Isolation and Characterization of Mesenchymal Progenitor Cells From Human Orbital Adipose Tissue

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Abstract

Adipose-derived stem cells (ASCs) have gained importance due to their myriad potential clinical applications. We hypothesize that progenitor cells also exist besides those conventionally isolated from the stromal vascular fraction (SVF).Central and medial orbital adipose tissues obtained from patients during eyelid surgery were digested with collagenase for 3 or 16 hours at 37°C with or without shaking. After centrifugation, the remaining cell pellet was resuspended and filtered to yield flow through in SVF and retained cells (RC) on the filter. Single cells from RC and SVF were cultured on 5% coated Matrigel in serum-free modified embryonic stem cells medium (MESCM) for 10 passages. The progenitor status was evaluated by the expression of a number of markers by qPCR and immunofluorescence staining as well as their plasticity for endothelial and tri-lineage differentiation.Type I collagenase digestion for 3 hours under shaking was significantly less effective in releasing progenitor cells than collagenase A digestion for 16 hours without shaking. Following filtration, cells in SVF and RC, of which the latter were tangled in collagen IV-containing matrix, expressed different markers of progenitor cells. Cells from SVF and RC could be expanded for 10 passages on coated Matrigel in MESCM and exhibited similar or better potential to differentiate into vascular endothelial cells, chondrocytes, osteocytes, and adipocytes than SVF cells expanded on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).Different progenitor cells can be isolated and expanded from orbital adipose tissues. Further characterization of their mesodermal or neuroectodermal origin might enhance clinical outcome when used as a source of autologous stem cells for ocular surface regeneration.

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