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HESC- and hiPSC-derived Schwann cells are Molecularly and Functionally Equivalent

ABSTRACT: hESC- and hiPSC-derived Schwann cells are Molecularly and Functionally Equivalent

PROVIDER: PRJNA860642 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20336600_1.fastq.gz	Fastqsanger.gz
	SRR20336600_2.fastq.gz	Fastqsanger.gz
	SRR20336595_1.fastq.gz	Fastqsanger.gz
	SRR20336595_2.fastq.gz	Fastqsanger.gz
	SRR20336596_1.fastq.gz	Fastqsanger.gz

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hESC- and hiPSC-derived Schwann cells are Molecularly and Functionally Equivalent

Project description:Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key model of human cells and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial, we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally, both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent.

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Three congruent human Schwann cell models of CMT1A reveal a converged phenotype

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Gene expression profile of hESC/hIPC-derived keratinocytes

Project description:To induce the differentiation, undifferentiated human pluripotent stem cells (both hESC andh iPSC; T0 time point) were transferred into 20% O2 atmosphere environment and treated with mTESR1 basal media supplemented with 1 μM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction). To select for the cells that acquired early ectodermal fate, cells were harvested and re-plated onto freshly prepared 3D human dermal fibroblast ECM at a density of 5-10x10^3 cells per cm2 and grown in DMEM:Ham F12 (3:1) (Life Technologies) supplemented with 1 μM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection). To enrich for putative epidermal progenitors, rapid-adhesion to type IV collagen-coated dishes was used, and the rapidly-adhering cells were cultured in DK SFM supplemented with 1 μM ATRA for 7 days (Enrichment). After that, the cells were cultured in EpiLife medium (Life Technologies) for a further 7 days (Expansion) before final harvest (T3 time point) and analysis. We analyzed here gene expression profiles of undifferentiated hESC/hiPSC (T0), hESC/hIPC-derived keratinocytes (T3) and primary normal human keratinocytes from skin biopsy (NHK). We found that hESC/hIPC-derived keratinocytes are similar to NHK. Biological triplicates of undifferentiated (T0) hESC (KCL034) and hiPSC lines (iKCL004, iKCL011) were compared with hESC/hiPSC-derived keratinocytes (T3), primary human keratinocyes (NHK) and fibroblasts (BJ).

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Project description:We obtained skin fibroblasts from CMT1A and control patients, and generated hiPSCs which were subsequently differentiated into cd49d+ human Schwann cells. We utilized microarray technology to explore the gene expression profiles of cd49d+ Schwann cells CMT1A hiPSCs, control hiPSCs, and control human embryonic stem cells in order to identify potentially disregulated pathways contributing to CMT1A pathogenesis. Patient-specific human induced pluripotent stem cells (hiPSCs) hold great promise for disease modeling of genetic disorders. Often the findings from hiPSC-based studies are validated with genetically-corrected hiPSCs generated by precise genome editing technologies, however, alternatives that have not yet been employed are validation with embryonic stem cells harboring the same disease mutation or utilizing another reprogramming approach from somatic cells of same patients. Here we report that disease-relevant phenotypes found in Charcot-Marie-Tooth 1A (CMT1A)-hiPSC-derived Schwann cells were further confirmed by two additional congruent CMT1A models as an alternative to gene correction. We have devised a defined and relatively fast protocol for the direct derivation and prospective isolation of Schwann cells from hiPSCs, leading us to uncover a phenotype of dysregulated immune signaling in CMT1A-hiPSCs-Schwann cells. Our study illustrates the promise of applying hiPSC technology to one of the most common hereditary neuropathies for gaining new insights into human disease pathogenesis and treatment, and these results demonstrate the feasibility of verifying disease phenotypes by utilizing the malleability of cellular fates.

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Comparing isogenic pairs of hESC and hiPSC lines reveals genetic background and reprogramming method as primary sources of transcriptional variation

Project description:The equivalency of human induced pluripotent stem cells (hiPSCs) with human embryonic stem cells (hESCs) remains controversial. Here, we devised a strategy to assess the contribution of clonal growth, reprogramming method and genetic background to transcriptional patterns in hESCs and hiPSCs. Surprisingly, transcriptional variation originating from two different genetic backgrounds was dominant over variation due to the reprogramming method or cell type of origin of pluripotent cell lines. Moreover, the few differences we detected between isogenic hESCs and hiPSCs neither predicted functional outcome, nor distinguished an independently derived, larger set of unmatched hESC/hiPSC lines. We conclude that hESCs and hiPSCs are transcriptionally and functionally highly similar and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional comparisons of pluripotent cell lines, explaining some of the previously observed expression differences between unmatched hESCs and hiPSCs. Expression profiling of human embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and fibroblasts, mostly in triplicates.

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