Isolation and transplantation of autologous peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting.

Peripheral stem cells were mobilized and collected in 26 pediatric patients with malignant diseases. A total of 47 leukaphereses were performed in the 26 patients. The mean number of nucleated cells collected was 4.5 +/- 2.6 x 10(8)/kg and the number of CD34+ progenitors collected was 6.7 +/- 6.8 x 10(6)/kg. CD34-positive selection was performed using a two-step method of magnetic-activated cell sorting (MACS) in 24 patients or a combination of an immunoaffinity column and MACS in two patients. The purity of the positively selected CD34+ progenitors was 98.8 +/- 0.7% and the number of isolated CD34+ cells was 6.5 +/- 5.9 x 10(6)/kg. Thus, the mean recovery of CD34+ cells was 93 +/- 10%. In 22 of the 26 patients, high-dose chemotherapy was performed with subsequent reinfusion of the highly purified CD34+ cells. In all 22 patients, a normal hematopoietic reconstitution was seen with a mean time of 12.4 +/- 2.7 days to reach >0.5 x 10(9)/l neutrophils (range 8-19 days). The time to reach independence from platelet transfusion was 31.6 +/- 17.0 days (range 16-78 days). There were no transplant-related deaths. In summary, we have shown that mobilized peripheral CD34+ progenitors can be highly purified with a good recovery, and that reinfusion of these cells after high-dose chemotherapy results in a rapid, complete and sustained engraftment. We conclude that this method can be used for purging in any CD34-negative malignancies and for autologous T and B cell depletion in the treatment of autoimmune diseases with high-dose immunoablative therapy.

Establishment and characterization of a cell line (Wa-2) derived from an extrarenal rhabdoid tumor.

A new human cell line (Wa-2) derived from an extrarenal rhabdoid tumor has been established. The cell line grows as a monolayer consisting of round- and spindle-shaped cells. Injection of cells into nude mice results in the growth of solid tumors within 2 wk of inoculation. These solid tumors have the microscopic appearance similar to that of the original tumor from which the cell line was derived. Moreover, the tumor cells have all the features of rhabdoid tumors, including the intracytoplasmatic hyaline globules, large prominent nucleoli, and inclusion bodies made up of whorls of fibrillary material. Transplanted tumor cells stain positive for vimentin, cytokeratin, actin, and desmin and negative for myoglobin and neuron-specific enolase. Karyotyping of the cell line at different passages and cells derived from the transplant tumors consistently revealed a diploid number of chromosomes with a reciprocal translocation between chromosomes 18 and 22 [t(18;22) (q21;p11.2)]. In fibroblasts derived from the patient, no translocation could be found. Culturing the cells in the presence of 1-beta-D-arabinofuranosylcytosine induces differentiation, characterized by the outgrowth of neuron-like processes and the morphological appearance of cells similar to neuroblasts. Southern blot analysis showed no amplification of the N-myc oncogene. Since no published cell line derived from rhabdoid tumors exists, this cell line should be helpful to further elucidate the biology and histological origin of the malignant rhabdoid tumor.