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Szu-Yu Chen, Megha Mahabole, Elan Horesh, Sara Wester, Jeffrey Goldberg, Scheffer Tseng; Isolation and Characterization of Stem Cells from Human Orbital Adipose Tissues. Invest. Ophthalmol. Vis. Sci. 2013;54(15):735.
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Adipose stem cells (ASCs) have gained importance due to their potential clinical applications. They are conventionally isolated from SVF after collagenase digestion, centrifugation, and filtration. Because collagenase does not remove the basement membrane matrix, we question whether some progenitor cells that are preferentially associated with the basement membrane might have been excluded from from filtration.
Orbital adipose tissues obtained from patients after blepharoplasty were digested with 1 mg/ml collagenase A in DMEM/10%FBS or a serum-free modified ESC medium (MESCM) for 16 h at 37 oC. After centrifugation at 300x g for 5 min to remove floating cells (FC), the remaining cell pellet was resuspended and filtered through a 250 µm mesh to yield cells retained on the filter (RC) and flowing through (SVF). Single cells from FC, RC, and SVF were cultured on 5% coated matrigel or immobilized nHC-HA/PTX3 purified from amniotic membrane in MESCM for 8 days. Expression of ESC markers (Oct4, Nanog, Sox2, and nestin) and angiogenic markers (CD34 and CD31) was determined by qPCR or immunostaining.
Compared to SVF, qPCR showed significantly higher expression of Oct4, Nanog, and Sox2 in RC of two patients and in FC of the third patient (P<0.01). Expression of Nestin transcript was consistently significantly highest in RC in all 3 patients. qPCR and immunostaining showed that cells in SVF were mostly CD34+/CD31-, those in RC were CD34+/CD31+, and those in FC were CD34-/CD31- in all three patients. Furthermore, RC cultured on 5% matrigel or immobilized nHC-HA/PTX3 in MESCM consistently showed significantly higher expression of both ESC and CD31 than in SVF and FC (P<0.01).
Our preliminary data strongly suggests that there are other progenitor cells that are currently not isolated by the conventional method in orbital adipose tissues. Further characterization and expansion of these progenitor cells is warranted to see if they exert better therapeutic actions.
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