The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

The formation of definitive endoderm (DE) is a fundamental step for the development of the gastrointestinal tract, respiratory tract and endocrine organs. We present a CRISPR-based pooled screening approach to identify genes which contribute to DE induction from hiPSCs in vitro. CRISPR-based pooled genetic screens in mammalian cell culture enable researchers to identify genes required for a cellular phenotype of interest in an unbiased way. To enable a CRISPR-based forward genetic screen for identifying regulatory genes required for DE differentiation from hiPSCs, we performed pooled screens using a human genome-scale CRISPR knockout library. In addition, we performed a transcriptional activation screen using a lentiviral CRISPRa library to identify the downstream targets of the TGF/nodal/activin signaling pathway, which is a key signaling pathway for DE specification. We identified several signaling pathways including TGFβ, Erk, JNK, and CREB pathways are involved with DE differentiation. We suggest that CRISPR-based pooled genetic screens are a useful tool to identify key signaling pathways and genes required for in vitro differentiation processes and are served as a platform to improve differentiation protocols.