisation in all cases and in all likelihood the predominant mechanism(s) will vary according to the mobilising agent employed. Nevertheless, mobilising agents are proposed to act to a greater or lesser extent by perturbing the function of the various cell adhesion molecules (CAM) or ligands which restrict HPC to the BM. In accord with this notion, studies by other have shown that antibody induced perturbation of VLA‐4 function results in mobilisation of HPC in primates.How therefore do cytokines, the most commonly mobilising agents, result in HPC release? The most frequently employed agent to induce HPC release is granulocyte colony‐stimulating factor (G‐CSF). We have previously demonstrated that in vitro exposure of human CD34+ cells (HPC) to G‐CSF, in common with several cytokines including IL‐3, GM‐CSF and SCF, results in transient increases in the affinity of the β1 integrins VLA‐4 and VLA‐5 for their ligands fibronectin (FN) and VCAM‐1. Thus in addition to the well documented mitogenic effects of cytokines these molecules also regulate the adhesive properties of HPC. Data will therefore be presented which examine the regulation of integrin function on human HPC and its relevance to the phenomenon of mobilisation. In addition we will also review recent observations in man and in a murine model which suggest an unanticipated link between bone turnover and BSC release. Collectively these studies provide some novel insights into the phenomenon of mobilisation but also suggest that the actual mechanisms involved are more complex than is currently appreciated.

Haematotoxicity testing in vitro using human cord blood haemopoietic cells

Exposure to benzene and lead results in human and experimental animals in myelotoxicity. Haemopoietic progenitor cells are considered the target cells for these effects. Currently, experimental animals are in widespread use to predict haematotoxicity. In vitro techniques allow use of human cells. This will highly improve the predictive power of myelotoxicity testing compared with experiments on syngeneic animals (mainly rat and mouse). Furthermore the number of animals required for product development and approval will be reduced. In vitro assays for studying haemopoietic progenitor cells are well developed. We examined the effect of two benzene metabolites and lead nitrate on the in vitro growth potential of human haemopoietic progenitor cells derived from human cord blood. The effects of lead nitrate and benzene metabolites such as catechol and hydroquinone on colony formation of cord blood haemopoietic progenitors were investigated in semi-solid (agar) assays (CFU-GM = Colony Forming Unit of granulocytes and/or macrophages). Toxic agents were added as single agents at the start of the cultures. Cells were exposed during the 14-day culture period. The in vitro exposure of the haemopoietic cells resulted in a dose-dependent depression of the CFU-GM numbers. The dose that caused a 50% decrease in colony formation (IC 50) was 5 μm for catechol and 20 μm for hydroquinone. An interindividual variability was seen in the response to Pb exposure. In some cord blood samples Pb inhibited colony growth only at higher doses (IC 50 > 100 μ m). In a large number of samples, haemopoietic cells were affected at much lower concentrations of Pb exposure (IC 50 < 10 μ m). Effects in vitro are seen at concentration levels that are found in human blood in situ.

The expression of the endothelial cell antigen CD34 in demyelinating disease

In this study, the antigenic expression of CD34, a 110 kDa glycoprotein which is expressed on human haemopoietic progenitor cells and vascular endothelium, has been assessed in a variety of neuropathological conditions, including infectious and demyelinating disease. Using immunoperoxidase staining on paraffin sections, the immunohistochemical results show that CD34 antigen is expressed widely on human CNS endothelium in grey and white matter, in the eye including retina, and in the anterior and posterior lobes of the pituitary. In demyelinating disease CD34 antigen expression was not detected in acute lesions, whereas strong expression was observed in old lesions. CD34 endothelial positivity was observed in areas of gliosis, vasogenic oedema, vascular disease and in Alzheimer's and Parkinson's disease pathology. A general pattern emerged, with CD34 antigen reactivity predominantly negative in areas of inflammation with demyelination but positive in adjacent non-inflamed tissue, irrespective of myelin pathology. We conclude that perivascular inflammation is a key factor in the absence of immunoreactivity of CD34 in the CNS in demyelinating disease.

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.

Differential effects of recombinant human interleukin-13 on the in vitro growth of human haemopoietic progenitor cells.

Effects of recombinant human interleukin (IL)-13 on in vitro haemopoiesis from non-adherent mononuclear cells (NAMC) or highly enriched CD34+ cells of human cord blood (CB) were studied. IL-13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34+ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemagglutinin-stimulated human peripheral blood leucocyte-conditioned medium (PHA-LCM) in a dose-dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA-LCM, demonstrated an increased MK colony number in the presence of IL-13, especially in combination with IL-3. However, IL-13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte-macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL-3, GM-CSF, Epo, G-CSF or in combination was significantly inhibited by the addition of IL-13. On the other hand, high concentrations (100 ng/ml) of IL-13 were needed to cause a slight inhibition on the growth of BFU-E-derived colonies under the same methylcellulose culture. These results indicate that IL-13, alone and synergistically with the effect of IL-3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells.

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl and hemopoietic factors.

Gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells.

gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.