Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

1. Prepare 0 to 2 days Old Newborn Mice for Skin Dissection

1. Euthanize mouse pups using a CO₂ chamber for at least 20 min. Leave pups on ice up to an hour until dissection. P0-P2 mice are highly recommended as cells prepared from older mice have reduced progenitor capacity that results in lower graft yields. Prepare one dish of 70% ethanol (for step 1.3) and three dishes of wash solution (for step 3) when ready to perform skin dissection. Thaw Dispase Solution and leave it at 4 °C. Cervical dislocate pups after CO₂ overexposure to ensure euthanasia.
2. Place euthanized pups in a culture dish on ice and transfer to a sterile flow hood.
3. Wash pups by briefly immersing in 70% ethanol and place onto sterile culture dish.

2. Dissect Mouse Skin

1. Cut off each limb and tail at the base of the torso with sterile scissors.
2. Grasp the body firmly between a pair of curved forceps and make an incision along the dorsal skin from head to tail using a scalpel without penetrating the underlying fascia.
3. Carefully peel the skin away from the midline of the mouse.
4. Grasp the exposed mouse firmly with the long side of the curved forceps and insert another pair of curved forceps underneath the skin at the posterior end of the mouse and gently pull skin over hips toward the ventral half of the mouse.
5. Carefully peel skin completely off the mouse in one smooth motion and discard the carcass.

3. Wash Skin and Incubate with Dispase Solution

1. Place the skin in the first dish of wash solution with dermis-side down. Spread skin out and agitate with forceps. Leave skin in the first dish of wash solution while dissecting the next skin.
2. After placing the next dissected skin in the first dish of wash solution, transfer the prior skin to the second dish of wash solution. Keep skins in the second wash solution until all skins are collected. Transfer all of the skins to the final wash, agitate briefly.
3. Add 10 ml ice cold Dispase to a sterile 10 cm culture dish. Transfer skin to the Dispase Solution and flatten skin dermis-side down.
4. Float skin for 8-16 hr at 4 °C (alternative option: 1 hr at 37 °C).

4. Separate Dermis and Epidermis

1. Transfer skin to a new sterile culture dish and flatten skin dermis-side down. Carefully hold down the dermis from one corner with a scalpel.
2. Grasp the epidermis with forceps and slowly peel away from the dermis. Epidermis should peel away as one piece. The epidermis will be white and thin and the dermis should be brownish, thicker, and gelatinous.
3. Separate the two tissues into separate sterile culture dishes.
   1. To make a dermal specific gene knockdown/overexpression graft, take dermis and mince into very small pieces with two scalpels. Discard epidermis. On day of infection (step 7), dissect another set of mouse skins to prepare fresh, untreated epidermal cells to combine with lentiviral-infected dermal cells.
   2. To make an epidermal specific gene knockdown/overexpression graft, slice each epidermis into 6 – 8 smaller pieces. Discard dermis. On day of infection (step 7), dissect another set of mouse skins to prepare fresh, untreated dermal cells to combine with lentiviral-infected epidermal cells.

5. Dissociate Primary Mouse Dermal Cells (mDCs) from Minced Tissue

1. Dissociate mDCs by incubating dermal pieces with freshly made Collagenase Solution at 37 °C for 1 hr. Use 10 ml of Collagenase Solution per mouse pup.
2. Gently mix solution containing dermis every 10 min. Check the extent of dissociation under a microscope; the digested cell suspension should be mostly single cells. The solution should change from clear to cloudy as dermal cells are dissociated from the dermis.
3. Add FBS to Collagenase Solution containing dermis to a 10% final volume to reduce the activity of collagenase.
4. Pass the cell suspension through a 70 μm cell strainer into a new 50 ml tube to remove large undigested clumps and whole follicles. Add 10 ml of DMEM/10%FBS per 30 ml of the flow-through to further dilute the collagenase.
5. Centrifuge tubes containing digested dermis at 30 x g on a tabletop centrifuge for 5 min to further remove whole follicles.
6. Carefully transfer the supernatant to a new 50 ml tube. Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.
7. Resuspend cells with DMEM/10%FBS (1 ml per pup) and count cells. Plate cells with Amniomax C-100 media for infection (step 7), or to combine with lentivirus-infected KC for grafting (step 9). Typical yield is 20 x 10⁶ cells per pup.

6. Dissociate Primary Keratinocytes (KC) from Minced Tissue

1. Dissociate KC by incubating epidermal pieces with freshly thawed 0.05% Trypsin-EDTA at 37 °C for 15 min. Use 7 ml of Trypsin-EDTA per mouse pup.
2. Gently swirl solution containing epidermis every 5 min.
3. Add equal volume of Neutralizing Solution to Trypsin-EDTA containing epidermis to reduce the activity of trypsin.
4. Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min. Remaining undigested tissue should float on top.
5. Carefully pour off most of the supernatant with undigested tissue. Remove remaining supernatant with a 10 ml pipette without disturbing cell pellet. Resuspend cell pellet with PBS. In case KCs are not pelleted completely, pour off most of the supernatant and repeat centrifugation until most of the cells are pelleted.
6. Strain cell suspension through 70 μm cell strainer into a new tube to remove remaining tissue or clumps.
7. Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.
8. Resuspend cells in CnT-07 media or PBS (1 ml per pup) and count cells. Use CnT-07 for plating cells for infection (step 7), use PBS to combine with lentivirus-infected mDCs for grafting (step 9). Typical yield is 3-10 x 10⁶ cells per pup.

7. Infect Primary Cells with Lentivirus Containing shRNA or cDNA

On day of infection dissect another set of mouse skins to prepare fresh, untreated primary KCs or mDCs to combine with virus-infected cells on day of grafting (step 9).

1. 1. For mDCs, plate 4 x 10⁶ mDCs per 10-cm plate with AmnioMAX-C100 media and incubate at 37 °C + 5% CO₂ for next day infection at 50% cell density. Usually 3-4 plates of mDCs are used to generate one graft, and a total of 10-12 plates are needed to generate three grafts in one experiment. Alternatively, let mDCs attach and spread for a minimum of 2 hr at 37 °C + 5% CO₂ and perform infection the same day of cell plating. mDCs will have fibroblast-like morphology.
   2. For KC, plate 12 x 10⁶ KC per 10-cm plate with CnT-07 media and incubate at 37 °C + 5% CO₂ for next day infection. Alternatively, let KC attach and spread for a minimum of 2 hr at 37 °C + 5% CO₂ and perform infection on the same day. Usually 3-4 plates of KC are used to generate one graft. A total of 10-12 plates are needed to generate three grafts for one experiment. KCs will have cobblestone morphology.
2. Preincubate cells for 5-10 min with 8 μg/ml Polybrene Solution at 37 °C + 5% CO₂.
3. Exchange media and add lentivirus + 8 μg/ml Polybrene Solution. Lentiviral titer will vary depending on the construct used and should be determined before infection.
4. Centrifuge the plates at 200 x g on a tabletop centrifuge for 1 hr at 32 °C to infect cells with lentivirus.
5. Remove lentivirus-containing media and add fresh media. For KC, wash cells once with PBS before adding back fresh media. Usually infected cells recover overnight at 37 °C + 5% CO₂. Alternatively, allow at least two hours of recovery at 37 °C + 5% CO₂ before trypsinizing the cells for grafts. Continue to culture a small portion of infected cells to check for infection efficiency as described in the Representative Results section.

8. Prepare Infected Cells for Grafting

1. Isolate fresh primary mDCs or KCs from skin using steps 4-6. Resuspend cells in ice cold sterile PBS. Count cells.
2. 1. For dermal specific gene knockdown/overexpression graft, dissociate infected mDCs from plates using 0.05% Trypsin-EDTA at 37 °C for 8 min.
   2. For epidermal specific gene knockdown/overexpression graft, dissociate infected KC from plates using 0.125% Trypsin-EDTA at 37 °C for 15 min.
3. Neutralize trypsin activity using DMEM + 10%FBS (mDCs) or Neutralizing Solution (KC). Transfer the trypsinized cells to a 50 ml tube.
4. Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.
5. Resuspend cells in ice cold sterile PBS. Count cells.
6. Combine the virus-infected cells with the freshly prepared untreated counterpart cell type. For one graft, combine 7-10 x 10⁶ mDCs with 7-10 x 10⁶ KCs in ice cold sterile PBS and pellet the cell slurry in a tabletop centrifuge at 4 °C. Resuspend cells in 50 -100 μl ice cold sterile PBS. Keep cells on ice until nu/nu mouse chambers are ready.

9. Create Wound Bed and Fix Chamber onto Back of nu/nu Mouse

A day before grafting, sterilize surgical tools with autoclave and sterile silicon chambers by soaking in 70% ethanol. Replace the 70% ethanol with sterile PBS before using the chambers.

1. Anesthetize nu/nu mouse (7-12 week old females) using ketamine (8 mg/ml)/xylazine (1.6 mg/ml) via intraperitoneal injection (100 μl/10 g body weight) or other preferred method. Apply eye lubricant. Place heating pad under mouse.
2. Disinfect nude mouse back skin with iodine and clean with alcohol swabs.
3. Add 1-3 drops 0.25% Bupivicaine to incision site topically. Pinch the nude mouse back skin at base of neck with forceps.
4. Cut a small circular hole (~1 cm in diameter) in pinched skin with sterile scissors.
5. Place sterile chamber onto wound bed and cover chamber edges with surrounding skin.
6. Secure chamber into place with sutures along chamber edges (6 stitches per chamber) as shown in Figure 2A.
7. When chambers are ready, gently swirl cell slurry and draw up into 23 gauge needle attached to 1 ml syringe. If cell slurry has condensed at the bottom, gently resuspend with 1 ml pipette tip before drawing into the needle.
8. Puncture chamber with needle and slowly drip cells onto wound.
9. Place mice into clean, autoclaved cages. House 2 mice per cage and add antibiotics and Tylenol (3 mg/ml) to drinking water. In our experience, two female mice per cage have not resulted in adverse trauma to the chamber, graft, or animals.
10. Place warming pad under half the cage and observe mice until anesthesia wears off.

10. Remove Chamber After 7-9 Days

1. Anesthetize chambered nu/nu mouse as in step 9.1. Apply eye lubricant.
2. Disinfect skin around chamber with iodine and clean with alcohol swabs.
3. Carefully cut stitches and remove from chamber.
4. Gently remove chamber from mouse. If new skin graft sticks to the chamber, lift part of the chamber and gently separate graft from chamber using forceps. Hold graft down during removal of the chamber. Graft should look dull and opaque as in Figure 2B.
5. Apply a non-adherent pad with a thin film of antibiotics on the open wound and suture into place (2 stitches).
6. Wrap bandage around mouse to secure the pad and protect the wound. Suture bandage into place.
7. The silicon chambers are cleaned by scrubbing with mild soap and thoroughly rinsed with deionized water. Soak the chambers in 70% ethanol overnight, air dry, and store.

11. Examine Mouse for Hair Growth

1. Remove bandages and pad after 3 days of chamber removal. Graft should be dry and opaque to brown in color.
2. Check mice periodically for hair growth. Hair should begin to show at 16 days after graft.

Procedure Outline

Day 1 (2-3 hr): Sacrifice newborn pups, remove skin, and leave on dispase at 4 °C overnight.

Day 2 (2-3 hr): Isolate primary cells from skin and plate at recommended cell density.

Day 3 (3-4 hr): Infect cells with lentivirus. Remove skin from another set of newborn pups and leave on dispase at 4 °C overnight.

Day 4 (6-8 hr): Isolate primary cells from skin, count, and leave on ice. Recover lentivirus-infected cells by trypsinization and centrifugation, count, and combine 7-10 x 10⁶ dermal cells and keratinocytes into 50-100 μl of PBS per graft. Create wound bed on mouse, attach chamber, and apply cell mixture to wound bed.

Alternative Procedure Outlines

Alternative procedure outlines will shorten the procedure from four days to three.

1. Combine Day 1 and Day 2 by treating mouse skins in dispase at 37 °C for one hour (estimated time 5-7 hr).
2. Combine Day 2 and Day 3 by infecting cells with lentivirus after plating cells for two hours (estimated time 5-7 hr).