Ex vivo expansion of CD34+/CD41+late progenitors from enriched peripheral blood CD34+cells

In our experience, patients with neuroblastoma who undergo transplantation with CD34+ cells following high-dose chemotherapy have prolonged delays in platelet recovery. In vitro expansion of megakaryocyte (MK) cells may provi de a complementary transplant product able to enhance platelet production i n the recipient. We investigated the ability of a combination of various he matopoietic growth factors to generate ex vivo MK progenitors. Immunoselect ed CD34+ cells from peripheral blood stems cells (PBSCs) were cultured in m edia with or without serum, supplemented by IL-3, IL-6, IL-11, SCF, TPO, Fl t-3 ligand, and MIP-1 alpha. In terms of MK phenotypes, we observed a maxim al expansion of CD61+, CD41+, and CD42a of 69-, 60-, and 69-fold, respectiv ely, i.e., 8-10 times greater than the expansion of total cell numbers. Whe reas the absolute increment of CD34+ cells was slightly elevated (fourfold) we showed increases of 163-, 212-, and 128-fold for CD34+/CD61+, CD34+/CD4 1+, and CD34+/CD42a+ cells, respectively. We obtained only a modest expansi on of CFU-MKs after only 4 days of culture (fourfold) and similar levels of CFU-MKs were observed after 7 days (fivefold). Morphology and immunohistoc hemistry CD41+ analyses confirmed expansion of a majority of CD41+ immature cells on days 4 and 7, while on day 10 mature cells began to ap-pear. Thes e results show that primarily MK progenitors are expanded after 4 days of c ulture, whereas MK precursor expansion occurs after 7 days. When we compare d the two culture media (with and without serum) we observed that increases of all specific phenotypes of the MK lineage were more elevated in serum-f ree culture than in medium with serum. This difference was especially marke d for CD34+/CD61+ and CD34+/ CD41+ (163 vs 42 and 212 vs 36, respectively). We contaminated CD34+ cells with a neuroblastoma cell line and we observed no expansion of malignant cells in our culture conditions (RT-PCR for tyro sine hydroxylase positive at day 4 and negative at day 7), With our combina tion of hematopoietic growth factors we are able to sufficiently expand ex vivo MK late progenitor cells to be used as complementary transplant produc ts in neuroblastoma patients who undergo transplantation with CD34+ cells. It is possible that these committed MK late progenitors could accelerate sh ort-term platelet recovery in the recipient until more primitive progenitor cells have had time to engraft.