Bone marrow (BM) mesenchymal stem cells (MSCs) are cells capable of expanding and differentiating in vitro into nonhematopoietic cells. Neurotrophic cytokines, such as human epidermal growth factor (hEGF) and bovine fibroblast growth factor (bFGF) can induce differentiation into neural cells (NCs). When BM MSCs were cultured with hEGF and bFGF, RNA expression of neuronal specific markers Nestin, MAP-2, and tyrosine hydroxylase (TH) were observed. We tested a new cytokine combination to generate mature NCs. The plastic-adherent cells were collected and then split when they were 90% confluent from an enriched mononuclear cell layer. At passage 3, MSCs were cultured in neural differentiation media (dbcAMP, IBMX, FGF-8, BDNF, hEGF, and bFGF in NEUROBASAL media plus B27). Cells were counted on day 6. Immunofluorescent staining and reverse transcriptase (RT)-PCR were performed to evaluate the expression of neural markers. On day 6, 66% of cells developed dendrites and presented typical neural cell morphology. Some cells were positive for early neural markers Nestin and beta-tubulin III. Cells expressing mature neuronal markers (NF, NeuN, Tau, Nurr1, GABA, oligodendryte GalC, and glial GFAP) were also seen. By adding hEGF, bFGF, dbcAMP, IBMX, BDNF, and bFGF-8 into NEUROBASAL media plus B27, BM MSCs were directed toward becoming early and mature NCs.
SummaryWT1 gene expression has been proposed as a useful marker of minimal residual disease in leukaemia. Its utility in paediatric haematopoietic stem cell transplantation (HSCT) has not been studied. We studied the prognostic value of WT1 expression in peripheral blood prior to HSCT in 36 children with acute myeloid leukaemia (AML). Samples were obtained 2 weeks pre-transplant to determine the level of WT1 expression. WT1 expression was normalized using K562 cells as a control and a relative value of 0AE5 was chosen as the cut-off point between high and low WT1 expression. The median level of pre-transplant WT1 expression in the 36 patients was 0AE09 (range 0AE0001-11AE0), with 11patients having WT1 ‡ 0AE5 and 25, WT1 < 0AE5. After HSCT, 76% of patients with high pre-transplant WT1 expression relapsed, in contrast to 0% of the patients with low WT1 expression. Those with high WT1 expression had significantly lower 5-year event-free survival (EFS) (18%, 95% CI 0-40%) as compared to those with low WT1 expression (68%, 95% CI 50-86%, P = 0AE007). Multivariate analysis showed that pretransplant WT1 level is the only significant prognostic factor for the difference in EFS. Our finding suggests that elevated WT1 gene expression before HSCT in paediatric AML predicts relapse and poor long-term EFS. A larger prospective study is warranted to compare the value of high WT1 expression and other markers of minimal residue disease in predicting clinical outcomes after HSCT.
MIBG scores >/= 3 following induction therapy identifies a subset of NB patients who are likely to relapse following three cycles of high-dose therapy with peripheral blood stem-cell rescue, local radiotherapy, and 13-cis-retinoic acid. Alternative therapeutic strategies should be considered for patients with a poor response to induction therapy.
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