JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Published: May 3rd, 2013

DOI:

10.3791/50185

1Department of Dermatology and Institute for Medical Engineering, Technische Universität München, 2Department of Dermatology and Allergology, Technische Universität München

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

  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 populati.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

  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: an update. Curr. Opin. Genet. Dev. 16, 518 (2006).
  3. Blanpain, C., Horsley, V., Fuchs, E. Epithelial stem cells: turning over new leaves. Cell. 128, 445 (2007).
  4. Hunt, D. P., Jahoda, C., Chandran, S. Multipotent skin-derived precursors: from biology to clinical translation. Vurr. Opin. Biotechnol. 20, 522 (2009).
  5. Toma, J. G., et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol. 3, 778 (2001).
  6. Fernandes, K. J. L., et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat. Cell Biol. 6, 1082 (2004).
  7. Wenzel, V., et al. Naïve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo. Bio. Open. 1, (2012).
  8. Livak, K. J., Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25, 402 (2001).
  9. Biernaskie, J. A., McKenzie, I. A., Toma, J. T., Miller, F. D. Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nature Protocol. 1, 2803 (2006).
  10. Toma, J. G., McKenzie, I., Bagli, D., Miller, F. D. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells. 23, 727 (2005).
  11. Fernandes, K. J., et al. Analysis of the neurogenic potential of multipotent skin-derived precursors. Electrophoresis. 201, 32 (2006).
  12. Hill, l. K. L., et al. Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function. Exp. Hematol. 38, 246 (2010).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved