| Budget Amount *help |
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2012: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2011: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
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| Research Abstract |
First, we generated a set of gene-engineered STO feeder cells that confer to resistance to several commercially available drugs. The STO cells were transfected with pcBIH (carrying bleomycin resistance gene (ble) and hygromycin B phosphotransferase gene (Hyg), pcBIP (carrying ble and puromycin resistance gene (puro)) or pcBSN (carrying ble and neomycin resistance gene (neo)). The resulting stably transfectants (termed SHB for pcBIH, SPB for pcBIP and SNB for pcBSN) exhibited bleomycin/Hygromycin, bleomycin/puromycin or puromycin/neomycin, as expected. The morphology of these cells passaged over 18 generations was indistinguishable from that of parental STO cells. Of isolated clones successfully supported the growth of mouse ES cells in an undifferentiated state, when co-culture was performed. Next, we examined the possibility that STO cells could phagocytose Streptococcus mutans (a bacteria causing tooth decay), which always contaminates cultures of primarily isolated human deciduous dental pulp cells (HDDPCs). There was no bacterial contamination in the cultures containing STO cells supplemented with mitomycin C (MMC).HDDPCs can be reprogrammed using 4 reprogramming factors, and dental-pulp-stem-cell(DPSC) specific promoter was transfected to HDDPC-derived iPS. For teratoma formation,recombinant HDDPC-iPS cells were injected subcutaneously into immune compromised mice. The teratoma was dispersed into primary culture, and we tried to select DPSC under the basic medium supplemented neomycin. Unfortunately, we were unable to gain DPSC because of the uncertainty of promoter expression and differentiation in vivo. We will review our transfection procedure and try to use the materials like scaffold in the future.
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