Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.     

Effect of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and malignant blood cells to oxazaphosphorines

The ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and several cultured human malignant blood cell lines to analogues of "activated" cyclophosphamide, namely, 4-hydroperoxycyclophosphamide and mafosfamide, and to phosphoramide mustard was quantified with and without concurrent exposure to an inhibitor of aldehyde dehydrogenase activity, namely, disulfiram, cyanamide, diethyldithiocarbamate, or ethylphenyl(2-formylethyl)phosphinate. Inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of 4-hydroperoxycyclophosphamide and mafosfamide toward all of the hematopoietic progenitors; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Potentiation of the cytotoxic action of mafosfamide toward cultured human malignant blood cells was minimal. Spectrophotometric assay revealed little NAD-linked aldehyde dehydrogenase activity present in the cultured human tumor cell lines as compared to that found in normal mouse liver or oxazaphosphorine-resistant L1210 cells. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major intermediate in cyclophosphamide bioactivation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that human multipotent hematopoietic progenitor cells contain the relevant aldehyde dehydrogenase activity, the relevant activity is retained upon differentiation to progenitors committed to the megakaryocytoid, granulocytoid/monocytoid, and erythroid lineages, and the relevant activity may be lost or diminished upon transformation of hematopoietic progenitors to malignant cells.     

Parameters affecting the ex vivo cytotoxic drug sensitivity of human hematopoietic cells

The drug sensitivity of normal cells provides a baseline for determining the therapeutic index, and therefore the effectiveness, of cytotoxic drugs, yet little is known about the factors that affect normal cell chemosensitivity. Some parameters are known to have a profound effect on tumor cell sensitivity. The purpose of this study was to determine how cytotoxic drug sensitivity of hematopoietic cells isolated from cancer patients was affected by various parameters. These included previous chemotherapy (yes or no), sex, age, tumor type (leukemias or solid tumors), sample source (blood, bone marrow, serous effusions, or tumor biopsies) and predominant cell lineage (lymphoid, myeloid, macrophage, or mixed). Mononuclear cells isolated from blood, bone marrow, serous effusions, and tumor biopsies were incubated for four days with a median of 16 drugs. The differential staining cytotoxicity assay, an ex vivo apoptotic drug sensitivity test in which cell survival is determined morphologically, was used to assess normal hematopoietic and tumor cell response to cytotoxic drugs. One hundred forty-six specimens yielded hematopoietic cell chemosensitivity results with 3-36 drugs. Compared with tumor cells, there was far less interpatient variation in chemosensitivity of hematopoietic cells. Mean hematopoietic cell drug sensitivity showed little variation due to previous chemotherapy, sex, age, tumor type, and sample source or cell lineage. We therefore concluded that cytotoxic drug sensitivity of hematopoietic cells from a variety of sources could be used for assessment of therapeutic index. Drug therapeutic index results are a valuable tool in identifying novel cytotoxic agents and individually tailored chemotherapy regimens.     

The insulin-like growth factor system in hematopoietic cells

The insulin-like growth factor (IGF) system regulates proliferation and differentiation of hematopoietic cells. IGFs exert their effects through specific receptors on growing and differentiating blood cells as they emerge from their small pool of ancestral stem cells. The IGF system is complex as both stimulating and inhibiting effects occur by interaction of IGFs and IGF-binding proteins (IGFBPs). IGFs stimulate erythrocytes and lymphocytes but also promote leukemic hematopoietic cell proliferation. IGF-I appears to be correlated with hemoglobin levels in anemia and could also be of benefit for patients with bone marrow aplasia after transplantation. Hypersensitivity to IGF-I has been implicated as an underlying cause of polycythemia vera. Loss of imprinting of IGF-II is found in acute myeloid leukemia and myelodysplastic syndrome. Apoptosis of hematopoietic cells is significantly reduced by IGF-I involving an intriguing signal transduction pathway. IGFs could therefore, although not classical hematopoietic growth factors, be of benefit for patients with diverse hematopoietic disorders.     

Murine stromal cells producing hyper-interleukin-6 and Flt3 ligand support expansion of early human hematopoietic progenitor cells without need of exogenous growth factors.

Expansion of primitive hematopoietic progenitor cells (HPC) is a major challenge in stem cell biology. Stimulation by growth factors (GF) is essential for proliferation of HPC, while the role of stromal cell coculture for maintenance of progenitor/stem cell potential is unclear. We evaluated the potential of a murine stromal cell layer providing hematopoietic GF to support expansion of human CD34(+) cells. Murine MS-5 cells were transfected with the cDNA encoding huFlt3 ligand and the interleukin6/sinterleukin-6R fusion protein hyper-IL-6. Expansion of CFC and week6 CAFC was at least as efficient in transfected clones compared to control cocultures supported with exogenous GF. Cell numbers reached 17.5- to 62.3- (day 14) and 17.4- to 92.4-fold (day 21) of input cells. Expansion of CFU-GM/Mix was 4.0- to 12.8-fold (day 14) and 4.9- to 11.7-fold (day 21). Primitive week6 CAFC were expanded up to 6.5-fold (day 14) and 6.2-fold (day 21) without exogenous GF. When direct contact of HPC and stromal cells was inhibited, a loss of CFC and much more of CAFC potential was observed with unaffected overall cell proliferation. Here, we show the generation of GF producing murine stromal cells which efficiently support early hematopoiesis without exogenous GF. Direct stromal cell-HPC contact is advantageous for maintenance of differentiation potential.     

The rho-kinase inhibitors Y-27632 and fasudil act synergistically with imatinib to inhibit the expansion of ex vivo CD34(+) CML progenitor cells.

Evidence from cell line-based studies indicates that rho-kinase may play a role in the leukaemic transformation of human cells mediated by the BCR/ABL tyrosine kinase, manifest clinically as chronic myeloid leukaemia (CML). We therefore employed two separate inhibitors, Y-27632 and fasudil, to inhibit the activity of rho-kinase against ex vivo CD34(+) cells collected from patients with CML. We compared the effects of rho-kinase inhibition in those cells with the effects of direct inhibition of BCR/ABL using the specific inhibitor imatinib. We found that inhibition of rho-kinase inhibited the effective proliferation, and reduced survival of CML progenitor cells. When combined with imatinib, rho-kinase inhibition added to the anti-proliferative and pro-apoptotic effects of the BCR/ABL inhibitor. Our studies may indicate therapeutic benefit in some cases for the combination of rho-kinase inhibitors with imatinib.     

Cell-mediated lysis of human hematopoietic progenitor cells

Several techniques are available for the serological analysis of antigenic determinants on human hematopoietic progenitor cells (HPC). However, techniques for the recognition of cellularly defined antigens on such progenitor cells have not yet been described. We therefore developed an in vitro cellular cytotoxicity assay, with bone marrow cells as target cells. In this assay specific cytotoxic T lymphocyte (CTL) lines are used as effectors for cell-mediated cytolysis of bone marrow mononuclear cells that express the antigens for which the CTLs were primed in a mixed lymphocyte culture. As a model we used CTL lines against HLA-A2 or -B7 determinants. By using effector-target ratios varying from 1:2 to 4:1, 4 hr of incubation of these CTL lines with bone marrow mononuclear cells from HLA-A2 or -B7 positive donors resulted in a specific dose-dependent growth inhibition up to 100% of myeloid (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) HPC. In contrast no inhibition of HPC was observed using mononuclear bone marrow cells from HLA-A2 or -B7 negative individuals as target cells. Experiments in which cell-cell contact was prevented showed that the antigen-specific lysis of HPC was dependent on intimate cell-cell contact between effector-CTLs and bone marrow target cells. Our results show that this cell-mediated cytotoxicity assay can be used as a sensitive and specific tool for the study of cellularly defined antigens on human hematopoietic progenitor cells.     

Granulocyte-macrophage colony-stimulating factor modulation of the inhibitory effect of transforming growth factor-beta on normal and leukemic human hematopoietic progenitor cells.

Experiments were undertaken to investigate the molecular basis of primitive hematopoietic progenitor cell regulation in both the long-term culture system and in methylcellulose, particularly with a view to characterizing factors either able or unable to influence the behaviour of primitive leukemic cells from patients with chronic myeloid leukemia (CML). Long-term cultures of CML cells with or without irradiated normal marrow feeder layers were initiated from peripheral blood cells of CML patients with high white blood cell counts. Three weeks later the effect of exogenously added transforming growth factor-beta 1 (TGF-beta 1) on progenitor cycling status was examined. A single addition of 5 ng/ml TGF-beta 1 was able to reversibly arrest the otherwise uninterrupted turnover of primitive leukemic erythroid and granulopoietic progenitors for a period of up to 7 days both in the presence and absence of a normal adherent cell population. When TGF-beta 1 was incorporated into methylcellulose cultures, its ability to inhibit colony formation by CML progenitors showed the same differential activity on primitive cell types exhibited by normal progenitors. Dose-response curves for analogous populations of normal and leukemic cells were indistinguishable. Increasing the concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose colony assays decreased the sensitivity displayed by normal clonogenic cells to TGF-beta 1 and no differences were detectable when CML cells were used in such regulator competition experiments. These findings support a general model of primitive hematopoietic cell regulation in which entry into S-phase is determined at the intracellular level by multiple convergent pathways that may deliver either positive or negative signals from activated cell surface receptors for distinct extracellular factors. The present study shows for the first time that primitive CML progenitors exposed to TGF-beta 1 in vitro can be transiently blocked in a noncycling state for several days without loss of viability and that the mechanisms responsible for the emergence and maintenance of a clonal population of CML cells in vivo do not appear to involve changes in their sensitivity to TGF-beta 1. It is thus unlikely that the heightened proliferative activity exhibited by primitive CML progenitors both in vivo and in long-term culture can be explained by an abnormality in the intracellular mechanisms normally activated by TGF-beta 1 receptor-ligand binding. We suggest that primitive CML cells are either defective in their ability to see (or activate) endogenously produced TGF-beta 1, or are defective in their responsiveness to another, undefined, regulator.     

Fibroblast growth factor overexpressing breast carcinoma cells as models of angiogenesis and metastasis

Summary Progression of breast cancer from an estrogen-dependent, slowly growing tumor amenable to tamoxifen treatment to an aggressive, metastatic, estrogen-independent phenotype has been mimicked by the transfection of MCF-7 breast carcinoma cells with fibroblast growth factors 1 or 4. FGF-transfected cells are aggressively tumorigenic in ovariectomized or tamoxifen-treated nude mice, conditions under which the parental cells would not produce tumors. When detection of metastasis was enhanced bylacZ transfection, the FGF-transfected MCF-7 cells were reliably metastatic to lymph nodes and frequently metastatic to lungs, in further contrast to parental cells. An antiangiogenic drug, AGM-1470, given to mice bearing tumors produced by FGF-transfected MCF-7 cells, produced a decrease in tumor size. The decreased tumor size was not as marked as that produced by treatment with pentosan polysulfate, an agent which would abrogate all autocrine or paracrine effects of the transfected FGF. Thus, increased angiogenesis may be a component of the phenotypic change produced by the FGF transfection, but other autocrine or paracrine effects may also be important.Since a clonal FGF-4 andlacZ doubly-transfected cell line, MKL-4, progressively lost expression of the transfectedlacZ gene in individual cells, we performed successive rounds of fluorescence-activated cell sorting to select high-expressing cells. High-expressing cell populations thus obtained rapidly lost expression of ß-gal activity in continued culture. High ß-gal expressing clonal cell lines of MKL-4 cells established by either one or two rounds of low-density cloning also lostlacZ expression with continued culture. Southern analysis of DNA fromlacZ transfected cell lines showed the transfected sequences to be present and grossly intact in both high and low expressing populations. However, Northern analysis revealed that high-expressing populations of MKL-4 cells contained the mostlacZ mRNA, implying that in the unstable MKL-4 cell line, individual cells are down-regulating mRNA levels oflacZ. StablelacZ expression has been obtained in other FGF-transfected and parental MCF-7 cell lines using the same expression vector. Thus, the MKL-4 cell line is down-regulating mRNA encoding the transfected gene through a mechanism not dependent on the CMV promotor utilized in the expression vector. This evidence suggests thatlacZ expression is not a benign modification in certain cells.     

The trk oncogene abrogates growth factor requirements and transforms hematopoietic cells

Oncogenes may cause transformation by altering the requirements of cells for growth factors which normally regulate growth. This type of transformation is particularly evident in the ability of oncogenes of the tyrosine protein kinase gene family to abrogate the requirement of hematopoietic cells for growth factors such as interleukin-3 (IL-3). We have used this property to study the effect of the human trk oncogene on hematopoietic cells. This oncogene was generated by a genetic rearrangement that fused a non-muscle tropomyosin gene to a tyrosine protein kinase with structural features characteristic of growth factor receptors. The results presented here demonstrate that a replication-defective murine retrovirus expressing the human trk oncogene can abrogate the growth factor requirements of hematopoietic cells through a non-autocrine mechanism. In these trk-transformed cells, many of the cellular proteins which are phosphorylated on tyrosine residues following IL-3 stimulation are constitutively phosphorylated, with the notable exception of a 140 kDa membrane, IL-3-binding protein. The 140 kDa protein becomes phosphorylated only following stimulation with IL-3 in these trk oncogene-transformed hematopoietic cells.     

Platelet factor 4 enhances the adhesion of normal and leukemic hematopoietic stem/progenitor cells to endothelial cells

Platelet factor 4 (PF4) is a growth regulator of hematopoietic stem/progenitor cells (HSPCs), but its role in modulating the adhesive property of normal and leukemic cells remains unclear. We used CD34(+) cord blood cells, KG1a cell line, human umbilical vein endothelial cells (HUVECs) and a transformed HUVECs ECV-304 cells to study the effect of PF4 on cell adhesion. When CD34(+) cord blood cells were cultured either in fibronectin-coated (FN) culture plate or over the layer of HUVECs for 2h, a concentration-dependent increase of the number of adhered cells was observed in the culture containing PF4. FACS analysis revealed that the treatment of PF4 resulted in an increased expression of CD49d and CXCR-4 on CD34(+) cells. Moreover, when CD34(+) cells were expanded in the presence of PF4, the adhesive ability to culture plate of CD34(+) cells was significantly increased. To elucidate the mechanism of action of PF4, KG1a cells were incubated with or without PF4 for 2h on pre-established layer of ECV-304 cells. The percentage of CD49d(+) KG1a cells increased about 1.56 +/- 0.4 fold, and that of CD54(+) ECV-304 increased about 1.7 +/- 0.6 fold. Furthermore, the mRNA expression of CD49d and CD54 was upregulated when KG1a or ECV-304 cells were incubated with PF4. The adhesion capacity of KG1a cells was reduced after incubation with the blocking monoclonal antibodies against CD49d and CD54, respectively. Our data demonstrate that PF4 is able to enhance the adhesive ability of normal and leukemia HSPCs.     

Fibroblast growth factor 2 up regulates telomerase activity in neural precursor cells

During brain development, neuronal and glial cells are generated from neural precursors on a precise schedule involving steps of proliferation, fate commitment and differentiation. We report that telomerase activity is highly expressed during embryonic murine cortical neurogenesis and early steps of gliogenesis and progressively decreases thereafter during cortex maturation to be undetectable in the normal adult brain. We evidenced neural precursor cells (NPC) as the principal telomerase-expressing cells in primary cultures from E15 mouse embryo cortices. Their differentiation either in neurons or in glial cells lead to a down regulation of telomerase activity that was directly correlated to the decrease of telomerase core protein (mTERT) mRNA synthesis. Furthermore, we show that FGF2 (fibroblast growth factor 2), one of the main regulators of CNS development, induces a dose-dependant increase of both the proliferation of NPC and telomerase activity in primary cortical cultures without affecting the mTERT mRNA synthesis compared to that of glyceraldehyde-3-phosphate dehydrogenase (mGAPDH). Finally, we evidenced that AZT (3'-azido-2', 3'-dideoxythymidine), known to inhibit telomerase activity, blocks in a dose dependant manner the FGF2-induced proliferation of NPC. Altogether, our results are in favor of an important role of telomerase activity during brain organogenesis. Oncogene (2000).     

The effects of transforming growth factor beta 3 on the growth of highly enriched hematopoietic progenitor cells derived from normal human bone marrow and peripheral blood

The effects of transforming growth factor beta 3 (TGF-beta 3) on growth in semisolid cultures of enriched hematopoietic progenitors derived from normal human marrow and blood were evaluated. Conditioned media from the Mo-T cell line (MoCM) were the source of colony-stimulating factors used to optimally stimulate primitive progenitors. To assess whether a proportion of granulocyte/monocyte (GM) progenitors were prevented from cycling, all sizes of GM aggregates were evaluated from 3 to 20 days. The activity of TGF-beta 3 on the growth of erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) was similar to that observed for TGF-beta 1. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or 72 h after initiation of culture, did not significantly affect the total number of marrow GM aggregates at 3, 7, 14, and 20 days, but TGF-beta 3 (1,000 pmol/liter), added initially, reduced the total number of blood GM aggregates. This suggests that some blood GM progenitors might be blocked from cycling but that the great majority of marrow GM progenitors are not blocked. Whether TGF-beta 3 (10, 100, and 1,000 pmol/liter) was added initially or after 72 h of stimulation by MoCM, there was a dose-dependent reduction of marrow and blood GM colony size even when the total number of colonies was unaffected. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or at 72 h, reduced in a dose-dependent manner the size of marrow and blood-derived BFU-E. TGF-beta 3 (1,000 pmol/liter) was more likely to reduce the total number of marrow and blood BFU-E, and this increased sensitivity of the erythroid lineage may prevent the development of this population in colonies derived from multipotential colony-forming unit-granulocyte/erythroid/monocyte (CFU-GEM). The results suggest that the main effect of TGF-beta 3 and TGF-beta 1 is to slow the rate of proliferation of hematopoietic progenitors rather than to prevent them from beginning proliferation. This results in a reduction in colony size which prevents the identification of primitive versus mature progenitor on the basis of standard criteria of colony size.     

Autocrine/paracrine platelet-derived growth factor regulates proliferation of neural progenitor cells

Growth factors play an important role in regulating neural stem cell proliferation and differentiation. This study shows that platelet-derived growth factor (PDGF) induces a partial differentiation of neural stem/progenitor cells (NSPCs) in the absence of other mitogens in vitro. NSPCs thus acquire an immature morphology and display markers for both neurons and glia. In addition, these cells do not readily mature in the absence of further stimuli. When NSPC cultures treated with PDGF were exposed to additional differentiation factors, however, the differentiation proceeded into neurons, astrocytes, and oligodendrocytes. We find that NSPC cultures are endowed with an endogenous PDGF-BB production. The PDGF-BB expression peaks during early differentiation and is present both in cell lysates and in conditioned medium, allowing for autocrine as well as paracrine signaling. When the NSPC-derived PDGF was inhibited, progenitor cell numbers decreased, showing that PDGF is involved in NSPC expansion. Addition of a PDGF receptor (PDGFR) inhibitor resulted in a more rapid differentiation. Neurons and oligodendrocytes appeared earlier and had more elaborate processes than in control cultures where endogenous PDGFR signaling was not blocked. Our observations point to PDGF as an inducer of partial differentiation of NSPC that also sustains progenitor cell division. Such an intermediate stage in stem cell differentiation is of relevance for the understanding of brain tumor development because autocrine PDGF stimulation is believed to drive malignant conversion of central nervous system progenitor cells.