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We report a method to isolate naïve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.
Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.
1. SKP Isolation from Primary Fibroblast Cultures
Human fibroblasts GMO3349C and GMO8398A were obtained from the Coriell Institute for Medical Research (Camden, NJ) and were used in this study.
We show that a population of cells that selectively expand to generate SKP spheres under controlled growth condition consisting of EGF and FGF2 are present in primary dermal fibroblast cultures (Figure 1) as we reported recently 7.
Fibroblast cultures from PPDs 15 to 25 that typically correspond to the primary fibroblasts strains available from cell banks were used in this study. Fibroblast cultures submitted to the double treatment consisting of cold tempera.......
Using the method described herein, naïve dermal stem cells can be isolated from primary dermal fibroblast cultures. Using this approach, we recently reported the isolation and characterization of adult stem cells from fibroblast cultures derived from patients with a rare genetic syndrome, Hutchinson-Gilford progeria syndrome 7. As show herein those precursor cells express stem cell markers are capable of self-renewal and can be directed to differentiate into different cellular lineages including fib.......
We have nothing to disclose.
This work was supported by the Alexander von Humboldt Foundation (5090371), the Christine Kühne Center for Allergy Research and Education (CK-CARE), and the Bayerischen Staatsministerium (to K.D.).
....Name | Company | Catalog Number | Comments |
Name of the reagent | Company | Catalogue number | Comments (optional) |
DMEM high glucose | Invitrogen | 31966-047 | |
DMEM low glucose | Invitrogen | 21885-108 | |
fetal bovine serum | Invitrogen | 10270-106 | |
L-glutamine | Invitrogen | 25030-024 | Final conc.: 200 mM |
Penicillin/ Streptomycin | Invitrogen | 15140-122 | Final conc.: 10 mg/ml /10 mg /ml |
trypsin solution (0.25%) | Invitrogen | 25200-056 | |
F-12 Nutrient Mixture (Ham) | Invitrogen | 21765-029 | |
FGF2 | BD Biosciences | 4114-TC-01M | Final conc.: 40 ng/ml |
EGF | BD Biosciences | 236-EG-200 | Final conc.: 20 ng/ml |
PDGFBB | Invitrogen | PHG0043 | Final conc.: 5 ng/ml |
TGF-b1 | Invitrogen | PHG9204 | Final conc.: 2.5 ng/ml |
25 cm2 flask | Omnilab | FALC353109 | |
PBS w/o CaMg | Invitrogen | 14190-169 | |
B27 | Invitrogen | 17504-044 | |
Methanol | Roth | 8388.2 | |
Vectashield mounting medium | Vector Inc. | H-1200 | |
RNeasy Minikit | Qiagen, Valencia, CA | 74104 | |
Omniscript Reverse Transcriptase | Qiagen | 205113 | |
SsoFast EvaGreen Supermix | BioRad | 172-5201 | |
Fungizone | Invitrogen | 15290-018 | Final conc.:1 mg/ml |
Table 2. Specific reagents and equipment. |
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