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 |
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Authors: | Wang Yan-Y I Yang Yong Chen Qingyong Yu Jianping Hou Yongzhong Han Lizhen He Jun Jiao Demin Yu Huihui |
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Institution: | Department of Pharmaceutical Engineering, College of Life Science, Guizhou University, Guiyang 550025, China;Department of Biomedical Engineering, Hangzhou Dianzi University, Hangzhou 310018, China;Department of Pulmonary Diseases, the 117th Hospital, Hangzhou 310013, China;Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada |
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Abstract: | A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18INK4c (p18) p27Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators pl8, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1+c-kit+ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1+c-kit+ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of pl8, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of pl8, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability. |
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Keywords: | p 18INK4C p27Kip1 MAD 1 hematopoietic stem cell |
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