The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 103 enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1(lo)LIN-Sca-1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 ± 19, 170 ± 30, and 740 ± 70 per 103 cells, respectively), and stem cells with competitive long-term repopulating potential (≤1 per 43 cells). Thy-1(lo)Sca-1+H-2K(hi) cells (TSH(FU)) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, ≤1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 ± 8, 0 and 15 ± 4 per 103 cells, respectively). Recipients of 103 TLS cells transiently recovered leukocytes to ≤2,000/μL in 12 days, but sustained engraftment required 25 days. Platelets recovered to ≤200,000/μL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 103 TSH(FU) cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 103 TLS or TSH(FU) cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-, 83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSH(Fu) cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSH(FU) cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 103 TSH(FU) cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 103 TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSH(FU) stem cells depleted of progenitors.
|Number of pages||12|
|State||Published - 1 Nov 1996|