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Protocols for Cryopreservation of Intact Hair Follicle That Maintain Pluripotency of Nestin-Expressing Hair-Follicle-Associated Pluripotent (HAP) Stem Cells

  • Satoshi Kajiura
  • Sumiyuki MiiEmail author
  • Ryoichi Aki
  • Yuko Hamada
  • Nobuko Arakawa
  • Katsumasa Kawahara
  • Lingna Li
  • Kensei Katsuoka
  • Robert M. Hoffman
  • Yasuyuki Amoh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1453)

Abstract

Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair-follicle-associated pluripotent (HAP) stem cells. Cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells are described in this chapter. Intact hair follicles from green fluorescent protein (GFP) transgenic mice were cryopreserved by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in DMEM with fetal bovine serum (FBS). After 4 weeks culture, cells from the upper part of the hair follicles grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week culture, the growing cells formed hair spheres, each containing approximately 1 × 102 HAP stem cells. The hair spheres contained cells which could differentiate to neurons, glial cells, and other cell types. The formation of hair spheres by the thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. These results suggest that the cryopreservation of the whole hair follicle is an effective way to store HAP stem cells for personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.

Key words

Hair follicle HAP stem cell Nestin Pluripotent Hair spheres Cryopreservation 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Satoshi Kajiura
    • 1
  • Sumiyuki Mii
    • 1
    Email author
  • Ryoichi Aki
    • 1
  • Yuko Hamada
    • 1
  • Nobuko Arakawa
    • 1
  • Katsumasa Kawahara
    • 4
    • 5
  • Lingna Li
    • 2
  • Kensei Katsuoka
    • 1
  • Robert M. Hoffman
    • 2
    • 3
  • Yasuyuki Amoh
    • 1
    • 2
    • 3
  1. 1.Department of DermatologyKitasato University School of MedicineSagamiharaJapan
  2. 2.AntiCancer, Inc.San DiegoUSA
  3. 3.Department of SurgeryUniversity of California, San DiegoSan DiegoUSA
  4. 4.Department of PhysiologyKitasato University School of MedicineSagamiharaJapan
  5. 5.Department of Cellular and Molecular PhysiologyKitasato University Graduate School Medical ScienceSagamiharaJapan

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