In vitro expansion of CD34+ cells from peripheral blood of myeloma and lymphoma patients

We studied the feasibility of in vitro expansion of CD34+ cells from patients with multiple myeloma (MM) or follicular non Hodgkin lymphoma (NHL). CD34+ cells were selected from peripheral blood (PB) using avidinbiotin immunoadsorption columns: purified CD34+ cells from three MM and five NHL patients were expanded. First, CD34+ cells (2 MM, 4 NHL) were grown for 14 days in 5 ml of IMDM plus 12.5% horse serum (HS), 12.5% fetal calf serum (FCS) and a commonly used combination of cytokines: IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (10 ng/ml each) and EP (4 UI/ml). In these conditions, at day 14, average increase in CD34+, CFU-GM and total cell numbers were, respectively: x 6.0 x 23 and x 2,113 fold with 20 to 35% of granulocytic cells. In terms of CD34+ cell, CFU-GM and total cell outputs, MM cultures were comparable to NHL cultures, but MM cultures seemed to produce less granulocytic cells than NHL cultures. Next, in vitro expansion of PB CD34+ cells was tested in culture media suitable for clinical use. Two cultures (1 MM, 1 NHL) were carried out for 14 days in 20 ml of X-Vivo 10 medium, 2% human serum, IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (6 ng/ml each) and EP (2 UI/ml). Increase in CD34+, CFU-GM and total cell numbers in these conditions were, respectively: x 5.7 and x 19.7, x 11.9 and x 40.9, x 424 and x 408 fold, with at least 75% of granulocytic cells in both cultures. We conclude that, although further improvements are necessary, in vitro expansion of PB CD34+ cells can presumably be carried out successfully for MM patients as well as for NHL patients, including in conditions suitable for clinical use.     

In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.     

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice

Previous studies demonstrated that biological N1-oxidation occurred for some 9-alkyl-/9-aralkyladenines, but not for others, when mammalian hepatic microsomal incubates were used as enzyme source. In order to understand the mechanisms controlling the metabolic fate of these compounds, the relationship between N1-oxidation and certain physicochemical characteristics of these substrates was studied. It was found that there was no marked link between N1-oxidation and the computer predicted pKa values of the substances studied. However, a computer predicted LogP value in the range 1.3-4 seems to be the most favourable for N1-oxidation. The 1H-NMR and 13C-NMR spectroscopic characteristics of the substrates, which reflect certain electronic characteristics of the purine moiety, also showed a correlation with their N1-oxidation. The electronic effects of the substrates in relation to their metabolism was investigated using computer modelling techniques; the results showed that different substituents at the 9-position of adenine may modify the electronic characteristics of the purine moiety thus affecting their metabolism. The conformation of the substrates may also be an important controlling factor for their N1-oxidative metabolism.     

Extrathymic T cell differentiation in vitro from human CD34+ stem cells

Recent experiments have extended our understanding of how sensory information in premotor networks controlling motor output is processed during locomotion, and at what level the efficacy of specific sensory-motor pathways is determined. Phasic presynaptic inhibition of sensory transmission combined with postsynaptic alterations of excitatory and inhibitory synaptic transmission from interneurons of the premotor networks contribute to the modulation of reflex pathways and to the generation of reflex reversal. These mechanisms play an important role in adapting the operation of central networks to external demands and thus help optimize sensory-motor integration.     

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.     

A neutralizing anti-TGF-beta1 antibody promotes proliferation of CD34+Thy-1+ peripheral blood progenitors and increases the number of transduced progenitors

To reduce the time of computation of a motor unit potential (MUP), the shape of intracellular action potential (IAP) and (or) MU anatomy are generally simplified. A method of MUP presentation is suggested. It provides accuracy of the MUPs calculated for any distance and size of rectangular electrodes together with considerably reduced computational load and time. No simplification of the IAP shape or location of muscle fibres of different diameters and lengths is required. The MUP generated by N temporally and spatially dispersed single fibre action potentials is considered to be the output signal of a linear time-shift invariant system for potential generation. The input signal is the first temporal derivative of the IAP. The common impulse response (CIR) is the sum of potentials produced at the electrode by N pairs of dipoles propagating from the motor end-plates to the ends of the corresponding fibres. The potentials of each dipole at the rectangular plate electrode are determined analytically. Thus, the MUP is calculated as a single convolution between the input signal and CIR for a rectangular electrode of any size.     

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.     

Effects of cytokines on platelet production from blood and marrow CD34+ cells

The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+ CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF- or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.     

Cytokine mediated expansion of human umbilical cord blood CD34+ cells: comparison of the use of partially purified and pure CD34+ target cells

The study compares an occurrence rate of congenital malformations in newborn infants of mothers with insulin dependent diabetes (IDDM) and newborns of healthy mothers and mothers with pregnancy diabetes (GDM). This paper evaluates the influence of stage of advancement (a class) of diabetes in the mother and its control during early pregnancy on a rate of congenital malformations in the fetus. We have taken a group of 170 neonates of mothers with IDDM. The control group was 56 newborn infants of mothers with GDM and 26,368 newborn infants of healthy women. We found 11.2% of congenital malformations in newborn infants of mothers with IDDM, compared to 1.8% of ones in the newborn infant population of mothers with GDM and 2.2% in the population of healthy mothers. The occurrence rate of congenital malformations in offspring of diabetic mothers with IDDM was 5-times higher than in the general population of newborn infants of healthy and also mothers with GDM. A risk of major birth defect occurrence in the fetus was directly proportional to the grade of the blood glucose level control in mothers during the I trimester of pregnancy, but the presence of diabetic angiopathy (classes D-H) had a significant influence on the occurrence rate of major birth defects in the fetus only in metabolic imbalance cases.     

RhG-CSF-mobilized CD34+ peripheral blood progenitors are myeloperoxidase-negative and noncycling irrespective of CD33 or CD13 coexpression

OBJECTIVE: The purpose of this study was to differentiate between the catabolic effects of interleukin-1 (IL-1) when infused into the central nervous system or into the periphery. SUMMARY BACKGROUND DATA: After injury and infection, IL-1 has been found in the central nervous system. Chronic intracerebrovascular infusion of IL-1 produces protein catabolism, anorexia, and fever. However, IL-1 may act directly on liver and bone marrow to elicit metabolic responses. Although IL-1 is thought to be involved in a number of metabolic responses associated with injury-inflammation, the sites of action are unclear. METHODS: Rats were implanted with chronic infusion pumps and received diluent or three doses of IL-1 infused subcutaneously for 6 days, and a variety of response variables were measured. In a second study, doses were adjusted so that similar systemic catabolic responses were obtained from peripheral and intracerebroventricular infusion of IL-1. The acute-phase responses then were compared in the two groups of animals receiving IL-1 by different routes. RESULT: Subcutaneously infused IL-1 elicited catabolic responses in a dose-response manner. Similar catabolic responses were achieved by infusing one tenth of the dose of IL-1 given subcutaneously into the central nervous system. Although similar systemic responses (protein catabolism, anorexia, fever, and weight loss) were observed with both routes of infusion, the subcutaneous infusion produced a much greater lymphocytosis, elevation in acute-phase reactants, and fall in serum iron and albumin. CONCLUSIONS: The IL-1 appears to have different effects depending on the site of production and site of action. Regionalization of signal proteins such as IL-1 should be taken into consideration when devising specific anticytokine treatment strategies.     

Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38- cells

In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45(+) cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 10(7) unfractionated cells, approximately 10(5) purified CD34+ cells, or 5 x 10(3) purified CD34+CD38- cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 x 10(4)/mouse) failed to engraft, whereas CD34- cells (10(7)/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38- cells produced in vivo showed a second-order relation to CD34+ graft size, and mice engrafted with purified CD34+CD38- grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38- cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38- cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38- cells in vivo when cotransplanted with purified CD34+CD38- grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.     

G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor

Vi bacterial polysaccharide is a homopolymer of alpha 1-4 N-acetyl polygalacturonic acid with variable O-acetylation at position C-3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose-dependent binding to the murine monocyte cell lines WEHI-274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B-cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP-1. Stimulation with Vi led to up-regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi-bound molecules from cell surface biotinylated A20 and WEHI-274.1 revealed two bands with MW of about 32,000 and 36,000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.     

In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential

Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38- cells. CD34+CD38- cells cocultivated on the murine stromal line S17 generated predominantly CD19(+) B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the immunophenotypically uncommitted CD19- cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38- cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38- cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38- cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.     

Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors

Adhesive interactions with the extracellular matrix of the bone marrow (BM) stroma are of critical importance in the regulation of hematopoiesis. In part, these interactions are presumed to play an important role in retaining CD34+ hematopoietic progenitor cells (HPCs) within the BM environment, in close proximity with BM stromal cells and the cytokines they produce. Evidence of a more direct role for cell adhesion in the regulation of hematopoiesis is provided by recent data showing that adhesive interactions can also provide important costimulatory signals. We have previously shown that normal CD34+ HPCs express high levels of fibronectin (Fn) receptors very late antigen-4 (VLA-4) and VLA-5 in a low-affinity state, which do not allow HPCs to strongly adhere on immobilized Fn, and that cytokines such as interleukin-3, granulocyte-monocyte colony-stimulating factor, and stem cell factor transiently activate these receptors, providing HPCs with an adhesive phenotype on Fn. Thus, knowledge of the functional states of adhesion receptors is critical to our understanding of the physiological mechanisms responsible for the regulation of normal hematopoiesis. Herein, we show that combinations of cytokines that synergize to stimulate the proliferation of CD34+ HPCs result in additive stimulation of the adhesion of these cells to Fn. Thus, the activation level of Fn receptors expressed by normal CD34+ HPCs is highly correlated with their proliferative state, suggesting a functional link between these two events. Therefore, we propose a 2-step model with an initial activation of VLA-4 and VLA-5 generated by cytokine receptors that is followed by a secondary signal resulting from Fn binding to VLA-4 and VLA-5, which may cooperate with those generated by cytokine receptors.     

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.     

Proliferation and differentiation of myelodysplastic CD34+ cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis involving hyperproliferative and ineffective hematopoiesis associated with morphologic evidence of marrow cell dysplasia resulting in refractory cytopenia(s), and an increased risk of transformation into acute myeloblastic leukemia (AML). The administration of colony-stimulating factor(s) (CSFs) to patients with MDS increased blood neutrophil concentrations, in most patients, and was also expected to be beneficial and to prevent infections. However, the progression to AML during the treatment with CSFs was suspected in some patients. Therefore, extensive in vitro studies were expected to lead to the establishment of criteria for selection of patients who are likely to benefit from CSF's as well as to establish the overall value of the different types of CSFs therapy. For this purpose, in vitro colony assays provide an excellent tool for investigating the biologic characteristics of MDS progenitor cells. However, conditions of the culture must be such that each progenitor can express its full potential for proliferation and differentiation. Because of the above, MDS progenitor cells cannot be used because they carry an impairment in proliferation and differentiation. To address this problem, one needs to know how many cells are being handled and the maximum numbers of colonies and clusters expected. CD34, a stem cell phenotype, is at present one of the best markers of progenitor cells, and can be used for purposes of purification. Using a defined number of CD34+ cells, it was feasible to make direct investigations on MDS progenitor cells. In this review the properties of MDS progenitor cells are described, in association with proliferation and differentiation, with special emphasis on the phenotypic subpopulations of MDS CD34+ cells.