Biopharma Insights Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.
Total time
~5–7 days (isolation to expanded stem cell culture)
Model organism
Mouse (mammary tissue)
Steps
1
Isolate primary mammary cell populations from tissue
Extract and dissociate mammary gland tissue from adult mice to obtain primary cell populations. This step prepares material for downstream FACS purification and reprogramming.
▶ 01:07
2
Purify mammary epithelial cells by FACS and seed cultures
Use fluorescence-activated cell sorting to isolate pure mammary epithelial cell populations, then plate them at defined density for subsequent reprogramming experiments.
▶ 04:07
3
Induce YAP expression and culture mammary stem cells
Introduce transient YAP expression into purified epithelial cells to reprogram them into expandable stem/progenitor cells (yMaSCs), then perform serial subculturing to maintain stem cell identity.
▶ 05:05
4
Generate and culture ductal organoids from yMaSCs
Culture reprogrammed yMaSCs under conditions that promote three-dimensional ductal structure formation (yDucts), demonstrating functional differentiation capacity of regenerated stem cells.
▶ 07:33
5
Validate reprogramming efficiency and stem cell properties
Characterize yMaSCs and yDucts through molecular and morphological analysis to confirm successful reprogramming of differentiated cells into functional tissue-specific stem cells.
▶ 09:57