Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: differential activity of cultured bone marrow and enriched stem cell populations.
In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.
Applied Immune Sciences, Santa Clara, CA 95054-1114.
K M
KM
Knobel
M A
MA
McNally
A E
AE
Berson
D
D
Rood
K
K
Chen
L
L
Kilinski
K
K
Tran
T B
TB
Okarma
J S
JS
Lebkowski
D000818:Animals; D001854:Bone Marrow Cells; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D011828:Radiation Chimera; D013601:T-Lymphocytes / Q000166:cytology; D018800:Thy-1 Antigens / Q000032:analysis; D013997:Time Factors; D014407:Tumor Cells, Cultured
D018800:Thy-1 Antigens