Effects of different culture systems on proliferation and differentiation of epidermal stem cells in rats

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Author:
LI Xiao-jian(Department of Burns,Guangzhou Red Cross Hospital, Jinan University, Guangzhou 510220, China)
SHEN Yan()
LI Yan-cang()
ZENG Yao-ying()
DAI LI-bing()
YANG Xiao-hong()
Journal Title:
Chinese Journal of Biomedical Engineering
Issue:
Volume 18, Issue 01, 2012
DOI:
10.3760/cma.j.issn.1674-1927.2012.01.003
Key Word:
Epidermal stem cells;Chitin;Cells,cultured;Cell differentiation;Cell proliferation

Abstract: Objective To explore the effects of different culture systems on proliferation and differentiation of epidermal stem cells and to establish an optimal culture system which can regulate and control the proliferation and differentiation of epidermal stem cells.Methods The rat epidermal stem cells obtained by enzyme digestion and type Ⅳ collagen rapid adherence were cultured in different culture systems (culture on dish,co-culture with biological chitin scaffold material,and culture with chitin membrane as the carrier in nude mice).The growth of epidermal stem cells was then observed.After 4 weeks,the colonyformation rates of epidermal stem cells were compared among two systems (co-culture with biological chitin scaffold material and culture on dish).Immunohistochemistry was used to study the proliferation and differentiation of the epidermal stem cells with chitin membrane as a carrier in nude mice at 4 weeks after implantation.Results In routine culture dish,the epidermal stem cells began to proliferate and clone at around day 3 and fused into patches at around day 12.However,the proliferation gradually decreased and the time to fusing into patches gradually became longer after passage,until terminal differentiation and loss of proliferation after passages 3 to 4.In co-culture with biological chitin scaffold materials,the epidermal stem cells grew in a chessboard-like colony after 2 weeks,and a great number of colonies could be seen on the biological chitin scaffold materials,with plenty of proliferating cells adhering to the colonies.Under scanning electronic microscope (SEM),the biological chitin scaffold materials were found to mainly consist of fibers (10 μm in diameter) arranged in two crisscrossing layers with massive colonies of epidermal stem cells between the X-shaped holes.The epidermal stem cells cultured with biological chitin scaffold materials had a significantly higher colony forming efficiency as compared with that in culture dish after 4 weeks [ (12.6±2.7)% vs (5.7± 1.1)%,P<0.05 ].The epidermal stem cells implanted into the nude mice showed vigorous proliferation with formation of “epidermal nests” at 4 weeks after implantation.Structure of skin appendages could be seen around the nests of epidermal stem cells.Conclusions Epidermal stem cells can proliferate in the culture dish in vitro but remain proliferating only for a relatively short time.Co-culture with biological chitin scaffold materials may allow for longer-lasting proliferation of the epidermal stem cells.Massive proliferation can be found in the epidermal stem cells implanted into nude mice.

  • [1]Rheinwald JG,Green H.Serial cultivation of strains of human epidermal keratinocytes:the formation of keratinizing colonies from single cells.Cell,1975,6:331-343.
  • [2]Rheinwald JG,Green H.Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.Cell,1975,6:317-330.
  • [3]Boyce ST,Ham RG.Cultivation,frozen storage,and clonal growth of normal human epidermal keratinocytes in serum-free media.J Tissue Cult Methods,1985,9:83-93.
  • [4]Boyce ST,Hansbrough JF.Biologic attachment,growth,and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate.Surgery,1988,103:421-431.
  • [5]Lam PK,Chan ES,Yen RS,et al.A new system for the cultivation of keratinocytes on acellular human dermis with the use of fibrin glue and 3T3 feeder cells.J Burn Care Rehabil,2000,21:1-4.
  • [6]Papini S,Cecchetti D,Campani D,et al.Isolation and clonal analysis of human epidermal keratinocyte stem cells in long-term culture.Stem cells,2003,21:481-494.
  • [7]Park KE,Jung SY,Lee SJ,et al.Biomimetic nanofibrous scaffolds:preparation and characterization of chitin/silk fibroin blend nanofibers.Int J Biol Macromol,2006,38:165-173.
  • [8]Sun TT,Cotsarelis G,Lavker RM.Hair follicular stem cells:the bulge-activation hypothesis.J Invest Dermatol,1991,96:77S-78S.
  • [9]Liang L,Bickenbach JR.Somatic epidermal stem cells can produce multiple cell lineages during development.Stem Cells,2002,20:21-31.
  • [10]Lin Y,Li H,Huang J,et al.Following the fate of murine epidermal stem cells in a syngeneic dermal equivalent in vivo.Burns,2005,31:1007-1012.
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