Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs
Figure 7
hiPSCs transduced with the reporter vector containing miRNA targets indicate differentiation-specific reporter expression in neural lineages.
EBs generated from pooled hiPSCs were differentiated into neural lineages using Noggin and SB431542. EBs were then transferred onto fibronectin coated 6-well plates and further differentiated in N2 medium. (A) Total RNA was isolated using QIAGEN's RNeasy Mini kit from the hiPSCs on day 0 (undifferentiated population: Undiff.) and day 30 (differentiated population: Diff.) after induction of differentiation. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. GAPDH was used as an internal control. (B) Neural tube-like rosettes observed after the differentiation. (C) Dark pigmented melanocyte-like cells surrounding neural tube-like structures.