Are mesenchymal stromal cells from children resistant to apoptosis?

OBJECTIVES: Mesenchymal stromal cells (MSC) represent a novel cellular candidate in the field of transplantation and tissue regeneration. Their clinical application requires their in vitro expansion. The aim of this study was to assess the effect of conditions that would favour apoptosis, and of long-term expansion, on the characteristics of MSC from children. MATERIALS AND METHODS: Bone marrow mononuclear cells were cultured for 10 passages (P1-P10). Expression of CD105, CD146, CD95 and apoptosis by 7-amino-actinomycin D staining were evaluated. CFU-F and cell doubling time (DT) were assessed in every passage. Cell-cycle study was performed at P2 and P6. RESULTS: CFU-F decreased from 38 +/- 3.7 at P2 to 9.6 +/- 3.2 per 10 MSC/cm(2) at P10 and DT increased from 1.93 +/- 0.1 (P2) to 6.1 +/- 2.45 days (P10). A low percentage of apoptotic (dead) cells was detected at P2 and this did not change until P10. Cells at P2 were at G(0)/G(1) phase, but in advanced passages more cells were in an active state. Induction of apoptosis (addition of anti-Fas agonist antibody) using standard culture conditions, showed a minor effect on MSC survival. Serum deprivation of MSC (up to 72 h) revealed no substantial apoptotic effect while cells retained their tri-lineage differentiation capacity. CONCLUSIONS: We conclude that MSC from children retain their functional characteristics throughout serial passages and remain stable under conditions that usually cause apoptosis. These features render MSC, especially those of early passages, optimal candidates for use in clinical applications.

Study of oncogenic transformation in ex vivo expanded mesenchymal cells, from paediatric bone marrow.

OBJECTIVES: Mesenchymal stromal cells (MSCs) have attracted considerable interest in both the scientific and clinical fields. In order to obtain a sufficient cell number for application, their in vitro expansion is necessary, but during this process their characteristics may be altered and cells may acquire oncogenic properties. We have investigated properties of MSC that may be related to oncogenesis, a critical parameter that has to be evaluated prior to MSC clinical use. MATERIALS AND METHODS: We studied the expression of p53, p16, RB, H-RAS and human telomerase reverse transcriptase (hTERT) in MSCs from bone marrow of children diagnosed with idiopathic thrombocytopenic purpura (ITP) and autoimmune neutropenia. The same cells were seeded in soft agar to confirm their anchorage dependence and were karyotypically analysed. Finally, MSCs were subcutaneously transplanted into SCID mice and their ectopic osteogenic as well as tumorigenic potential was evaluated. RESULTS: We have shown that MSCs derived from bone marrow of children with ITP and autoimmune neutropenia do not undergo transformation, the cells expressed normal levels of p53, p16, RB and H-RAS. Expression of hTERT was undetectable, chromosome content remained stable, and their anchorage dependence was confirmed. In an in vivo model, when MSCs were subcutaneously transplanted into SCID mice, no tumorigenesis was observed. CONCLUSIONS: These findings suggest that MSCs from bone marrow of children do not have oncogenic properties and, therefore, represent validate candidates for applications in regenerative medicine.

Could cord blood be a source of mesenchymal stromal cells for clinical use?

BACKGROUND: Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. METHODS: Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated. RESULTS: Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. DISCUSSION: These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.

Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases.

BACKGROUND: Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. METHODS: BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. RESULTS: MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. DISCUSSION: MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

kappa-opioids induce a reversible inhibition of CFU-GM from CD133(+) cord blood cells.

BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined.

Mutational analysis of the cell cycle inhibitor Kip1/p27 in childhood leukemia.

BACKGROUND: Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. METHODS: Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. RESULTS: Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. CONCLUSIONS: Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia.

Does Hypericum perforatum L. extract show any specificity as photosensitizer for HL-60 leukemic cells and cord blood hemopoietic progenitors during photodynamic therapy?

UNLABELLED: Autologous bone marrow transplantation is a therapeutic modality that increases the survival rates for children with malignancies with poor prognosis but relapse rates are high and attributed partially to the existence of residual malignant cells. Photodynamic treatment (PDT) has been developed among purging strategies. We investigated the effect of the methanolic extract (ME) and its polar methanolic fraction (PMF) of Hypericum perforatum L., as a new photosensitizer for the leukemic cell line HL-60 and cord blood (CB) hemopoietic progenitors as well as the subcellular localization of the photosensitizer. METHODS: ME and PMF were prepared after extraction of the dry herb with methanol (ME), followed by liquid-liquid extraction with petroleum ether (PMF). Cells were incubated with the extracts before irradiation with Nd-Yvo Laser. Various concentrations of PMF or ME as well as irradiation doses were tested. Following irradiation, cell viability was determined by trypan blue in continuous liquid cultures for HL-60 cells and in clonogenic assays for CB cells. The subcellular localization of the photosensitizer was determined by confocal microscopy. RESULTS: Laser photoirradiation in the presence of both PMF and ME induces the killing of HL-60 cells. This effect is dose dependent. No CFU-GM and BFU-E growth was observed from CB mononuclear cells under the tested experimental conditions. Confocal microscopy revealed that the extracts localize mainly in the cytoplasm of the cells. CONCLUSIONS: PDT with both PMF and ME induces the killing of HL-60 leukemic cells and the optimal conditions of treatment were determined. This effect of PDT/PMF was also exerted on CB progenitor cells indicative of the non-selective uptake of the photosensitizer by malignant cells. Though this suggests that PDT/PMF cannot be helpful in autologous bone marrow purging, these novel extracts can however be beneficial in the PDT treatment of tumors given their photostability, low toxicity and low cost.

Adhesion molecules, endogenous granulocyte colony-stimulating factor levels and replating capacity of progenitors in autoimmune neutropenia of childhood.

AIM: To investigate the role of granulocyte colony-stimulating factor (G-CSF) and adhesion molecules and the response of bone marrow to peripheral cytopenia in autoimmune neutropenia of childhood (AIN). METHODS: Thirty-five children with AIN, 25 with acute leukaemia in remission, 10 of whom developed chemotherapy-associated neutropenia, and 28 non-neutropenic age-matched children were studied. The methods included haemopoietic progenitor cells' colony growth, replating of colony-forming unit-granulocyte macrophage (CFU-GM) of the 7th day and ELISA for the detection of serum levels of cytokines and adhesion molecules. RESULTS: In cases of severe autoimmune neutropenia, haemopoietic progenitors showed increased proliferative capacity compared to the control group (p = 0.03). Intercellular adhesion molecule-1 (ICAM-1), E-selectin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels inversely correlated with neutrophil counts (r = -0.8, p < 0.001 for ICAM-1, r = -0.5, p = 0.04 for E-selectin, r = -0.58, p = 0.01 for TNF-alpha, r = -0.62, p = 0.04 for IL-1beta). Serum ICAM-1, TNF-alpha and IL-1beta levels correlated positively with G-CSF levels (r = 0.47, p = 0.03 for ICAM-1, r = 0.65, p = 0.01 for TNF-alpha, r = 0.67, p = 0.04 for IL-1beta). Serum G-CSF levels were widely distributed and did not correlate with neutrophil counts (r = -0.44, p = 0.09). In secondary neutropenias the respective levels were lower than those in autoimmune neutropenia. CONCLUSIONS: Haemopoietic progenitors show increased proliferative capacity in cases of severe autoimmune neutropenia. G-CSF seems to act as an inducer of endothelium activation. The degree of neutropenia correlates with serum ICAM-1, E-selectin, TNF-alpha and IL-1beta levels, indicating the existence of an activated endothelium and presumably of a latent, low-grade, inflammatory process in severe autoimmune neutropenia.

The role of L-carnitine on a restricted number of myeloid leukemia progenitor cells: generation of atypical cell types.

AIM: Since cellular maturation largely depends on lipid metabolism, we examined whether L-carnitine (L-C), a substance involved in these biochemical pathways, is able to promote differentiation of the promyelocytic cell line HL-60. METHODS: Differentiation was assessed by marker analysis, morphology, immunohistochemistry, proliferation and cellular activity assays. RESULTS: L-C increases HLA-DR and CD14 surface antigens, while morphologic and marker analysis of the treated cells reveals the presence of monocytes, neutrophiles and few dendritic cells. What is important, however, is the induction of cells that have an atypical to this pathway allure staining positive for the neurofilament 3A10 monoclonal antibody, specific for nerve cells and the anti-p75 (Nerve Growth Factor Receptor) monoclonal antibody. The events described concern active and, at the same time, not proliferative senescent cells. CONCLUSIONS: L-C exerts its differentiation action on a certain fraction of the leukemic population yielding a non-negligible number of atypical for the myeloid lineage cells. These findings complement earlier and recent reports that describe the generation of cells of a different lineage irrelevant to their parent line of differentiation indicating that the hemopoietic pool appears to be the source of any kind of cell types according to the stimulus provided. Thus, in the context of the plasticity theory it appears that the HL-60 cell line also possess the potential to differentiate towards unexpected pathways.

Aspects of childhood cancer during the Byzantine period.

The main trends in the diagnosis and management of childhood cancer during the Byzantine period (330-1453 CE) are investigated. Therapeutic modalities reflected the influences from Ancient Greek and Greco-Roman medicine. Medical treatment included a great variety of regimens, and surgery was not unknown. The attitudes toward cancer suggest that people of that time did not believe in a superstitious origin of the disease. Even though most of these remedies and many procedures are nowadays out of use, the physicians of the Byzantine period preserved the scientific medical thought of antiquity, improved it, and set the basis of current achievements. Medical terms introduced during the Byzantine period are still used. The texts have been studied in their original languages, that is, Ancient and Byzantine Greek, and Latin.

Mutations of retinoblastoma gene (Rb-1) as a prognostic factor in children with acute leukemia and neuroblastoma.

Rb-1 is a tumor suppressor gene encoding for a nuclear phosphoprotein acting as a cell cycle regulator, normally expressed in hematopoietic cells and more often inactivated by point mutations with predominance for exons 20-24. The aim of this study is to correlate the retinoblastoma-1 (Rb-1) gene mutations with the prognosis and progression of childhood acute leukemia and neuroblastoma. Bone marrow slides from 26 children with leukemia (18 acute lymphoblastic leukemia [ALL] and 8 acute myeloid leukemia [AML]) and 4 children with neuroblastoma were studied. Exons 20, 21, and 22 were amplified using the polymerase chain reaction technique. Single strand conformational polymorphism (SSCP) and heterodoublex analysis were performed to detect mutations. In ALL cases, two samples in exon 20 (11.11%), one in exon 21 (5.56%), and four in exon 22 (22.22%) had altered conformation. All but one of these cases were classified as high-risk leukemia patients who either relapsed or never achieved remission. Two of the AML cases who did not achieve remission and one of the neuroblastoma cases with concomitant bone marrow infiltration had altered conformation as well. The SSCP and heterodoublex analysis showed that all but one who did not belong to the high-risk group had the same altered conformation. These data suggest that Rb-1 gene could possibly be used as an independent prognostic factor for the acute leukemia of childhood and result in the intensification of chemotherapy. In solid tumors with bone marrow involvement it could play a role as a marker of aggressive disease.

Soluble transferrin receptor levels and soluble transferrin receptor/log ferritin index in the evaluation of erythropoietic status in childhood infections and malignancy.

UNLABELLED: Soluble transferrin receptor (sTfR) is a new diagnostic tool for determining iron status and erythropoietic activity. The increased concentrations of sTfR in patients with iron deficiency reflect the hyperplasia of erythroid precursors. The objective of this study was to evaluate sTfR and sTfR/log ferritin index (sTfR-F) values in healthy children (n = 64), full-term neonates (n = 18), children with iron deficiency (n = 16), hemolytic anemia (n = 7), beta-thalassemia traits (n = 18), respiratory infections (n = 41) and malignancies (n = 13), and to compare these parameters for the different subgroups with those of healthy children. The sTfR levels were increased in children with iron deficiency in the same way as in adults (p < 0.0001) and in cases of increased erythropoietic activity, such as during the neonatal period (p < 0.0001), and of hemolytic anemias (p = 0.006). The index was significantly increased in iron deficiency (p < 0.0001) and decreased in neonates (p = 0.011). Children carriers of beta-thalassemia were found to have increased sTfR values (p = 0.015), but not sTfR/log ferritin index (p = 0.491), a finding suggesting that use of both parameters is necessary for distinguishing between those with and those without iron deficiency. In children with upper respiratory infection, the sTfR levels were close to normal, while the index was found to be low. In order to evaluate the iron status in infections, we further subdivided the children into two groups according to the value of ferritin, with the cut-off point at 35 microg/L. Children with ferritin level above 35 microg/L experienced normal sTfR levels but very low index, a finding which could enable the use of these two parameters for distinguishing patients with infection without concomitant iron deficiency. In the group of malignancies under chemotherapy both indices were low (p = 0.005, p < 0.0001) mainly due to myelosuppression. CONCLUSION: The interpretation of both sTfR and sTfR/log ferritin index is useful in the evaluation of iron status and erythropoietic activity, especially in children with heterozygous beta-thalassemia, infection and malignancies.

Bone marrow purging by photodynamic treatment in children with acute leukemia: cytoprotective action of amifostine.

In order to evaluate the combined effect of Amifostine and Merocyanine 540 during photoirradiation in neoplastic cells, bone marrow cells from children with acute leukemia (AL), age-matched controls as well as HL-60 cell line were studied. Cell suspensions were incubated with Amifostine, then with MC 540 and they were subsequently exposed to different irradiation doses by Argon Laser 514 nm. Cell survival was estimated by trypan blue supravital stain following a 24-h incubation. The leukemic cell line was studied in continuous liquid cell cultures for 4 weeks. The survival of normal bone marrow progenitors has been estimated by colony formation assay in methylcellulose cultures. Our results showed that Amifostine enhances the photokilling effect of MC 540 on leukemic cells and significantly protects bone marrow nucleated and committed progenitors (BFU-E and CFU-GM) from children with AL under chemotherapy. In conclusion, Amifostine seems to be a promising cytoprotective agent in the clinical use of purging with MC 540 mediated phototherapy.

Ondansetron and tropisetron in the control of nausea and vomiting in children receiving combined cancer chemotherapy.

Ondansetron (Zofron, Glaxo) and tropisetron (Navoban, Sandoz) are selective serotonin (5HT3) antagonists that have proven very effective in the prevention of vomiting and nausea in adults and children receiving cancer chemotherapy. This study compared the efficacy of the two agents in the prevention of vomiting and nausea in children receiving chemotherapy for solid tumors and blood malignancies. A total of 23 children were studied in 205 chemotherapeutic cycles (116 one-day regimens and 89 multiple-day regimens). In 102 chemotherapeutic cycles the children received ondansetron as an antiemetic agent in a dose of 5 mg/m2 30 min before chemotherapy was given and then 4 mg/m2 every 8 h i.v. (group A) and in 103 cycles they received tropisetron in one dose of 0.2 mg/kg 24 h-1 i.v. (max dose 5 mg) 30 min before cytotoxic drugs administration every day they received chemotherapy (group B). The response was defined as complete in the absence of nausea and vomiting per 24 h of chemotherapy, as partial given the presence of 1-4 events of vomiting and/or nausea less than 5 h per 24 h, and as failure if there were more than 4 events of vomiting and/or nausea for more than 5 h per 24 h of chemotherapy. The response of the two groups was studied independently and depending on the degree of emetogenicity of the chemotherapeutic agents, which were divided into mildly, moderately, and highly emetogenic. The comparison of the two groups not taking into consideration the emetogenicity of the chemotherapeutic agents showed that ondansetron was more effective in 1-day regimens (P = .023), whereas the two agents were equally effective in multiple-day regimens (P = .2). The statistical analysis depending on the emetogenicity of the chemotherapeutic agents showed increased efficacy of ondansetron in mild (P = .017) and moderately emetogenic chemotherapeutic agents, whereas there was no difference in the highly emetogenic drug group. Ondansetron is found to be more effective than tropisetron in controlling acute nausea and vomiting in children receiving mild and moderately emetogenic chemotherapeutic drugs, although there is no difference in the efficacy of both antiemetic agents when highly emetogenic drugs are administered.

Phenotypic characteristics of cord blood hemopoietic cells.

Bone marrow transplantation is limited due to the lack of HLA-matched donors and to the frequent occurrence of GvHD. Hemopoietic transplants using cord blood cells are being increasingly used in pediatric patients. In this study, the immunophenotypic characteristics of cord blood cells have been investigated by one or two-color flow cytometric analysis. The CB cells were characterized by a low proportion of CD3+ T-cells, increased CD4/CD8 and CD45RA/CD45RO ratios, minimal expression of HLA-DR, increased proportion of CD5CD19 double positive B-cells, while CD3- CD8+ and CD3- CD7+ subsets, not usually found in adult PB, were detected. These data reflect the immaturity of CB cells as assessed by immunophenotypic analysis suggesting that it could be a valuable alternative source of transplantable hematopoietic progenitor cells and might alleviate some of the problems associated with bone marrow or peripheral blood transplantation.

Erythropoietin (rHuEPO) administration to premature infants for the treatment of their anemia.

In an attempt to stimulate erythrocyte production and thereby decrease the requirement for red blood transfusions, recombinant human erythropoietin (rHuEPO) was administered to 16 premature infants with birth weights less than 1000 g and to 18 with birth weights of 1000-1300 g; two corresponding groups, who did not receive rHuEPO, were used as control groups. The rHuEPO was administered subcutaneously in a dose of 300 IU/kg three times a week for 6-8 weeks. The erythropoietin decreased the red blood requirement in both groups of infants, and the increment of hemoglobin following rHuEPO administration was not statistically significant. No correlation was observed between gestational age, number of transfusions, and reticulocyte percentage. The effect of rHuEPO was higher in the group of infants with birth weights of 1000-1300 g than in those of less than 1000 g. No significant side effects were observed during rHuEPO administration.

High sensitivity of leukemic peripheral blood lymphocytes to triethyllead action.

In a previous study we reported that triethyllead (Et3Pb+) inhibits cell proliferation of normal human lymphocytes. To further characterize this interaction, we studied herein the effects of Et3Pb+ on the cell viability of normal and leukemic human lymphocytes and analysed the expression and dynamics of the monomer/polymer equilibrium of tubulin in these cells. Short- and long-term cell culture experiments demonstrated significantly different dose-dependent effects of Et3Pb+ on cell viability of leukemic compared to normal lymphocytes. Indeed, in the presence of increasing concentrations of Et3Pb+ (10(-12)-10(-5) M), primary cultures of chronic lymphocytes (CLL) and acute lymphoblastic (ALL) leukemic human Lymphocytes were much more sensitive to Et3Pb+ treatment when compared to normal peripheral blood lymphocytes (PBL). The IC50 values were approximately 5 x 10(-6) M for PBL and 8 x 10(-10) M for both CLL and ALL respectively, when cells were preincubated for 3 h with this agent. These experiments revealed a 1000-fold higher responsiveness of leukemic cells to Et3Pb+ treatment. Quantitative immunoblot analysis showed that leukemic cells express up to 4-fold higher total tubulin amounts. However, the proportion of polymerized tubulin in leukemic compared to normal lymphocytes increased only slightly (up to 1.4-fold). These findings reveal a significant decrease in the polymeric to total tubulin ratio in leukemic lymphocytes, indicating important modifications in tubulin dynamics and reorganization of the microtubular structures. Our results demonstrate that leukemic cells are much more sensitive than normal lymphocytes to Et3Pb+ action. This effect may be due to the altered monomer/polymer dynamic equilibrium of tubulin shown in leukemic cells. It is, therefore, worthwhile exploring future applied uses of Et3Pb+ as a potential suppressor of leukemic cell growth.

Merocyanine 540 mediated photolysis of normal bone marrow, committed hemopoietic progenitors and neoplastic cells. implications for bone marrow purging.

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.