Unknown,Transcriptomics,Genomics,Proteomics

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Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium

ABSTRACT: Limbal epithelial stem cell (LESC) deficiency represents a significant clinical problem especially in bilateral cases. Induced pluripotent stem cells (iPSC) may be a promising source of LESC, allowing standardized and continual propagation and banking. The objective of this study was to generate iPSC from human limbal epithelial cultures and differentiate them back into limbal epithelial cells using substrata mimicking the natural LESC niche. Using Yamanaka’s episomal vectors limbal-derived iPSC were reprogrammed from LESC cultured from donor corneoscleral rims and from human skin fibroblasts. A clone from limbal-derived iPSC expressed stemness markers, had a diploid karyotype, and produced teratomas in nude mice representing three germ layers. Compared to parental LESC, this clone had fewer specific gene methylation changes revealed using the Illumina Infinium Methylation 450k Beadchips than compared to skin fibroblasts. The expression of putative LESC markers was examined by quantitative RT-PCR and immunostaining in limbal-derived and fibroblast-derived iPSC cultured on denuded human amniotic membrane or denuded cornea. Limbal-derived iPSC had markedly stronger expression of PAX6, ABCG2, Np63, keratins 14, 15, 17, and N-cadherin than fibroblast-derived iPSC. On denuded corneas, limbal-derived iPSC showed the expression of differentiated corneal keratins 3 and 12. The data suggest that iPSC differentiation to a desired lineage may be facilitated by their generation from the same tissue. This may be related to preservation of parental tissue epigenetic methylation signatures in iPSC and use of biological substrata similar to the natural niche of parental cells. The data pave the way for generating transplantable LESC from limbal-derived iPSC. Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip

ORGANISM(S): Homo sapiens

SUBMITTER: Jie Tang

PROVIDER: E-GEOD-53918 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Limbal stem cells including epithelial and stromal/Mesenchymal stem cells that contribute to sustained corneal homeostasis, maintain their ability to act as self-renewal progenitor cells by virtue of their limbal niche and intercellular communication. Extracellular vehicles (EVs), including exosomes (Exos), are important paracrine mediators through their cargo transfer for intercellular communication in various stem cell niches. Previously we have shown the differential cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos, in limbal epithelial cells (LEC) in normal (N) and diabetic (DM) limbal niche. In the present study, to have a comprehensive knowledge of reciprocal LEC-LSC crosstalk, we investigated the proteomics and miRNA profile of exosomes derived from LEC and their regulatory roles in LSC in N and DM limbus. Our study showed wound healing and proliferation rates in primary N-LSC were significantly enhanced upon treatment by normal LEC-derived Exos (N-Exos), but not by diabetic Exos (DM-Exos). Further, N-Exos treated LSC showed downregulation of keratocyte markers, ALDH3A1 and lumican, but not keratocan, and upregulation of MSC markers, CD105, CD90, and CD73 compared to the DM-Exos treated LSC. Using next generation sequencing (NGS) and proteomics analysis, we revealed some miRNAs and proteins in the Exos that affect the cellular crosstalk and the function of the cornea. We also documented differences in DM vs. normal LEC-derived Exo’s cargos. Overall, DM-Exos have less effect on LSC proliferation, wound healing, and stem cell maintenance than N-Exos, likely by transferring their cargo proteins and/or regulatory miRNAs targeting cell cycle, ERK/MAPK, TGF-β, EMT, PI3K-Akt-mTOR signaling molecules. This suggests that the small RNA and protein cargo differences in DM vs. N LEC-derived Exos could contribute to the disease state. Our study revealed a complex contribution of Exos to health and diabetic state of corneal homeostasis and suggests the potential of EV therapeutics for diabetic cornea regenerative medicine

Project description:Limbal stromal cells were reported to resemble mesenchymal stem cells (MSCs) with multipotential differentiation cability. However, little is known about their gene expression profiles compared to MSC derived from various sources. In this study, the gene expression profile of limbal stromal cells was compared to bone marrow, adipose stromal cells and foreskin fibroblasts. In addition, we also explored the gene expression changes of ex vivo expanded limbal stromal cells when cultured in two different systems. Expanded limbal stromal cells were divided into two groups; each cultured separately on a matrigel-coated plate in DMEM/F12 medium supplemented with bFGF and LIF and the other on a normal plate in DMEM medium supplemented with 10% fetal bovine serum (FBS). Cryopreserved bone marrow mesenchymal cells, adipose stromal cells and foreskin fibroblasts were cultured-expanded until confluent. Total RNA was extracted from all the samples and subjected to microarray experiments with an Agilent platform by using Human GE 8x60k microarrays. Data analysis was carried out with GeneSpring software. A total of 871 genes were upregulated when the limbal stromal cells were cultured in the matrigel system, whereas 58 genes were consistently differentially expressed in limbal stromal cells compared to other lineages. Besides the long intergenic non-coding RNA and unknown genes, these genes represent gene ontology for cellular components, molecular function and biological process. Samples derived from the same source were closely clustered by Hierachical clustering analysis. The limbal stromal cells have a distinct molecular signature compared to MSCs from other lineages. The culture system affected the gene expression profile of limbal stromal cells tremendously. Derived limbal stromal cells were cultured using two different methods, one with matrigel and the other with FBS. Their gene expression profiles were compared. The gene expression profile of limbal stromal cells that were cultured with FBS also was compared to the gene expression profiles of bone marrow mesenchymal stem cells, adipose stromal cells and foreskin fibroblasts.

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Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium

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