Biopharma Insights Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.
Total time
~2–4 weeks (from initial fibroblast plating to clonal iPSC expansion and characterization)
Model organism
Human primary fibroblasts
Steps
1
Prepare reagents for reprogramming protocol
Prepare modified mRNA encoding reprogramming factors, mature microRNA-367/302 mimics, and culture media. Ensure all reagents are thawed and ready for cell transfection.
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2
Plate human primary fibroblasts for reprogramming
Seed human primary fibroblasts at appropriate density on culture plates in feeder-free conditions on Day 0. Establish baseline cultures for reprogramming induction.
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3
Initiate reprogramming with mRNA and microRNA mimics
On Day 1, transfect fibroblasts with modified reprogramming factor mRNAs and microRNA-367/302 mimics. Monitor cells for morphological changes and early reprogramming markers.
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4
Assess reprogramming efficiency and pluripotency
Evaluate reprogramming efficiency through colony formation analysis and confirm pluripotency marker expression (TRA-1-60) using immunofluorescence or flow cytometry. Expand clonal iPSC colonies.
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