To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized ''replication domains'' punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400-to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes, ID1, BCL2L1 and HM13, expressed in human ES cells, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

SUMMARY Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo-, but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro, do not fully eliminate PcG-mediated repression on endocrine-specific genes, likely contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.