The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

Indigenous Tibetan people have lived on the Tibetan Plateau for millennia. There is a long-standing question about the genetic basis of high-altitude adaptation in Tibetans. We conduct a genome-wide study of 7.3 million genotyped and imputed SNPs of 3,008 Tibetans and 7,287 non-Tibetan individuals of Eastern Asian ancestry. Using this large dataset, we detect signals of high-altitude adaptation at nine genomic loci, of which seven are unique. The alleles under natural selection at two of these loci [methylenetetrahydrofolate reductase (MTHFR) and EPAS1] are strongly associated with blood-related phenotypes, such as hemoglobin, homocysteine, and folate in Tibetans. The folate-increasing allele of rs1801133 at the MTHFR locus has an increased frequency in Tibetans more than expected under a drift model, which is probably a consequence of adaptation to high UV radiation. These findings provide important insights into understanding the genomic consequences of high-altitude adaptation in Tibetans.high-altitude adaptation | Tibetans | genome-wide association study | mixed linear model | polygenic selection G enetic adaptation to a novel environment is a fundamental process for the survival and adaptation of a species. In humans, one of the most recent examples is adaptation to high altitude, such as the Tibetan highlands. The Tibetan Plateau (TP; also known as the Qinghai-Tibet Plateau in China) has an average elevation of ∼4,000 m above sea level, where the oxygen concentration is ∼40% lower (1) and UV radiation is ∼30% stronger (2) than at sea level. The indigenous Tibetan people have developed a distinctive set of physiological characteristics to adapt to the extreme environmental conditions in the highlands (1). Previous population-based genetic studies have reported evidence that genetic variants at the EPAS1 and EGLN1 loci have been under positive natural selection (3-7). These genetic variants are associated with phenotypic variation of hemoglobin concentration (HGB) in Tibetans (3-5). The EPAS1 gene, which encodes the hypoxia inducible factor-2α (HIF-2α) subunit of HIF complex, is a transcription factor involved in body response to hypoxia (8, 9). EGLN1 encodes PHD2, which is a major oxygen-dependent negative regulator of HIFs (10, 11). Apart from these two known genes that have biological relevance to hypoxia adaptation (3-7, 12), several other candidate gene loci (e.g., PPARA and HBB) have been highlighted in recent studies (3,4,(13)(14)(15). Genetic adaptation to high altitude, however, is likely to be a complex process, with a large number of genes involved in response to not only hypoxia but also, other extreme environmental conditions, such as low temperature, high UV radiation, and insufficient food supply. If the strength of natural selection at these gene loci has been small to moderate, these loci would not be detected in previous studies (3-7) of small sample size (typically n < 150). In this study, we perform a largescale genome-wide study to detect genetic signals of high-altitude adaptation in 3...

CRISPR/Cas9 -mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering.