A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Bangalore, Megha Prakash; Panicker, Mitradas M.

doi:10.1016/j.stemcr.2014.05.004

Cited by 19 publications

(10 citation statements)

References 46 publications

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“…All the lines showed typical Embryonic stem cells (ES) like morphology in co-culture with inactivated MEF feeder layer cells. iPSCs cultured in standard stem cell medium exhibited blue fluorescence, characteristics of pluripotent stem cells in primed state as reported previously (Muthusamy et al, 2014). The iPSCs were immunopositive for pluripotency markers (OCT4, SOX2, SSEA4) ( Fig.…”

Section: Development Of Ipsc Derived Neurons Of Sca12supporting

confidence: 79%

Transcriptomic Dynamics of a non-coding trinucleotide repeat expansion disorder SCA12 in iPSC derived neuronal cells: signatures of interferon induced response

Kumar

Dhapola

et al. 2017

Preprint

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Abstract:Spinocerebellar ataxia type-12 (SCA12) is a neurological disorder that exhibits a unique progressive tremor/ataxia syndrome induced by triplet (CAG) repeat expansion in 5' UTR of PPP2R2B. SCA12 is one of the most prominent SCA-subtype in India and till date no appropriate disease models have been described. Our aim was to establish human iPSC derived neuronal cell lines of SCA12 and study transcriptomic level alterations induced by CAG expansion. For translational application, peripheral blood transcriptomics of SCA12 patients was also performed. Lymphoblastoid cell lines of three SCA12 patients were reprogrammed to iPSCs and then re-differentiated into pan-neuronal lineage. RNAsequencing based comparative transcriptomics was performed for disease and control cell lineages. Microarray based transcriptomic profiling of peripheral blood of SCA12 patients was performed in a case/control (n=15/9) design. We have successfully created human neuronal cell lines of SCA12 patient as exhibited by their molecular profiling. Differential expression analysis of RNA-Seq data has shown enrichment for type-I interferon signaling and other relevant cellular processes in SCA12-neurons. At the splice-isoform level, we observed an upregulation of expanded CAG containing non-coding transcript of PPP2R2B.Peripheral blood transcriptomics analysis and targeted validation of RNA-Seq data has allowed us to identify inflammatory signatures as potential markers of molecular pathology in SCA12. Our study has allowed us to establish first iPSC based neuronal cell lines of SCA12.We have identified pro-inflammatory signatures in SCA12-neurons suggestive of a dsRNA mediated activation of interferon signaling and that corroborates with the emerging evidence of neuronal atrophy due to neuro-inflammation in common neurodegenerative diseases. This study involved development of an iPSCs derived neuronal cells of SCA12 and look through signatures of neurodegeneration by whole RNA sequencing. This model sheds light upon key role of RNA mediated induced response in Interferon signaling for neurodegeneration.peer-reviewed)

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Section: Development Of Ipsc Derived Neurons Of Sca12supporting

confidence: 79%

Transcriptomic Dynamics of a non-coding trinucleotide repeat expansion disorder SCA12 in iPSC derived neuronal cells: signatures of interferon induced response

Kumar

Dhapola

et al. 2017

Preprint

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“…6i ). One property of the naïve mESCs state is lower number of lipid droplets 35 . So, the decreased number of lipid droplets per cells during reprogramming is not unexpected.…”

Section: Discussionmentioning

confidence: 99%

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming

Pei

Yue

Zhang

et al. 2015

Sci Rep

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Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.

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“…Utilization of the micropatterned substrates to probe these mechanisms has strong potential to provide deeper insight in the biophysical and microenvironmental signaling involved in epigenetic reprogramming. The microscale approach is complementary to other imaging modalities for dissecting complex reprogramming cultures (e.g., on cell surface markers (Paull et al, 2015;Smith et al, 2010;Tanabe et al, 2013), metabolic markers (Muthusamy et al, 2014), or dye uptake (Hirata et al, 2014;Smith et al, 2010)) and could be implemented in a variety of formats (e.g., microfluidic (Luni et al, 2016), µShear (Singh et al, 2013)). Enhanced capabilities to produce iPSCs on micropatterned substrates move both allogeneic and autologous PSC-based regenerative medicine closer to the clinic and use in precision medicine (Andrews et al, 2014;Inoue et al, 2014;Yaffe et al, 2016).…”

Section: Mechanisms Of Extracellular Accumulation Of Secreted Factorsmentioning

confidence: 99%

Micropatterned substrates to promote and dissect reprogramming of human somatic cells

Carlson-Stevermer

Harkness

Prestil

et al. 2017

Preprint

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Reprogramming of human somatic cells to induce pluripotent stem cells (iPSCs) generates valuable precursors for disease modeling and regenerative medicine. However, the reprogramming process can be inefficient and noisy, creating many partially reprogrammed cells in addition to fully reprogrammed iPSCs. To address these shortcomings, we developed a micropatterned substrate that allows for dynamic live-cell microscopy of thousands of cell subpopulations undergoing reprogramming. Micropatterning facilitated a change in shape, size and clustering of nuclei to promote somatic identity erasure. Increased proliferation, cell density and decreased intercellular YAP signaling accompanied these nuclear changes. A combination of eight nuclear characteristics could be used to track reprogramming progression and distinguish partially reprogrammed cells from those that were fully reprogrammed.Micropatterned substrates constitute a new tool for facile iPSC production and can be used in high-throughput to probe and understand the subcellular changes that accompany human cell fate transitions.

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A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

Cited by 19 publications

References 46 publications

Transcriptomic Dynamics of a non-coding trinucleotide repeat expansion disorder SCA12 in iPSC derived neuronal cells: signatures of interferon induced response

Transcriptomic Dynamics of a non-coding trinucleotide repeat expansion disorder SCA12 in iPSC derived neuronal cells: signatures of interferon induced response

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming

Micropatterned substrates to promote and dissect reprogramming of human somatic cells

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