SummaryThe plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems.

Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.

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