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Y. Chen, S. Chen, S. C. G. Tseng; Isolation and Expansion of Human Amniotic Membrane-Derived Mesenchymal Cells as Feeder Layers for Expanding Human Limbal Epithelial Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6062. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mesenchymal stem cells (MSCs) are found in human placenta and amniotic membrane. Isolation and expansion of human amniotic mesenchymal stromal cells (hAMSCs) has not been optimized and it remains unknown whether hAMSCs contain MSCs or can serve as feeder layers for expanding epithelial progenitor cells.
Three enzymatic isolation methods, i.e., dispase followed by collagenase (DC), collagenase alone (C) and combined collagenase and hyaluronidase (CH) were compared for the purity of isolating hAMSCs from the amniotic membrane. The expansion medium was optimized by modifying the DMEM-based medium with different supplements such as F12, FBS, EGF, cholera toxin (CTX), etc, so that expression of α-smooth muscle actin (α-SMA) could be suppressed. The capability of expanded hAMSCs serving as feeder layers was examined in a clonal assay for expanding human limbal epithelial progenitor cells (HLECs) using NIH 3T3 as the control.
DC and CH achieved high purity in isolating hAMSCs with a significantly lower rate of contamination by human amniotic epithelial cells (0.24%, 0.56% respectively) as compared to C (14.02%). Around 76% of hAMSCs cultured in DMEM with 10%FBS, 5% FBS, or an equal volume of F12 with various combination of EGF and CTX continued to differentiate into α-SMA-expressing myofibroblasts by Day 5 (D5) and accompanied by rapid retardation of cell proliferation. In contrast, myofibroblast differentiation was completely prevented at D5 by including ITS, hydrocortisone and DMSO; some subpopulation still expressed α-SMA by D11. Cells could be continuously expanded up to 13 passages. HLECs generated lower CFE with homogenous small cells when Passage 8 hAMSCs were used as feeder layers, but yielded higher CFE and heterogenous large cells on 3T3 fibroblasts.
Using DC or CH isolation method, further optimization is possible to expand hAMSCs based on refinement of supplements in the medium. The resultant hAMSCs might exhibit better MSC potential and serve as a superior feeder layer for expanding HLECs.
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