Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

Summary

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Abstract

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

Introduction

Adult stem cells (SCs) are essential for maintaining tissue homeostasis by replacing dying cells and repairing damaged tissues upon injury. These SCs are defined by their ability to undergo continual self-renewal and to differentiate into various cell lineages ^1-3. The best studied systems, which are dependent upon adult SCs for their replenishment, include the hematopoietic system, the intestine and the skin ^1,2,4.

During embryogenesis, the skin begins as a single layer of epidermal cells. Morphogenesis of the hair follicle (HF) starts when mesenchymal cells populate the skin and form an underlying collagenous dermis ....

Protocol

This study was performed in strict accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of Israel's Ministry of Health. All of the animals were handled according to the approved institutional animal care protocol IL-02302-2015 of the Technion Israel Institute of Technology.

1. Experimental Preparation

1. Preparation of chelated Fetal Bovine Serum
   Note: Epithelial cells are very sensitive to calcium so it is crucial to control the exposure of these cells to calcium. In order to ensure that cells are not exposed to calcium during the isolation process chelation is used to rem....

Results

This protocol describes in detail the enrichment and isolation of two types of populations: bulge SCs and epidermal keratinocytes. Figure 2 illustrates the major steps of the protocol. Utilizing skin removed from the dorsal back of 8 week old mice, we enriched bulge SCs using the CD34 marker, which is only expressed in HFSCs; and Sca-1, which labels epidermal keratinocytes. Figure 3 shows different patterns of CD34 and Sca-1 expression within the α6^+.......

Discussion

The protocol described here is well established for isolating HFSCs from the dorsal skin of adult mice but can be equally applied for isolation of other populations within the HF structure, based on the selection of markers ^{2,16,23,28,29}. This method is notably advantageous over other methods of cell isolation, such as tissue dissociation, in that a specific cell type can be selected and harvested from a mixture of heterogeneous cell populations. Furthermore, the method described here is fast and reliable and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane	Primal Critical Care	66794-017-10
Carbon dioxide	-	-
Electro Shaver	Oster	Golden A5	Shaver from any other company could be used
70% ethanol	Gadot Lab	830000051	96% ehtanol diluted with distilled water
Dissection mat			Dissection tools from any provider can be used
Forceps	Dumont	11251-10	Foreceps from any other company could be used
Scissors	Dumont	14094-11	Scissors from any other company could be used
Needles/Pins	-	-
Scalpel	Albion	10	Ensure that the scalpel has a blunt end
Tissue culture dish 60 mm x 15 mm	Sigma-Aldrich	CLS430166
PBS	-		In house PBS without Calcium and Magnesium
0.25% Trypsin/EDTA	Biological Industries	03-050-1A	Trypsin obtained from a different company might have a different activity and duration of the trypsin digest has to be adjusted accordingly
Pipettes 10 ml	Sigma-Aldrich	Corning, 4488
Ice	-	-
50 ml sterile centrifuge tubes	Minplast Ein-shemer	35050-43
70 µm Cell strainer	Fisher	22362548
40 µm Cell strainer	Fisher	22362549
Staining buffer	-
Centrifuge	Eppendorf 5804 R	5805 000.017
FACS tubes with Cell strainer caps	Falcon	352235
FACS tubes	Falcon	352063
Integrin β1	eBioscience	25-0291	1:400
Integrin α6	eBioscience	15-0495	1:600
Sca I	eBioscience	11-5981	1:200
CD34	eBioscience	9011-0349	1:300
DAPI	Sigma-Aldrich	D9542	50 ng/ml
Dry Chelex	BioRad	142-2842
Beaker	Pyrex	-
Distilled H2O	-	-
Stir bar	-	-
NHCl	BioLab	1903059
Fetal bovine serum (FBS)	Beit Haemek Biological Industries	400718	FBS obtained from a different company can be used
1 L glass bottle	Ilmabor	Boro 3.3
Bottle top filter	Autofil	1102-RLS