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In This Article

  • Summary
  • Abstract
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Protocol

1. SKP Isolation from Primary Fibroblast Cultures

  1. Fibroblast cultures either from cell banks or directly obtained from skin biopsies are maintained in culture in fibroblast growth medium DMEM containing 15% fetal calf serum, 2 mM glutamine, 10 mg/ml penicillin, and 10 mg/ml streptomycin.

Human fibroblasts GMO3349C and GMO8398A were obtained from the Coriell Institute for Medical Research (Camden, NJ) and were used in this study.

  1. Cultures from population doublings (PPDs) 20 to 35 were used for SKP cultures at a confluency of 80%. One 10 cm tissue culture dish (BD Falcon) contains approximately 1.5x1....

Representative Results

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.......

Discussion

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.......

Disclosures

We have nothing to disclose.

Acknowledgements

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.).

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Materials

NameCompanyCatalog NumberComments
Name of the reagentCompanyCatalogue numberComments (optional)
DMEM high glucoseInvitrogen31966-047 
DMEM low glucoseInvitrogen21885-108 
fetal bovine serumInvitrogen10270-106 
L-glutamineInvitrogen25030-024Final conc.: 200 mM
Penicillin/ StreptomycinInvitrogen15140-122Final conc.: 10 mg/ml /10 mg /ml
trypsin solution (0.25%)Invitrogen25200-056 
F-12 Nutrient Mixture (Ham)Invitrogen21765-029 
FGF2BD Biosciences4114-TC-01MFinal conc.: 40 ng/ml
EGFBD Biosciences236-EG-200Final conc.: 20 ng/ml
PDGFBBInvitrogenPHG0043Final conc.: 5 ng/ml
TGF-b1InvitrogenPHG9204Final conc.: 2.5 ng/ml
25 cm2 flaskOmnilabFALC353109 
PBS w/o CaMgInvitrogen14190-169 
B27Invitrogen17504-044 
MethanolRoth8388.2 
Vectashield mounting mediumVector Inc.H-1200 
RNeasy MinikitQiagen, Valencia, CA74104 
Omniscript Reverse TranscriptaseQiagen205113 
SsoFast EvaGreen SupermixBioRad172-5201 
FungizoneInvitrogen15290-018Final conc.:1 mg/ml
   Table 2. Specific reagents and equipment.

References

  1. Jahoda, C. A., Whitehouse, J., Reynolds, A. J., Hole, N. Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. Exp. Dermatol. 12, 849 (2003).
  2. Watt, F. M., Celso, L. o., C, V., Silva-Vargas, Epidermal stem cells:....

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Adult Stem CellsSKPsSkin derived PrecursorsPrimary Fibroblast CulturesMultipotent Precursor CellsDermal PrecursorsNestinVimentinFibronectinStem Cell MarkersTissue BanksGenetic DiseasesDisease Modeling

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