Lung Epithelial Stem Cell

UNIT 2G.1Isolation and Clonal Assay of Adult LungEpithelial Stem/Progenitor Cells

Ivan Bertoncello1 and Jonathan McQualter1

1Lung Regeneration Laboratory, The Department of Pharmacology, University ofMelbourne, Victoria, Australia

ABSTRACT

Adult mouse lung epithelial stem/progenitor cells (EpiSPC) can be defined in vitroas epithelial colony-forming units that are capable of self-renewal, and which when co-cultured with lung mesenchymal stromal cells (MSC) are able to give rise to differentiatedprogeny comprising mature lung epithelial cells. This unit describes a protocol for theprospective isolation and in vitro propagation and differentiation of adult mouse lungEpiSPC. The strategy used for selection of EpiSPC and MSC from adult mouse lung byenzymatic digestion and flow cytometry is based on the differential expression of CD45,CD31, Sca-1, EpCAM, and CD24. The culture conditions required for the differentiation(co-culture with MSC) and expansion (stromal-free culture with FGF-10 and HGF) ofEpiSPC are described. Curr. Protoc. Stem Cell Biol. 16:2G.1.1-2G.1.12. C© 2011 by JohnWiley & Sons, Inc.

Keywords: lung epithelium � stem cells � colony-forming assay

INTRODUCTION

This unit describes isolation and culture of adult mouse lung epithelial stem/progenitorcells (EpiSPC). EpiSPC are defined as colony-forming units (CFU) that have the capacityfor self-renewal and are able to generate progeny of differentiated lung epithelial cells.Basic Protocol 1 describes a cell-fractionation method using flow cytometry for isolatingEpiSPC and mesenchymal stromal cells (MSC) from enzymatically digested adult mouselung tissue, based on their differential expression of CD45, CD31, Sca-1, EpCAM, andCD24 (McQualter et al., 2010). Basic Protocols 2 and 3 describe culture methods for thedifferentiation, maintenance, and propagation of adult lung EpiSPC, respectively. Whenco-cultured with MSC in an organotypic three-dimensional Matrigel culture, EpiSPCgenerate colonies comprising mature differentiated lung epithelial cells. In stromal-freecultures, EpiSPC undergo clonal proliferation when supplemented with FGF-10 andhepatocyte growth factor (HGF), and can be enzymatically dissociated and passagedusing this culture setup, demonstrating their high proliferative capacity and ability toself-renew.

BASICPROTOCOL 1

ISOLATION OF EPITHELIAL STEM/PROGENITOR CELLS ANDMESENCHYMAL STROMAL CELLS FROM ADULT MOUSE LUNG

This protocol is used for the dissociation of adult mouse lung tissue and the enrichmentof cell fractions containing EpiSPC and MSC. A combination of mechanical and en-zymatic dissociation is used to prepare a single-cell suspension of lung cells, followedby a pre-enrichment step using discontinuous density gradient centrifugation to removehigh-density cells such as erythrocytes, mature myeloid cells, and cellular debris. Flowcytometry is then used to identify and sort subpopulations of cells enriched for eitherEpiSPC or MSC, based on the expression of defined cell-surface markers (McQualteret al., 2009, 2010).

Current Protocols in Stem Cell Biology 2G.1.1-2G.1.12Published online January 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/9780470151808.sc02g01s16Copyright C© 2011 John Wiley & Sons, Inc.

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Supplement 16

Isolation andClonal Assay of

Adult LungEpithelial

Stem/ProgenitorCells

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Supplement 16 Current Protocols in Stem Cell Biology

Materials

Collagenase, type 1 (Worthington)Phosphate buffered saline (PBS; Sigma, cat. no. P-3813)Adult mice (strain C57BL/6; 6 to 12 weeks; 18 to 22 g; male or female)PBS with 2% (v/v) fetal bovine serum (FBS; JRH Biosciences, batch tested)Nycoprep density gradient medium (1.077 g/cm3, 265 mOsm (see recipe)Fluorochrome-conjugated antibodies against:

EpCAM (clone G8.8)CD24 (clone M1/69)Sca-1 (clone E13-161.7)CD45 (clone 30-F11)CD31 (clone 390)

Viability dye (4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), orFluoroGold (Fluorochrome LLC, http://www.fluorochrome.com)

Dissecting equipment15- and 50-ml conical polypropylene centrifuge tubes (e.g., BD Falcon)60-mm-diameter Petri dishesThermomixer (Eppendorf)18-G and 21-G needles5-ml and 20-ml syringes40-μm cell strainer (BD Biosciences)Refrigerated centrifuge (with capacity for 5-ml, 15-ml and 50-ml tubes)Sysmex KX-21N cell counter (Sysmex Corporation; http://www.sysmex.com/) or

hemacytometer counting chamber and trypan blue (UNIT 1C.3)Sterile mixing cannulas (Unomedical, cat. no. 500.11.012;

http://www.unomedical.com)5-ml FACS tubes with 35-μm cell strainer caps (BD Biosciences), sterileFlow cytometer

Additional reagents and equipment for euthanasia of mice (Donovan and Brown,2006), determining viable cell concentration (UNIT 1C.3), and flow cytometry(Robinson et al., 2010)

Dissociate lung tissue1. Prepare 1 mg/ml collagenase type I solution in sterile PBS and preheat to 37◦C,

allowing 3 ml per whole mouse lung.

2. Euthanize mouse by cervical dislocation (Donovan and Brown, 2006), open mouseabdominal cavity, and sever major arteries/vessels behind intestines to exsanguinateanimal.

3. Open thoracic cavity, carefully dissect out the lungs, place into a 15-ml tube con-taining 10 ml chilled PBS, and agitate to rinse out excess blood.

If preparing multiple lungs, pool lungs and rinse into a 50-ml tube (up to five lungs pertube).

This protocol has been optimized for the isolation of distal lung cells. All upper airwaysare carefully removed at this point.

4. Transfer lungs into a second 60-ml tube of fresh chilled PBS and rinse lungs well bygentle agitation.

The lungs should become pale when adequately rinsed. Rinse a third time if necessary.

5. Transfer the lungs into a sterile 60-mm-diameter Petri dish and finely mince thelungs using fine dissecting scissors or a single-sided razor blade.

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Current Protocols in Stem Cell Biology Supplement 16

Prepare single-cell suspension6. Transfer the minced tissue into a 15-ml conical centrifuge tube and add the preheated

collagenase type I solution (3 ml per lung) to the tube.

If processing multiple lungs, use a 50-ml centrifuge tube (up to five lungs per tube).

7. Place the tube in a Thermomixer (Eppendorf) and agitate (750 rpm, 37◦C) for30 min.

8. Remove the tube from the Thermomixer and triturate with an 18-G needle attachedto a 5-ml syringe until chunks of tissue are mostly dissociated.

If processing multiple samples, use a 20-ml syringe.

9. Return tube to the Thermomixer and agitate (750 rpm, 37◦C) for a further 15 to 30min until most of the lung tissue fragments are digested.

When tissue digestion is complete, you should be unable to see chunks of pink lungtissue, although clumps of extracellular matrix will remain visible as white strands in thesuspension.

10. Following digestion, remove the tube from the Thermomixer and triturate with a21-G needle attached to a 20-ml syringe to generate a single-cell suspension.

11. Strain the tissue digest through a 40-μm cell strainer into a clean, sterile 50-mlcentrifuge tube.

12. Resuspend the tissue digest in 50 ml of PBS containing 2% fetal bovine serum andcentrifuge 5 min at 400 × g, 4◦C.

13. Remove supernatant and resuspend the cell pellet in 50 ml of PBS containing 2%fetal bovine serum.

To maximize cell recovery, supernatant can be collected in a 50-ml tube and recentrifuged.

14. Centrifuge tube(s) 5 min at 400 × g, 4◦C.

15. Remove supernatant(s) and resuspend the cell pellet(s) in PBS containing 2% fetalbovine serum (5 ml per lung).

16. Count the cells and calculate cell concentration.

A Sysmex KX-21N automated cell counter can be used to count cells, or alternativelya hemacytometer can be used with trypan blue (UNIT 1C.3) to exclude nonviable cells.Average cell yield per lung is 2.2 × 107 cells.

Prepare low-density cell fraction17. Transfer the cell suspension (5 ml) into a 15-ml centrifuge tube and underlay the

suspension with 3 ml of Nycoprep (1.077 g/cm3) using a sterile mixing cannulaattached to a 20-ml syringe.

If processing multiple lungs, the equivalent cell suspension from four lungs (20 ml) canbe transferred to a 50-ml centrifuge tube and underlayed with 10 ml of Nycoprep.

18. Centrifuge the gradients 15 min at 600 × g, 21◦C, with the brake off.

Gradient centrifugation will result in most high-density cells (i.e., erythrocytes, maturemyeloid cells, and cellular debris) passing through the Nycoprep, leaving an enrichedband of low-density cells at the Nycoprep interface.

19. Harvest low-density cells from the interface between the PBS layer and the Nycoprepsolution using a 10-ml pipet and transfer into a 50-ml centrifuge tube (Fig. 2G.1.1A).

If processing multiple lungs, the low-density cell fractions can be pooled at this point.Average low-density cell yield per lung is 6.9 × 106 cells.




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Via

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PI)

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1000 10K 20K 30K 101 102 103 104

CD24 (PacBlue)

Low-densitycell band

High-densitycell pellet

Nycoprep

FBS-2%FBS

Figure 2G.1.1 Cell fractionation strategy used to enrich and sort EpiSPC and MSC from enzymatically digestedlung tissue. (A) Photographic image of low-density cell band from density gradient centrifugation. (B) Gating strat-egy inclusion of PIneg viable cells, and (C) exclusion of CD45negCD31neg cells. (D) Gating strategy for sortingCD45negCD31negEpCAMnegSca-1hi MSC. (E) Gating strategy for sorting CD45negCD31negEpCAMhiCD24low EpiSPC.

20. Top up tube(s) containing the low-density cells to a total volume of 50 ml with PBScontaining 2% fetal bovine serum and centrifuge 5 min at 400 × g, 4◦C.

21. Discard supernatant(s) and resuspend the cell pellet(s) in 50 ml PBS containing 2%fetal bovine serum.

22. Centrifuge the tube(s) 5 min at 400 × g, 4◦C.

23. Discard supernatant(s) and resuspend the cell pellet(s) in 5 ml per lung of PBScontaining 2% fetal bovine serum.

24. Count the cells and calculate cell concentration (UNIT 1C.3).

Prepare cells for flow cytometry25. Aliquot 100,000 to 200,000 cells into a 5-ml FACS tube for each of the compensation

tubes and 500,000 cells for isotype control tubes. Set aside remaining cells in a 15-mlconical centrifuge tube (cells for sorting).

Compensation tubes should be prepared for each of the antibodies and fluorochromes tobe used in the sort strategy. An aliquot of unstained cells should also be prepared.

26. Wash all samples, including cells for sorting, with PBS containing 2% fetal bovineserum, centrifuge 5 min at 400 × g, 4◦C, and discard supernatant.

27. Resuspend cell pellets in compensation tubes in 50 μl of each optimally pretiteredantibody, and resuspend cells for sorting at 5 × 106 cells per 100 μl of optimallypretitered antibody combination.

Use PBS containing 2% fetal bovine serum to dilute antibodies. If streptavidin-conjugatedantibodies are used, PBS containing 0.5% (w/v) BSA should be used as a diluent to preventreactivity of streptavidin with serum biotin.

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28. Incubate all samples on ice in the dark for 20 min.

29. Wash cells in PBS containing 2% fetal bovine serum, centrifuge 5 min at 400 × g,4◦C, and discard supernatant.

Repeat steps 26 to 29 if secondary or tertiary antibody labeling steps are required.

30. Resuspend cells in compensation tubes in 300 μl and cells for sorting and isotypecontrol tubes at 10–15 × 106 cells/ml in PBS containing 2% fetal bovine serum andan appropriate viability dye.

Depending on the flow cytometer setup and laser configuration, DAPI (0.5 μg/ml), PI (1μg/ml), or Fluorogold (2 μM) may be used as a viability dye. Viability dyes should beincluded in all compensation, control, and sort tubes.

31. To remove any cell clumps which may block the flow cytometer, aliquot samplesinto 5-ml FACS tubes with cell strainer caps (35-μm, sterile).

To filter larger volumes of cells for sorting, a 40-μm nylon-mesh cell strainer can be used.

Set up for FACSDetailed protocols for flow cytometry are provided in Robinson et al. (2010).

32. Prepare collection tubes containing PBS with 2% fetal bovine serum.

Cells can be collected into microcentrifuge tubes (containing 200 μl of PBS/2% fetalbovine serum) or 5 ml FACS tubes (containing 1 ml of the PBS/serum).

33. Set up flow cytometer with a large (90 to 100-μm) nozzle and stabilize the flowstream under low pressure (30 to 40 psi).

34. Set the compensation settings using single-color control tubes.

When setting the compensation using single-color control tubes containing lung cellsstained with the appropriate antibodies and fluorochromes, it is important to take intoconsideration the autofluorescence of lung cells and avoid overcompensation (see CriticalParameters).

35. To sort lung EpiSPC and MSC, set sequential gates for selection of viable cells(Fig. 2G.1.1B) followed by exclusion of hematopoietic (CD45) and endothelial(CD31) cells (Fig. 2G.1.1C), prior to selection of EpCAMhiCD24low EpiSPC(Fig. 2G.1.1D) and/or EpCAMnegSca-1hi MSC (Fig. 2G.1.1E).

Isotype controls should be used to account for non-specific antibody staining in settinggates to identify and isolate antibody-positive cells.

36. Sort cells into collection tubes containing PBS containing 2% fetal bovine serum.

BASICPROTOCOL 2

ORGANOTYPIC CULTURE OF LUNG EPITHELIAL STEM/PROGENITORCELLS (DIFFERENTIATION CULTURE)

This protocol is used for the identification and characterization of EpiSPC. During culture,epithelial CFU (Epi-CFU) proliferate and differentiate to form complex lineage-restrictedor multipotent epithelial colonies comprising alveolar cells, airway cells, or cells of mixedlineage. The growth of these colonies requires seeding EpiSPC in a three-dimensionalextracellular matrix (Matrigel) in co-culture with lung-derived MSC (Fig. 2G.1.2A,B).These cultures can be re-fed and maintained over 2 weeks, after which cells begin to dieand colonies deteriorate.

This culture technique has been developed for EpiSPC sorted on the phe-notype CD45negCD31negEpCAMhiCD24low, but can also be used to identifyEpiSPC from heterogeneous cell fractions. MSC are sorted on the phenotype ofCD45negCD31negEpCAMnegSca-1hi.




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Figure 2G.1.2 (A) Schematic representation of organotypic culture setup for EpiSPC differentia-tion. (B) Image of 24-well tissue culture plate with culture inserts. (C) Whole-well image of coloniesafter 2 weeks in culture. (D) Bright-field and (E) dark-field images depicting representative colonysubtypes, including (i) airway, (ii) alveolar, and (iii) mixed colonies.

NOTE: All procedures are performed in a sterile class II biological hazard flow hood ora laminar-flow hood. All solutions, reagents, media and equipment used to process andculture EpiSPC must be sterile, and proper aseptic technique should be used.

Materials

Epi-CFU medium (see recipe)Matrigel (standard concentration; BD Biosciences)Adult lung EpiSPC (CD45negCD31negEpCAMhiCD24low) cells (Basic Protocol 1)Adult lung MSC (CD45negCD31negEpCAMnegSca-1hi) cells (Basic Protocol 1)

Refrigerated centrifugeMillicell-CM culture inserts (0.4-μm membrane, 12-mm diameter, hydrophilic

PTFE, Millipore)24-well flat-bottom tissue culture platesTriple-mix incubator (5% v/v O2, 10% v/v CO2, 85% v/v N2; see Critical

Parameters), humidified

Additional reagents and equipment for determining viable cell concentration(UNIT 1C.3)

Prepare organotypic cultures of lung EpiSPC1. Following flow cytometric cell sorting, wash the sorted EpiSPC and MSC separately

in 5 ml/tube Epi-CFU medium, centrifuge 5 min at 400 × g, 4◦C, and aspiratemedium.

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2. Resuspend individual cell pellets in Epi-CFU medium (1 to 2 ml per lung) and takean aliquot to determine cell concentration (UNIT 1C.3).

Cell counts can be performed using an automated cell counter or a hemacytometer(UNIT 1C.3).

3. After determining cell concentrations, take aliquots of EpiSPC and MSC and com-bine so that the final mixed cell suspension contains the desired number of cells forculture. Allow for 100 μl per well.

For optimal Epi-CFU growth supporting capacity, MSC (CD45negCD31negEpCAMnegSca-1hi) should be used at 2 × 106 cells/ml.

The concentration of sorted cells seeded for detection of EpiSPC depends on their level ofenrichment in the sorted fraction. Ideally, cells should be seeded at a concentration whichwill generate about 20 colonies per Millicell insert (∼500 cells). The colony-formingefficiency of CD45negCD31negEpCAMhiCD24low EpiSPC from adult (8- to 12-week old)C57Bl/6 mice is typically 1 in 23.

4. Centrifuge EpiSPC/MSC mix 5 min at 400 × g, 4◦C, and aspirate medium.

Prepare Matrigel suspension cultures5. Resuspend cell pellet in Matrigel diluted 1:1 with Epi-CFU medium. Allow 100 μl

per well.

Ensure matrix always stays on ice; otherwise, it will solidify.

6. Gently mix the Matrigel cell suspension.

It is important to avoid creating bubbles. This can be achieved by holding the tube in thecenter of a Vortex mixer to create a swirling motion.

7. Place 12-mm Millicell-CM inserts in 24-well culture plates.

8. Add 90 μl of Matrigel cell suspension on top of the filter membrane of a Millicell-CMinsert and place plate in incubator (37◦C) for 5 min to allow matrix to set.

Be careful to avoid creating bubbles in the suspension.

9. Remove plate from incubator and add 400 μl of Epi-CFU medium per well aroundthe insert.

400 μl is just enough to allow the medium to touch the bottom of the insert, allowingdiffusion of medium into the Matrigel without submerging the Matrigel culture. Thissemi-dry state of the Matrigel is essential for epithelial colony-formation.

10. Incubate cell cultures in a humidified 37◦C triple-mix incubator (5% v/v O2, 10%v/v CO2, 85% v/v N2) and change to fresh Epi-CFU medium three times weekly.

11. Visualize colony morphology by bright- or dark-field microscopy (Fig. 2G.1.2C-E).

The fractionation strategy described in Basic Protocol 1 enriches for a population of cellscontaining both lineage-restricted airway and alveolar CFU and mixed-lineage CFU,which can be identified based on colony morphology (Fig. 2G.1.2D,E).

BASICPROTOCOL 3

EXPANSION OF LUNG EPITHELIAL STEM/PROGENITOR CELLS INCULTURE

In this protocol, EpiSPC are seeded in a stromal-free Matrigel culture supplemented withFGF-10 and HGF (Fig. 2G.1.3), in which they generate spherical cystic colonies that canbe enzymatically dissociated and passaged weekly to maintain EpiSPC. This assay canalso be used to assess the self-renewal capacity of putative EpiSPC.




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Figure 2G.1.3 Schematic representation of stromal-free culture setup for EpiSPC expansion.

NOTE: All procedures are performed in a sterile class II biological hazard flow hood ora laminar-flow hood. All solutions, reagents, media and equipment used to process andculture EpiSPC must be sterile, and proper aseptic technique should be used.

Materials

Epi-CFU expansion medium (see recipe)Matrigel (standard concentration; BD Biosciences)Adult lung EpiSPC (CD45negCD31negEpCAMhiCD24low) cells (Basic Protocol 1)Phosphate buffered saline (PBS; Sigma, cat. no. P-3813)Enzymatic digestion cocktail (see recipe)

Refrigerated centrifugeMillicell-CM culture inserts (0.4-μm membrane, 12-mm diameter, hydrophilic

PTFE, Millipore)24-well flat-bottom tissue culture platesTriple-mix incubator (5% v/v O2, 10% v/v CO2, 85% v/v N2), humidified15-ml conical tubes (e.g., BD Falcon) and 2-ml microcentrifuge tubes21-G needles

Set up expansion culture of lung EpiSPC1. Following flow cytometric cell sorting, wash the sorted EpiSPC and MSC in 5 ml/tube

Epi-CFU medium, centrifuge 5 min at 400 × g, 4◦C, and aspirate medium.

2. Resuspend individual cell pellets in Epi-CFU medium (1 to 2 ml per lung) and takean aliquot to determine cell concentration (UNIT 1C.3).

Cell counts can be performed using an automated cell counter or a hemacytometer(UNIT 1C.3).

3. After determining cell concentrations, take an aliquot of Epi-SPC so that the finalsuspension contains the desired number of cells for culture (1000 cells per well).Allow for 100 μl per well.

4. Centrifuge EpiSPC cell suspension 5 min at 400 × g, 4◦C, and aspirate medium.

5. Resuspend cell pellet in Matrigel diluted 1:1 with Epi-CFU expansion medium.Allow 100 μl per well.

Ensure matrix always stays on ice otherwise it will solidify.

6. Gently mix the Matrigel cell suspension.

It is important to avoid creating bubbles. This can be achieved by holding the tube in thecenter of a Vortex mixer to create a swirling motion.

7. Place 12-mm Millicell-CM inserts in 24-well culture plates.

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8. Add 90 μl of Matrigel cell suspension atop of the filter membrane of a Millicell-CMinsert and place plate in incubator (37◦C) for 5 min to allow matrix to set.

Be careful to avoid creating bubbles in the suspension.

9. Remove plate from incubator and add 400 μl of Epi-CFU expansion medium perwell around the insert.

400 μl is just enough to allow the medium to touch the bottom of the insert, allowingdiffusion of medium into the Matrigel without submerging the Matrigel culture.

10. Incubate cell cultures in a humidified 37◦C triple–mix incubator (5% v/v O2, 10%v/v CO2, 85% v/v N2) and change to fresh Epi-CFU expansion medium three timesweekly.

Passage cells11. After 1 week in culture, aspirate medium and wash cultures twice, each time with

1 ml sterile PBS.

It is important to remove serum-supplemented medium because the serum will inhibitsubsequent enzymatic digestion.

12. Harvest epithelial CFU by adding 1 ml of enzymatic digestion cocktail to the top ofthe insert and break up Matrigel by trituration.

If clonal passaging is required, single colonies can be picked from the Matrigel andenzymatically digested rather than the bulk culture.

13. After Matrigel has been displaced from the insert, place enzymatic digestion cocktail(containing Matrigel and colonies) in a 2-ml microcentrifuge tube and incubate at37◦C for 30 min.

14. Using a 21-G needle, triturate the enzymatic digest to prepare a single-cell suspen-sion.

15. Wash twice, each time with 5 ml Epi-CFU expansion medium, centrifuge 5 min at400 × g, 4◦C, and aspirate medium.

16. Re-seed single-cell suspension by repeating steps 5 to 10.

Cells should be passaged weekly and can be split into multiple cultures to accommo-date the increase in cell concentration. The number of cells re-seeded depends on theprogressive enrichment of colony-forming cells after sequential passage.

REAGENTS AND SOLUTIONSFor culture recipes and steps, use sterile tissue culture–grade water. For other purposes, usedeionized, distilled water or equivalent in recipes and protocol steps. For suppliers, see SUPPLIERS

APPENDIX.

Enzymatic digestion cocktail

PBS (tissue-culture grade; Invitrogen, cat. no. 14040) containing:3 mg/ml collagenase (Type I)3 mg/ml dispasePreheat at 37◦C and use immediately

Epi-CFU expansion medium

Epi-CFU medium (see recipe) containing:50 ng/ml recombinant FGF-10 (R&D Systems, cat. no. 345-FG)30 ng/ml recombinant HGF (R&D Systems, cat. no. 2207-HG)Store up to 1 week at 4◦C




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Epi-CFU medium

α-MEM (Invitrogen, cat. no. 41061) containing:10% (v/v) fetal bovine serum (heat inactivated)1× penicillin/streptomycin (add from 100× stock)1× insulin/transferrin/selenium (Invitrogen; add from 100× stock)2 mM L-glutamine0.0002% (w/v) heparin [1/1000 dilution of 0.2% heparin sodium salt (Invitrogen,

cat. no. 07980) in PBS (Invitrogen, cat. no. 14040)]Store up to 4 weeks at 4◦C

Nycoprep 1.077 g/cm3, 265 mOsm

Combine the following:

300 ml Nycoprep Universal: (Nycodenz: 60% w/v solution), ready-made, ster-ile, endotoxin-tested, density = 1.310 g/cm3; 580 mOsm; 300 ml (Axis-Shield;http://www.axis-shield.com/)

300 ml sterile Tricine-NaOH (20 mM, pH = 7.2)676.6 ml sterile 0.65% NaCl (w/v)Density = 1.077 gm/cm3, Osmolarity = 265 mOsm, pH = 6.9Store at room temperature and use before manufacturer’s expiration date

COMMENTARY

Background InformationIdentification and characterization of adult

lung EpiSPC have been confounded by alack of specific markers and functional as-says for their prospective isolation, enumer-ation, and measurement of their prolifera-tive and differentiative potential (Weiss et al.,2008; Chen et al., 2009; Bertoncello andMcQualter, 2010). A number of studies haveutilized flow cytometry for isolation of can-didate lung stem/progenitor cell populations,including those based on the efflux of Hoechst33342 which has proven to be selective forenriching Sca-1pos mesenchymal stromal cells(MSC; Reynolds et al., 2007; Summer et al.,2007). We have also demonstrated that sort-ing directly on the basis of Sca-1 expressionenriches for MSC (McQualter et al., 2009).On the other hand, Kim et al. (2005) have re-ported a fractionation strategy in which sort-ing based on the co-expression of Sca-1 andCD34 resulted in the enrichment of a can-didate CCSPposPro-SPCpos bronchioalveolarstem cell (BASC) cell subpopulation that re-tained epithelial character after serial passagein vitro. This protocol describes a strategy forisolating CD45negCD31negEpCAMhiCD24low

lung EpiSPC, which are Sca-1low and clearlydistinct from CD45negCD31negEpCAMneg

Sca-1hi MSC or BASC (McQualter et al.,2010). The lack of concordance in the prop-erties of cells isolated in these studies could

be explained by technical differences in tissuedisaggregation (Raiser and Kim, 2009) and thelimitations of in vitro assays used to assessproliferative and differentiative potential.

The lack of knowledge of the intricate inter-actions between epithelial cells, mesenchymalcells, and the extracellular matrix has proven asignificant obstacle in recapitulating the nec-essary conditions in vitro required for the de-velopment of assays for the identification ofEpiSPC and the analysis of their organizationand regulation. The culture system describedin this unit utilizes a three-dimensional extra-cellular matrix (Matrigel), which allows theformation of a basement membrane for ep-ithelial cell polarization and lumen formation,and enables the organotypic rearrangement ofcells in culture recapitulating the physiologicalmicroenvironment of the lung. For that reason,this culture system can also be used to studyepithelial-mesenchymal interactions that areimportant for lung regeneration and repair.

Critical ParametersTo correct for spectral overlap between dif-

ferent fluorochromes during multicolor flowcytometric analysis and sorting, color compen-sation must be performed to correctly quan-tify the fluorescence intensity of each fluo-rochrome with which cells are labeled. Whensetting the level of compensation using cellsfrom dissociated lung tissue, it is important to

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Figure 2G.1.4 Bivariate dot plot with back gating of EpiSPC (red) and MSC (blue) ontoCD45negCD31neg cells (gray) showing the potential overlap of these populations when plottedas CD45 + CD31 versus Sca-1.

take into account the autofluorescence of thesecells. Alignment of autofluorescent cells withnonautofluorescent cells will result in over-or under-compensation and inaccurate assign-ment of cell phenotype. Correct compensationassumes that the positive and negative popula-tions have equal autofluorescence (Alexanderet al., 2009; Alvarez et al., 2010).

When applying the gating strategy usedto sort cells by flow cytometry it is impor-tant to understand that very rarely are theboundaries between populations absolute. Wehave previously isolated lung MSC basedon the CD45negCD31negSca-1pos phenotype,which also comprised a minor population ofepithelial CFU (McQualter, et al., 2009).However, subsequent introduction of Ep-CAM to the sort strategy demonstratedthat all epithelial CFU could be removedfrom the MSC fraction by gating onthe CD45negCD31negEpCAMnegSca-1hi phe-notype. Figure 2G.1.4 shows that whenEpiSPC and MSC are back-gated onto ourtraditional plot of CD45+CD31 versus Sca-1, the two subsets marginally overlap, whichwould explain why gates previously set forMSC using the CD45negCD31negSca-1pos phe-notype may also include a minor fraction ofEpiSPC (McQualter et al., 2010).

This protocol describes a method in whichEpiSPC are cultured within Matrigel atop aMillicell-CM insert with medium suppliedonly at the basal surface to allow diffusion ofnutrients into the Matrigel culture. It is impor-tant that the Matrigel layer not be submergedby medium, as this prevents colony formation.The Millicell-CM inserts chosen for this as-say contain a special Biopore membrane (hy-drophilic PTFE), which helps limit overgrowthof the stromal layer, and is transparent, allow-ing microscopic visualization.

The Epi-CFU described in these protocolshave been grown under physiological low oxy-gen tension (5% v/v O2, 10% v/v CO2, 85%v/v N2), which has been shown to be optimalfor growth of stem/progenitor cells at clonaldensity in vitro (Wion et al., 2009). However,Epi-CFU can be grown under standard oxygentension (10% v/v CO2 in air), but the cloningefficiency may be lower.

TroubleshootingIt is the authors’ experience that the dif-

ferentiation state of MSC used in organ-otypic co-cultures is critical for supporting thegrowth of EpiSPC. It is important to use freshCD45negCD31negEpCAMnegSca-1hi MSC, asexpansion of these cells in culture results intheir differentiation and inhibits their abilityto support growth of EpiSPC (manuscript inpreparation).

Anticipated ResultsUsing this protocol, the

CD45negCD31negEpCAMhiCD24low cellfraction isolated comprises an enrichedbut heterogeneous population of lineage-restricted (airway or alveolar) epithelialprogenitors and multipotent (multi-lineage)stem cells (∼2000 cells per lung), whilethe CD45negCD31negEpCAMnegSca-1hi cellfraction represents a population of enrichedMSC (∼100,000 cells per lung; McQualteret al., 2010).

EpiSPC are cultured using two differenttechniques. Morphological characterization ofcolonies generated from EpiSPC grown inorganotypic differentiation cultures demon-strates the generation of large lobular cys-tic colonies with a clearly defined lumen(airway-CFU), small dense saccular colonies




2G.1.12


(alveolar-CFU), and colonies of mixed phe-notype with distinct budding (multipotent-CFU, Fig. 2G.1.2). Under these conditions,MSC differentiate into lipid-filled fibroblastsand myofibroblasts. In stromal-free expansioncultures, EpiSPC generate smaller sphericalcolonies, which can be enzymatically pas-saged on a weekly basis.

Time ConsiderationsTemporal analysis of colony formation

in this organotypic assay system (BasicProtocol 2) results in the emergence ofcolonies after 5 days in culture, and their con-tinued expansion and differentiation over a2-week period. In stromal-free expansion cul-tures (Basic Protocol 3), optimal colony for-mation for serial propagation and re-seeding ofCFU is achieved when colonies are harvestedafter 1 week. After this point, colonies begindifferentiate and deteriorate, and the recloningefficiency of dissociated colonies is substan-tially reduced.

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McQualter, J.L., Brouard, N., Williams, B., Baird,B.N., Sims-Lucas, S., Yuen, K., Nilsson,S.K., Simmons, P.J., and Bertoncello, I. 2009.Endogenous fibroblastic progenitor cells in theadult mouse lung are highly enriched in thesca-1 positive cell fraction. Stem Cells 27:623-633.

McQualter, J.L., Yuen, K., Williams, B., andBertoncello, I. 2010. Evidence of an epithe-lial stem/progenitor cell hierarchy in the adultmouse lung. Proc. Natl. Acad. Sci. U.S.A.167:1414-1419.

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Lung Epithelial Stem Cell