Stem Cell Research and Diabetes

Experimental Procedure

Undifferentiated ES-D3 murine embryonic stem lines were cultured on a feeder layer of mouse embryonic fibroblasts. Then the ES-D3 cells were transferred onto gelatin-coated flasks for 30 minutes to remove the feeder layer. The ES-D3 cells were seeded at 1x10^6 cells per well with a supplemental medium overnight. Then the ES-D3 were exposed to another supplemental medium for 6 days. Next RNA samples were reverse transcribed with transcription step, followed by denaturing step and then by 30 cycles of denaturing, 30 seconds of annealing, and 30 seconds of extending, followed by 7 minutes for elongation. The PCR products that were produced were separated by electrophoresis. The cells were fixed for 30 seconds at room temperature in 4% paraformaldehyde, then washed three times in PBS. Cells were blocked for 20 minutes in PBS. The cells were then rinsed three times in PBS and incubated for 45 minutes. After washing cells were incubated with the DAPI for ten minutes. Differentiated cells were seeded at 1x10^6 cells per well in a 24 well culture plate and incubated overnight in culture media and were grown for 24 hours in DMEM/F-12 medium without insulin. Cells were then

washed twice and pre-incubated at 37◦C for 1 hour with Krebs-Ringer bicarbonate HEPES buffer (KRBH).

Results

Conclusions

Embryonic stem (ES) cells are a potential source for insulin producing cells (IPCs), but existing differentiation protocols are of limited efficiency.In summary, these studies show that small molecules induce definitive endoderm and pancreatic cells in monolayer cells at the same time in the differentiation process. Our differentiation system represents an efficient protocol to direct mouse ES cells into the pancreatic lineage by generating IPCs and is applicable to further strategies for the improvement of in vitro differentiation into functional insulin-producing cells.

Special Comments/Discussion

This study that was conducted could...