Characterization of Putative Stem Cell Phenotype in Human Limbal Epithelia - 《stemcells》2004-2005文献干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 24 下一页

返回列表

查看: 288224\|回复: 237	go Characterization of Putative Stem Cell Phenotype in Human Limbal Epithelia [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, and

b Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
$ f( F2 [- r7 k) Y! ?# u6 ?, a7 ~
Key Words. Cornea ? Limbus ? Epithelium ? Stem cells ? Stem cell phenotype9 f  C' D  Z3 d9 ~( [
3 M8 I) j0 U, \# [( [6 y0 z8 h
De-Quan Li, M.D., Ph.D., Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, NC-205, Houston, Texas 77030, USA. Telephone: 713-798-1123; Fax: 713-798-1457; e-mail: dequanl@bcm.tmc.edu4 w9 E! g* I( R3 _9 N

ABSTRACT

Recent data indicate that not only embryonic stem cells (SCs) are pluripotent, but there are also tissue-specific SCs residing in many adult tissues, including blood, liver, brain, skeletal muscle, intestine, skin, and cornea. SCs derived from these adult tissues have the ability to regenerate these tissues, and they offer great therapeutic potential for treating diseased and damaged tissues and serving as gene delivery vehicles . Widely accepted criteria for defining SCs are: A) slow cycling or long cell cycle time during homeostasis in vivo; B) poorly differentiated with primitive cytoplasm; C) high capacity for error-free self renewal, and D) activation to proliferate by wounding or placement in culture .! o* l8 p+ s. s
! \4 u( m, |: s$ @( O; r
The ocular surface is an ideal region to study epithelial SC biology because of the unique spatial arrangement of SCs and transient amplifying cells. The compartmentalization of the corneal epithelial SCs within the limbus provides a valuable opportunity to study the behavior of adult SCs . The supporting data for the limbal location of corneal epithelial SCs include: A) the limbal basal cells lack the corneal epithelial differentiation-associated keratin pair K3  and K12 ; B) the limbal basal epithelium contains slow-cycling cells identified as the "label-retaining cells" following pulse-chase labeling of all cells with a DNA precursor, such as -thymidine or bromodeoxyuridine (BrdU) , and the limbal basal epithelium exhibits high proliferative potential in culture ; C) experimental studies and clinical observations show abnormal corneal epithelial wound healing with conjunctivalization, vascularization, and chronic inflammation when the limbal epithelium is partially  or completely defective , and D) limbal cells are essential for the long-term maintenance of the central corneal epithelium, and they can be used to reconstitute the entire corneal epithelium in patients with limbal SC deficiencies . Collectively, these data leave little doubt that corneal epithelial SCs reside in the limbus.
0 @6 k% ~& f! k. O$ a3 Y7 f
To date, no direct methods have been established to identify the corneal SCs because of the lack of specific molecular markers, although a variety of SC-associated markers have been proposed. The major markers proposed for epithelial SCs in ocular or non-ocular tissues in the past decade can be categorized into at least three groups: A) nuclear proteins such as the transcription factor p63; B) cell membrane or transmembrane proteins including integrins (integrin ?1, 6, 9), receptors (epidermal growth factor receptor , transferrin receptor CD71), and drug resistance transporters (ABCG-2), and C) cytoplasmic proteins such as cytokeratins (CK) (cytokeratin 19), nestin, and -enolase. In addition, a variety of differentiation markers have also been proposed to distinguish the SCs from differentiated cells. These include cytokeratins K3 and K12, involucrin, intercellular adhesive molecule E-cadherin, and gap junction protein connexin 43, etc. This study was conducted to evaluate currently proposed molecular markers related to SC properties with the intention to characterize a putative SC phenotype in human limbal epithelia. While no single marker can identify adult SCs to date, characterization of a putative SC phenotype may shed light on the understanding of SC features.% o, c& u9 {+ u( c9 C5 \# I  Z2 z

MATERIALS AND METHODS

A New Look at Limbal Structure and Ultrastructure
: h7 t" }0 H( ?& |' u7 L2 c
Previously reported studies  evaluated limbal structure in radially oriented tissue sections (Fig. 1A). In order to completely evaluate the complex architecture of the limbal palisades of Vogt, we also evaluated cross-sections of the superior limbus (Fig. 1B). The palisades of Vogt in the superior limbus are structured with papilla-like columns, with smaller densely packed basal cells, larger less densely packed cells within the columns, and flattened squamous cells in the apical layers. There are also blood vessels, nerves, and connective tissue between the epithelial columns (Fig. 1B).
% m% Y$ p% H' T: H+ l
Transmission electron microscopy (TEM) shows that the basal cells of central corneal epithelium are columnar cells with a low nucleus/cytoplasm (N/C) ratio (Fig. 2A). The nucleus has loose chromatin and a pronounced nucleolus with a number of coiled DNA (heterochromatin). The cytoplasm of these cells contains a large number of ribosomes and tonofilaments. The basal epithelial cells are connected to the Bowman’s membrane by hemidesmosomes (Fig. 2B). In contrast, the basal cells of limbal epithelium are smaller with a larger N/C ratio (Fig. 2E). Their nucleus has more euchromatin as open DNA and barely detectable nucleolus. In contrast to the central cornea, the cytoplasm of the limbal basal cells contains more tonofilaments, and there is no underlying Bowman’s membrane. The limbal basal cells have basal invaginations through the basement membrane to connect with the underlying matrix, which contains collagen, fibrils, dilated capillaries, and some macrophages (Fig. 2F). This structure provides an ideal environment for cell nutrition. The peripheral corneal structure is between the central cornea and limbus. Their basal epithelial cells have an intermediate size between corneal and limbal epithelial cells, and the nuclei have less coiled DNA than corneal cells. They interdigitate with the basement membrane by basal infoldings (Figs. 2C, 2D).5 U' w, y" W  k( D

Figure 2. Transmission electron microscopic images of the central cornea (A, B), peripheral cornea (C, D) and limbus (E, F). The basal cells (B) of central corneal epithelium are large columnar cells with low N/C ratio. The nucleus has loose chromatin and a pronounced nucleolus with coiled DNA, and the cytoplasm contains ribosomes and tonofilament. The basal cells (F) in limbal epithelia are smaller with a large N/C ratio, and they have barely detectable nucleolus with open DNA. The morphology of peripheral corneal epithelia is between central corneal and limbal epithelia (C, D). There is a Bowman’s membrane (A, C) under the central and peripheral corneal basal epithelia with hemidesomosomes (B) or basal infoldings (D). The basal limbal epithelia (E) connect to basement membrane with invaginations, and the underneath stroma contains collagen and dilated capillaries. BM = Bowman’s membrane; Cap = capillaries; Coil = coiled DNA; Col = collagen; Fold = basal infoldings; He = hemidesmosome; Inv = invaginations; N = nucleolus; Ri = ribosomes; To = tonofilaments.8 f4 U% u/ ^9 l2 K
9 x: g- N' ~; u3 v7 \0 F& H$ d
Immunostaining of SC-Associated Markers in the Cornea and Limbus
% V6 L5 o" G/ [! g/ ?
Nuclear protein p63 was recently proposed as an SC marker to identify epidermal SCs as well as limbal epithelial SCs . Using the mAb clone 4A4, which reacts with all p63 isoforms , the p63 protein was immunodetected primarily in the nuclei of limbal epithelial basal layer, but not in most limbal suprabasal and corneal epithelial cells (Fig. 3). Immunohistochemical staining with the same antibody revealed more clearly that within limbal basal layer, patches of p63 negative cells were interspersed with more numerous p63 positive cells (Fig. 4). ABCG2, a member of the ATP binding cassette (ABC) transporters, has been proposed as a universal marker for SCs . Using mAb clone BXP-21 , the ABCG2 transporter protein was primarily immunodetected in the cell membrane and cytoplasm of certain limbal basal epithelial cells, but not in most limbal suprabasal cells and corneal epithelial cells (Fig. 3). There were ABCG2 negative cells interspersed with the ABCG2 positive cells in the basal layer (Fig. 4). Interestingly, integrin 9 was also immunodetected at the cell membranes and cytoplasm of certain limbal basal epithelial cells but not in limbal suprabasal and cornea cells (Figs. 3, 4). This is a similar expression pattern to p63 and ABCG2.
' w# [. t  d5 t( z8 t0 O2 ~
Figure 3. Immunofluorescent staining of proposed SC-associated markers, p63, ABCG2, integrin 9, integrin ?1, EGFR, K19, enolase-, CD71, and integrin 6 on frozen sections of limbus (left panels) and cornea (right panels). Hoechst 33342 staining was used as counterstaining. Magnification: x200.

Figure 4. Immunohistochemical staining for p63, ABCG2, and integrin 9 on limbal frozen sections. Only certain basal cells of limbal epithelia expressed nuclear p63, ABCG2, and integrin 9. Arrows indicate the positive stained cells.  o- c6 o$ \& p! R  G

Integrin ?1 and 6 were previously proposed as putative SC markers for epidermal keratinocytes . Our results showed that integrin ?1 was abundantly expressed by the cell membranes of corneal and limbal epithelia with a much higher level of expression by the limbal basal cells (Fig. 3). In contrast, integrin 6 antibody strongly stained the cell membranes of suprabasal layers of corneal and limbal epithelia. It did not stain the basal cell layers of the limbal epithelia (Fig. 3). The EGFR antibody stained the cell membranes of limbal basal epithelia much stronger than the suprabasal cells, and it also strongly stained most corneal epithelial cells (Fig. 3), a pattern similar to integrin ?1. Transferrin receptor CD71 mAb strongly stained the cell membranes of certain basal cells in the limbus and most corneal epithelial cells (Fig. 3). Cytokeratin 19 (K19), a proposed marker for skin hair follicle SCs , was expressed in the cytoplasm of basal epithelial cells more strongly than the suprabasal cells in limbus. K19 was also expressed by most corneal epithelial cells. Enolase- was largely expressed by limbal basal epithelial cells, although some limbal suprabasal and corneal cells were stained (Fig. 3). The immunostaining patterns of these proposed SC-associated markers by corneal and limbal epithelial cells are summarized in Table 2.

Table 2. Localization of sc-associated markers
* z7 b3 o; C. y1 h6 f
Immunostaining of Differentiation Associated Markers in the Cornea and Limbus

Cytokeratin 3 (K3), a corneal specific marker , was strongly expressed by all corneal epithelia and the superficial limbal epithelial cells. Involucrin antibody strongly stained only superficial epithelial cells in the cornea and limbus. Neither antibody stained the basal layer of the limbal epithelium (Fig. 5).  k+ A' I4 Y6 Q2 |/ H; i2 z, h

Figure 5. Immunofluorescent staining for differentiation associated markers, K3, involucrin, connexin 43, E-cadherin, and nestin on frozen sections of limbus (left panels) and cornea (right panels). Hoechst 33342 was used as a counterstaining. Magnification: x200.. R; v3 L6 @- _8 m# ?

The absence of gap junction components, such as connexin 43 and 50, was recognized as a feature of SCs . The connexin 43 antibody stained membranes of suprabasal epithelia, but not the basal layer of the limbus (Fig. 5). E-cadherin had a similar expression pattern to connexin 43. This antibody only stained suprabasal epithelia of the cornea and limbus, but not the basal layer of the limbal epithelia (Fig. 5). Interestingly, nestin, a neuron SC marker, was expressed in the cytoplasm of superficial corneal and limbal epithelia. The limbal basal cells were totally nestin negative (Fig. 5). These immunostaining patterns suggest that connexin 43, E-cadherin, and nestin may serve as differentiation markers for corneal epithelia. The localization of these differentiation associated markers in corneal and limbal epithelia is summarized in Table 3.( |. m) \* E% m% ?# v

Table 3. Localization of differentiation associated markers

Gene Expression of Molecular Markers in Corneal and Limbal Epithelia) g9 ~5 n7 J2 Z5 A6 R9 z, [. v
, S4 R/ v0 Y4 E" m9 Z
With the housekeeping gene GAPDH as an internal control, semi-quantitative RT-PCR disclosed a differential expression pattern of SC associated markers and differentiation markers by human corneal and limbal epithelia. The expressions of Np63, ABCG2, and integrin 9 transcripts were markedly higher by limbal epithelia than corneal epithelia (Fig. 6). Although some bands of Np63 and integrin 9 were clearly visible in one corneal sample (Fig. 6), this may have resulted from sample overlapping of the peripheral cornea and limbus when they were separated by a trephine. There was no significant difference in integrin ?1, EGFR, K19, and enolase- mRNA between limbal and corneal epithelia (data not shown). The mRNA of corneal specific differentiation markers, K3 and K12, were abundantly expressed by the corneal epithelia but expression was weak or undetectable in the limbal epithelia (Fig. 6). Expression of connexin 43 mRNA was also barely detectable in limbal epithelia, but was abundant in corneal epithelia (Fig. 6).

Figure 6. Semi-quantitative RT-PCR for SC-associated markers, p63 (440 bp), ABCG2 (379bp), and integrin 9 (123 bp), and differentiation associated markers, K3 (145bp), K12 (150 bp), and connexin 43 (154 bp) expressed by corneal (C1, C2, C3) and limbal (L1, L2, L3) epithelia from three fresh human corneal and limbal tissues. A 100 bp DNA ladder is shown in the first left lane. GAPDH, a housekeeping gene, was used as an internal control.

In Situ Hybridization of p63 Riboprobe in Corneal and Limbal Epithelia
6 L: x. O2 B6 X4 u' k" i
To confirm the p63 expression pattern, in situ hybridization with -labeled sense and antisense p63 RNA riboprobes was performed on cornea and limbal tissues. The radioautograms showed that strong p63 mRNA signals were only in the basal layer of limbal epithelia, but not in the suprabasal limbal and entire corneal epithelia (Figs. 7C, 7D), when the sections were hybridized with antisense riboprobes. In contrast, there was no signal in control sections hybridized with p63 sense riboprobes (Figs. 7A, 7B).6 x0 N2 n1 s( Q* K7 }- [9 \. @5 R

Figure 7. In situ hybridization of human limbal paraffin sections with -labeled p63 riboprobes. The strong p63 signals were only located in the basal layer of limbal epithelia when hybridized with antisense p63 riboprobe (C, x200 and D, x400). There was no signal detected in control sections which were hybridized with sense riboprobe (A, x200 and B, x400).9 P* V+ S( V/ U! U, |$ A- v+ ?
. D  \/ d* Z, u' r8 |& ?  U
DISCUSSION

We thank Dr. Mary Ann Stepp for kindly providing the integrin 9 antibody.4 {& @( Q* {4 K2 `
7 G  K; F, u2 I; O
This study was supported by NIH Grants, EY014553 (D.Q.L.) and EY11915 (S.C.P.), National Eye Institute, Bethesda, MD, a grant from Lions Eye Bank of Texas, an unrestricted grant from Research to Prevent Blindness, a post-doctoral research fellowship from Fight For Sight, the Oshman Foundation, and the William Stamps Farish Fund.
- J$ L# p$ f/ I' X
REFERENCES" _3 |' A' l3 ^- [: Y* J2 ?& j& Y# t
9 P' f  H, L5 X5 K3 a, e. y
Hall PA, Watt FM. Stem cells: the generation and maintenance of cellular diversity. Development 1989;106:619–633./ A& A5 Q  v4 n# n, m% ~* D. m# h) M! x

Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990;110:1001–1020.- z8 t( A4 }! N6 `" W/ \
: B* g: t8 G! F; C; M. Q/ N+ ?
Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function? Cell 2001;105:829–841.8 l, U- R" s7 |

Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.

Lavker RM, Sun TT. Epidermal stem cells: properties, markers, and location. Proc Natl Acad Sci USA 2000;97:13473–13475.+ z4 V8 s" Y( k. B( |9 h6 ^- i
" }: M9 p, {0 J, }" t5 `0 ~
Watt FM. Epidermal stem cells as targets for gene transfer. Hum Gene Ther 2000;11:2261–2266.
" {1 F8 i! G; O1 k
Cotsarelis G, Kaur P, Dhouailly D et al. Epithelial stem cells in the skin: definition, markers, localization and functions. Exp Dermatol 1999;8:80–88.7 M! Y3 n3 c) q8 n( m8 p% m1 R; S/ O

Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol 2000;44:415–425.
( a+ a! J* @5 P3 \1 F$ Z4 M
Tseng SC. Concept and application of limbal stem cells. Eye 1989;3:141–157.
1 g) a# U: ~& f
Schermer A, Galvin S, Sun TT. 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 1986;103:49–62.

Kurpakus MA, Maniaci MT, Esco M. Expression of keratins K12, K4, and K14 during development of ocular surface epithelium. Curr Eye Res 1994;13:805–814.

Cotsarelis G, Cheng SZ, Dong G et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201–209.& c! {- F0 K8 z% q- z# `" B

Ebato B, Friend J, Thoft RA. Comparison of limbal and peripheral human corneal epithelium in tissue culture. Invest Ophthalmol Vis Sci 1988;29:1533–1537.0 `  J; t: u) v  S& w

Kruse FE, Tseng SC. A tumor promoter-resistant subpopulation of progenitor cells is larger in limbal epithelium than in corneal epithelium. Invest Ophthalmol Vis Sci 1993;34:2501–2511.( W" z% D6 S" b* L% F4 D

Pellegrini G, Golisano O, Paterna P et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 1999;145:769–782.3 ]2 h3 H2 K6 B  ?8 N
; e% ~9 i8 s/ [& U) k
Chen JJ, Tseng SC. Corneal epithelial wound healing in partial limbal deficiency. Invest Ophthalmol Vis Sci 1990;31:1301–1314.
8 J( L7 Z& u! }# g3 F
Chen JJ, Tseng SC. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32:2219–2233.2 S( l& W4 H: Z% L# T0 h* M, h
  I9 n/ h2 I7 W( |/ b- k
Huang AJ, Tseng SC. Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32:96–105.0 p- E9 _( r4 H' D* s
/ W7 {5 J. ^" |
Jenkins C, Tuft S, Liu C et al. Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy. Eye 1993;7:629–633.

Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86–93.4 M4 s8 N( T7 G+ @7 f
7 \1 i3 J( I1 [0 t: t4 G3 [9 V, f
Li DQ, Tseng SC. Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. J Cell Physiol 1995;163:61–79.3 [9 i- N% w/ B1 D# x/ Z" c6 b

Liu Z, Carvajal M, Carraway CA et al. Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia. Cornea 2001;20:81–85.

Yoshino K, Tseng SC, Pflugfelder SC. Substrate modulation of morphology, growth, and tear protein production by cultured human lacrimal gland epithelial cells. Exp Cell Res 1995;220:138–151.

Li DQ, Lokeshwar BL, Solomon A et al. Regulation of MMP-9 production by human corneal epithelial cells. Exp Eye Res 2001;73:449–459.

Goldberg MF, Bron AJ. Limbal palisades of Vogt. Trans Am Ophthalmol Soc 1982;80:155–171.( e, t, B5 X5 o+ ~3 p' D( k3 L" r* N

Gipson IK. The epithelial basement membrane zone of the limbus. Eye 1989;3:132–140.
8 A, F* L% G. e$ U
Pellegrini G, Dellambra E, Golisano O et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 2001;98:3156–3161.4 Z  G; E% g( W, G; z/ X

Yang A, Schweitzer R, Sun D et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999;398:714–718.1 n' n# @4 x, O% C$ Q0 ~( \8 V
" W: b7 i* `/ t3 r* ^2 O
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.9 r3 O" v2 o/ \- G

Maliepaard M, Scheffer GL, Faneyte IF et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 2001;61:3458–3464.

Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713–724.

Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 1998;95:3902–3907.0 b% X8 t. `% T7 V7 k
0 X" q$ N) |' H1 }! K
Michel M, Torok N, Godbout MJ et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 1996;109:1017–1028.
/ _3 e: p- O5 {
Matic M, Petrov IN, Chen S et al. Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity. Differentiation 1997;61:251–260.
4 \  `- V  ]5 g) r$ S2 C
Yang A, Kaghad M, Wang Y et al. p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998;2:305–316.1 u4 d) e8 X# H" T2 a" R: W

Barbareschi M, Pecciarini L, Cangi MG et al. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol 2001;25:1054–1060.
- w- t8 \3 k! s! k9 D- v2 Q
Parsa R, Yang A, McKeon F et al. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 1999;113:1099–1105.

Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.
8 Q9 h1 ~( Z0 [
Stepp MA, Zhu L, Sheppard D et al. Localized distribution of alpha 9 integrin in the cornea and changes in expression during corneal epithelial cell differentiation. J Histochem Cytochem 1995;43:353–362.

Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell 1995;80:83–93.. X) a0 p+ F/ y1 m, [; T/ X
  e( }7 _2 `+ c) B4 t
Watt FM. Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 1998;353:831–837.
/ B7 h7 k: ^4 T/ |" U5 l
Kaur P, Li A. Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. J Invest Dermatol 2000;114:413–420.
$ b% P, l0 ~: N* x  `. N8 E5 o
Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 2000;97:10960–10965.
* g  X% }- y2 K* y  i
Zieske JD, Wasson M. Regional variation in distribution of EGF receptor in developing and adult corneal epithelium. J Cell Sci 1993;106:145–152.
  w! }' O" h2 ?- z) V! R& K0 V
Zieske JD, Bukusoglu G, Yankauckas MA. Characterization of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci 1992;33:143–152.

Chung EH, DeGregorio PG, Wasson M et al. Epithelial regeneration after limbus-to-limbus debridement. Expression of alpha-enolase in stem and transient amplifying cells. Invest Ophthalmol Vis Sci 1995;36:1336–1343.

Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585–595.  y0 C& l) h' S' T
5 s0 A3 a" q( Y& j
Scott RA, Lauweryns B, Snead DM et al. E-cadherin distribution and epithelial basement membrane characteristics of the normal human conjunctiva and cornea. Eye 1997;11:607–612.6 a6 L2 E& f8 D+ }
+ i; f/ R- \" o, H
Gumbiner BM. Regulation of cadherin adhesive activity. J Cell Biol 2000;148:399–404.4 x; b/ N6 p) k5 E; z% a

Liu CY, Zhu G, Converse R et al. Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12. J Biol Chem 1994;269:24627–24636.
+ O$ ~' A  d$ ^+ f2 L! G
Watt FM, Green H. Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol 1981;90:738–742.

Banks-Schlegel S, Green H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia. J Cell Biol 1981;90:732–737.(Zhuo Chena, Cintia S. de )

收藏0 分享0 顶0 踩0