Abstract
Objective To isolate and culture bone marrow mesenchymal stem cells (MSC) from chronic myelogenous leukemia (CML) patients and examine their functional characteristics; To investigate their biological characteristics like proliferation, apoptosis and tumorigenicity;To observe their effects on proliferation and apoptosis of K562 cells.
Methods Bone marrow was extracted from the anterior superior iliac spines of 20 patients with CML. MSC were isolated and cultured in vitro and subcultured for three to five generations. Cell morphology was observed under microscope. The ultrastructure was observed with electron microscope. The immunophenotype was detected by flow cytometry (FCM). Different agents were used to induce the MSC to differentiate into osteocyte and adipocyte, and Von Kossa staining, oil-red staining was used to examine the ability of differentiation. The growth curve, cell cycle and apoptosis were investigated. MSC were cultured in soft agar for 2 weeks to observe the clone growth. BALB/C nude mice were inoculated with MSC to observe the tumorigenicity. MSC and K562 cells were cocultured in vitro and coinjected into subcutaneous of BALB/C nude mice, to observe the effects of MSC on proliferation and apoptosis of K562 cells.
Results Fibroblast-like, positive in CD44, CD73 and CD90,and negative in CD34, CD45 and HLA-DR, the CML derived MSC could differentiate into osteocyte and adipocyte. The MSC showed normal ultrastructure. After 2 weeks’ culture, no clone was formed from the MSC. Four weeks after, no tumor was seen in the mice inoculated with MSC. The population doubling time of P3 MSC is (32.61±1.54)h. The population doubling time of P4 MSC is (32.59±1.23)h, The population doubling time of P5 MSC is (32.41±0.75)h. To investigate the cell cycle of the third passage MSC by FCM, there were(93.67%±1.66%)cells in phase G0/G1,(6.33%±1.66%)in phase S+G2+M. There weren’t any apoptosis observed. K562 cells adhensively cultivated with MSC were accelerated and the population doubling time decreased([29.59±0.46]h vs [37.49±2.19]h, P<0.05),cells in G0/G1 decreased ([16.43%±1.67%] vs [32.23%±3.35%], P<0.01),cells in phase S increased ([69.63%±3.09%] vs [59.37%±4.40%], P<0.05),and those in G2/M increased([13.93%±1.45%] vs [8.40%±1.05%], P<0.01),compared with that cultivated in suspension. The apoptosis in both conditions were not observed. Coinjected with MSC, K562 cells developed tumors in 100% of the mice by (12.00±0.82)d compared to (15.50±0.58)d when implanted alone, indicating a statistically earlier onset of tumor growth in presence of MSC (P<0.01). The tumor volume and tumor weight were also increased when K562 cells coinjected with MSC compared with implanted alone (K562+MSC vs K562):tumor volume([75.70±7.30]mm2 vs [37.38±2.39]mm2, P<0.01),tumor weight([0.64±0.08]g vs [0.32±0.06]g, P<0.01).
Conclusion MSC from the bone marrow of CML patients can be isolated and cultured;MSC derived from CML seem to have the abilities of powerful proliferation in vitro and multiple differentiation and have no tumorigenicity. The MSC can accelerate the proliferation of K562 cells both in vitro and vivo.
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