Ex vivo expansion of T, NK and CD34+ cells from umbilical cord blood / 中国实验血液学杂志

Umbilical cord blood stem cell transplantation (CBSCT) has made significant progress in treatment of lethal congenital or malignant disorders. Both the incidence and severity of GVHD from CBSCT were lower than that from bone marrow and peripheral blood stem cell transplantation, particularly for adult patients, but these advantages were also associated with higher rates of relapse. The immune-mediated effect of natural killer and cytotoxic T cells against residual tumor cells were shown to prevent relapse and to induce remission after bone marrow transplantation. To explore possibility of ex vivo expansion of T, NK and CD34(+) cells from umbilical cord blood, cord blood was expanded ex vivo with different combinations of cytokines, T and NK cells proliferation and differentiation were observed. CB MNCs were separated in Ficoll-Isopaque column and cultured in IMDM for 14 days with different recombinant cytokines. Cultured cells were collected and analyzed for progenitor/stem cell immunophenotyping at day 0, 3, 7, and 14 by using flow cytometry. The results indicated that all test groups cultured with different combinations of SCF, IL-3, IL-6, IL-7, IL-2 showed significant expansion of UCB MNC, compared with the group without cytokines. All test groups showed expansion effects on CD34(+) cells, CD34(+) percentage went up from 1.6% in fresh CB to the highest 11.9% in group D (SCF + IL-3, IL-6, IL-2). The CD34(+) cells peak displayed at day 7 of culture in group A and D, while in other two groups B and C appeared at day 14 of culture. The expansion multiple of CD34(+) cells in all test groups at day 7 of culture were from 10 to 50. The average value of CD3(+) T cell in fresh UCB was 18.7 +/- 4.3%, the CD3(+) T cells decreased sharply in the medium without any interleukin, while obvious increase were observed in the other test groups containing different combinations of cytokines. The maximal expansion multiple of CD3(+) T cells reached 2 times of the fresh UCB level. CD56(+) cells amounted to 3.6 +/- 1.9% of fresh UCB, CD56(+) cell number increased significantly only in medium containing IL-2. It is concluded that T cells, NK cells as well as stem/progenitor cells can be expanded in the same time from CB-MNC with the combinations of cytokines.

Establishment of a transplantable human myeloid BALB/c nude mouse model / 中国实验血液学杂志

To establish a mouse model bearing transplantable human chronic myeloid leukemia for hematopoietic stem cell transplantation to treat leukemia, 4 - 5-week-old female BALB/c nude mice were given cyclophosphamide 2 mg/mouse at day -2, -1, and then the human chronic myeloid leukemia K562 cells were engrafted into the mice at day 0 by injection via tail vein or peritoneal cavity. PB and BM cells were collected, the CD45, CD13, and CD33 antigens were delected by using FCM, the bcr/abl fusion gene mRNA was examined by RT-PCR. The results showed that transplantable leukemic mice could be yielded from 4 - 5-week-old nude mice either by injection through tail vein or peritoneal cavity when the total number of inoculated tumor cells was more than 2 x 10(5) per mouse, whether being pretreated with 2 mg CTX/mouse or not. The transplanted mice could survive 30 - 60 day with leukemia. In conclusion, the mouse model bearing leukemia can be established by inoculation 2 x 10(5) K562 cells into immunodeficient BALB/c nude mice.