Hair Follicle Stem Cell Isolation and Expansion   

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Original research article

A brief version of this protocol appeared in:
Stem Cells
Jan 2011


Stem cells are widely used for numerous clinical applications including limbal stem cell deficiency. Stem cell derived from the bulge region of the hair follicle have the ability to differentiate into a variety of cell types including interfollicular epidermis, hair follicle structures, sebaceous glands and corneal epithelial cells when provided the appropriate cues. Hair follicle stem cells are being studied as a valuable source of autologous stem cells to treat disease. The protocol described below details the isolation and expansion of these cells for eventual clinical application. We used a dual-reporter mouse model to visualize both isolation and eventual differentiation of these cells in a limbal stem cell-deficient mouse model.

Keywords: Holoclones, Clonal expansion, Hair follicle stem cells, Bulge, Stem cell isolation


Stem cells are widely used for a multitude of translational and clinical applications. One such clinical application is for the treatment of limbal stem cell deficiency (LSCD). LSCD occurs when there is dysfunction or loss of the limbal stem cell population, which is critical for maintaining a healthy ocular surface, due to congenital or acquired pathologies. The primary treatment strategy for LSCD is cultivating autologous epithelial cell sheets from a limbal biopsy of the patient’s healthy eye (Pellegrini et al., 1997; Shortt et al., 2007). The limitation of this strategy is that it is only applicable for patients that have unilateral LSCD. Those that have bilateral LSCD, must rely on an allogenic limbal biopsy from an immunologically related living donor or cadaveric tissue. Due to the need of systemic immunosuppressive therapy and the limited availability of donor tissue, the therapeutic success rate is decreased. Several research groups have been examining the use of cultivated oral mucosal cells for the treatment of LSCD and have achieved some success. However, these cells often fail to express the corneal epithelial differentiation marker, Keratin 12 (Inatomi et al., 2006) and often result in the development of peripheral neovascularization (Nakamura et al., 2004; Nishida et al., 2004; Ma et al., 2009). Due to these limitations, there was a need for an alternative source of autologous stem cells. Thus we focused on the use of hair follicle stem cells as they harbor multiple sources of stem cells that have been used in regenerative medicine (Cotsarelis et al., 1990; Purba et al., 2014). The hair follicle contains mesenchymal stem cells in the dermal papilla and connective tissue sheath, which can give rise to several cell lineages (Lako et al., 2002; Jahoda et al., 2003; Richardson et al., 2005). Additionally, the bulge region of the hair follicle contains stem cells, which can generate the interfollicular epidermis, hair follicle structures and sebaceous glands (Cotsarelis et al., 1990; Taylor et al., 2000; Cotsarelis, 2006). The hair follicle stem cells (HFSC) derived from the bulge region express of variety of cytokeratins including cytokeratin 15 (Krt15) (Tiede et al., 2007; Kloepper et al., 2008; Larouche et al., 2008), which has been successfully used for the purification and enrichment of HFSC (Blazejewska et al., 2009). HFSC have been successfully used in the treatment of a mouse model of LSCD (Meyer-Blazejewska et al., 2011) and research continues to focus on other therapeutic applications and the eventual translation to humans (Purba et al., 2014). Continued research efforts into these areas rely on a standard method for isolating and expanding the bugle-derived HFSC.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Call, M., Meyer, E. A., Kao, W. W., Kruse, F. E. and Schloetzer-Schredhardt, U. (2018). Hair Follicle Stem Cell Isolation and Expansion. Bio-protocol 8(10): e2848. DOI: 10.21769/BioProtoc.2848.

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