Developmental-stage-dependent transcriptional response to leukaemic oncogene expression

10.1038/ncomms8203Nature Communications6720

Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.Ministry of Education (MOE)National Research Foundation (NRF)Published versionThis study was supported by National Research Foundation (NRF), Singapore, under its Competitive Research Program (CRP) [NRF-CRP21-2018-0008] and Ministry of Education (MOE) for its MOE Academic Research Fund Tier 3 (STEM) [MOET32020-0001]

H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome

In mammals, genome-wide chromatin maps and immunofluorescence studies show that broad domains of repressive histone modifications are present on pericentromeric and telomeric repeats and on the inactive X chromosome. However, only a few autosomal loci such as silent Hox gene clusters have been shown to lie in broad domains of repressive histone modifications. Here we present a ChIP-chip analysis of the repressive H3K27me3 histone modification along chr 17 in mouse embryonic fibroblast cells using an algorithm named broad local enrichments (BLOCs), which allows the identification of broad regions of histone modifications. Our results, confirmed by BLOC analysis of a whole genome ChIP-seq data set, show that the majority of H3K27me3 modifications form BLOCs rather than focal peaks. H3K27me3 BLOCs modify silent genes of all types, plus flanking intergenic regions and their distribution indicates a negative correlation between H3K27me3 and transcription. However, we also found that some nontranscribed gene-poor regions lack H3K27me3. We therefore performed a low-resolution analysis of whole mouse chr 17, which revealed that H3K27me3 is enriched in mega-base-pair-sized domains that are also enriched for genes, short interspersed elements (SINEs) and active histone modifications. These genic H3K27me3 domains alternate with similar-sized gene-poor domains. These are deficient in active histone modifications, as well as H3K27me3, but are enriched for long interspersed elements (LINEs) and long-terminal repeat (LTR) transposons and H3K9me3 and H4K20me3. Thus, an autosome can be seen to contain alternating chromatin bands that predominantly separate genes from one retrotransposon class, which could offer unique domains for the specific regulation of genes or the silencing of autonomous retrotransposons

Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

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cGMP-grade human iPSC-derived retinal photoreceptor precursor cells rescue cone photoreceptor damage in non-human primates

10.1186/s13287-021-02539-8STEM CELL RESEARCH & THERAPY121CompletedComplete

A bio-functional polymer that prevents retinal scarring through modulation of NRF2 signalling pathway.

10.1038/s41467-022-30474-6Nat Commun1312796