Abstract
Parathyroid hormone (PTH) induces activation and increases the number of osteoblasts. An increase the stem cells number (Lin-Sca1+cKit+) was observed in mice after 4 weeks of PTH treatment, that suggest osteoblasts participation in regulation of hematopoiesis (Calvi, et al., 2003, Nature, 425, 841). Long-term bone marrow culture (LTBMC) was used for the study of PTH influence on hematopoietic progenitors of different stages of maturation. Rat PTH (1–34) (final concentration, 10−7M) was supported during whole culture period. Cell number, colony-forming units in culture (CFU-GM, CFU-C-21 days) and cobblestone area forming cells (CAFC) were measured during 10 weeks. The number of mature cells in culture did not changed during PTH treatment. The number of studied progenitors did not changed significantly during 3 and 6 weeks of PTH treatment. The number of CFU-GM, CFU-C-21 days and CAFC 14 days, CAFC 21 days, CAFC 28 days increased 7 –10 fold in suspension fraction of LTBMC after 10 weeks of PTH treatment.
Table 1. Number of different precursor cells in suspension fraction of LTBMC after PTH treatment.
To evaluate the possibility of stem cells expansion by using of PTH treated adherent cell layers (ACL) of LTBMC, the PTH-treated for 4 and 8 weeks cultures, were irradiated with 40 Gy and seeded with 2 × 106 bone marrow cells depleted of adherent cells. Following 24 hours the number of survived CAFC 8 – 28 was analyzed. On PTH-treated ACL the number of CAFC 21 increased 2,7 ± 0,4 fold as compared with fresh bone marrow or culture with non-treated ACLs. The number of CAFC 28, which characterizes the number of marrow repopulating cells, increased 2 fold only after cultivation on ACL treated with PTH for 8 weeks.
Table 2. CAFC number in bone marrow cultivated 24 hours on PTH treated ACL of LTBMC
The data suggest possibility to increase stem cell expansion ex vivo on pharmacologically manipulated microenvironment.
Time of cultivation, weeks Specificity of progenitor Number of precursors, per 100000 cells Control Control PTH treatment 3 CFU-GM 50 ± 5.2 77 ± 5.6 6 CFU-GM 33 ± 4 31 ± 5.9 10 CFU-GM 20 ± 4.8 147.5 ± 3.5 3 CFU-C-21 55 ± 5.9 76.6 ± 11.2 10 CFU-C-21 11.5 ± 1.7 102.0 ± 2.0 3 CAFC-7 21.6 20.3 6 CAFC-7 25.9 26.9 10 CAFC-7 0.9 3.0 3 CAFC-21 1.01 0.69 6 CAFC-21 0.43 0.48 10 CAFC-21 0.28 3.11 3 CAFC-28 0.32 0.55 6 CAFC-28 0.12 0.14 10 CAFC-28 0.1 0.82 Age of CAFC (days) Number of CAFC per 2 x 10^6 bone marrow cells not treated PTH (4 weeks) PTH (8 weeks) CAFC-7 424.1 804.5 647.5 CAFC-14 262.5 529.8 295.2 CAFC-21 93.5 272.7 226.2 CAFC-28 38.3 30.1 79.2
Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro