Other recent protocols that provide methods to purify NPCs, neurons and glia from differentiated cultures are likely to be very useful when comparing the differentiation efficiencies of different iPSC lines, when seeking to remove tumorigenic cells from cultures destined for transplantation, and in the isolation of specific cell types of interest.
It is, in addition, important to ensure that differentiated cultures are pure and, depending on whether cell renewal is required or to be avoided, retain or lack appropriate stem cells.
To assess culture purity and CXCR2 expression, 4 day differentiated cultures were washed in cold HBSS and fixed in 4% paraformaldehyde and blocked with 10% goat serum.
Differentiated cultures were first studied after 7DIV in standard NS medium containing 1% FBS but without growth factors, and on poly-D-Ornithine treated coverslips.
In agreement with qRT-PCR data, ADAR2 protein expression levels in differentiated cultures were significantly higher compared to undifferentiated hESCs, without a significant difference between neuronal and EBs cultures (Figure 3B).
Our differentiated cultures were predominantly occupied by myotubes, showing a mature sarcomeric cross-striated pattern.
Platelets released into the differentiated cultures were assessed by collecting the supernatants and identified by flow cytometry.
To examine the roles of Nurr1 and Foxa2, differentiated cultures were treated with silencing small hairpin RNAs (shRNAs) for these factors.
For relative plasma membrane potential measurement, differentiated cultures were dissociated on day 21 (after treatment as above from day 16) using 5× TrypLE Select and resuspended in 2.5 nM TMRM Life Technologiess) in warm assay medium.
Differentiated cultures were subjected to expression analysis by real-time PCR of two well-known markers of epidermal suprabasal layers: keratin 1 (KRT1) and involucrin (IVL) as well as markers of proliferating basal cells: keratin 14 (KRT14) and PCNA.
