In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63α+ and small in the size (8 μm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin−/p63α−/vimentin+ cells with the size as small as 5 μm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.
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References
1.
LavkerR.M., TsengS.C., SunT.T.Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res, 78:433. 2004.
2.
SchermerA., GalvinS., SunT.-T.Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol, 103:49. 1986.
3.
ChenW.Y., MuiM.M., KaoW.W., LiuC.Y., TsengS.C.Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium. Curr Eye Res, 13:765. 1994.
4.
LiuC.-Y., ZhuG., ConverseR., KaoC.W.-C., NakamuraH., TsengS.C.G., MuiM.-M., SeyerJ., JusticeM.J., StechM.E.et al.Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12. J Biol Chem, 269:24627. 1994.
5.
MaticM., PetrovI.N., RosenfeldT., WolosinJ.M.Alterations in connexin expression and cell communication in healing corneal epithelium. Invest Ophthalmol Vis Sci, 38:600. 1997.
6.
CotsarelisG., ChengS.Z., DongG., SunT.-T., LavkerR.M.Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell, 57:201. 1989.
7.
RomanoA.C., EspanaE.M., YooS.H., BudakM.T., WolosinJ.M., TsengS.C.Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry. Invest Ophthalmol Vis Sci, 44:5125. 2003.
8.
De PaivaC.S., PflugfelderS.C., LiD.Q.Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Stem Cells, 24:368. 2006.
9.
EbatoB., FriendJ., ThoftR.A.Comparison of central and peripheral human corneal epithelium in tissue culture. Invest Ophthalmol Vis Sci, 28:1450. 1987.
10.
KruseF.E., TsengS.C.G.A tumor promoter-resistant subpopulation of progenitor cells is present in limbal epithelium more than corneal epithelium. Invest Ophthalmol Vis Sci, 34:2501. 1993.
11.
LindbergK., BrownM.E., ChavesH.V., KenyonK.R., RheinwaldJ.G.In vitro preparation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci, 34:2672. 1993.
12.
PellegriniG., GolisanoO., PaternaP., LambiaseA., BoniniS., RamaP., De LucaM.Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol, 145:769. 1999.
Di IorioE., BarbaroV., RuzzaA., PonzinD., PellegriniG., DeL.M.Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration. Proc Natl Acad Sci USA, 102:9523. 2005.
15.
WatanabeK., NishidaK., YamatoM., UmemotoT., SumideT., YamamotoK., MaedaN., WatanabeH., OkanoT., TanoY.Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2. FEBS Lett, 565:6. 2004.
16.
De PaivaC.S., ChenZ., CorralesR.M., PflugfelderS., LiD.-Q.ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells, 23:63. 2005.
17.
BudakM.T., AlpdoganO.S., ZhouM., LavkerR.M., AkinciM.A., WolosinJ.M.Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells. J Cell Sci, 118:1715. 2005.
18.
SteppM.A., ZhuL., SheppardD., CranfillR.Localized distribution of alpha 9 integrin in the cornea and changes in expression during corneal epithelial cell differentiation. J Histochem Cytochem, 43:353. 1995.
19.
ChenZ., De PaivaC.S., LuoL., KretzerF.L., PflugfelderS.C., LiD.Q.Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells, 22:355. 2004.
20.
HayashiR., YamatoM., SugiyamaH., SumideT., YangJ., OkanoT., YanoY., NishidaK.N-cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche. Stem Cells, 25:289. 2006.
21.
FuchsE., TumbarT., GuaschG.Socializing with the neighbors: stem cells and their niche. Cell, 116:769. 2004.
22.
XieT., LiL.Stem cells and their niche: an inseparable relationship. Development, 134:2001. 2007.
23.
LiL., XieT.Stem cell niche: structure and function. Annu Rev Cell Dev Biol, 21:605. 2005.
24.
DuaH.S., ShanmuganathanV.A., Powell-RichardsA., TiqheP.J., JosephA.Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. Br J Ophthalmol, 89:529. 2005.
25.
ShorttA.J., SeckerG.A., MunroP.M., KhawP.T., TuftS.J., DanielsJ.T.Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cells, 25:1402. 2007.
26.
Schlotzer-SchrehardtU., DietrichT., SaitoK., SorokinL., SasakiT., PaulssonM., KruseF.E.Characterization of extracellular matrix components in the limbal epithelial stem cell compartment. Exp Eye Res, 85:845. 2007.
27.
EspanaE.M., RomanoA.C., KawakitaT., Di PascualeM., SmiddyR., TsengS.C.Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Invest Ophthalmol Vis Sci, 44:4275. 2003.
28.
KawakitaT., EspanaE.M., HeH., YehL.K., LiuC.Y., TsengS.C.Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells. Invest Ophthalmol Vis Sci, 45:3507. 2004.
BarrandonY., GreenH.Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA, 84:2302. 1987.
31.
MellerD., TsengS.C.G.Conjunctival epithelial cell differentiation on amniotic membrane. Invest Ophthalmol Vis Sci, 40:878. 1999.
32.
ShanmuganathanV.A., FosterT., KulkarniB.B., HopkinsonA., GrayT., PoweD.G., LoweJ., DuaH.S.Morphological characteristics of the limbal epithelial crypt. Br J Ophthalmol, 91:514. 2007.
33.
LiW., SabaterA.L., ChenY.T., HayashidaY., ChenS.Y., HeH., TsengS.C.A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Invest Ophthalmol Vis Sci, 48:614. 2007.
34.
McKeonF.p63 and the epithelial stem cell: more than status quo?Genes Dev, 18:465. 2004.
KawakitaT., ShimmuraS., HigaK., EspanaE.M., HeH., ShimazakiJ., TsubotaK., TsengS.C.Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets. Invest Ophthalmol Vis Sci, 50:4611. 2009.
37.
KawakitaT., EspanaE.M., HeH., YehL.K., LiuC.Y., TsengS.C.Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells. Invest Ophthalmol Vis Sci, 45:3507. 2004.
38.
KawakitaT., ShimmuraS., HorniaA., HigaK., TsengS.C.Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell. J Cell Mol Med, 12:1303. 2008.
39.
KawakitaT., EspanaE.M., HeH., YehL.-K., LiuC.-Y., TsengS.C.G.Epidermal differentiation promoted by high calcium serum free condition in mouse corneal/limbal epithelial culture. Invest Ophthalmol Vis Sci, 45:3507. 2005.
40.
SpurrS.J., GipsonI.K.Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone. Invest Ophthalmol Vis Sci, 26:818. 1985.
41.
GipsonI.K.The epithelial basement membrane zone of the limbus. Eye, 3,Pt 2:132. 1989.
42.
YeungA.M., Schlotzer-SchrehardtU., KulkarniB., TintN.L., HopkinsonA., DuaH.S.Limbal epithelial crypt: a model for corneal epithelial maintenance and novel limbal regional variations. Arch Ophthalmol, 126:665. 2008.
43.
TuoriA., UusitaloH., BurgesonR.E., TerttunenJ., VirtanenI.The immunohistochemical composition of the human corneal basement membrane. Cornea, 15:286. 1996.
44.
LiW., HeH., KuoC.L., GaoY., KawakitaT., TsengS.C.Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. Invest Ophthalmol Vis Sci, 47:2381. 2006.
45.
KosterM.I., KimS., MillsA.A., DeMayoF.J., RoopD.R.p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev, 18:126. 2004.
46.
SenooM., PintoF., CrumC.P., McKeonF.p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell, 129:523. 2007.
47.
BarbaroV., TestaA., DiI.E., MavilioF., PellegriniG., DeL.M.C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells. J Cell Biol, 177:1037. 2007.
48.
O'RourkeJ., YuanR., DeWilleJ.CCAAT/enhancer-binding protein-delta (C/EBP-delta) is induced in growth-arrested mouse mammary epithelial cells. J Biol Chem, 272:6291. 1997.
49.
HuttJ.A., O'RourkeJ.P., DeWilleJ.Signal transducer and activator of transcription 3 activates CCAAT enhancer-binding protein delta gene transcription in G0 growth-arrested mouse mammary epithelial cells and in involuting mouse mammary gland. J Biol Chem, 275:29123. 2000.
50.
ZhaoX., DasA.V., ThoresonW.B., JamesJ., WattnemT.E., Rodriguez-SierraJ., AhmadI.Adult corneal limbal epithelium: a model for studying neural potential of non-neural stem cells/progenitors. Dev Biol, 250:317. 2002.
DravidaS., PalR., KhannaA., TipnisS.P., RavindranG., KhanF.The transdifferentiation potential of limbal fibroblast-like cells. Brain Res Dev Brain Res, 160:239. 2005.
53.
UmemotoT., YamatoM., NishidaK., YangJ., TanoY., OkanoT.Limbal epithelial side-population cells have stem cell-like properties, including quiescent state. Stem Cells, 24:86. 2006.
54.
PolisettyN., FatimaA., MadhiraS.L., SangwanV.S., VemugantiG.K.Mesenchymal cells from limbal stroma of human eye. Mol Vis, 14:431. 2008.
55.
KellnerJ.C., CoulombeP.A.Preview. SKPing a hurdle: Sox2 and adult dermal stem cells. Cell Stem Cell, 5:569. 2009.
56.
SamuelS., WalshR., WebbJ., RobinsA., PottenC., MahidaY.R.Characterization of putative stem cells in isolated human colonic crypt epithelial cells and their interactions with myofibroblasts. Am J Physiol Cell Physiol, 296:C296. 2009.
57.
ButlerJ.M., NolanD.J., VertesE.L., Varnum-FinneyB., KobayashiH., HooperA.T., SeandelM., ShidoK., WhiteI.A., KobayashiM.et al.Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 6:251. 2010.
58.
PellegriniG., TraversoC.E., FranziA.T., ZingirianM., CanceddaR., De LucaM.Long-term restoration of damaged corneal surface with autologous cultivated corneal epithelium. Lancet, 349:990. 1997.
59.
SchwabI.R., ReyesM., IsseroffR.R.Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea, 19:421. 2000.
60.
TsaiR.J.F., LiL.-M., ChenJ.-K.Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med, 343:86. 2000.
61.
SangwanV.S., VemugantiG.K., IftekharG., BansalA.K., RaoG.N.Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea, 22:478. 2003.
62.
NakamuraT., InatomiT., SotozonoC., KoizumiN., KinoshitaS.Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease. Acta Ophthalmol Scand, 82:468. 2004.
63.
NakamuraT., InatomiT., SotozonoC., AngL.P., KoizumiN., YokoiN., KinoshitaS.Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease. Ophthalmology, 113:1765. 2006.
64.
ShimazakiJ., AibaM., GotoE., KatoN., ShimmuraS., TsubotaK.Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology, 109:1285. 2002.
65.
De LucaM., PellegriniG., GreenH.Regeneration of squamous epithelia from stem cells of cultured grafts. Regen Med, 1:45. 2006.
66.
RamaP., MatuskaS., PaganoniG., SpinelliA., DeL.M., PellegriniG.Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med, 363:147. 2010.
67.
LiW., HayashidaY., HeH., KuoC.L., TsengS.C.The fate of limbal epithelial progenitor cells during explant culture on intact amniotic membrane. Invest Ophthalmol Vis Sci, 48:605. 2007.
68.
GrueterichM., EspanaE., TsengS.C.Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane. Invest Ophthalmol Vis Sci, 43:63. 2002.
69.
Hernandez GalindoE.E., TheissC., SteuhlK.P., MellerD.Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane. Exp Eye Res, 76:303. 2003.
70.
Hernandez GalindoE.E., TheissC., SteuhlK.P., MellerD.Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane. Invest Ophthalmol Vis Sci, 44:2959. 2003.
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