Highly Efficient Neural Differentiation of CD34-Positive Hair-Follicle-Associated Pluripotent Stem Cells Induced by Retinoic Acid and Serum-Free Medium

  • Mohsen Sagha
  • Nowruz NajafzadehEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1453)


Neural differentiation of hair-follicle-associated pluripotent (HAP) stem cells residing in the bulge area is a promising autologous source for stem cell therapy. In the present chapter, we describe the identification and enrichment of CD34+ HAP stem cells by magnetic-activated cell sorting (MACS), and induce them to differentiate into neuronal and glial cells using defined neural-induction media. The different neural cell populations arising during in vitro differentiation from HAP stem cells are characterized by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry assay.

Key words

Hair follicle Stem cells Neural differentiation Magnetic-activated cell sorting CD34+ hair-follicle-associated-pluripotent (HAP) stem cell 



This work was supported by grants from the Ardabil University of Medical Sciences.


  1. 1.
    Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61(7):1329–1337CrossRefPubMedGoogle Scholar
  2. 2.
    Cotsarelis G (2006) Epithelial stem cells: a folliculocentric view. J Invest Dermatol 126:1459–1468CrossRefPubMedGoogle Scholar
  3. 3.
    Blanpain C, Lowry WE, Geoghegan A et al (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118:635–648CrossRefPubMedGoogle Scholar
  4. 4.
    Lenoir MC, Bernard BA, Pautrat G et al (1988) Outer root sheath cells of human hair follicle is able to regenerate a fully differentiated epidermis in vitro. Dev Biol 130:610–620CrossRefPubMedGoogle Scholar
  5. 5.
    Morris RJ, Liu Y, Marles L et al (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417CrossRefPubMedGoogle Scholar
  6. 6.
    Li L, Mignone J, Yang M, Matic M, Penman S, Enikolopov G, Hoffman RM (2003) Nestin expression in hair follicle sheath progenitor cells. Proc Natl Acad Sci U S A 100:9958–9961CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hoffman RM (2014) Nestin-expressing hair follicle-accessible-pluripotent (HAP) stem cells for nerve and spinal cord repair. Cells Tissues Organs 200:42–47CrossRefPubMedGoogle Scholar
  8. 8.
    Amoh Y, Li L, Katsuoka K et al (2009) Multipotent nestin-expressing hair follicle stem cells. J Dermatol 36:1–9CrossRefPubMedGoogle Scholar
  9. 9.
    Najafzadeh N, Nobakht M, Pourheydar B et al (2013) Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury. Neural Regen Res 8:3365PubMedPubMedCentralGoogle Scholar
  10. 10.
    Sieber-Blum M, Grim M (2004) The adult hair follicle: cradle for pluripotent neural crest stem cells. Birth Defects Res C Embryo Today 72:162–172CrossRefPubMedGoogle Scholar
  11. 11.
    Amoh Y, Li L, Yang M et al (2004) Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells. Proc Natl Acad Sci U S A 101:13291–13295CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Füllgrabe A, Joost S, Are A et al (2015) Dynamics of Lgr6+ progenitor cells in the hair follicle, sebaceous gland, and interfollicular epidermis. Stem Cell Reports 5:843–855CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Najafzadeh N, Sagha M, Tajaddod SH et al (2014) In vitro neural differentiation of CD34+ stem cell populations in hair follicles by three different neural induction protocols. In Vitro Cell Dev Biol Anim 51:192–203CrossRefPubMedGoogle Scholar
  14. 14.
    Cotsarelis G (2006) Gene expression profiling gets to the root of human hair follicle stem cells. J Clin Invest 116:19–22CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Sieber-Blum M, Grim M, Hu YF et al (2004) Pluripotent neural crest stem cells in the adult hair follicle. Dev Dyn 231:258–269CrossRefPubMedGoogle Scholar
  16. 16.
    Amoh Y, Li L, Katsuoka K et al (2005) Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc Natl Acad Sci U S A 102:5530–5534CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Nobakht M, Najafzadeh N, Safari M et al (2009) Bulge cells of rat hair follicles: isolation, cultivation, morphological and biological features Isolation. Yakhteh Med J 12:51–58Google Scholar
  18. 18.
    Aebi S, Kroning R, Cenni B et al (1997) All-trans retinoic acid enhances cisplatin-induced apoptosis in human ovarian adenocarcinoma and in squamous head and neck cancer cells. Clin Cancer Res 3:2033–2038PubMedGoogle Scholar
  19. 19.
    El Seady R, Huisman MA, Löwik CW et al (2008) Uncomplicated differentiation of stem cells into bipolar neurons and myelinating glia. Biochem Biophys Res Commun 376:358–362CrossRefPubMedGoogle Scholar
  20. 20.
    Amoh Y, Mii S, Aki R et al (2012) Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper, middle and lower part of the vibrissa hair follicle. Cell Cycle 11:3513–3517CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Guan K, Chang H, Rolletschek A et al (2001) Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res 305:171–176CrossRefPubMedGoogle Scholar
  22. 22.
    Schulz TC, Noggle SA, Palmarini GM et al (2004) Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells 22:1218–1238CrossRefPubMedGoogle Scholar
  23. 23.
    Woodbury D, Reynolds K, Black IB (2002) Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 69:908–917CrossRefPubMedGoogle Scholar
  24. 24.
    Liu F, Zhang C, Hoffman RM (2014) Nestin-expressing stem cells from the hair follicle can differentiate into motor neurons and reduce muscle atrophy after transplantation to injured nerves. Tissue Eng 20:656–662Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Research Laboratory for Embryology and Stem Cells, Department of Anatomical Sciences and PathologySchool of Medicine, Ardabil University of Medical SciencesArdabilIran

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