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First published online 24 July 2007
doi: 10.1242/jcs.03478

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Clonal analysis of nestin^– vimentin⁺ multipotent fibroblasts isolated from human dermis

Fu Guo Chen^*, Wen Jie Zhang^*, Dan Bi, Wei Liu, Xian Wei, Fan Fan Chen, Lian Zhu, Lei Cui and Yilin Cao

Department of Plastic and Reconstructive Surgery, Shanghai 9th People's Hospital, Shanghai Stem Cell Institute, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, National Tissue Engineering Center of China, Shanghai 200011, China

View larger version (26K): [in this window] [in a new window]   Fig. 1. Characterization of dermal fibroblasts. (A) Morphology of dermal fibroblasts under light microscopy. (B) Cumulative population doubling curve generated from cell counting after each passage (n=5, see Materials and Methods). Bar, 250 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Immunophenotyping of dermal fibroblasts. Dermal fibroblasts (passage three, n=3) were characterized using flow cytometry analysis for the expression of the following markers: CD13 (62.1±7.26%), CD29 (53.2±14.68%), CD49d (38.4±7.44%), CD105 (30.9±7.12%), Stro-1 (8±2.14%), CD34 (1.7±0.67%), CD45 (0.7±0.23%), CD106 (0.8±0.18%) and CD133 (0.5±0.3%). Isotype-matching IgG-FITC and IgG-PE were used to determine non-specific signals.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Adipogenic induction of dermal fibroblasts. (A) Dermal fibroblasts were cultured in adipogenic-inducing (left panel) medium or in regular culture medium (right panel) for 3 weeks. Oil Red O staining is positive in the adipogenic-induced cells. (B) RT-PCR shows PPAR-2 and leptin mRNA expression in cells with adipogenic induction for 3 weeks. No expression was found in non-induced cells. Bars, 100 µm.

View larger version (121K): [in this window] [in a new window]   Fig. 4. Osteogenic induction of dermal fibroblasts. (A) After 1 week of osteogenic induction, positive staining of alkaline phosphatase (ALP) was observed in the induced group (left panel) but not in the non-induced group (right panel). (B) After 4 weeks of osteogenic induction, intensive calcium deposition was detected by Alizarin Red staining (ARS) in the induced group (left panel), and only very weak staining was observed in the non-induced group (right panel). (C) RT-PCR detects ALP mRNA expression after 1 and 2 weeks of induction, and osteocalcin (OCN) mRNA expression after 2 weeks of induction. No expression of ALP or OCN was observed in non-induced cells. Bars, 50 µm (A); 100 µm (B).

View larger version (101K): [in this window] [in a new window]   Fig. 5. Chondrogenic induction of dermal fibroblasts. (A) After 2 weeks of chondrogenic induction in plate culture, positive staining of Safranin O was observed in the induced group (left panel) but not in the non-induced group (right panel). (B) After 3 weeks of pellet culture, immunohistochemical staining showed positive staining of collagen type II (Col II) in the induced groups (left panel) but not in the non-induced group (right panel). (C) RT-PCR shows the mRNA expression of aggrecan after 1 week of induction and collagen type II mRNA expression after 2 weeks of induction. Bars, 250 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Differentiation potential of dermal fibroblast clones. Single-cell-derived clones were expanded for over 25 doublings and then induced in differentiation media for different time intervals as described in the Materials and Methods. Adipogenesis was evaluated by Oil Red O staining. Osteogenesis was revealed by alkaline phosphatase staining (ALP) and Alizarin Red staining (ARS). Chondrogenesis was revealed by Safranin O staining and immunostaining of type II collagen (Col II). Forty-seven clones were analyzed and numbers and percentages of different lineage types are listed. A/O/C, adipogenic/osteogenic/chondrogenic potential.

View larger version (145K): [in this window] [in a new window]   Fig. 7. Three multipotent dermal fibroblast clones exhibit trilineage potential: Clone 96A14, 96A29 and 96A33. (Left panel) Oil Red O staining after 3 weeks of adipogenic induction (upper-right corner, high magnification of one positive cell). (Middle panel) Type II collagen staining after 3 weeks of chondrogenic induction (upper-right corner, cell pellet). Red arrow shows the typical lacuna structure. (Right panel) Alizarin Red staining after 4 weeks of osteogenic induction (upper-right corner, alkaline phosphatase staining). Bars, 100 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 8. One multipotent dermal fibroblast clone exhibits neurogenic and hepatogenic differentiation potentials. (A) After 10 days of neurogenic induction, cells change to a neuron-like shape and express neurofilament-M (NF-M), neurotensin receptor 3 (NTR3) and neuron-specific class III-tubulin (III-tubulin) in Clone 96A14, but not in Clone 96A33 or Clone 96A29 (data not shown). (B) After 3 weeks of hepatogenic differentiation, cells express albumin (ALB), CK18 and hepatocyte nuclear factor-3 beta (HNF-3) in Clone 96A14. No hepatocyte markers are positive in Clone 96A33, even though a slight morphological change is observed. Neither morphological change nor hepatocyte markers are observed in Clone 96A29 (data not shown). Bars, 100 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 9. Differentiation potential of lineage-committed cells. Tripotent clones were osteogenically induced for 3 weeks, followed by adipogenic induction for another 3 weeks. Parent cells show positive staining for Alizarin Red (ARS) after osteogenic induction (A), and positive staining for Oil Red O after adipogenic induction (B), whereas osteogenically induced cells fail to exhibit Oil Red O staining after switching to adipogenic medium for 3 weeks (C). Bars, 100 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 10. Immunofluorescent staining of single-cell-derived multipotent dermal fibroblasts (MDFs). Cells were stained for vimentin (top panels) and nestin (bottom panels). Epidermal cells (EPCs) and P19 embryonal carcinoma cells (mouse neuron cell line) were used as negative and positive controls, respectively. Nuclei of all the cells are red because of the propidium iodide counterstain. Bars, 20 µm.