Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Summary

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Abstract

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells¹. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells², melanocyte stem cells³ and neural crest like stem cells^4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles^4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury⁸. Furthermore, peripheral nerves have been repaired with stem cell grafts⁹, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination¹⁰. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.

Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

Protocol

1. Preparation of Tissue Culture Plates

1. Coat each well with enough Poly-D-Lysine (PDL) to cover the bottom of the well. Allow the plates to dry in the hood.
2. After the wells are dry, rinse with sterile water, and aspirate. Allow the plates to dry in the hood.
3. When dry, coat with fibronectin (that was dissolved in biowhittaker water overnight at 37 °C at a concentration of 1 mg in 6 ml).
4. Add NCSC medium [95 ml DMEM/F12, 1 ml Penn/Strep (P/S), 1 ml N2, 2 ml B27, 100 μl mercaptoethanol (2ME; 50 mM stock), bFGF (20 ng/ml medium), IGF-1 (20 ng/ml of medium) and EGF(20 ng/ml of medium)] before fibronectin dry in the plates,

Discussion

The cell isolation and culture methods described are reproducible and robust. We have generated NCSCs from dozens of individuals across a broad age range. Although it is best to process the tissue right after tissue harvest, we found that scalp tissues can be safely stored in media on ice for overnight transportation with minimal impact on cell viability.

It is important to treat the scalp tissue with antibiotics and use aseptic technique during hair follicle isolation to avoid potential micro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	11965-092
DMEM/F12	Invitrogen	11330-32
Heat-inactivated FBS	Hyclone	SH30071.03
B27 supplement	Invitrogen	17504044
N2 supplement	Invitrogen	17502048
bFGF	Invitrogen	PHG0026
EGF	R&D system	236-EG-01M
IGF-I	R&D system	291-G1-050
0.05% Trypsin/EDTA	Invitrogen	25300-054
Dispase	Invitrogen	17105041
Penicillin-Streptomycin	Invitrogen	15070063
Table 1.