When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

Tat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip), observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009). Although adult heterozygous (Tip60(+/-)) mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60(+/-) adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007). These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60α and Tip60β, functions to suppress the cell-cycle in adult cardiomyocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.

Elucidating the molecular basis of cell differentiation will advance our understanding of organ development and disease. We have previously established a protocol that efficiently produces cells with hepatocyte characteristics from human induced pluripotent stem cells. We previously used this cell differentiation model to identify the transcription factor hepatocyte nuclear factor 4 α (HNF4A) as being essential during the transition of the endoderm to a hepatic fate. Here, we sought to define the molecular mechanisms through which HNF4A controls this process. By combining HNF4A chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) analyses at the onset of hepatic progenitor cell formation with transcriptome data collected during early stages of differentiation, we identified genes whose expression is directly dependent upon HNF4A. By examining the dynamic changes that occur at the promoters of these HNF4A targets we reveal that HNF4A is essential for recruitment of RNA polymerase (RNA pol) II to genes that are characteristically expressed as the hepatic progenitors differentiate from the endoderm.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Polyglutamine (polyQ) diseases are caused by expansion of translated CAG repeats in distinct genes leading to altered protein function. In spinocerebellar ataxia type 1 (SCA1), a gain of function of polyQ-expanded ataxin-1 (ATXN1) contributes to cerebellar pathology. The extent to which cerebellar toxicity depends on its cognate partner capicua (CIC), versus other interactors, remains unclear. It is also not established whether loss of the ATXN1-CIC complex in the cerebellum contributes to disease pathogenesis. In this study, we exclusively disrupt the ATXN1-CIC interaction in vivo and show that it is at the crux of cerebellar toxicity in SCA1. Importantly, loss of CIC in the cerebellum does not cause ataxia or Purkinje cell degeneration. Expression profiling of these gain- and loss-of-function models, coupled with data from iPSC-derived neurons from SCA1 patients, supports a mechanism in which gain of function of the ATXN1-CIC complex is the major driver of toxicity.