Isolation and amplification of residural normal stem cells from bone maurow of clonal hemopoietic disorders

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We analyzed the clonality of hemopoietic progenitor cells in bone marrow failure including myelody splastic syndromes (MDS) and aplastic anemia using X-chromosome inactivation and the RFLP of phosphogly cerate kinase (PGK) gene or the heterogeneity of short tandem repeat of CAG sequence in human androgen receptor gene (HUMARA) in female patients. CD34-positive bone marrow cells were cultured in methylcellrose medium supplemented with EPO,GM-CSF and SCF for 14 days. Individual colonies were picked-up under an inverted microscope. Extracted DNA was divided into two parts. One was digested with methylation-sensitive enzyme Hha 1 and the other remained undigested. The resulting DNA were amplified by PCR using PGK or HUMARA primers. PCR products were electrophoresed in polyacrylamide gel. In the present study we demonstrated residual non-clonal progenitor pupulations in bone marrow of most MDS patients. According to the results of residual normal progenitors in MDS we treated the patients with chemotherapy and confirmed the recovery of nonclonal hemopoiesis at remission of the disease. These results strongly suggested that isolation and amplification of residual normal progenitors permit us to develop a new treatment strategy for MDS.