Isolation and characterization of cutaneous epithelial stem cells

SUMMARY

During homeostasis, adult mammalian skin turnover is maintained by a number of multipotent and unipotent epithelial progenitors located either in the epidermis, hair follicle or sebaceous gland. Recent work has illustrated that these various progenitor populations reside in regionalized niches and are phenotypically distinct from one another. This degree of heterogeneity within the progenitor cell landscape in the cutaneous epithelium complicates our ability to target, purify and manipulate cutaneous epithelial stem cell subpopulations in adult skin. The techniques outlined in this chapter describe basic procedures for the isolation and purification of murine epithelial progenitors and assessing their capacity for ex vivo propagation.

1. Introduction

The perpetual renewal of mammalian skin is known to be maintained by permanently residing stem cells that are able to sustain three principal differentiated lineages: the interfollicular epidermis (IFE), sebaceous gland (SG) and hair follicle (HF) (1, 2). In addition, recent studies identified Merkel cell mechanoreceptors residing in specialized epithelial structures termed touch domes in the hairy skin as a fourth lineage maintained by keratinocyte progenitors (3). While it has long been accepted that skin homeostasis is dependent on the ability of stem cells to replenish the turnover of these mature epithelial lineages, it is the work over the last decade that has significantly enhanced our understanding the location and function of multiple stem or progenitor niches in the skin. These findings have dramatically changed our view of the cutaneous epithelial stem cell landscape rendering a highly compartmentalized epithelium maintained by multiple classes of phenotypically distinct regional niches (2).

In some cases progenitor niches have been labeled using mouse genetics approaches and characterized under normal conditions to be long-lived and able to sustain the cellular input to certain epithelial structures including the interfollicular epidermis (4, 5), sebaceous gland (6, 7) and hair follicle (8-11). In other cases, antibodies against cell surface proteins have been utilized to mark and isolate epithelial progenitors located in the IFE (3, 5, 12) and HF (13-16). These efforts have facilitated our understanding of the relative proliferative capacity of progenitor pools as well as their capacity to regenerate IFE, HF, SG or Merkel lineages in surrogate assays. Collectively, these studies have illustrated the role of epithelial progenitors during skin homeostasis as well as their ability to respond to skin insult.

As new biomarkers have been implemented to better define the profile of progenitor cell subsets in the IFE and HFs, the individual cell of interest becomes less frequent. This can be a major technical challenge to functional studies such as skin and hair reconstitution and clonogenic studies where a significant number of cells may be required. In this chapter, we will outline some basic methods for isolation and functional assessment of keratinocyte clonogenicity, multipotency and self-renewal capabilities from freshly isolated single cell suspensions of murine epidermal keratinocytes that have been subjected to FACS sorting. In particular, we will focus on clonogenic and skin and hair reconstitution assays. Methodologies to establish cultures of epidermal keratinocytes at clonal densities have been established for more that 3 decades and were developed by Rheinwald and Green (17). While there have been many modifications added this method over the years (18), we observe the highest success rates when maintaining Rheinwald and Green's principle of using a feeder layer of mitotically-arrested mouse 3T3 fibroblasts. The development of the hair reconstitution assay (19, 20) revealed the shortcomings of in vitro assays, which typically do not account for stem cell potency. Importantly, we feel the ability to conduct skin and hair reconstitution assays from freshly isolated FACS-sorted keratinocyte subsets provides a robust platform to identify and distinguish unipotent, bipotent and multipotent epithelial progenitors.

2. Materials

2.1. Skin cell isolation solutions

1. 0.25% trypsin/1 mM EDTA stock solution (Invitrogen).

2. 1X PBS, pH = 7.6 (Invitrogen), sterilized.

3. Fibroblast growth medium: DMEM (Invitrogen) supplemented with 10% Donor Bovine Serum (Invitrogen) and 2% Penicillin-Streptomycin (Invitrogen).

4. Collagenase Type I (Worthington Biochemical), 10 mg/ml stock solution in PBS.

5. DNAse I (Worthington Biochemical), 20,000 units/ml stock solution in PBS.

6. 70 μm cell strainer (Fisher Scientific).

7. Hank's Balanced Salt Solution (HBSS) (Invitrogen).

8. Betadine 1% solution in water.

9. 70% EtOH solution.

2.2. Antibodies

1. α6 integrin (CD49f, BD Biosciences) (see Note 1).

2. Sca-1 (Ly6G, BD Biosciences)

3. CD34 (RAM 34, BD Biosciences)

4. CD200 (OX-2, BD Biosciences)

2.3. Clonogenic assay

1. Complete FAD growth medium: 3 parts DMEM (Invitrogen), 1 part Ham's F12 Supplement (Invitrogen), 10% Defined Fetal Bovine Serum (HyClone), 10 ng/ml EGF (Peprotech), 0.5 mg/ml hydrocortisone (Sigma-Aldrich), 10⁻¹⁰ M cholera enterotoxin (Sigma-Aldrich), 5 mg/ml insulin (Sigma-Aldrich), 1.8 × 10⁻⁴ M adenine (Sigma-Aldrich), 100 U/ml penicillin (Invitrogen) and 100 mg/ml streptomycin (Invitrogen).

2. Cnt-57 serum free medium (CELLnTEC).

3. 3T3 fibroblasts (ATCC) mitotically arrested with either Mitomycin c (Sigma) or γ-radiation.

4. 0.25% trypsin/1 mM EDTA stock solution (Invitrogen).

5. Nunclon 6-well dishes (Fisher Scientific).

6. 0.5 mM EDTA (Fisher Scientific), a.k.a. Versene.

7. Rhodamine B, 1% solution in H₂O (Sigma).

2.4. Skin reconstitution assay

1. Silicon culture chambers- Upper F2U #30-268; Lower F2L #30-269 (Renner Gmbh).

2. Surgical instruments including forceps, curved scissor, stapler and staple remover (all from Temin); sterile drapes, alcohol swabs, and anesthetics.

3. Immunodeficient mice Nude mice (NCR nude, male, 7-9 weeks old, supplied by Taconic or we prefer NSWNU-M (homozygote females) (see Note 2).

4. Small heating pad.

3. Methods

3.1. Epidermal keratinocyte isolation

1. Under a biological cabinet, submerge euthanized eight-week old mice in 1% Betadine for 2 min and wash in sterile H₂O. Submerge mice in 70% EtOH for 1 min and wash in sterile H₂O.

2. Under a biological cabinet, surgically excise the dorsal skin from using sterile forceps and scissors and float skin dermis side up in sterile PBS in a sterile Petri dish. Scrape away the subcutaneous fat and muscle using a sterile scalpel and forceps using multiple dishes with new PBS as necessary (see Note 3).

3. Float skins epidermis side up in 0.25% trypsin/1 mM EDTA in a 10 cm Petri dish for 1.5 – 2 hours (see Note 4).

4. Aspirate trypsin and recover skins in 10 ml fibroblast growth medium. Gently detach epidermis from dermis using a scalpel and mince epidermal scrapings into small pieces.

5. Transfer scrapings into a sterile 100 ml bottle and add 30 ml fibroblast growth medium and a stir bar. Recover epidermal cells with gentle stirring for 30 min and filter cells through a 70 μm cell strainer into a 50 ml Falcon tube. Spin cells for 10 min @ 1000 rpm. Wash cells in 10 ml PBS, spin and resuspend cells in 10 ml fibroblast growth medium and spin for 10 min @ 1000 rpm.

6. For antibody labeling (see below), resuspend cell pellets at 5 - 10 × 10⁶ cells/ml in fibroblast growth medium. Typically 10 - 12 × 10⁶ viable basal keratinocytes are harvested from a single dorsal skin.

3.2. Preparation of highly inductive dermal fibroblasts

1. Surgically excise the dorsal skin from euthanized postnatal day 1-2 mice using sterile forceps and scissors (see Note 5).

2. In a dry Petri dish lay skins flat dermis side down with no folded edges. Slowly pour in ice-cold 0.25% trypsin/1 mM EDTA and avoid getting the tops of the skins wet. Incubate skins overnight at 4°C.

3. Remove skins, one at a time, from trypsin and place on dry p150 plate, dermis side up. Flatten it again and use fine forceps to separate epidermis from dermis, starting at one edge of skin and flipping the dermis up and off the epidermis, which should stay on the plate.

4. Transfer dermis one at a time to a plate containing 10 ml of media on ice.

5. For 8 dermises, use 0.5 mL collagenase stock solution (10 mg/mL in H₂O) plus 12 mL HBSS in a 50-100 ml sterile beaker. Transfer dermises into the beaker and mince into small pieces using sharp scissors.

6. Transfer to a 250 ml flask with a magnetic stir bar. Stir @ 37^oC for 30 min (Optional: For the last 5 min add 20 μl of DNAse I (stock at 20000 U/ml in PBS)

7. Dilute 3 - 4 fold with media and filter through sterile gauze or 70 μm filter. Rinse the flask with 5 ml media and pass through the filter.

8. Spin the cells at 1500 rpm for 5 min at 4C

9. Resuspend and wash the pellet once in HBSS

10. Count cells, use @ 8 × 10⁶ fresh dermal cells per graft, cryopreserve or plate at 1 × 10⁶ cells per 10 cm dish for later use.

3.2. Antibody labeling and FACS analysis

1. Select an appropriate panel of antibodies for the target cells of interest (Please see Table 1 for examples of published progenitor marker profiles). When possible, select antibodies directly conjugated to fluorescent dyes.

Table 1.

Markers of epithelial progenitor cells in adult mouse skin

Progenitor location	Markers	References
IFE	Lrig1+	5
IFE	α6brightCD71dim	12
IFE & HF Infundibulum	α6+CD34−Sca-1 +	16
IFE Touch Dome	α6+CD34−Sca-1+CD200+	3
Junctional zone	Lrig1 +	5
HF Isthmus & Infundibulum	α6+MTS24(Plet-1)+	15
Sebaceous duct	Blimp1 +	7
HF Isthmus	α6lowCD34−Sca-1−	16
	Lgr6+	11
HF Bulge	α6dim+brightCD34+	13, 14
	Krt15+	21
Lower HF bulge & hair germ	Lgr5+	10
Hair germ	P-cadherin+	22

2. Count cells and aliquot equal amounts into experimental and control (single-stained and unstained tubes) in complete growth medium.

3. Incubate antibodies at concentrations according to manufacturer guidelines for 30 min to 1 hour in complete growth medium on ice (see Note 9). Spin and wash cells, resuspend in growth medium supplemented with DAPI or an alternative nuclear stain.

4. Conduct sorting (see Note 10). Typically, 10 – 20 × 10⁶ viable α6⁺ basal keratinocytes can be sorted from a single dorsal skin.

3.3. Clonogenic analysis

Clonogenic assays typically require fewer cells compared to skin and hair reconstitution assays, and harvested cells from a single animal will usually suffice. Cells can be grown with a confluent layer of mitotically arrested 3T3 fibroblasts in complete FAD growth medium or defined serum free medium in the absence of fibroblasts, although the clonogenic efficiency is generally lower. Serum free medium can also be used in control experiments to facilitate assays that quantify the number of adherent cells shortly after plating (12 - 16 hours).

1. Preparing the 6-well plates one day in advance will allow the fibroblasts to fully attach, spread and condition the growth medium. Plate 1 × 10⁶ mitotically arrested 3T3 fibroblasts in 3 ml complete FAD growth medium per well.

2. The next day harvest mouse keratinocytes from a single mouse dorsal skin and FACS sort desired keratinocyte subpopulations as described above. The FACS instrument can be optimized towards purity and accuracy in counting since cell numbers will be in excess. Propidium iodide or DAPI should be used to exclude dead cells. Many of the dead cells are post-mitotic suprabasal cells that are sensitive to the 70% ethanol washes during cell harvesting. Use either the FACSAria Automated Cell Deposition Unit (ACDU) function or manually seed 1 × 10³ (a range of 0.5 to 2 ×10³ cells can be used) sorted keratinocytes per well. 3. Incubate cells for 2 weeks at 32°C in a humidified incubator with 5% CO₂ and change the medium every 48 hours.

4. After 2 weeks, aspirate off the culture medium and replace with 3 ml Versene per well. After 1-2 min at room temperature, detach the feeder layer by repeated pipetting of Versene over the plate (keratinocytes will not detach). Gently wash plates 2 times with PBS (take care not to detach colonies).

5. Stain wells with Rhodamine B for 1 hour (see Note 6) at room temperature (just enough Rhodamine solution to cover the cells is sufficient). Aspirate off Rhodamine solution and gently wash wells 3 times with PBS (take care not to detach colonies).

6. Aspirate off the final wash and allow wells to completely dry by turning plates upside down. Afterwards, plates can be imaged and colonies may be manually counted. Typically the total number of colonies as well as the number of colonies greater than 4 mm in diameter are counted and compared between keratinocyte subpopulations.

3.4. Skin and hair reconstitution assay

1. Clip hair with electric clippers, if necessary, and clean the dorsal skin with 1% Betadine and place anesthetized mice on heating pad.

2. Use scissors to make a small incision on the back of the mouse (approximately 1 cm in diameter). Better areas for chamber placement are interscapular or suprapelvic. Do not make incisions directly on the spinal protrusion.

3. Assemble the upper and lower grafting chambers together and insert through the incision so that the rims of the chamber are under skin (see Note 7).

4. Secure the chamber to the skin with surgical stapler clips (two staples is usually enough).

5. Allow the chamber to adhere to the dorsal surface overnight prior to implanting cells.

6. Mix the desired number of epidermal cells and 2 × 10⁶ dermal cells together as a slurry in HBSS. We have successfully implanted 1 × 10⁵ to 6 x 10⁶ epidermal cells per graft. Spin cells at 1000 rpm for 5 min, resuspend the pellet in 100 μl HBSS and store on ice until use.

7. Gently mix cell suspensions before pipetting entire aliquot into chamber of hat, through the hole on top.

8. Replace each mouse in individual cages (on belly and away from the spout of the water bottle).

9. After one week (see Note 8), anesthetize mice and remove staples and gently tug on chamber to release it from mouse's back. Use tweezers to loosen skin around edge of chamber. Grafted area may be moist and oozy, leave it be and replace mouse in cage, as before.

10. Chambers are retained, cleaned (soak overnight in soapy water) and autoclaved for reuse.

11. Grafts are usually biopsied at 5 - 10 weeks post-grafting. Hair usually appears after approximately 2 - 3 weeks.