In vitro characterization of the hematopoietic system in pediatric patients with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) has been recognized as a hematologic neoplasia that originates at the level of a primitive lymphoid stem/progenitor cell. To date, however, the biology of the hematopoietic system in this disorder is still not fully understood. In the present study, we have determined the progenitor cell content (including myeloid, erythroid and multipotent progenitors) in 14 children with ALL and followed the proliferation kinetics of these cells in Dexter-type long-term marrow cultures. We have also characterized some aspects related to the composition and function of the hematopoietic microenvironment developed in vitro. All patients included in this study showed extremely reduced levels of progenitor cells (median of 6.2% of the levels found in normal marrow). Proliferation of these cells in long-term cultures was markedly deficient, since they showed very low numbers - compared to normal cultures - and reached undetectable levels after only a few weeks. Regarding the microenvironment developed in vitro, whereas normal marrow samples contained a median of 8 fibroblastic progenitors/10(5) marrow cells and the stromal cell layers developed in culture contained a median of 341000 adherent cells per well, ALL marrow samples showed no fibroblastic progenitors and the numbers of adherent cells were 21% of those in normal cultures. Interestingly, the levels of TNFalpha and IL-6 in ALL culture supernatants were significantly increased, compared to normal cultures. Bone marrow samples from all 14 children were also analyzed once they reached a complete clinical and hematological remission. Myeloid, erythroid and multipotent progenitor cell levels were significantly increased, compared to patients at diagnosis, and proliferation of myeloid progenitors in long-term cultures was also improved. In contrast, proliferation of erythroid progenitors showed no difference to that in cultures from patients at diagnosis. The numbers of fibroblastic progenitors and adherent cells were significantly increased, compared to patients at diagnosis, and TNFalpha and IL-6 levels returned to normal. In summary, in the present study, we have demonstrated significant in vitro alterations of the hematopoietic system, both in terms of its composition and function, in pediatric patients with ALL. Importantly, most of these alterations are corrected, at least partially, after chemotherapy.     

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34⁺ Lin⁻ cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.     

Dye-mediated photolysis of normal and neoplastic hematopoietic cells

The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.     

Enhancement of the proliferation of murine fetal liver erythroid progenitors by infection with Harvey sarcoma virus

We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.     

Positive and negative effects of tumor necrosis factor on colony growth from highly purified normal marrow progenitors.

The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on colony growth were studied using highly enriched progenitor cells from normal human bone marrow. Supplementation of TNF to culture resulted in a dose-dependent suppression of granulocyte colony-stimulating factor (G-CSF) induced granulocytic colony formation and also erythropoietin (Epo) induced erythroid burst formation. However, the number of erythroid bursts, stimulated by interleukin-3 (IL-3) plus Epo, increased when TNF was added at comparable concentrations. Further, TNF enhanced eosinophilic colony growth induced by IL-3 or granulocytic-macrophage colony-stimulating factor (GM-CSF). In GM-CSF cultures TNF (100-1000 U/ml) also induced granulocytic and macrophage colonies. The addition of neutralizing antibodies against G-CSF, GM-CSF, or interleukin-6 (IL-6) to culture did not abrogate the observed effects of TNF, so that stimulation of myeloid colony growth was unlikely to result from the secondary induction of G-CSF or GM-CSF. TNF therefore exerts favourable effects on hematopoietic progenitors responsive to the more primitive colony-stimulating factors (IL-3, GM-CSF) and potent negative effects on precursors reactive to the single lineage G-CSF and Epo. These contrasting effects of TNF suggest that TNF, when available to marrow progenitors at similar tissue concentrations, may drive hematopoiesis within the progenitor cell compartment into selected directions.     

In vitro proliferation and expansion of hematopoietic progenitors from patients with diffuse large B-cell lymphoma before and after chemotherapy

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells. Our results demonstrated that bone marrow-derived HPC from patients with DLBCL, both before and right after chemotherapy, possessed reduced proliferation and expansion potentials in vitro, as compared to their normal counterparts. Interestingly, in patients analyzed 18 months after treatment the proliferation and expansion levels were similar to those of normal HPC, indicating a complete restoration of the hematopoietic function. Although the reason for these observations is not clear, our results suggest the possibility that primitive CD34+ progenitor cells present in bone marrow, which show deficient proliferation and expansion potentials in vitro, are involved in the origin/progression of DLBCL.     

Impaired response of myelodysplastic marrow progenitors to stimulation with recombinant haemopoietic growth factors.

The regulation of haemopoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring and comparing the in vitro response of marrow progenitors from 18 MDS patients to stimulation with recombinant haemopoietic growth factors (HGFs), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) and interleukin-3 (IL-3). A similar pattern of colony growth was detected with all three HGFs in most MDS patients, exhibiting subnormal growth of GM-CFU and markedly poor to absent growth of BFU-E and CFU-GEMM. A common severe impairment in the growth of all colony types with all three HGFs was observed in five patients, four of whom presented with pancytopenia. The stimulation of MDS marrow progenitors with a five-fold higher than control saturating dose of HGFs induced a significant increase in the frequency of one, two, or all three colony types in cultures of 14 patients, whereas colony numbers in control (n = 8) marrow cell cultures were not significantly changed. All four of the non-responders were pancytopenic and three exhibited markedly impaired colony growth. Supersaturating GM-CSF, G-CSF and IL-3 increased GM-CFU numbers in six, three, and three patients, respectively. The values for BFU-E were three, six, and seven and for CFU-GEMM two, one, and five. The enhancement of MDS marrow colony numbers by supersaturating HGFs which exert their effects directly or via the action of marrow accessory cells, suggests that the progenitor cell growth abnormalities in these disorders may involve a defect in the capacity of accessory and/or progenitor cells to respond to stimulation with specific haemopoietic growth regulators.     

Clonal growth of haemopoietic progenitor cells from myelodysplastic marrow in response to recombinant haemopoietins.

The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been analysed. GM progenitors from two of six patients who grew normal numbers of colonies in response to conditioned medium + erythropoietin (5637CM + Epo) showed defective responses to either GMCSF and/or IL-3. Of all the recombinant factors tested (IL-3, IL-1, GCSF, GMCSF, MCSF), GMCSF was the strongest stimulator of myeloid clonal growth, inducing normal numbers of GM colonies from marrow of six patients (two of whom were neutropenic). Erythroid colonies were low in 5637CM + Epo-supplemented cultures of marrow from all but one patient and remained poor in the presence of any of the haemopoietins. tested. Supraoptimal doses (for normal marrow) of these haemopoietins improved colony growth in only one patient (GM colonies in response to IL-3). Combinations of factors were also largely ineffective at raising myeloid or erythroid colony numbers. These data indicate that the defective response of MDS progenitor cells to growth factors is not amenable to experimental manipulation of recombinant factor levels or combinations. Clonal assays might suggest a role for GMCSF therapy in a subpopulation of neutropenic MDS patients but their potential now needs to be evaluated in association with clinical trials.     

Effects of recombinant human granulocyte colony stimulating factor and granulocyte-monocyte colony stimulating factor on in vitro hemopoiesis in the myelodysplastic syndromes.

We evaluated the effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) and granulocyte-monocyte colony stimulating factor (rhGM-CSF) on the in vitro proliferative, differentiative, and regenerative responsiveness of marrow cells from myelodysplastic syndrome patients (MDS) in comparison to those from normal individuals. Our studies showed decreased primary clonogenicity of myeloid (CFU-GM) and erythroid (BFU-E) hemopoietic progenitor cells from the MDS patients. rhGM-CSF had more potent stimulatory effects than rhG-CSF for MDS marrow CFU-GM growth; no enhanced cellular proliferation in the MDS patients was observed in liquid culture with either rhGM-CSF or rhG-CSF. Decreased myeloid clonal cell self-generation and/or recruitment occurred in the MDS patients upon exposure to either rhG-CSF or rhGM-CSF. rhG-CSF demonstrated more potent granulocytic differentiation effects than rhGM-CSF both for normals and MDS patients using marrow enriched for immature myeloid cells with lesser differentiation noted for MDS. Cytogenetic abnormalities, present with or without additional normal karyotypes in native marrow of four MDS patients, persisted after culture with rhG-CSF, indicating induced differentiation of both normal and abnormal clones. Although proliferative and differentiative effects were seen with both factors these data show MDS marrow cells in vitro to have predominantly differentiative responsiveness to rhG-CSF and proliferative responsiveness to rhGM-CSF.     

Amifostine does not preferentially stimulate the growth of residual polyclonal progenitor cells in myelodysplastic syndromes

Amifostine is a phosphorylated aminothiol that has besides anti-oxidative and cytoprotective properties, also survival- and growth-promoting effects on hematopoietic progenitor cells. Clinical studies have demonstrated that infusions with amifostine are able to increase erythro-, myelo-, and thrombopoiesis in some patients with myelodysplastic syndromes (MDS). Since clonal and non-clonal progenitors can coexist in early phase MDS, we have studied if amifostine exerts a selective growth-promoting effect on clonal or non-clonal cells. For this purpose, purified CD34(+) marrow progenitors from nine female MDS patients were grown in short- and long-term cultures. Clonality was studied on individual colonies using polymorphisms in the human androgen receptor assay (HUMARA) locus. Three patients had growth of residual non-clonal progenitors at baseline. Continuous exposure to 100nM amifostine exerted a growth-promoting effect on progenitors in 50% of the patients. HUMARA patterns of individual colony-forming unit granulocyte macrophage (CFU-GM; 5/9) and 5 week long-term culture-initiating cells (LTC-IC; 2/9) were compared without and with amifostine exposure. We did not observe preferential stimulation of clonal or non-clonal progenitors. Based on these results, the stimulation of committed and immature progenitor growth in MDS by amifostine, is non-selective and does not favor nor suppress the growth of residual non-clonal cells.     

In vitro growth of myeloid and erythroid progenitor cells from myelodysplastic patients in response to recombinant human granulocyte-macrophage colony-stimulating factor

Marrow progenitor cells from 14 myelodysplastic (MDS) patients and 17 normal donors were assayed in semisolid cultures supplemented with increasing doses of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or medium conditioned by 5637 bladder carcinoma cells (5637CM). At doses of supplements shown to be optimal for colony formation in cultures of normal marrow, myeloid (day 14) colony numbers were subnormal in 10 of 14 MDS marrows cultured in 5637CM and in 8 of 14 cultures containing rhGM-CSF (2.5 ng/ml). However, a high dose of rhGM-CSF (20 ng/ml) raised myeloid colony numbers in cultures of many MDS marrows, so that 9 of 14 now yielded colonies within the normal range; increased levels of 5637CM failed to do this. Erythroid colony growth was poor in 13 of 14 MDS marrow cultures supplemented with erythropoietin in addition to 5637CM or rhGM-CSF. High concentrations of rhGM-CSF did not increase erythroid growth. These data suggest that myeloid progenitors from the MDS clone may have a decreased responsiveness to hemopoietins which can be overcome at high concentrations of growth factors.     

In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.     

In vitro hematopoiesis in patients with malignant lymphoma during active disease and at complete clinical remission after chemotherapy

Malignant lymphomas are neoplastic diseases of lymphoid cells, which usually originate in the lymph nodes. During the last two decades, significant progress has been made in the characterization of chromosomal and molecular alterations in these malignancies. To date, however, the composition and function of the hematopoietic system in this group of hematological disorders is still not fully understood. In the present study, we have determined the progenitor cell content in 10 patients with diffuse large-cell lymphoma (DLCL) and characterized the proliferation of these cells in long-term marrow cultures. We have also addressed some issues regarding the composition and function of the hematopoietic microenvironment in this malignancy. All the patients included in this study showed normal hematological parameters in peripheral blood, both before and after chemotherapy, however, significant hematopoietic alterations were consistently observed. As compared to normal subjects, lymphoma patients showed a 35% reduction in progenitor cell numbers, including myeloid, erythroid and multipotent progenitors. The in vitro proliferation of these cells was also deficient, since their levels in long-term marrow cultures were significantly lower than those observed in normal bone marrow cultures. Fibroblastic progenitors were reduced by >50% and this correlated with a deficient adherent cell layer development in culture. A reduction was also seen in the levels in culture supernatant of the stimulatory cytokines Stem Cell Factor and Interleukin-6. Interestingly, all the hematopoietic alterations mentioned above were still present in patients at complete clinical remission after chemotherapy. Thus, in the present study we have demonstrated significant in vitro deficiencies in the composition and function of the hematopoietic system in patients with diffuse large-cell lymphoma, both during active disease and at the time of complete clinical remission.     

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.     

Maintenance of Philadelphia-chromosome-positive progenitors in long-term marrow cultures from patients with advanced chronic myeloid leukemia.

We investigated the marrows of 19 patients with advanced Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) in long-term marrow culture (LTMC) to determine the frequency of loss of clonogenic leukemic cells in vitro. Sixteen patients were in first chronic phase at a median of 24 months from diagnosis and had received prior therapy with busulphan and/or hydroxyurea. The effect of interferon therapy on loss of Ph+ clonogenic cells in LTMC was also investigated. Of 16 patients who had not previously received interferon, complete loss of Ph+ progenitors was documented by 4-5 weeks in the LTMCs from two (12.5%). Ph+ progenitors persisted at 4-5 weeks in the LTMC derived from 12 patients. Marrows from nine patients treated with interferon were also established in LTMC. Cultures from four patients did not yield colonies with detectable metaphases at 3-5 weeks, while Ph+ clones were present in the cultures initiated with marrows from five patients. Mean hematopoietic colony yields from the adherent layer at 2-4 weeks, and from the supernatant layer at 1-3 weeks, of cultures derived from interferon-treated patients were significantly lower than in LTMCs of patients not treated with interferon (p less than 0.05). The results indicate that in previously treated patients with late chronic phase CML there is a low frequency of conversion of Ph-negative hematopoiesis in long-term culture. Interferon therapy is associated with impaired progenitor yields in LTMC and does not improve selective loss of Ph+ progenitors.     

Stimulatory effects of neopterin on hematopoiesis in vitro are mediated by activation of stromal cell function

The pteridine neopterin (NP) was shown to be produced by monocytes and is known to be a useful marker of immunological activation, although, its biological activity is still unclear. Recently, we found that intravenous administration of NP increased the numbers of blood leukocytes, and granulocyte-macrophage progenitor cells (CFU-GM) in the bone marrow and spleens of mice. In order to elucidate the mechanism whereby NP stimulates hematopoiesis, the effects of NP on hematopoietic stem cell proliferation and differentiation in vitro were studied using a long-term bone marrow culture (LTMC) system with cloned stromal cell line, MS-5. Adding NP to the LTMC increased the numbers of cells in total, CFU-GM and colony-forming unit in spleen (CFU-S). NP also increased the number of CFU-GM in a soft agar culture system, but it did not enhance CFU-GM colony formation when target bone marrow cells were semi-purified (T, B and adherent cell-depleted bone marrow cells) and cultured in this system, suggesting that NP did not directly affect the proliferation of hematopoietic progenitors. Conditioned medium obtained from NP-treated stromal cells had much greater colony-stimulating activity than that obtained from untreated stromal cells. Furthermore, NP treatment stimulated the production of IL-6 and GM-CSF by stromal cells. All these findings suggest that NP stimulates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.     

Mechanism of diethyldithiocarbamate modulation of murine bone marrow toxicity

Sodium diethyldithiocarbamate (DDTC) has been shown to modulate the myelosuppression that commonly occurs following treatment with anticancer drugs in mice. In order to investigate the mechanism of action of this myeloprotector, murine long-term bone marrow cultures were treated with DDTC alone or were preceded by the anticancer drug cis-diammine(cyclobutanedicarboxylato)platinum(II) (CBDCA), and the granulocyte/macrophage colony-stimulating activity of the supernatants was measured. The supernatants harvested from DDTC-treated cultures enhanced proliferation of granulocyte/macrophage progenitor cells almost 4-fold compared to cultures treated with no drug or with CBDCA alone. Pretreatment of cultures with CBDCA neither enhanced nor inhibited DDTC-induced colony-stimulating activity. Similar results were obtained by using marrow stromal cell cultures free of hematopoietic cells. Thus, DDTC may hasten bone marrow recovery by augmenting stromal cell production of a factor(s) with hematopoietic colony-stimulating activity.     

In vitro growth response to G-CSF and GM-CSF by bone marrow cells of patients with acute myeloid leukemia.

The in vitro growth response of bone marrow and blood cells to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) was studied in 18 acute myeloid leukemia (AML) patients using semisolid and suspension cultures. In 80% of the cases growth of leukemic progenitor cells was stimulated by GM-CSF and/or G-CSF, as judged by colony or cluster formation. In acute promyelocytic leukemia [t(15;17)], G-CSF stimulated and maintained the leukemic progenitors only transiently but fully stimulated the residual normal granulocyte/macrophage colony-forming units (CFU-GM). In some cases of M2 and M4 leukemia, G-CSF enhanced markedly the production of mature but cytochemically abnormal neutrophils. In some cases of M1 leukemia, neither CSF stimulated leukemic progenitors but instead stimulated only residual normal granulopoiesis. Spontaneous colony formation was observed in 20% of cases and was correlated with high-grade leukemic growth in vivo and a poor response to chemotherapy. The differing effects of the CSFs upon leukemic cells and residual normal granulopoiesis may have some implications for the clinical use of GM-CSF and G-CSF to overcome infectious complications.     

In vitro hemopoiesis of marrow cells from myelodysplastic patients: effects of mitogen-stimulated spleen cell-conditioned medium and vitamin A and D derivatives

We determined the ability of pokeweed mitogen-stimulated human spleen cells to support the growth and proliferation of hemopoietic progenitors from normal and myelodysplastic syndrome (MDS) patients, in the presence and absence of the maturation-inducing agents 13-cis-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (vit D). The addition of various concentrations of vit D or RA to culture plates containing MDS patients' marrow cells stimulated myeloid colony growth. A higher number of myeloid colony-forming units-cell was noted in cultures containing vit D or RA in addition to pokeweed mitogen spleen-conditioned medium compared to these substances and placenta conditioned medium. When colony-forming units-cell from MDS patients was incubated with 2 X 10(-8) M vit D the percentage of monocyte-macrophage colonies was increased and the number of granulocyte colonies was decreased. Neither vit D nor RA affected the number of cells per colony. Our findings suggest that pokeweed mitogen spleen-conditioned medium provides a more potent source than placenta-conditioned medium for humoral factors that stimulate the growth of hemopoietic progenitors from MDS patients and that the use of pokeweed mitogen spleen-conditioned medium may improve studies of hemopoiesis in MDS patients.     

Multipotent and committed CD34+ cells in bone marrow transplantation.

In order to study the role of CD34+ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34+ cells were also performed to evaluate their in vitro hematopoietic function. CD34+ cells were detectable in bone marrow cells on day 14. More than 80% of CD34+ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34+ cells. In normal bone marrow cells, CD34+, CD33+ cells amounted to about 40% of CD34+ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34+, CD33- cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34+, CD33+ cells as committed progenitors and CD34+, CD33- cells as multipotent stem cells have distinctive biological behaviors in BMT.