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Objective: The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro., Materials and Methods: We cultured murine lineage antigen-negative (Lin(-)) bone marrow cells in transwells above the UG26-1B6, urogenital ridge-, and EL08-1D2, embryonic liver-derived cell lines. We, also, performed real-time quantitative PCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines., Results: Lin(-) murine bone marrow cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in noncontact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of competitive repopulating HSC., Conclusions: We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells responsible for preserving HSC is Wnt5a., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objective: We have previously shown that all-trans retinoic acid (ATRA) enhanced the maintenance of early human hematopoietic progenitor cells (HPCs) in the presence of an irradiated stromal cell line AFT024. In this study, we examined the effects of ATRA on the stromal cell component with particular reference to cellular proliferation and gene expression., Methods: Irradiated AFT024 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum and were incubated with ATRA at 1 mumol/L up to 21 days. The cells were examined in terms of immunostaining for proliferative cell nuclear antigen (PCNA) and BrdU incorporation, apoptosis assay, cell cycle analysis, and gene expression using semiquantitative reverse-transcriptase polymerase chain reaction., Results: In the control experiments, AFT024 cells lost their confluence in culture after 15-Gy irradiation and were arrested in G2/M phase on days 7 and 21. ATRA restored the cellular confluence with an increase in proliferation on day 21 (BrdU incorporation: 20.6-fold; PCNA staining: 51.7-fold) with reversal of cell cycle arrest (S phase: 2.7-fold increase; G2/M phase: 2.0-fold decrease). There was no effect on apoptosis as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. ATRA significantly upregulated the expression of cell cycle genes for checkpoint transition, including cyclin A2, B2, and aurora kinase B, as well as genes associated with a putative role in HPC maintenance, including osteopontin, HoxA5, enhancer of zeste homolog 2, and peroxisome proliferator-activated receptor gamma., Conclusion: We concluded that ATRA induced cellular proliferation of irradiated AFT024 cells and expression of a number of genes whose relevance to HPC homeostasis would have to be further examined.

Objective: We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach., Methods: MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo)., Results: zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, p < 0.05). zChd+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DR(mo) compared with zChd+RS(mo) embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene., Conclusion: Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.

Objective: We have previously shown that hematopoietic progenitor cells (HPCs) from umbilical cord blood (UCB) can be maintained in a cytokine-supplemented stroma-noncontact (SNC) system. Here, we tested if all-trans retinoic acid (ATRA), known to improve expansion of murine hematopoietic stem cells, would enhance human HPC maintenance in a SNC culture system., Methods: CD34+CD38-Lin- cells from UCB were cultured in transwells above AFT024 in the presence of Flt-3 ligand (FLT) and thrombopoietin (TPO), with or without ATRA. Total nucleated cells (TNC), colony-forming units (CFUs), long-term culture-initiating cells (LTC-ICs), myeloid-lymphoid initiating cells (ML-ICs) and SCID repopulating cells (SRCs) were evaluated 1 to 5 weeks after culture., Results: All-trans retinoic acid (1 mumol/L) reduced expansion of CD34+CD38-Lin- TNC and CFUs after 2 to 5 weeks of culture. However, it significantly increased LTC-IC expansion after 1 to 3 and, even more so, 5 weeks of culture. ATRA also increased recovery of more primitive ML-ICs and SRCs. Increased HPC recovery appeared dependent on the presence of stromal cells, as LTC-IC expansion was significantly reduced when ATRA was added to stroma-free cultures., Conclusion: All-trans retinoic acid increases expansion of early HPCs in a stromal cell-dependent fashion.

Objective: Assays that can evaluate the potential of individual human hematopoietic stem cells (HSC) are still lacking. We previously developed the myeloid-lymphoid initiating cell (ML-IC) assay that enumerates single CD34(+) cells that generate long-term culture-initiating (LTC-IC) and NK-initiating (NK-IC) daughter cells, or single primitive progenitors with multilineage potential. When transplanted in vivo, umbilical cord blood (UCB) has greater repopulating ability than bone marrow (BM) or mobilized peripheral blood (MPB). Whether the greater in vivo repopulating ability is due to an increased frequency of HSC in UCB and generative potential of UCB, BM, and MPB CD34(+) cells is not known., Materials and Methods: Single UCB, BM, and MPB CD34(+)CD38(-)Lin(-) or CD34(+)CD38(-)CD33(-) cells were plated in ML-IC assay and after 2 to 4 weeks, progeny was evaluated for frequency and generative potential of ML-IC. We also tested whether the ML-IC assay could be used to define if increased numbers of primitive progenitors generated by different cytokines in expansion cultures are mediated by recruitment of quiescent cells or by increasing their generative potential., Results: The frequency of ML-IC in BM, UCB, and MPB was similar, but the generative potential of UCB ML-IC was significantly higher. Substitution of Flt3-L, SCF, and IL-7 with Flt3-L and thrombopoietin significantly increased the generative potential of ML-IC, whereas Flt3-L, SCF, and hyper-IL-6 increased both ML-IC frequency and generative potential., Conclusion: The ML-IC assay demonstrates that the greater repopulating ability of UCB is due to the higher generative ability of HSC in UCB. Furthermore, the ML-IC assay can discriminate between cytokine-mediated expansion of hematopoietic progenitors by enhancing generation of immature daughter cells or by recruiting otherwise quiescent cells.

Objective: beta1-integrins mediate hematopoietic stem and progenitor cell homing and retention in the bone marrow (BM) and inhibit hematopoietic proliferation and differentiation. Having no intrinsic kinase activity, integrins recruit intracellular kinases, such as the focal adhesion kinase (FAK) or the related proline-rich tyrosine kinase 2 (PYK2), to initiate signal transduction. Phosphatidylinositol-3-kinase (PI3K), which is involved in beta1-integrin signaling in many cell types, is physically and functionally associated with FAK in anchorage-dependent cells. Because PYK2 is the principal focal adhesion kinase expressed in primary human CD34+ cells, we assessed its functional relationship with PI3K in CD34+ cells in response to integrin engagement., Methods: beta1-integrins on primary mobilized peripheral blood CD34+ cells and CD34+ KG1A cells were engaged by adhesion to fibronectin (FN) or by cross-linking with an anti-beta1 integrin antibody, respectively. PI3K activity and PYK2 phosphorylation were then assessed in the presence or absence of the PI3K inhibitor, wortmannin. Association between PI3K, PYK2, and the beta1-integrin subunit were also evaluated in co-immunoprecipitation experiments., Results: beta1-integrin engagement induced PI3K activation, which was required for, and temporally preceded, PYK2 phosphorylation, indicating that PI3K lies upstream of PYK2 in CD34+ cells. Furthermore, although PYK2 and PI3K were constitutively associated, interaction of the PYK2/PI3K complex with beta1-integrins required prior integrin engagement and PI3K activation., Conclusion: Activation of PI3K following beta1-integrin engagement on human CD34+ cells results in subsequent phosphorylation of PYK2, and is required for the recruitment of the PI3K/PYK2 complex to beta1-integrins at the cell surface.

Objective: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells., Methods: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays., Results: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors., Conclusion: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.

Objective: Recent studies have shown that cells from bone marrow (BM), muscle, and brain may have greater plasticity than previously known. We have identified multipotent adult progenitor cells (MAPC) in postnatal human and rodent BM that copurify with mesenchymal stem cells (MSC). BM MAPC proliferate without senescence and differentiate into mesodermal, neuroectodermal, and endodermal cell types. We hypothesized that cells with characteristics similar to BM MAPC can be selected and cultured from tissues other than BM., Materials and Methods: BM, whole brain, and whole muscle tissue was obtained from mice. Cells were plated on Dulbecco modified Eagle medium supplemented with 2% fetal calf serum and 10 ng/mL epidermal growth factor (EGF), 10 ng/mL platelet-derived growth factor (PDGF-BB), and 1000 units/mL leukemia inhibitory factor (LIF) for more than 6 months. Cells were maintained between 0.5 and 1.5 x 10(3) cells/cm(2). At variable time points, we tested cell phenotype by FACS and evaluated their differentiation into endothelial cells, neuroectodermal cells, and endodermal cells in vitro. We also compared the expressed gene profile in BM, muscle, and brain MAPC by Affimetrix gene array analysis., Results: Cells could be cultured from BM, muscle, and brain that proliferated for more than 70 population doublings (PDs) and were negative for CD44, CD45, major histocompatibility complex class I and II, and c-kit. Cells from the three tissues differentiated to cells with morphologic and phenotypic characteristics of endothelium, neurons, glia, and hepatocytes. The expressed gene profile of cells derived from the three tissues was identical (r(2) > 0.975)., Conclusions: This study shows that cells with MAPC characteristics can be isolated not only from BM, but also from brain and muscle tissue. Whether MAPC originally derived from BM are circulating or all organs contain stem cells with MAPC characteristics currently is being studied. Presence of MAPC in multiple tissues may help explain the "plasticity" found in multiple adult tissues.

Objective: The study of hematopoietic stem cells (HSC) is limited by lack of specific markers for HSC. Rhodamine 123 (Rho) is one of the substrates of P-glycoprotein (Pgp), and the presence of active Pgp can be shown by the efflux of Rho. Rho can also be used to measure the mitochondrial transmembrane potential (energy state) of a cell. We reasoned that selection of hematopoietic progenitors using a combination of Rho efflux and phenotypic markers might be superior to use of phenotypic markers alone., Materials and Methods: We used the myeloid-lymphoid initiating cell (ML-IC) assay as functional measure of primitive progenitors. Umbilical cord blood CD34(+)CD33(-)CD38(-), CD34(+)CD33(-)CD38(-)Rho(-), and CD34(+)CD33(-)CD38(-)Rho(-)c-kit(+) cells were sorted singly onto AFT024 feeders to assess their capacity to become ML-IC., Results: The frequency of ML-IC in CD34(+)CD33(-)CD38(-)Rho(-) cells was significantly higher (15 +/- 0.4%) than that in CD34(+)CD33(-)CD38(-) cells (6.2 +/- 0.9%, p < 0.05). However, the frequency of long-term culture-initiating cells (LTC-IC) (17 +/- 3% vs 12 +/- 1.5%) and natural killer culture-initiating cells (NK-IC) (25 +/- 3% vs 20 +/- 4%) was similar in the two populations. Following the treatment of CD34(+)CD33(-)CD38(-)Rho(-) cells with verapamil, which blocks Pgp function, no increase in ML-IC was detected compared with CD34(+)CD33(-)CD38(-) cells (6 +/- 0.7%), suggesting that differences in the energy state, which is reflected by Rho staining after verapamil treatment, cannot be used as a criterion to identify human HSC. Further selection of CD34(+)CD33(-)CD38(-)Rho(-) cells based on expression of c-kit significantly increased the frequency of ML-IC, LTC-IC and NK-IC by 1.75-, 1.3-, and 1.8-fold, respectively., Conclusion: Combining the function of Pgp and phenotypic features of hematopoietic progenitors enriches the frequency of cord blood ML-IC to greater than 25%. Use of such enriched populations will allow us to characterize the biological behavior of human HSC.

The BCR-ABL oncogene is essential to the pathogenesis of chronic myelogenous leukemia, and immune mechanisms play an important role in control of this disease. Understanding of the molecular pathogenesis of chronic myelogenous leukemia has led to the development of several novel therapies, which can be broadly divided into therapies based on 1) inhibition of the BCR-ABL oncogene expression, 2) inhibition of other genes important to the pathogenesis of chronic myelogenous leukemia, 3) inhibition of BCR-ABL protein function, and 4) immunomodulation. We have systematically reviewed each of these novel therapeutic approaches in this article.

Objective: The majority of studies assessing ex-vivo expansion of primitive hematopoietic cells only address production of myeloid progeny whereas it may be more appropriate to maintain or expand progenitors that retain capacity for multilineage differentiation. In this study, we assessed the capacity of the murine fetal liver cell line AFT024 to expand primitive myeloid progenitors (LTC-IC) and lymphoid progenitors (NK-IC) from umbilical cord blood (CB) and mobilized peripheral blood (PB) CD34(+)lin(-)38(-) cells., Methods: Sorted cells were established in expansion cultures in direct contact with the feeder or in a transwell above the feeder (noncontact culture) and various combinations of Flt-3L (FL), stem cell factor, interleukin 7, thrombopoietin (Tpo), and macrophage inflammatory protein-1alpha added. Frequency of LTC-IC and NK-IC was assessed at day 0 and following 2 and 5 weeks expansion culture., Results: CB contained significantly more LTC-IC and NK-IC at day 0 and showed an enhanced capacity for expansion compared to PB. The combination of FL and Tpo showed maximal expansion of CB LTC-IC and NK-IC at 5 weeks in both contact and noncontact conditions. In contrast, expansion of PB LTC-IC and NK-IC was maximal at 2 weeks and required multiple cytokines., Conclusions: These results demonstrate that AFT024 can expand primitive hematopoietic progenitors from CB and PB and expanded cells retain the capacity for myeloid and lymphoid differentiation. These findings emphasize the importance of assessing multi-lineage differentiation capacity following ex-vivo expansion. Elucidation of specific factors necessary for ex-vivo expansion will contribute to the development of a clinically applicable system.

Objective: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF)., Materials and Methods: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells., Results: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells., Conclusions: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.

Abnormal circulation and unregulated proliferation of chronic myelogenous leukemia (CML) progenitors is related, at least in part, to BCR/ABL induced abnormalities in beta1 integrin-mediated adhesion and signaling. The BCR/ABL oncogene has several potential interactions with cytoskeletal elements that are important for normal integrin signaling. In the present study, we evaluated whether abnormalities in beta1 integrin-cytoskeletal interactions were present in primary CML progenitors and contributed to defective integrin function. beta1 integrin-cytoskeletal interactions were studied in CML and normal CD34+ primary hematopoietic progenitors as well as BCR/ABL-transfected or mock-transfected M07e cells. In normal CD34+ progenitors, antibody-mediated cross-linking of beta1 integrins resulted in their redistribution into caps via a process requiring receptor-cytoskeletal interactions. CML CD34+ cells demonstrated significantly impaired beta1 integrin capping. This defect was related to the presence of the BCR/ABL gene, because capping also was impaired in BCR/ABL-transfected M07e cells. Defective receptor capping was not seen for non-integrin receptors. In addition, CML CD34- and M07eBCR/ABL cells also demonstrated increased actin polymerization and altered actin cytoskeletal organization. Further studies suggested that impaired beta1 integrin capping and defective integrin-mediated adhesion and proliferation inhibition in CML cells were related to abnormally enhanced integrin-cytoskeletal association and restricted receptor mobility. Finally, interferon alpha, which restores integrin-mediated adhesion and signaling in CML progenitors, also enhanced integrin capping in CD34+ cells. These studies suggest that p210BCR/ABL induces abnormal association of integrin receptors with the cytoskeleton and restricted receptor mobility and provide new insights into mechanisms underlying abnormal integrin function in CML progenitors.

The mechanism of localization, migration, and regulation of hematopoiesis at different stages of ontogeny is not well understood, but may relate to the specific adhesive interactions between hematopoietic stem cells and their microenvironment at different ontogenic stages. We studied the expression of cell adhesion molecules (CAM) on fetal liver (FL), umbilical cord blood (UCB) and adult bone marrow (ABM) CD34+ cells, and the adhesion of committed progenitors (CFC) from all three sources to ABM stromal layers and purified extracellular matrix proteins. Compared to ABM CFC, significantly more UCB CFC and fewer FL CFC adhered to ABM stroma. Adhesion of FL CFC to fibronectin (FN), the 75 kD RGD containing FN fragment and the 33-66 kD COOH-terminal heparin binding FN fragment was also significantly less than that of ABM CFC. Like ABM CFC, the adhesion of FL CFC was mediated through alpha4beta1 and alpha5beta1 integrins. Of note, more FL CD34+ cells expressed alpha5 integrins and the number of alpha4, alpha5 and beta1 integins per cell (mean channel frequency) was similar or higher for FL CD34+ cells than ABM CD34+ cells. Further, treatment of FL CFC with a beta1 integrin activating antibody (8A2), increased adhesion of FL CFC to FN to the same level as that of 8A2 treated ABM CFC. This suggests that the alpha4beta1 and alpha5beta1 integrins on FL CD34+ cells may be present in a low avidity/affinity state. We also show that unlike ABM, FL CD34+ cells expressed alpha2 and that approximately 20% FL CFC adhered to collagen IV. Further, alpha2beta1 integrin on FL CFC was functional since their engagement, either by adhesion to collagen IV or through blocking alpha2 antibodies, transmitted proliferation inhibitory signals. In contrast to alpha4b and alpha5beta1 integrin dependent adhesion, alpha2beta1 dependent adhesion of FL CFC to collagen IV was not enhanced after treatment with 8A2. The reason for this is not clear but suggests that alpha2 integrins on FL CFC are maximally activated. This novel adhesive interaction with collagen IV, reminiscent of that described for CML progenitors, may have a role in the extramedullary localization of FL hematopoiesis or its developmental stage-specific regulation by its microenvironment. Studies to evaluate these possibilities are underway.

FACS-selected CD34+ HLA-DR- cells (DR- cells) may provide a source of benign stem cells suitable for autografting in chronic myelogenous leukemia (CML) and other hematological malignancies. However, DR- cell selection depletes the majority of committed hematopoietic progenitors, which may be important for early engraftment. Furthermore, only a small number of DR- cells may be selectable in certain patients. These impediments to the use of DR- cells for autografting may be overcome through the development of ex vivo culture systems that support expansion and initial differentiation of primitive progenitors. Because 2-week culture of DR- cells in a stroma "noncontact" system supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein 1-alpha (MIP-1alpha) expands both long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs), we adapted this system to a clinically applicable method for expanding LTC-ICs and CFCs ex vivo. In initial small-scale studies, DR cells were grown in stroma conditioned medium (SCM) supplemented with IL-3 with or without additional growth-promoting cytokines and the chemokines PF-4 and BB10010, all approved for clinical use. An IL-3 dose-dependent expansion of committed progenitors and LTC-ICs was observed when DR- cells were cultured in tissue culture plates in SCM+IL-3 for 2 weeks. Similar CFC expansion along with increased (5-fold) LTC-IC expansion was observed following addition of PF-4 to SCM+IL-3 cultures. The addition of stem cell factor (SCF), but not of IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, IL-1, and IL-7, increased CFC and LTC-IC expansion beyond the levels observed with SCM+IL-3 alone. We next evaluated the suitability of this culture system for scale-up. Culture of 2-6 x 10(5) DR- cells in gas-permeable bags with SCM+IL-3 resulted in similar CFC and LTC-IC expansion as seen in small-scale cultures. In addition, we observed that progenitors capable of differentiating to natural killer (NK)-cells were maintained under these conditions. Finally, we found that BCR/ABL mRNA-negative CFCs and LTC-ICs present in DR- cells selected from steady-state CML marrow could be expanded in large-scale SCM+IL-3 cultures. We conclude that culture of DR- cells for 2 weeks in SCM+IL-3 culture, with or without PF-4 or SCF, results in significant CFC and LTC-IC expansion and lymphoid NK progenitor maintenance. This culture system is readily adaptable to the expansion of primitive progenitors for autotransplantation.

We studied the phenotypic and functional properties of colony-forming cells (CFCs), primitive long-term culture initiating cells (LTC-ICs) and lymphoid precursors present in human fetal liver (FL) and compared these with their adult bone marrow (BM) counterparts. FL (7-14-week) cells were selected by fluorescence-activated cell sorting based on increasing CD34 antigen expression (34+, 34++, or 34 ), CD38 antigen expression (CD34++/ CD38+, or CD38-), and HLA-DR antigen expression (CD34++/ HLA-DR+ or HLA-DR-). 13 +/- 0.6% of FL CD34-positive cells were 34 . Significantly more FL CD34++/ cells were CD38- (49 +/- 2.4%) and HLA-DR-(72 +/- 6.7%) than BM CD34++ cells (6.8 +/- 0.7% CD38- and 13.3 +/- 3.2% HLA-DR-). FL and BM CFCs were CD34+/++, CD38+, and HLA-DR+. However, significantly more FL CFCs were erythroid (40%) than adult BM CFCs (15%), and FL colonies were larger (8111 +/- 738 cells/CFC) than BM colonies (3466 +/- 272 cells/CFC, p < 0.001). As is seen in adult BM, FL LTC-ICs were CD34++/ CD38-. In contrast to BM LTC-ICs, FL LTC-ICs were almost exclusively CD34++/ HLA-DR+. In addition, a single FL LTC-IC gave rise to >30 CFCs at 5 weeks compared with only 5 +/- 0.9 CFCs per LTC-IC from BM. Finally, we demonstrate that the FL CD34++/ /CD38-/HLA-DR+ population, which contains 3.7% LTC-ICs, also contains primitive lymphoid progenitors capable of differentiating into natural killer (NK) cells. In conclusion, the phenotype of primitive human FL progenitors such as LTC-IC and primitive NK progenitors is CD34++/ /CD38-/HLA-DR+, suggesting that this population may contain FL hematopoietic stem cells. The phenotypic characterization of FL primitive LTC-ICs and NK progenitors will facilitate further studies of the functional properties of these progenitors.

Adhesion receptors can serve as primary signal transduction molecules that convey information into cells that can affect cell proliferation and differentiation. Since hematopoietic progenitors adhere to marrow stroma and fibronectin via the alpha 4 beta 1 integrin and CD44, we examined the role of these receptors in the transfer of proliferation-regulatory signals to progenitors. Actively proliferating colony-forming cells (CFCs) present in cultured CD34+ cells were incubated with mouse monoclonal antibodies against the alpha 4, beta 1, or CD44 receptors and crosslinking was performed with a secondary goat-anti-mouse antibody. The effect on CFC proliferation was examined with a 3H thymidine suicide assay. Compared with controls (39 to 51% kill), crosslinking the alpha 4 or beta 1 integrins significantly reduced CFC proliferation (12 to 26% kill, p = 0.01), indicating that proliferation-inhibitory signals are transmitted through the VLA-4 integrin. Cytochalasin D, a compound that prevents actin polymerization, prevented not only alpha 4 receptor capping, but also the inhibition of CFC proliferation observed following alpha 4 crosslinking. However, crosslinking of the CD44 receptor with the antibodies Hermes-3 and 50B4, which inhibit adhesion of CFC to fibronectin, failed to cap the CD44 receptor in the majority of CD34+ cells. Furthermore, crosslinking of the CD44 receptor with these antibodies also failed to inhibit proliferation of CFCs. These studies demonstrate that adhesion receptor crosslinking of the alpha 4 beta 1 integrin, together with subsequent changes in F-actin polymerization, negatively regulates hematopoietic progenitor proliferation in a manner independent of the shape change associated with adhesion.

Significantly more primitive human hematopoietic progenitors are maintained and differentiate when cultured separately from the stroma by a microporous membrane ("stroma-noncontact" cultures) than when cultured in direct contact with stromal layers ("stroma-contact" cultures). This suggests that diffusible stroma-derived factors may be sufficient for the in vitro induction of progenitor proliferation and differentiation. To further characterize stroma-derived factors that maintain long-term bone marrow culture initiating cells (LTBMC-IC), we compared the hematopoietic supportive capacity of human marrow stroma with that of the murine marrow stroma-derived fibroblast cell line M2-10B4 as well as two embryonic fibroblast cell lines, the human FHS-173-WE and the murine NIH-3T3 cell lines. We demonstrate that LTBMC-IC, present in human CD34+/HLA-DR- (DR- cells), are maintained equally well and give rise to similar numbers of committed progenitors, that is--colony-forming cells (CFC)--when cultured in contact with human marrow stroma or any cell line feeder (stroma-contact cultures). LTBMC-IC, cultured in marrow stroma or M2-10B4 stroma-noncontact cultures, were maintained significantly better and gave rise to significantly more CFC than when cultured in human marrow stroma or M2-10B4 contact cultures. However, LTBMC-IC maintenance and differentiation in FHS-173-WE or NIH-3T3 noncontact cultures was significantly less than in human marrow stroma or M2-10B4 noncontact cultures. These studies indicate that systematic comparison of diffusible growth stimulatory factors in conditioned media from M2-10B4 cells and FHS-173-WE may lead to the characterization of growth regulatory factors required for in vitro maintenance and differentiation of human primitive LTBMC-IC. Since diffusible factors from the M2-10B4 cell line can support human hematopoiesis, our observations may also have important implications for in vitro stem cell expansion protocols.