Method for inducing cells to less mature state
A cellular, mature technology that can be applied to embryonic cells, animal cells, vertebrate cells, etc., and can solve problems such as low-efficiency reprogramming, impractical use of iPS cell therapy, and low rate of induction of pluripotency.
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Embodiment 1
[0221] Example 1: MUCl* promotes growth and cell death resistance.
[0222] MUCl* promotes colony growth (colony expansion) of fibroblasts. Full-length MUC1 (SEQ ID NO: 1), MUC1* 1110 Single cell colonies of 3Y1 cells transfected with either (SEQ ID NO:5) or empty vector were plated in DMEM medium containing 10% fetal bovine serum, penicillin / streptomycin and G148 (600 μg / ml). Cells were grown for 9 days and then fixed in 4% paraformaldehyde for 15 min at room temperature. Petri dishes were washed with water and then stained with 1% crystal violet in 70% methanol for 20 min at room temperature. Petri dishes were washed three times with water and allowed to dry overnight at room temperature and photographed. figure 1 A shows that the amount of crystal violet adsorbed (an indicator of cell number) is much higher where MUCl* single cell colonies #3 and #44 are growing. In contrast, cells transfected with full length MUCl (single cell colonies #8 and #17) showed no increase in...
Embodiment 2
[0223] Example 2: Through resistance to trastuzumab in cells (Copeptin) (by culturing at 1ug / ml development of resistance in ), the anti-MUCl* Fab blocks cell death resistance. Fessler et al. reported in 2009 that the Cells also resist Doxorubicin and cyclophosphamide. As reported, these drug-resistant cancer cells achieved drug resistance by overexpressing MUC1*. The following experiments show that blocking PSMGFR of the MUCl* extracellular domain partially reverses acquired resistance in cancer cells. Parental cells (BT474) or resistant cells (BTResl ) were plated in 96-well plates at a density of 10,000 cells / well, 4 wells / condition. the following day, in the presence or absence of Addition of anti-MUCl* Fab, control Fab or no Fab to cells (Paclitaxel Sigma T7191 ). Two days later, cells were resuspended in 50 μl of trypsin and counted in the presence of trypan blue. Based on the trypan blue uptake rate, the percent cell death rate was calculated. In each co...
Embodiment 3
[0224] Example 3: MUCl* functions as a growth factor receptor and is activated by dimerization of its extracellular domain using artificial (anti-MUCl* antibody) or its natural ligand NM23 (NME). MUC1* positive ZR-75-30 cells, 6000 / well, or control (MUC1 negative) HEK293 cells, 4000 / well, were spread in a 96-well plate. The following day, 0-hour cell counts were performed, and various concentrations of anti-MUCl* antibody or Fab with a small amount (0.1%) of serum were added to the medium every 24 or 48 hours. After several days of incubation, cells were resuspended in trypsin and counted, and percent normalized growth was calculated. Stimulation to ZR-75-30 cells showed a bell-shaped curve, as demonstrated for ligand-induced growth stimulation, but HEK293 cells did not ( figure 1 C). In a similar experiment using MUC1*-positive T47D breast cancer cells stably transfected with siRNA targeting MUC1 or control siRNA, growth stimulation only occurred in control transfected cell...
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