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Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

ABSTRACT: Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD.

SUBMITTER: Nieto-Nicolau N

PROVIDER: S-EPMC8178265 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Nieto-Nicolau Núria N Martínez-Conesa Eva M EM Fuentes-Julián Sherezade S Arnalich-Montiel Francisco F García-Tuñón Ignacio I De Miguel María P MP Casaroli-Marano Ricardo P RP

Journal of cellular and molecular medicine 20210505 11

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standar ...[more]

PMID: 33951289

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Project description:PurposeHuman corneal endothelial cells (hCECs) have been considered unable to regenerate in vivo, resulting in corneal decompensation after significant loss of hCECs. adipose-derived mesenchymal stem cell (ASC)-derived exosomes can regenerate tissues and organs. In this study, we investigated whether ASC-derived exosomes could protect and regenerate CECs.MethodsWe performed cell viability and cell-cycle analyses to evaluate the effect of ASC-derived exosomes on the regeneration capacity of cultured hCECs. Transforming growth factor-β (TGF-β) and hydrogen peroxide (H2O2) were used to induce biological stress in CECs. The effect of ASC-derived exosomes on CECs was investigated in vivo. ASC-derived exosomes were introduced into rat CECs using electroporation, and rat corneas were injured using cryoinjury. Next-generation sequencing analysis was performed to compare the differentially expressed microRNAs (miRNAs) between ASC-derived and hCEC-derived exosomes.ResultsASC-derived exosomes induced CEC proliferation and suppressed TGF-β- or H2O2-induced oxidative stress and senescence. ASC-derived exosomes protect hCECs against TGF-β- or H2O2-induced endothelial-mesenchymal transition and mitophagy. In an in vivo study, ASC-derived exosomes promoted wound healing of rat CECs and protected the corneal endothelium against cryoinjury-induced corneal endothelium damage. Next-generation sequencing analysis revealed differentially expressed miRNAs for ASC-derived and hCEC-derived exosomes. They are involved in lysine degradation, adherens junction, the TGF-β signaling pathway, the p53 signaling pathway, the Hippo signaling pathway, the forkhead box O (FoxO) signaling pathway, regulation of actin cytoskeleton, and RNA degradation based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.ConclusionsASC-derived exosomes promoted wound healing and regeneration of endothelial cells by inducing a shift in the cell cycle and suppressing senescence and autophagy.

Project description:Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

Dataset Information

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

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Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

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