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Reprogramming of Somatic Cells into Stem (iPS) Cells

Induced pluripotent stem cells (iPS cells )are stem cell-like cells derived from reprogrammed somatic cells. iPS cells reprogramming is achieved by simultaneously expressing 2-4 ES cell specific transcriptional factors including Oct4, Sox2, Myc and Klf4/Lin28 using viral vectors (lentivirus or retrovirus). The reprogramming process takes 15-30 days. First, somatic cells such as fibroblast cells are infected with a mixture of viral particles expressing the 2-4 stem cell factors. The infected cells are then seeded on mouse embryonic fibroblast (MEF) feeder cells and cultured in ES cell medium. After 10-15 days, ES like colonies of cells will appear, can be picked and expanded. Reprogramming can also be done without using MEF feeder cells. The above materials are obtained from Dr. Chiou’s laboratory.

Separation of iPS Cells from MEF

Note: All solutions and equipment that come in contact with the cells are sterile and work is done in a laminar flow hood.

Materials:
Culture medium :10% FBS-DMEM
Dissociation reagent: trypsin

1. Remove and discard the spent cell culture media from the culture vessel (10cm dish).
2. Wash cells using balanced salt solution 1x PBS(Phosphate buffered saline) and rock the vessel back and forth several times.
3. Remove and discard PBS from the culture vessel.
4. Add pre-warmed trypsin 1 ml for a 15 cm-dish cells to dissociate cells from the culture dish. Gently rock the container to get complete coverage of the cell layer, and incubate the culture vessel at 37。C incubator for approximately 1 minute.
5. Observe the cells under the microscope for detachment.
6. When ≥ 90% of the cells have detached, add 7 to 8 ml of pre-warmed culture medium and disperse it by pipetting over the cell layer surface several times.
7. Transfer the cells to a 15-mL tube, then seed them back to the original dish and incubate the culture vessel at 37。C incubator for 25 to 30 minutes. Note that the actual incubation time and actual volume of medium vary with the sedimentation condition of iPS cells and MEF.
8. After 30 minutes, collect the supernatant filled with non-sedimented iPS cells some for cell number counting and retain most of the supernatant for further use.

Formation of Embryonic Body (EB) from iPS Cells

Formation of embryonic body(EB) is achieved by static suspension culture in a Petri-dish because the culture systems maintain a balance between iPS cell aggregation essential for EB formation and prevention of EB agglomeration. The desired cell concentration is 3*10^6 per 15 cm dish. Add prepared iPS cells to a Petri-dish for the formation of EB.

Culturing of iPS Cells

1. Coating 0.1% gelatin on the culture dish and incubate for 30 minutes.
2. Add medium into culture dish and incubate for 10 minutes.
3. Add 20ul embryonic body(EB) in 2ml medium.
4. Culture EB cells until they attach on the plate with gelatin.
5. Do the coculture experiment!