Stem Cell Biology

Islet Stem Cells Protocols

Differentiation–selection
Cell Proliferation Analysis and BrdU Incorporation
Insulin Content and Secretion Assays

Differentiation–selection

  • To direct the differentiation, besides eliminating LIF from the medium, combine the reduction in serum concentration (from 15% to 3%) with the addition of selected factors. Use 2 µg/ml anti-sonic hedgehog (S8321) and 10 mmol/l nicotinamide (N0636).
  • Co-culture cells with embryonic pancreases from embryonic day 17.5, in which the soluble factors liberated by the forming islets induce differentiation. Embryos from time-mated pregnant OF1 mice are liberated in Hank’s balanced salt solution (H6648, H8264, H9269, H9394). Whole pancreases were collected by microdissection, washed in Hank’s solution several times and transferred to Petri dishes, where D3 cells were grown.
  • Plant pancreatic rudiments of seven to eight fetuses on each Petri dish. Pancreatic rudiments and mES cells were separated by nylon filters with a pore size of 0.2 µm (Z290823). Place the pancreatic rudiments in the bottom of the plate, then the nylon filter, and place the embryoid bodies on top of the filter. This ensures that the ES cells and the pancreatic rudiments are never in physical contact.
  • To begin the differentiation, count 5 million cells for 7 days to form embryoid bodies (EB, 3% FBS (F2442)) and plat for 7 days (P; 10% FBS). During this 14-day period, the cells need to be treated with the above-mentioned factors, except in the case of anti-sonic hedgehog (S8321) and co-culture with pancreatic rudiments, which are present only during the 7 days of EB formation. Afterwards, the differentiated cultures are grown in D3 cell culture medium, without LIF and penicillin/streptomycin, supplemented with 800 µg/ml G 418 (A1720), to select Nkx6.1-positive cells. These cells were cultured for an additional 20 to 30 days.

Cell Proliferation Analysis and BrdU Incorporation

  • After trypsinisation of undifferentiated stem cells, cells were counted and cultured using the differentiation protocols previously described. After 7 days of EB formation and 7 days of plating, the plates were trypsinised and counted, so that the number of population doublings could be determined. Using neomycinselected cells, cell count was done approximately 2 days after neomycin was added, when only Nkx6.1-positive cells survived, and then counted again 20 days later, to calculate the number of population doublings. For BrdU staining, the cells were incubated with 10 µmol/l BrdU (B5002) for 24 hours. Then, the cells were fixed with 4% paraformaldehyde (P6148), and DNA was denatured using 4 mol/l HCl (H9892). The rest of the protocol is a standard immunocytochemistry protocol, described below. Mouse anti-BrdU antibody 1:1000 (B8434) was used as the primary antibody, and anti-mouse FITC 1:300 (F5636) was used as the secondary antibody.

Insulin Content and Secretion Assays

  • To determine total insulin content, 250,000 to 1,000,000 cells need to be incubated for 24 hours with ethanol/HCl buffer at 4 °C. For insulin secretion, plate cells in 12-well dishes at a density of 100,000 to 250,000 cells per well and allow them to grow overnight in the same medium as the neomycin-selected cells. Wash cells twice for 10 min. each in fresh phosphate buffer solution (P7059) and incubated for 4 hours in 0.5 ml of fresh modified Krebs buffer with 3 mmol/l glucose. Afterwards, incubate cells (95% O2, 5% CO2) at 37 °C for 1 hour in 0.25 ml of the fresh modified Krebs buffer with 3 mmol/l glucose, followed by a 1-h incubation period with 22 mmol/l glucose. Mouse islets were used as positive controls (from 5 islets to 50), obtained from 2-month old OF1 mice. At the end of the incubation period, the buffer was removed and studied for insulin content. Insulin was determined by RIA using the Coat-a-Count kit.

Protocols taken in part from:

  • Leon-Quinto, T. et al. Diabetologia 2004.

 

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