Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.
Total time
~7-10 days (MEF generation 2-3 days, reprogramming 4-7 days, FACS isolation 1-2 hours)
Model organism
Mouse embryonic fibroblasts (C57BL/6 or similar strain)
Steps
1
Generate mouse embryonic fibroblasts from embryos
Isolate embryonic tissue and culture to obtain primary MEFs for reprogramming experiments. This provides the starting cell population required for the reprogramming protocol.
▶ 01:19
2
Reprogram MEFs using Oct-4, Sox-2, Klf-4, c-Myc
Introduce pluripotency transcription factors into MEFs via forced expression to initiate reprogramming into induced pluripotent stem cell state. This generates a heterogeneous population containing rare reprogramming intermediates.
▶ 03:17
3
Label cells with surface marker antibodies
Incubate reprogrammed cell cultures with fluorescently-conjugated antibodies against Thy-1.2, Ssea-1, and Epcam to identify distinct reprogramming intermediates. This enables downstream FACS-based isolation of specific cell populations.
▶ 05:14
4
Isolate reprogramming intermediates via FACS sorting
Use fluorescence-activated cell sorting to physically separate and collect rare intermediate populations based on surface marker expression patterns. Sorted cells enable downstream analysis of reprogramming progression.
▶ 06:50
5
Analyze FACS profiles and reprogramming progression
Visualize and interpret representative FACS plots and microscopy images showing surface marker expression during reprogramming stages. This confirms successful isolation of intermediates and characterizes reprogramming kinetics.
▶ 08:26