Epidermis Stem Cell (EpSC) Preparation from neonate skin (Timing 1h)
1.1.1 Wear sterile gloves while handling animals Use neonate Balb/c mice
1.1.2 Asphyxiate 10 pups (age 0-3d old) by CO2-Narcosis. (Timing 20min)
1.1.3 Place carcass on a sterile tray and wipe-clean gently (Figure-1a to e) using sterile cotton-pads soaked in 70%ethanol. (Timing 2min)
CAUTION\!Avoid skin-abrasion and tissue damage.
1.1.4 Wipe gently again using sterile cotton pads soaked in Betadine-solution \(Figure-1b); keep carcass packed in iodine-soaked cotton-pads for 10min
1.1.5 Rinse carcass with MiliQ autoclaved water and subsequently with 70%ethanol in a sterile container; shift carcass aseptically to Laminar Flow Hood and change gloves for processing in Laminar Flow
1.2 Skin excision \(Timing 30min)
1.2.1 Excise trunk skin in one piece2 using sterile dissection tools viz. surgical scissors, fine or toothed-forceps \(small size), and surgical blade \(size #15) mounted on a scalpel \(size #3) as shown in Figure-1f to n12
1.2.2 Cut limbs just above wrist- and ankle-joints leaving visible stumps (Figure-1 f to h); soak oozing blood with autoclaved-cotton.
1.2.3 Hold tail and lacerate skin by an incision near lower-sacrum (Figure-1l), insert scissor through cut-open skin and cut tissue smoothly along dorsal midline of carcass up to nose-tip; cut-remove the tail.
CAUTION\! Do not cut through the subcutaneous tissue into peritoneum.
1.2.4 Loosen skin gently from midline using a fine forceps and a toothed forceps to expose subcutaneous muscular integument \(Figure-1j to k).
1.2.5 Grip carcass with a toothed forceps and peel off skin gently beginning from hind-leg stumps (Figure-1l)
1.2.6 Hold dorsal skin flap and continue peeling off gently to ventral side using forceps till whole skin is excised (Figure-1m).
1.2.7 The excised skin specimen shall exclude tail and whisker pads (Figure-1n). It could be removed at this stage using scalpel blade and scissors for isolating specially the whisker follicles.
1.2.8 Collect skin specimens immediately in DPBS-1%Pen-strep (100X)-0.25mg/ml amphotericin B; wash tissue specimens with PBS-Antibiotics solution (Figure-1o to p).
1.2.9 Wash procedure: Rinse specimens immediately with PBS-2%Antibiotic mixture. (Timing 2min)
1.2.10 Wash with sterile milliQ water for 2min followed by rinsing with 70%ethanol for 1min, and collecting specimens again in fresh PBS-1%Antibiotic mixture for 1min.
1.2.11 Keep specimens in PBS-1%Antibiotic solution till removal of subcutaneous fat.
1.2.12 Remove fat by suitably placing specimens dermis side-up on a sterile culture dish and getting rid of subcutaneous fat with help of sterile surgical tools (forceps/scalpel/surgical blade) scrapping very gently and carefully using the side of scalpel-blade (Figure-1o).
CAUTION\! Do not scrap too hard as it may damage the sites of stem cell location viz. basal cell layer, hair follicle bulge. This critical step may require practice.
1.2.13 Skin tissue looks almost transparent after removing fat when examined against a light source \(Figure-1n to o).
1.3 Epidermis separation using Dispase
1.3.1 Place specimens epidermis-side up in 5ml Dispase solution in a sterile 60mm Petridish.
1.3.2 Spread tissue carefully allowing specimens to float serenely on Dispase.
1.3.3 Place specimens at 4°C to incubate (Figure-1p) overnight.
CAUTION\! Do not allow Dispase solution to leak on top of specimen in order to avoid disintegration of epidermis causing difficulty in separation of dermis and epidermis.
1.4 EpSC Isolation \(Timing 2h)
1.4.1 Arrange autoclaved materials viz. dissection-scissor, forceps, 15ml & 50ml disposable centrifuge tubes, 2cm2 pieces of nylon \(gauze 80 & 100micron), SMEM, SMEM+20%chelexed FBS, Collagen-fibronectin coated flasks under sterile condition in Laminar Flow Hood.
Note: Base of sterile culture dish can be used as sterile surface to rest dissecting tools.
1.4.1 Remove the overnight-Dispase-digested specimens placing epidermis-side-up on a sterile 60mm petridish.
1.4.2 Peel-off epidermis gently using sterile forceps (Figure-1q to r).
1.4.3 Transfer epidermis specimen into sterile 50ml centrifuge tube containing 5ml 0.25% trypsin-1mM EDTA and incubate for 30min at 25ºC in Laminar hood.
1.4.4 Agitate tissue slowly 1-10 times to dissociate cells; add 20 ml SMEM+20%FBS medium and agitate again to dissociate cells.
1.4.5 Filter patiently through an autoclaved nylon cloth filter (mesh size 100micron) using sterile 20ml syringe.
Note: Fill up 20ml sterile syringe with dissociated-cell suspension and filter \(in 100µl-aliquots) through a select small area on the nylon cloth mounted on sterile centrifuge tube.
CAUTION\! Do not rush the filtration process by unnecessary pressure on nylon cloth, which may damage cells.
1.4.6 Replace nylon cloth filter with 80micron mesh and filter again patiently as above.
1.4.7 Collect filtrate in a sterile 15ml conical centrifuge tube and spin to pellet cells at 500g for 10min at 4°C in swing-out bucket type rotor.
1.4.8 Discard supernatant and re-suspend the cell-pellet in 5ml fresh medium to spin at 300g for 10min at 4°C.
1.4.9 Aspirate supernatant and re-suspend pellet in 2ml GPM.
Note: Halt point: Processing can be halted for overnight period.
1.5.1 Determine cell density and seed 1×106 primary cells in 5ml GPM in 25cm2 flasks coated with fibronectin and collagen type-IV.
1.5.2 Allow cells to attach in 10min (Figure-2a).
1.5.3 Decant media carefully over the side of the flask.
1.5.4 Add GPM 5ml/flask slowly over the side of the flask.
CAUTION\! Else it may pull out or partially damage stem cells loosely placed on the coatings and result in considerable loss of stem cells.
1.5.5 Place flasks in CO2 incubator at 37°C and 5%CO2.
1.5.6 Initial growth is visible only after 4-5d of seeding (Figure-2b).
1.5.7 Replace media first after 4d of seeding with fresh medium thereafter every alternate day.
1.5.8 See first confluence of stem cell culture after 7d of seeding (Figure-2c); Cell-growth speeds up once the cell density becomes greater.
Troubleshooting: Delay in confluence
(1) Due to improper removal of Ca2+ from FBS or medium-conditioning of Ca2+-free SMEM in fibroblast culture.
(2) Due to unfit conditioned medium; place SMEM for conditioning only after attainment of 70% confluence in fibroblast culture as higher confluence can change the fitness of the conditioned-medium; harvest conditioned medium exactly after 48h of medium-conditioning; delay in freeze-storage at -20°C may also influence the fitness of conditioned medium for stem cell culture; freeze-store immediately at -20°C.
Note: Collection of Low-Ca2+ conditioned-medium long after 48h of conditioning may result in overgrowth; prolonged Calcium starvation may also result in detachment of fibroblast leading to suspension of cells in conditioned-medium and changing its suitability for stem cell culture.
1.6 EpSC passage and long-term culture
1.6.1 After achieving 80-90% confluence in primary culture, trypsinize \(1-2min) using trypsin-EDTA; inactivate trypsin with SMEM-20%FBS-chelexed.
1.6.2 Seed 5×105 cells in 5ml GPM for sub-culture in T25 flask (Figure-3).
Note: Coating of flask with fibronectin-collagen-type-IV not required for subculture.
1.7 EpSC Cryopreservation
1.7.1 Suspend 5×105 in 1ml freezing medium \(FBS-chelexed+10% DMSO-sterile)
1.7.2 Freeze serially through sequential temperature-decreasing-program (4°C for 3-4h, -20°C and -80°C for overnight, and then in liquid nitrogen finally).
CAUTION\! Transport cell-preparations in ice-box at 4°C during cryopreservation.
1.8 Mycoplasma testing: Indirect method using Hoechst33342 stain
1.8.1 Fix Cells with 4%PFA/PBS for 10min.
1.8.2 Wash with PBS and fix with methanol for 10min.
1.8.3 Wash three times with PBS-0.1%Tween20.
1.8.4 Block samples in 1%BSA/0.1%Tween 20/PBS (2h).
1.8.5 Incubate with Hoechst dye for 10min.
1.8.6 Wash with PBS and mount it with Vectashield mounting medium.
1.8.7 Spot and document mycoplasma in cells using fluoroscence microscope as per instructions from kit-manufacturer.
2 EpSC preparation from adult mouse skin \(Timing 1h)
2.1.1 Use adult Balb/c mice \(either sex; 6-12week old).
2.1.2 Prepare animals using sterile gloves and ensure that hair-growth cycle of skin in excision area (viz. inter-scapular) is in Telogen phase; it can be verified by hair-clipping on dorsal surface and observing no hair growth for 3d.
2.1.3 Sacrifice animals by cervical dislocation; and wash carcass using 70%ethanol and Betadine solution as described earlier in case of neonatal epidermis stem cell.
2.2.1 Excise the hairless area \(in Telogen phase) from inter-scapular region using sterile dissection tools viz. surgical scissors, fine or toothed-forceps \(small size), and surgical blade \(size #15) mounted on a scalpel \(size #3) and place skin specimens immediately in D-PBS-1%Pen-strep-0.25mg/ml amphotericin-B.
2.2.2 Shift to laminar hood and wash as previously.
2.2.3 Put the specimen epidermis-side down on a 60mm culture plate and scrap subcutaneous fat using surgical blade (size #15) mounted on a scalpel (size #3) and toothed forceps.
CAUTION\! Scrapping skin too hard may result in loss of important areas housing Stem cells viz basal cell layer, hair follicle bulge. This critical step may require practice.
Add 4ml Dispase solution and incubate at 4°C overnight or for 10h.
2.2.4 Remove Dispase and add 0.25%trypsin-1mM EDTA; keep at 37°C for 60min.
2.2.5 Peel off epidermis from dermis gently using fine and toothed forceps.
2.2.6 Collect epidermis specimens in flask containing 20ml GPM.
2.2.7 Stir 30min using magnetic stirrer to dissociate cells into suspension.
2.2.8 Filter cell-suspension using nylon mesh 100 micro-meter followed by 80micron as described earlier.
2.2.9 Collect cells in 15ml centrifuge tube and centrifuge at 500xg for 10min at 4°C.
2.2.10 Aspirate supernatant, add fresh GPM, and centrifuge (300xg, 5min, 4°C).
2.2.11 Discard supernatant and re-suspend pellet.
3 EpSC preparation from adult mouse tail-skin \(Timing 5h)
Procedure is primarily based on the method developed earlier for preparation of primary keratinocytes from adult mouse tail-skin 1,2. Isolate EpSC from tail skin at any age of the animal;
the advantage is less hair density in tail skin and low hindrance in separation of dermis & epidermis.
3.1.1 Wash mice with betadine and then with 70%ethanol for processing as earlier \(STEP # 1.1.4-1.1.5).
CAUTION\! Cut open tail ventrally through ventral fascia avoiding damage to ventral artery located in that area; start de-skinning from ventral side over to dorsal side so as to keep the epidermis flap intact yielding an uncut piece.
Note: It is easy to start de-skinning from ventral side and removing the cartilage adhered on dermis using scalpel; scaly skin does not hinder in epidermis isolation.
3.2.1 Cut the tail from base \(i.e. at the joint with trunk) and excise up to 2cm from the tip; cut the specimen into three pieces; and remove sub-cutaneous fat.
3.2.2 Place excised skin into PBS-antibiotic solution.
3.2.3 Wash sample as described previously (STEP # 1.2.9 -1.2.12).
3.2.4 Place dermal side down in dispase solution and allow to digest (see STEP 1.3.1-1.3.3).
4 EpSC preparation from adult mouse ear-skin \(Timing 5h)
Here also the procedure is primarily based on the method developed for preparation of primary keratinocytes from adult mouse ear-skin10. EpSC could be isolated from ear-skin at any age of the mouse. Advantage is again less hair density, low hindrance in separation of dermis & epidermis, and ease to get infection free skin.
4.1.1 Wash mice first with betadine and then with 70%ethanol \(see STEP # 1.1.4 -1.1.5).
4.2.1 Cut ear-pinnae from the head-joint.
CAUTION\! Avoid hairy pat at the joint; start to peel skin gently from ventral area over to dorsal side; this way of dissection eases the peeling-process otherwise starting to peel from dorsal area fragments the tissue and yields bad specimen.
4.2.2 Peel ear skin along the side of cartilage and place in PBS-antibiotic mixture.
4.2.3 Wash tissue specimen as described previously (STEP # 1.2.9-1.2.12).
4.2.4 Remove cartilage and subcutaneous fat using scalpel & blade.
CAUTION\!Remove cartilage very carefully as continuous scrapping may lead to loss of epidermis.
4.2.5 Place ear skin dermis side down in dispase solution allowing digestion as previously \(STEP #1.3.1-1.3.3)
5.1 Biomarker detection using Fluorescence based immunocytochemistry
5.1.1 Fix Cells with 4%PFA/PBS for 10min.
5.1.2 Wash with PBS and fix with methanol for 10min.
5.1.3 Wash three times with PBS-0.1%Tween-20 (PBST).
5.1.4 Block Samples in 1%BSA/0.1%Tween 20/PBS (2h).
5.1.5 Incubate with primary antibodies overnight at 4°C as per the kit-instructions.
5.1.6 Wash with PBS, and incubate sections with rabbit anti-mouse-Alexa flore-conjugated (1/200) (Invitrogen) and anti-mouse- Alexa flore conjugated (1:200) secondary antibodies for 2h at RT
5.1.7 Rinse with PBST and mount with Vectashield solution containing DAPI Antibodies:Beta-1-integrin rabbit polyclonal-Santacruz (1:200) and Anti BrdU mouse monoclonal-Santacruz (1:200).
5.1.8 Spot and document cells using fluoroscence microscope (Figure-4 to 6).
5.2 Detection using Western Blot
5.2.1 Lyse cells with ice-cold Celllytic-M-10mM NaF-1mM Na3VO4-1mM PMSF
5.2.2 Centrifuge cell-lysates at 15,000rpm for 5min at 48°C.
5.2.3 Determine protein content by Bradford method using BSA as standard.
5.2.4 Separate proteins (40mg) by 10%SDS-PAGE and transblot onto nitrocellulose paper.
5.2.5 Block membranes with 5%fat-free dry milk or 5%BSA for 1h in Tris-buffered-saline (TBS; 25mM Tris-HCl, pH 7.6, plus 150mM NaCl) containing 0.1%Tween 20 (TBS-T).
5.2.6 Incubate with primary monoclonal antibodies: keratin-10, -14, -15, -19, beta-1-integrin, p63, CD34 at 4°C overnight; dilute 1:1,000–1:5,000 in 1%skim milk or BSA in TBS-T. Wash membranes 3X with TBS-T with gentle shaking for better results
5.2.7 Incubate with secondary peroxidase-conjugated anti-mouse and anti-rabbit antibodies (diluted 1:1,000 in 1% fat-free dry milk in TBS-T) at RT for 1-2h (at 4°C for 4h) with gentle shaking.
5.2.8 Detect select proteins using an enhanced chemiluminescence kit (ECLPlus Western blotting kit, Pierce).
5.2.9 Quantify Western blots by densitometry using VersaDoc (BioRad USA)-Quantity-one (Figure-7).
5.3 LRC \(BrdU retaining cell) detection using DAB based immunocytochemistry
5.3.1 Fix cultured cells on chamber slide in fixation buffer for 15min.
5.3.2 Wash slides 2X in PBS for 5min each time.
5.3.3 Incubate slides with diluents buffer for 30min to permeabilize cells.
5.3.4 Wash slides twice.
5.3.5 Incubate slides for 10 min in 0.3% H2O2 in PBS to block endogenous peroxidase activity.
5.3.6 Rinse slides thrice 5min each in PBS.
5.3.7 Retrieve antigen using BD Retrievagen-A (Mix 9ml solution-1 and 41ml solution-2 and make it up to 500ml with distilled water as described in the kit).
5.3.8 Place the slide in coplin jar containing the working solution of BD-Retrievgen-A.
5.3.9 Place the entire setup in hot water at 89ºC for 10min.
5.3.10 Remove the coplin jar with slides; cover the jar tightly and allow solution to cool down slowly to room temperature over 20min.
5.3.11 Rinse slides thrice 2min each in PBS.
5.3.12 Incubate with ready-to-use-streptavidin-HRP for 30min at RT.
5.3.13 Rinse slides thrice 2min each in PBS.
5.3.14 Incubate slides for 5min with DAB substrate solution and cover the slide.
Note: Prepare DAB solution by adding 1drop of DAB chromogen to 1ml DAB buffer provided in kit.
5.3.15 Rinse slides thrice 2min each in double distilled water.
5.3.16 Dehydrate 5min each serially through 2 changes of 95%, 100% alcohol.
5.3.17 Clear in three changes of xyline and coverslip the specimen (Figure-8).
5.4 LRC detection in keratinocyte-isolates using FACS
5.4.1 Wash cells \(0.5×106cells/ml SMEM-medium) twice with cold PBS; and resuspend the pellet in residual supernatant \(failing to do so shall clump the cells while adding copious amount of cold 70%ethanol \(15ml)
5.4.2 Place Cells on ice for 30min and pellet to resuspend in residual supernatant.
5.4.3 Add copious amount of 4N HCl (15ml).
5.4.4 Allow cell-pellet to stand at RT for 30min.
5.4.5 Pellet Cells again and resuspend in PBS (106cells/ml).
5.4.6 Aliquot 1ml cell-suspension into polystyrene tube and pellet the cells.
5.4.7 Wash once with 0.5%tween-20 and mix pellet with 10ul of FITC-conjugated anti-BrdU antibody.
5.4.8 Vortex the contents and incubate at RT for 30min.
5.4.9 Wash cells twice with PBS and resuspend finally in 1ml PBS before analyses over BD-FACS-Calibur flowcytometer.
5.4.10 Sort cells using BD-CellQuest-Pro software; set gates using forward light scattter (FSC) and side light scatter (SSC). Detect anti-BrdU monoclonal antibody FITC-conjugated fluorescence on FL1-H channel (Excitation 488nm, Emission 530nm±30nm band pass). Acquire a total of 10,000 events for each sample and plot FL1-H data on log scale (Figure-9).
5.5 FACSaria based validation of cultured-EpSC using Hoechst dye \(DAPI) Exclusion Method
5.5.1 Use stem cell in culture \(3rd passage) after collagen-fibronectin-adherence selection.
5.5.2 Trypsinize and pellet cells at 300Xg/5min/4°C.
5.5.3 Wash twice in 1%BSA to avoid loss of cells occurring due to their stickiness onto the FACSAria-tube-lumen.
5.5.4 Resuspend 1×106cells/ml-Growth-Promoting-Medium-1mMHEPES-5µg/ml Hoechst-33342.
5.5.5 Incubate cell in Hoechst dye-medium mix at 37ºC for 90min.
5.5.6 Pellet cells and resuspend in 1ug/ml Propidium iodide containing Growth Promoting medium, and proceed to analyze using FACSAria in rectilinear sort gate as shown in Figure-10.
5.6 FACSAria based sorting of neonate mouse epidermis keratinocytes into ample-pure TA & stem-cell population and culture
Isolate EpSC from BALB/c neonate mouse skin as earlier \(see step 1 to 1.4.10).
5.6.1 Sacrifice neonatal mouse and excise skin.
5.6.2 Place tissue in 70% ethanol for 1min.
5.6.3 Wash extensively with Ca2+/Mg2+-free-PBS.
5.6.4 Incubate specimens in dispase overnight at 4ºC.
5.6.5 Peel epidermis from dermis next day.
5.6.6 Obtain single cell suspension by gently shaking the epidermis.
5.6.7 Pellet cells at 300Xg/5min/4°C and wash twice in 1%BSA to avoid loss of cells sticking onto the FACSAria-tube-surface.
5.6.8 Resuspend 5×106cells/ml-Growth-Promoting-Medium-1mMHEPES-5µg/ml Hoechst33342.
5.6.9 Incubate cell in Hoechst dye-medium mix for 90min at 37ºC.
5.6.10 Pellet cells, resuspended in 1ug/ml Propidium iodide containing Growth Promoting medium, and place on ice until sorting.
5.6.11 Sort cells on FACSDiva cell sorter (BectoneDickinson).
5.6.12 Acquire 50,000 events in list mode for each sample.
5.6.13 Gate out Debris and cells positive for PI.
5.6.14 Select pressure 20PSI and pressure difference 0.8.
5.6.15 Perform cell sorting on FACSAria-DiVa (Becten Dickinson).
5.6.16 Configure equipment so as to set non-rectilinear sort-gates as shown in Bitmap Histogram (Figure-11); set Gates (for Propidium iodide 370mW; for DAPI 190mW; Hoechst 355nm excitation & 450/50nm emission band pass filter; Propidium iodide 488nm excitation & 575/25nm emission band pass filter) on set parameters.
5.6.17 Sort keratinocytes into two populations (EpSC and TA cells)
5.6.18 Collect all data on set parameters using linear amplification in list-mode (Figure-11).
5.6.19 Culture the EpSC thus obtained in Growth Promoting Medium as described earlier (see step 1.5 to 1.6.2). TA cells fail to adhere to culture-surface in flasks.
5.7 FACSAria based sorting of adult mouse epidermis keratinocytes into ample-pure TA & stem-cell population and culture
Isolate EpSC from BALB/c adult mouse skin \(see step 2 to 2.2.12) and process for FACSAria based sorting as described in step 5.4.7 to 5.4.19 to get results as shown in Figure-12 & 13.