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Isolation of Human Oral Keratinocyte Progenitor/Stem Cells

Department of Oral and Maxillofacial Surgery, University of Michigan Health System, B1-208 TC, Box 0018, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0018, USA.

View larger version (122K): [in this window] [in a new window]   Figure 1. Immunohistochemical staining of non-cultured oral mucosa. (A) ß1 integrin, (B) PPAR. ß1 integrin staining was more intense in the basal layer and a few layers above the basal layer at the tops of the ridges than seen at the bases of the ridges. The differential PPAR expression was more distinct in the basal layer at the tops of the ridges. D11 EVPOME: (C) ß1 integrin, (D) PPAR. D11 EVPOME showed an expression pattern similar to that seen in native oral mucosa.

View larger version (33K): [in this window] [in a new window]   Figure 2. Characteristics of different cell sizes as determined by FACS analysis. (A) Representative FACS dot plot of cultured oral keratinocytes at passages 2 and 4. Shown are the 3 gates (Small, Medium, Large) for sorting cells by relative cell size in diameters in equal proportions (approximately 25%) (left panels). The subpopulation indicated by a black arrow in the dot diagrams was debris eliminated by the gate in the side-scatter vs. forward-scatter, as well as PI-fluorescence before cell-sorting. (right panels) ß1 integrin expression at passage 2 in each size of sorted cells. ß1 integrin was expressed 99.4 ± 0.6% in Large cells, 99.5 ± 0.4% in Medium cells, and 99.4 ± 0.5% in Small cells. (B) Representative PPAR expression at passages 4 (72.8% in Large cells, 57.3% in Medium cells, 44.0% in Small cells) and 6 (84.2% in Large cells, 83.9% in Medium cells, 49.5% in Small cells) in each size of sorted cells. Among the cell size groups, the different proportion of PPAR-positive cells was statistically significant (79.9 ± 25.5% in Large cells, 57.6 ± 34.7% in Medium cells, 37.4 ± 32.6% in Small cells) (overall p < 0.0001). Dotted line drawn at the top 3% of the total population of background control indicates the borderline showing positive FITC fluorescence when compared with background control. Note shift of the histograms to the right as the passage number increases, indicating that a larger percentage of cells is PPAR-positive. (C) Linear regression analysis with a linear mixed model, showing relationship between proportion cells in S + G2M phases and days in culture in each size group. Passage numbers ranged from 1 to 8. The longer Small cells were in culture, the more cycling cells increased. In contrast, the longer Medium and Large cells were in culture, the more non-cycling cells increased. (D) Linear regression analysis with a linear mixed model, showing relationship between proportions of cells in S + G2M phases and cell diameters in each size group. Passage numbers ranged from 1 to 8. Within Small cells, the smaller they were, the more quiescent. In contrast, within Medium and Large cells, the smaller they were, the more cycling occurred.

View larger version (37K): [in this window] [in a new window]   Figure 3. Progenitor/stem cell functional assays. (A) Colony-forming efficiency assay. Left bars show colonies consisting of 16-49 cells. Right bars show colonies consisting of greater than 50 cells. N = 10; Passage 3 (N = 1), Passage 4 (N = 5), Passage 6 (N = 3), Passage 8 (N = 1). Higher colony efficiency of both size colonies is seen with the Small cells. (B) Long-term growth potential. (1) Assumptive total output of cells after cell-sorting (N = 7). Type IV collagen-attached cells out of the 2.0 x 104 sorted Small cell subpopulation yielded cells up to 5.0 x 108 folds, and were significantly more productive. Out of 7, 3 failed further propagation in Medium and Large cells. (C) Long-term growth potential. (2) Days in culture after cell-sorting (N = 7). Large, 26.4 ± 26.6; Medium, 40.1 ± 49.7; Small, 121.1 ± 43.9 days (Mean ± SD). Small cell subpopulations significantly survived longer than Medium and Large cell subpopulations in vitro. (D) Long-term growth potential. (3) Cumulative population doublings after cell-sorting (N = 7). Small cells had an enhanced replicate lifespan. In contrast, the lifespan of Medium and Large cells diminished earlier than that of Small cells. In B, C, and D, each number indicated a different person’s sample.

View larger version (113K): [in this window] [in a new window]   Figure 4. EVPOME fabricated by size-sorted cells from unsorted passage 8 (hematoxylin-eosin staining). (A) Large cells show no evidence of an intact epithelial cell layer. (B) Medium cells show a thin monolayer of cells. (C) Small cells are the only ones that show an intact stratified epithelial layer of cells similar to D, an EVPOME produced by unsorted passage 2 cells.