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Human adipose-derived stem cells support the growth of limbal stem/progenitor cells.

ABSTRACT: The most efficient method to expand limbal stem cells (LSCs) in vitro for clinical transplantation is to culture single LSCs directly on growth-arrested mouse fibroblast 3T3 cells. To reduce possible xenobiotic contamination from 3T3s, primary human adipose-derived stem cells (ASCs) were examined as feeder cells to support the expansion of LSCs in vitro. To optimize the ASC-supported culture, freshly isolated limbal epithelial cells in the form of single cells (SC-ASC) or cell clusters (CC-ASC) were cultured using three different methods: LSCs seeded directly on feeder cells, a 3-dimensional (3D) culture system and a 3D culture system with fibrin (fibrin 3D). The expanded LSCs were examined at the end of a 2-week culture. The standard 3T3 culture served as control. Expansion of SC-ASC showed limited proliferation and exhibited differentiated morphology. CC-ASC generated epithelial cells with undifferentiated morphology in all culture methods, among which CC-ASC in 3D culture supported the highest cell doubling (cells doubled 9.0 times compared to cells doubled 4.9 times in control) while maintained the percentage of putative limbal stem/progenitor cells compared to the control. There were few cell-cell contacts between cultured LSCs and ASCs in 3D CC-ASC. In conclusion, ASCs support the growth of LSCs in the form of cell clusters but not in single cells. 3D CC-ASC could serve as a substitute for the standard 3T3 culture to expand LSCs.

SUBMITTER: Mei H

PROVIDER: S-EPMC5636133 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Human adipose-derived stem cells support the growth of limbal stem/progenitor cells.

Mei Hua H González Sheyla S Nakatsu Martin N MN Baclagon Elfren R ER Chen Felix V FV Deng Sophie X SX

PloS one 20171011 10

The most efficient method to expand limbal stem cells (LSCs) in vitro for clinical transplantation is to culture single LSCs directly on growth-arrested mouse fibroblast 3T3 cells. To reduce possible xenobiotic contamination from 3T3s, primary human adipose-derived stem cells (ASCs) were examined as feeder cells to support the expansion of LSCs in vitro. To optimize the ASC-supported culture, freshly isolated limbal epithelial cells in the form of single cells (SC-ASC) or cell clusters (CC-ASC) ...[more]

PMID: 29020119

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Project description:PurposeWe investigated whether human limbal niche cells generate mesenchymal stem cells.MethodsLimbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells.ResultsStromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63? and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63?.ConclusionsIn the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.

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Human adipose-derived stem cells support the growth of limbal stem/progenitor cells.

Publications

Human adipose-derived stem cells support the growth of limbal stem/progenitor cells.

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