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Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development
Journal Pre-proof Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development Zhi Hou Guo, Yi Ming Zeng, Jun Sheng Lin PII:
S0014-4835(19)30318-5
DOI:
https://doi.org/10.1016/j.exer.2020.107915
Reference:
YEXER 107915
To appear in:
Experimental Eye Research
Received Date: 5 May 2019 Revised Date:
20 December 2019
Accepted Date: 2 January 2020
Please cite this article as: Guo, Z.H., Zeng, Y.M., Lin, J.S., Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development, Experimental Eye Research (2020), doi: https://doi.org/10.1016/j.exer.2020.107915. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Research article
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Dynamic Spatiotemporal Expression Pattern of Limbal Stem Cell Putative Biomarkers During Mouse Development
5
Zhi Hou Guo1，2, Yi Ming Zeng3, Jun Sheng Lin1*
3
6 7 8 9 10 11 12 13 14 15
，
1. School
of
Medicine,
Huaqiao
University,
Quanzhou
362021,
Fujian,
China;
[email protected] (Z.H.G.); [email protected] (J.S.L); 2. Stem cell laboratory, The second affiliated Hospital of Fujian Medical University, Quanzhou 362000, China; [email protected] (Z.H.G.); 3. The second affiliated Hospital of Fujian Medical University, China; [email protected] (Z.Y.M); Corresponding to: Jun Sheng Lin* Email: [email protected]; Tel.: +86-595-2269-0889
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Abstract: Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to
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Keywords: Limbal stem cells; biomarkers; niche; spatiotemporal expression; stemness
the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim=p63>CK14>CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.
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Highlight:
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1. The differentiation of CECs is related to eye opening in mice. 2. Dynamic spatial expression pattern reveals a temporal restriction of LSCs putative biomarkers. 3. The order of Vim>p63>CK15>CK14 might represent the stemness degree in ocular surface during mouse development.
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1. Introduction Limbal stem cells (LSCs) are the basal cells of limbal epithelium with self-renewal, high-proliferation and differentiation potential, etc[1]. LSCs undergo a symmetric and/or asymmetric mitosis fashion to divide into the transient amplifying cells (TACs), which have limited stemness with the capable of detach from the basement membrane and centripetal migration. Subsequently, TACs anteriorly centripetally migrate and continuously divide into the terminally differentiated cells (TDCs) to maintain the homeostasis of corneal epithelium and to heal
up
the
wounded
area
during
physiological
and
pathophysiological
processes,
respectively[2-3]. The stemness of LSCs relies on the microenvironment of the LSCs niche, composed of the cells, extracellular matrix, and functional mediators, such as cytokines, growth factors, and exosomes, etc[4-6]. The isolation and identification of LSCs are mainly depended on the LSCs biomarkers. To date, several LSCs putative biomarkers, such as CK14[7], CK15[8], CK19[9], p63[10], ABCG2[9], etc. were reported. However, their specificity was still controversial. Lack of identification of specific LSCs biomarker is the bottleneck of basic research of the LSCs. Many reports have demonstrated that the microenvironment of stem cells niche was age-related. Zheng et al. found that the human periodontal ligament stem cells from young donors showed a stronger proliferation and differentiation capacity compared to those from aged donors, which were rejuvenated by exposure to the young extrinsic environment[11]. Okaley et al. reported that the functions of mouse hematopoietic stem cells, such as proliferation and differentiation, were influenced by the age-related hematopoietic stem cells niche cells[12]. Notara et al. comparatively analyzed the functions and morphology of the human corneas derived from the different age groups, they found that the surface area, degrees of arc occupied by limbal niche structures, and the colony forming efficiency of limbal epithelial cells (LECs) were reduced with aging. These indicated that the LSCs niche and proliferation were age-related[13]. The LSCs niche and the capacity of proliferation and differentiation were closely related to the stemness of LSCs[14-15]. Hence, the expression of LSCs biomarkers might be dynamic during development. As one of the common animal models, mice have controllable advantages in the study of LSCs biomarkers, such as the source of samples, age grouping and many other influencing factors compared with sampling of human limbus. The correlation between the mouse cornea development and both the LSCs niche morphology and the spatiotemporal expression patterns of LSCs biomarkers have not as far been investigated. By this study, we provide an insight into the spatiotemporal expression patterns of LSCs putative biomarkers during mouse development. The pre- and post-natal mouse corneas were stained
with
Haematoxylin-eosin
(HE),
4',6-diamidino-2-phenylindole
(DAPI),
and
immunohistochemistry (IHC). By combining the obtained data, the development process of mouse corneal and limbal epithelium was analyzed and the unique LSCs niche in mice that supports the stemness of LSCs was discussed. In addition, the time spectrum of the expression of putative biomarkers of mouse LSCs was revealed. These findings may help us understand the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness. Our data could be useful to determine a strategy for identifying LSCs at different physiological ages in the absence of a known lifelong LSCs-specific biomarker.
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2. Material and methods
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2.1 Ethics statement
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The postnatal 0d, 7d, 8d, 9d, 10d, 11d, 12d,13d, 14d, 28d, 56d, 70d and the pregnant ICR mice were housed，respectively, in the medical research facility at Huaqiao University. All experimental protocols and surgery followed the animal welfare of ethical regulations. The Institutional Animal Care and Use Committee (IACUC) of the Huaqiao University approved all animal protocols (A2018006). 2.2 Sample preparation Prenatal, obtained by caesarean section, and postnatal eyes were fixed with a pH 7.4 4% paraformaldehyde (AR1069, BOSTER) at 4℃ overnight. The tissues were dehydrated using an ethanol gradient (30 min each of 70%, 80%, 90%, 100% ethanol) and embedded in paraffin (39601095, Leica Biosystems). The paraffin-embedded tissue blocks were cut into serial 5 µm thickness using microtome (RM2235, Leica). These were further processed for HE, DAPI, and IHC staining. Three more pre- and post-natal mice were assayed at each timepoint. 2.3 HE staining Paraffin sections were deparaffinized using xylene for 10 min and rehydrated using an ethanol gradient (5 min each of 100%, 90%, 80%, 70% ethanol). Then washed with ddH2O and stained with hematoxylin (G1080, Solarbio) for 10 min, and washed with ddH2O following differentiation with 1% acid alcohol (G1861, Solarbio) for 5 s, washed with ddH2O for 10 min, and then counterstained with Eosin Y solution (G1140, Solarbio) for 2 min, washed with ddH2O for 5 min, dehydrated with 90%, 100% alcohols and cleared with xylene, and then mounted with polyvinylpyrrolidone mounting medium (C0185, Beyotime). 2.4 Immunohistochemistry staining Deparaffinization and rehydration were done with xylene and ethanol gradient (100%, 90%, 80%, 70% ethanol). Followed antigen retrieval with pH 6.0 sodium citrate buffer by heating (110℃, 5 min). Slides were blocked with 5% bovine serum albumin (E661003, Sangon Biotech) for 30min at room temperature (RT) and incubated with primary antibodies (Table 1) at 4℃ overnight, washed thrice with phosphate-buffered saline containing 0.05% Tween (PBST) for 5 min. They were then incubated with secondary antibodies (Table 1) for 1 h at RT, washed thrice with PBST for 5 min. DAPI (AR1177, BOSTER) was used for counterstaining. The slides were then washed and mounted with coverslips using the antifade mounting medium (P0126, Beyotime). The primary antibodies （Table 1） were detected by western blotting. The normal rabbit and mouse serum were used as primary antibodies negative controls for IHC staining.
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3. Results
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3.1 Morphological features of mouse limbus
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The HE staining was conducted for mouse limbus histological analysis. As our results (Fig. 1), the limbal epithelium was the thinnest. The epithelial cell layer was gradually increased from
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limbus to central cornea. The stroma showed about 3-4 times thickness of epithelium and the
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3.2 Spatial expression of LSCs putative biomarkers in mouse ocular surface
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Bowman's layer was not observed. Palisade of Vogt (POV) is a structure in the limbal epithelium which provided the microenvironment of LSCs niche to protect LSCs from the physical, chemical, and UV damaged so as to maintain the stemness of LSCs in human[16]. The limbal epithelium in our studied mice showed smoothly without detectable POV.
In order to investigate the expression pattern of putative biomarkers of LSCs, CK14, CK15, CK19, Vim, p63, and ABCG2 were used for IHC study. To accurately identify the LSCs, the corneal epithelial cells (CECs) biomarker (CK12) and conjunctival epithelial biomarker (CK13) were also used to exclude the influence of the CECs and conjunctival epithelial cells. As our 4-week aged mice corneas IHC staining results (Fig. 2), CK12 was specifically expressed in CECs whereas CK13 was in both the conjunctival epithelial cells and the superficial cells of the limbal epithelium. CK14 and CK15 were strongly expressed in both LECs and conjunctival epithelial cells. CK14 was also weakly expressed in basal cells of the corneal epithelium. Double IHC staining of CK13 and CK19 showed that the expression pattern of CK19 was highly similar to the CK13, which expressed in superficial cells of limbal epithelium and conjunctival epithelium. The expression of Vim was detected in the stroma of mouse ocular surface. However, it was not detectable in the epithelium. ABCG2 and p63 are mostly reported as stem cell biomarkers. We found that both of them were not only expressed in limbal basal epithelial cells but also in corneal basal epithelial cells (Fig. 3). 3.3 The development of mouse cornea We further isolated the corneas from the mice with different ages to investigate the histological characteristics of corneal development by HE and DAPI staining (Fig. 4, Fig. 5, Fig. A2.1, and Fig. A2.2). During E12-E16, the corneal epithelium was composed of the monolayer squamous cells (Fig. A2.1A-C and Fig. 4A-I). At E19, the corneal epithelium began to consist of the squamous and cuboidal epithelial cells (Fig. A2.1D and Fig. 4J-M). Underneath of epithelium, the stroma showed more cell layers, which were increased during E12-E19 (Fig. 4 and Fig. A2.1). The monolayer corneal endothelial cells emerged at E16 were gradually tight (Fig. 4G). Furthermore, the eyelids were first observable at E14, then underwent centripetal growth until closure during E14-E19 (Fig. 4 and Fig. A2.1). During P0-P7, the central corneal epithelium was similar to the limbal epithelium. However, the stromal cells at the central corneal region and limbal region showed tightly and loosely, respectively (Fig. A2.2A-B and Fig. 5A-F). At P14, the eyelids began to open, then the cell layers of central corneal epithelium were quickly increased. Whereas the limbal epithelium kept no obviously change (Fig. A2.2C and Fig. 5G-I). From P28 to P70, the squamous epithelial cells in superficial increased up to 3-4 layers, the cuboidal epithelial cells in suprabasal increased to 1-2 layers and the cell layers of the columnar epithelial cells in basal didn't increase (Fig. A2.2D-F and Fig. 5J-R). 3.4 Spatial expression of LSCs putative biomarkers during development To explore the spatial expression patterns of LSCs putative biomarkers during mouse cornea
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development, the mice corneas from designated ages were collected and the putative biomarkers
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4. Discussions
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(CK14, CK15, CK19, Vim, p63, Fzd7 and Actn1) were used for IHC staining. The primary antibodies showed a high specificity by western blotting (Fig. A1.1). The normal rabbit and mouse serum used as the primary antibodies for IHC staining showed good negative controls (Fig. A1.2). The expression of CK15 was emerged at E16. From E16 to E21, the expression of CK15 was detectable in epithelial cells overall the ocular surface and gradually enhanced (Fig. 6). At P7, CK15 was strongly expressed in both the superficial squamous epithelial cells and LECs. However, during P14-P70, CK15 was specifically detectable in LECs (Fig. 7A-B). It was found that the expression of CK15 was gradually weakened in CECs (P7-P13) until undetectable (P14), while it was persistently strongly expressed in LECs (Fig. 8A-B). The expression of CK14 was emerged at E12 (Fig. A2.3). From E12 to P7, CK14 was expressed in both the CECs and LECs, the expression was gradually enhanced (Fig. A2.3, A2.4). During P14-P70, the CK14 was weakly expressed in corneal epithelial basal cells and strongly expressed in LECs (Fig. A2.4). Additionally, the mice corneas on the time points of P8, P9, P10, P11, P12 and P13 were also collected for IHC staining to clarify the expression changes of CK14 in the CECs. The results (Fig. A2.5) showed that the expression of CK14 was gradually weakened in CECs from P7 to P14, while it was persistently strongly expressed in LECs. The expression of CK19 was emerged in superficial epithelial cells at E16 and gradually enhanced during prenatal. At P7, CK19 was strongly expressed in both the superficial squamous epithelial cells and LECs. During P14-P70, CK19 was strongly specifically detectable in the limbal superficial epithelial cells. We also found that the expression of CK19 was gradually weakened in corneal superficial epithelial cells (P7-P13) until undetectable (P14), while it was persistently strongly expressed in limbal superficial epithelial cells (Fig. A2.6-8). The expression of p63 was strongly detected in both CECs and LECs at E12, then gradually weakened from E12 to P70 (Fig. A2.9-11). Moreover, the expression of p63 showed stronger in corneal basal epithelial cells compared to the limbal basal epithelial cells (Fig. A2.10-11). At E12, Vim was strongly expressed in both CECs and LECs, then gradually weakened until undetectable at E19. During prenatal, Vim was strongly expressed in both corneal and limbal stroma tissues. During postnatal, the expression of Vim was gradually weakened in the corneal stroma, while it was persistently strongly expressed in the limbal stroma (Fig. A2.12-14). From E12 to P70, the expression of Fzd7 (Fig. A2.15-17) and Actn1(Fig. A2.18-20) were both strongly detectable in the epithelial cells overall the ocular surface. To determine the boundary of the cornea, limbus, and conjunctiva, CECs biomarker (CK12) and conjunctival epithelial cells biomarker (CK13) were also be used for IHC staining. The expression of CK12 was emerged in CECs at E19, then gradually enhanced from E19 to P70. During pre- to post-natal, CK12 was always specifically expressed in CECs (Fig. A2.21-23). The expression of CK13 was emerged in superficial epithelial cells at E19. The spatial expression patterns of CK13 was similar to CK19 during prenatal to postnatal (Fig. A2.24-26).
LSCs are the basal cells of limbal epithelium resided in the POV in human[17]. The limbal epithelium and stroma have the most cell layers and thickness in the human ocular surface[9].
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However, in our 4-week aged mouse model, the limbal epithelium and stroma have the least cell layers, which was consistent with the recent reports[18-19]. Lin et al.[20] and Yang et al.[21] reported that the morphology and thickness of human corneal and limbal epithelium were varied with the phases of development and growth. In our results, the limbal basal epithelium in 4 weeks old mouse was smooth and without POV structure (Fig. 1). To analyze whether the morphological differences were related to age, the mouse corneas of different ages were isolated for HE and DAPI staining (Fig. 4, Fig. 5, Fig. A2.1, and Fig. A2.2). The results showed that the mouse limbal epithelium and stroma were always the thinnest in ocular surface and no POV was observed in prenatal and postnatal. These were different from the well-known fact that the limbus in ocular surface with the thickest corrugated epithelium, known as the limbal POV, and without a distinctive Bowman's layer in human[9, 16]. As reported, the deep stromal location of limbal POV[16] and the distinctive Bowman's layer[22-23] provided the environment of protection from the potential light damage. Moreover, Grieve et al. demonstrated that the three-dimensional (3D) architecture of LSCs niche in the species of human, pig, and mouse appears associated with eye exposure to light[24]. Furthermore, the mouse was a nocturnal activity animal which adapted to live in low-light environments. Hence, the mouse limbus morphological feature observed in this study might be related to the adaptation of low-light environment. The cornea is composed of the epithelium, endothelium and stroma[25]. During the prenatal and neonatal (Fig. 4, Fig. 5, Fig. A2.1, and Fig. 2.2), the mouse cornea was composed of the monolayer of epithelial cells. At this stage, the development of mouse cornea was dominated by the stroma. The stroma was thickened with increasing of the cell layer, and the stromal cells were tight, providing the structural basis for the passage of light. As reported, the liquid-air interface promoted the differentiation of epithelial cells[26-27]. In our results, the eyelids of the mice were opened at P14, then the development of cornea was dominated by corneal epithelium (Fig. 5 and Fig. A2.2). It was found that after the eyelids opening, the CECs quickly go stratification in response to the liquid-air interface, suggesting that the rate of the differentiation of mouse CECs was increased. The corneal epithelium was thickened with the increasing of CECs layers. The thickening corneal epithelium and the tightening stroma, leading the ocular surface to be parallel with the posterior stroma from P14. This increased the transparency of mouse cornea, decreased the scattering of light, and promoted light transmission. All in all, our results supported that the liquid-air interface promotes the differentiation of CECs. To date, the LSCs specific biomarkers are still remained elusive[28-29]. The main method for the identification of LSCs is based on the co-expression of LSCs putative biomarkers and negative biomarkers[30]. In this study, the most promising LSCs putative biomarkers, CK14[7], CK15[31], CK19[9], Vim[32], p63[33], and ABCG2[34], expressed in human limbal basal cells were used for the spatial expression analysis of mouse LSCs. To exclude the CECs and conjunctival epithelial cells, the CECs differentiated biomarker (CK12) [9] and conjunctival epithelial cells biomarker (CK13) [35] were also used. In our 4 weeks old mice, the expression of these putative biomarkers was detectable not only in the LECs, but also in both the corneal basal cells (CK14, p63, ABCG2) and conjunctival epithelial cells (CK14, CK15, CK19, p63, ABCG2) (Fig. 2 and Fig. 3), indicating a unique spatial expression of mouse LSCs. Recently, Lee et al. reported the microenvironment, generated by bone marrow stem cells, for the regulation of normal
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hematopoietic and leukemia stem cells showed age-related. They found that the self-renewal of leukemia stem cells was higher in adult bone marrow than in neonatal bone marrow[36]. Sagga et al. comparatively analyzed the cell-cycle time of the limbal and corneal epithelia in young adult and aging mouse eyes, found that the cell cycle was significant faster in central corneal epithelium of aging eyes compared to young adult mice[37]. These indicated that age was an important factor influence of the stemness of LSCs. The unique spatial expression of mouse LSCs in our results might be age-related. To explore the spatial expression pattern of the putative biomarkers of LSCs during mouse development, the prenatal and postnatal of mice corneas were further collected for IHC staining under the same exposure time (Fig. 6-8 and Fig. A2.3-26). In current study, we found that the expression of p63 were detected throughout the ocular surface at all ageing groups, in which the highest expressed in cornea, the second in limbus, and the lowest in conjunctiva. This expression pattern of p63 was consistent with Hsueh et al. reported in rat[38] and Sonam et al. reported in Xenopus frogs[39], whereas contrary to Davies et al. reported in human[40]. The expression of CK15 were initially observed across the ocular surface in early mouse development, eventually confined to the limbal and conjunctival epithelium. It was contrast to the finding by Davies et al. reported in the developing human fetal and adult cornea[40]. CK14 was known as a LSCs putative biomarker specifically expressed in limbal epithelial basal cells in human[41]. However, in our result, the expression of CK14 were detectable across the ocular surface and then progressively weakened in CECs, which were consistence with Richardson et al. reported[42]. In our IHC results, the presents of Vimentin expressing cells were observed in stroma during postnatal stage, which supported previous reported in a variety of vertebrates, including chick[43], cat[44], Xenopus frogs[39], and rabbit[45]. However, we found the expression of Vimentin were observed in CECs and LECs during prenatal stage. The expression of CK19 was observed in corneal superficial epithelial cells only during prenatal and
early postnatal stage, whereas persistently in limbal superficial epithelial cells. This expression pattern of CK19 was contrast to the Sonam et al. reported in larval versus adult Xenopus frogs[39]. Moreover, we further analyzed the dynamic expression of the examined LSCs putative biomarkers in different timepoints during mouse development to understand the dynamic stemness of LSCs and the temporal restriction of LSCs putative biomarkers. Before E19, the expression of LSCs' putative
biomarkers (CK14, CK15, CK19, p63, Fzd7, Actn1 and Vim) were detectable in both CECs and LECs, while the differentiation biomarker, CK12, had not yet begun to express, suggesting that CECs still had some degree of stemness. At E19, the expression of CK12 was detectable in CECs, indicating that CECs began to loss stemness. Just at the same time point, the expression of Vim was progressively weakened and eventually disappeared in CECs while others examined LSCs putative biomarkers (CK14, CK15, CK19, p63, Fzd7, and Actn1) remained detectable. This finding suggested that Vim might represent higher potency of stemness than the others examined LSCs putative biomarkers. During E19-P14, the expression of CK14 and CK15 in CECs were enhanced in E19-P7 and weakened in P7-P14, whereas the expression of p63 was progressively weakened, meaning that p63 represented weaker stemness degree than Vim, but higher than CK14 and CK15. At P14, the mouse eyelids were opened, CK14 was weakly expressed in corneal basal epithelial cells, whereas the expression of CK15 was not detectable in CECs, which suggested that the expression of CK15 represents weaker stemness degree than p63, but higher than CK14. As
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reported, CK19 was used as human LSCs putative biomarker[9]. However, Donisi et al.
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5. Conclusion
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demonstrated that CK19 was a specific biomarker of conjunctival epithelial cells for the diagnosis of limbal stem cells deficiency (LSCD)[46]. In our results, the spatial expression patterns of CK19 was similar to the CK13 during mouse development (Fig. A2.6-8 and Fig. A2.24-26), indicating that the CK19 was more suitable served as a conjunctival epithelial cell biomarker than LSCs biomarker. Fzd7[47] and Actn1[48] have been reported to be the LSCs putative biomarkers expressed in the limbal epithelial basal cells. However, in this study, the expression of Fzd7 and Actn1 were always detectable in both CECs and LECs during mouse development (Fig. A2.15-20), therefore, whether they could serve as mouse LSCs biomarkers need to be confirmed by more experimental data. Above all, our results suggest that the expression of the examined LSCs putative biomarkers in limbal epithelial basal cells (Fig. 9A) were emerged in chronological order as follows: Vim=p63>CK14>CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells (Fig. 9B) were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development (Fig. 9) also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others LSCs interested gene expression products, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mice and revealed the time spectrum of the expression of LSCs in mice, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.
Stemness of the LSCs in mice could be maintained without the structure of POV. The differentiation of CECs is related to eye opening in mice. Furthermore, our data demonstrated that the dynamic spatial expression patterns of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs putative biomarkers in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness. Appendix A. Supplementary data The following is the Supplementary data to this article. Funding
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No.2016YFE0101700), Huaqiao University Grant (No. 13Y0391), and the fourth round of joint
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Declaration of Interest
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This research was funded by the National Key R&D Program of China (Grant key research project jointly with health and education in Fujian province (No. WKJ2016-2-13).
None.
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[9] U. Schlötzer-Schrehardt, F.E. Kruse, Identification and characterization of limbal stem cells, EXP EYE RES, 81 (2005) 247-264. [10] M. Saghizadeh, S. Soleymani, A. Harounian, B. Bhakta, S.M. Troyanovsky, W.J. Brunken, G. Pellegrini, A.V. Ljubimov, Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy, MOL VIS, 17 (2011) 2177-90. [11] W. Zheng, S. Wang, D. Ma, L. Tang, Y. Duan, Y. Jin, Loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment, Tissue Eng Part A, 15 (2009) 2363-71. [12] E.J. Oakley, G. Van Zant, Age-related changes in niche cells influence hematopoietic stem cell function, CELL STEM CELL, 6 (2010) 93-4. [13] M. Notara, A.J. Shortt, A.R. O'Callaghan, J.T. Daniels, The impact of age on the physical and cellular properties of the human limbal stem cell niche, Age (Dordr), 35 (2013) 289-300. [14] W. Li, Y. Hayashida, Y.T. Chen, S.C. Tseng, Niche regulation of corneal epithelial stem cells at the limbus, CELL RES, 17 (2007) 26-36. [15] T.F. Gesteira, M. Sun, Y.M. Coulson-Thomas, Y. Yamaguchi, L.K. Yeh, V. Hascall, V.J. Coulson-Thomas, Hyaluronan Rich Microenvironment in the Limbal Stem Cell Niche Regulates Limbal Stem Cell Differentiation, Invest Ophthalmol Vis Sci, 58 (2017) 4407-4421. [16] N. Di Girolamo, Moving epithelia: Tracking the fate of mammalian limbal epithelial stem cells, PROG RETIN EYE RES, 48 (2015) 203-25. [17] P. Ordonez, N. Di Girolamo, Limbal epithelial stem cells: role of the niche microenvironment, STEM CELLS, 30 (2012) 100-7.
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[18] J.T. Henriksson, A.M. McDermott, J.P. Bergmanson, Dimensions and morphology of the cornea in three strains of mice, Invest Ophthalmol Vis Sci, 50 (2009) 3648-54. [19] H. Zhang, L. Wang, Y. Xie, S. Liu, X. Deng, S. He, G. Chen, H. Liu, B. Yang, J. Zhang, S. Sun, X. Li, Z. Li, The measurement of corneal thickness from center to limbus in vivo in C57BL/6 and BALB/c mice using two-photon imaging, EXP EYE RES, 115 (2013) 255-62. [20] H.C. Lin, T.B. Tew, Y.T. Hsieh, S.Y. Lin, H.W. Chang, F.R. Hu, W.L. Chen, Using optical coherence tomography to assess the role of age and region in corneal epithelium and palisades of vogt, Medicine (Baltimore), 95 (2016) e4234. [21] Y. Yang, J. Hong, S.X. Deng, J. Xu, Age-related changes in human corneal epithelial thickness measured with anterior segment optical coherence tomography, Invest Ophthalmol Vis Sci, 55 (2014) 5032-8. [22] W.M. Townsend, The limbal palisades of Vogt, Trans Am Ophthalmol Soc, 89 (1991) 721-56. [23] M.F. Goldberg, A.J. Bron, Limbal palisades of Vogt, Trans Am Ophthalmol Soc, 80 (1982) 155-71. [24] K. Grieve, D. Ghoubay, C. Georgeon, O. Thouvenin, N. Bouheraoua, M. Paques, V.M. Borderie, Three-dimensional structure of the mammalian limbal stem cell niche, EXP EYE RES, 140 (2015) 75-84. [25] M.A. Dziasko, J.T. Daniels, Anatomical Features and Cell-Cell Interactions in the Human Limbal Epithelial Stem Cell Niche, OCUL SURF, 14 (2016) 322-30. [26] A. Ootani, S. Toda, K. Fujimoto, H. Sugihara, An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture, Biochem Biophys Res Commun, 271 (2000) 741-6. [27] C. Nossol, A.K. Diesing, N. Walk, H. Faber-Zuschratter, R. Hartig, A. Post, J. Kluess, H.J. Rothkotter, S. Kahlert, Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells
(IPEC),
HISTOCHEM CELL BIOL, 136 (2011) 103-15. [28] A.W. Joe, S.N. Yeung, Concise review: identifying limbal stem cells: classical concepts and new challenges, Stem Cells Transl Med, 3 (2014) 318-22. [29] J.R. Kasinathan, V.P. Namperumalsamy, M. Veerappan, G.P. Chidambaranathan, A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis, Microsc Res Tech, 79 (2016) 1165-1172. [30] U. Schlötzer-Schrehardt, T. Dietrich, K. Saito, L. Sorokin, T. Sasaki, M. Paulsson, F.E. Kruse, Characterization of extracellular matrix components in the limbal epithelial stem cell compartment, EXP EYE RES, 85 (2007) 845-860. [31] S. Yoshida, S. Shimmura, T. Kawakita, H. Miyashita, Den S, J. Shimazaki, K. Tsubota, Cytokeratin 15 can be used to identify the limbal phenotype in normal and diseased ocular surfaces, Invest Ophthalmol Vis Sci, 47 (2006) 4780-6. [32] Y. Hayashida, W. Li, Y.T. Chen, H. He, S.Y. Chen, A. Kheirkah, Y.T. Zhu, Y. Matsumoto, S.C. Tseng, Heterogeneity of limbal basal epithelial progenitor cells, CORNEA, 29 Suppl 1 (2010) S32-40. [33] P. Rama, S. Matuska, G. Paganoni, A. Spinelli, M. De Luca, G. Pellegrini, Limbal stem-cell
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therapy and long-term corneal regeneration, N Engl J Med, 363 (2010) 147-55. [34] Z. Chen, C.S. de Paiva, L. Luo, F.L. Kretzer, S.C. Pflugfelder, D.Q. Li, Characterization of putative stem cell phenotype in human limbal epithelia, STEM CELLS, 22 (2004) 355-66. [35] M. Poli, H. Janin, V. Justin, C. Auxenfans, C. Burillon, O. Damour, Keratin 13 immunostaining in corneal impression cytology for the diagnosis of limbal stem cell deficiency, Invest Ophthalmol Vis Sci, 52 (2011) 9411-5. [36] G.Y. Lee, S.Y. Jeong, H.R. Lee, I.H. Oh, Age-related differences in the bone marrow stem cell niche generate specialized microenvironments for the distinct regulation of normal hematopoietic and leukemia stem cells, Sci Rep, 9 (2019) 1007. [37] N. Sagga, L. Kuffova, N. Vargesson, L. Erskine, J.M. Collinson, Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice, STEM CELL RES, 33 (2018) 185-198. [38] Y.J. Hsueh, D.Y. Wang, C.C. Cheng, J.K. Chen, Age-related expressions of p63 and other keratinocyte stem cell markers in rat cornea, J BIOMED SCI, 11 (2004) 641-51. [39] S. Sonam, J.A. Srnak, K.J. Perry, J.J. Henry, Molecular markers for corneal epithelial cells in larval vs. adult Xenopus frogs, EXP EYE RES, 184 (2019) 107-125. [40] S.B. Davies, J. Chui, M.C. Madigan, J.M. Provis, D. Wakefield, N. Di Girolamo, Stem cell activity in the developing human cornea, STEM CELLS, 27 (2009) 2781-92. [41] E.C. Figueira, N. Di Girolamo, M.T. Coroneo, D. Wakefield, The phenotype of limbal epithelial stem cells, Invest Ophthalmol Vis Sci, 48 (2007) 144-56. [42] A. Richardson, E.P. Lobo, N.C. Delic, M.R. Myerscough, J.G. Lyons, D. Wakefield, N. Di Girolamo, Keratin-14-Positive Precursor Cells Spawn a Population of Migratory Corneal Epithelia that Maintain Tissue Mass throughout Life, STEM CELL REP, 9 (2017) 1081-1096. [43] E.R. Ritchey, K. Code, C.P. Zelinka, M.A. Scott, A.J. Fischer, The chicken cornea as a model of wound healing and neuronal re-innervation, MOL VIS, 17 (2011) 2440-54. [44] J.V. Jester, P.A. Barry, G.J. Lind, W.M. Petroll, R. Garana, H.D. Cavanagh, Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins, Invest Ophthalmol Vis Sci, 35 (1994) 730-43. [45] N. SundarRaj, J.D. Rizzo, S.C. Anderson, J.P. Gesiotto, Expression of vimentin by rabbit corneal epithelial cells during wound repair, CELL TISSUE RES, 267 (1992) 347-56. [46] P.M. Donisi, P. Rama, A. Fasolo, D. Ponzin, Analysis of limbal stem cell deficiency by corneal impression cytology, CORNEA, 22 (2003) 533-8. [47] H. Mei, M.N. Nakatsu, E.R. Baclagon, S.X. Deng, Frizzled 7 Maintains the Undifferentiated State of Human Limbal Stem/Progenitor Cells, STEM CELLS, 32 (2014) 938-945. [48] R. Sartaj, C. Zhang, P. Wan, Z. Pasha, V. Guaiquil, A. Liu, J. Liu, Y. Luo, E. Fuchs, M.I. Rosenblatt, Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers, Sci Rep, 7 (2017) 3793.
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Figure captions.
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Fig. 1 Morphological features of the cornea, limbus and conjunctiva in the 4 weeks old ICR mouse.
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Fig. 2 Double IHC staining of LSCs putative biomarkers on the 4 weeks old ICR mouse ocular
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Fig. 3 Double IHC staining of p63 and ABCG2 on the 4 weeks old ICR mouse ocular surface. The
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Fig. 4 DAPI staining of mouse ocular surface during the prenatal development. The development
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Fig. 5 DAPI staining of mouse ocular surface during the postnatal development. The development
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Fig. 6 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
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Fig. 7 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
(A) and (B) showed the boundaries of the cornea, limbus and conjunctiva in the mouse eye; (C) exhibited the thickest epithelium in mouse central cornea and the thinnest epithelium in limbus, which with no visible POV. Scale bar: 100 µm.
surface. The CK12 and CK13 were used for the identification of CECs and conjunctival epithelial cells. The detectable gene products of CK14, CK15, CK19, Vim, and p63 were not only limited to the limbal epithelial basal cells but also corneal epithelial basal cells and conjunctival epithelial cells. L: limbus, C: cornea, Cj: conjunctiva. Scale bar: 100 µm.
expression of LSCs putative biomarkers p63 and ABCG2 were detectable not only in limbal epithelial basal cells but also in corneal epithelial basal cells and conjunctival epithelial basal cells. L: limbus, C: cornea, Cj: conjunctiva. Scale bar: 100 µm.
was dominated by the stroma, the corneal and limbal epithelium were composed of the monolayer epithelial cells. The white arrow and dotted line indicated the emerged monolayer corneal endothelial cells at E16 (G). The left panel represents the magnified area of the green block within central cornea, and the right panel represents the magnified area of the red block surround limbus, respectively. Scale bar: middle panel, E12/E14=100 µm, E16/E19=200 µm; left and right panels, 20 µm.
was dominated by the stroma during neonatal. At P14, the eyelids were opened responding to the stimulation of the liquid-air interface. The development of cornea was then dominated by the epithelium, the speed of the differentiation of mouse CECs was increased and the epithelium was thickened with the increasing of CECs layers. The white dotted frame indicated the Eyelid (H and I). The left panel represents the magnified area of the green block within central cornea, and the right panel represents the magnified area of the red block surround limbus, respectively. Scale bar: middle panel, 500 µm; left and right panel, 100 µm.
during prenatal development. The expression of CK15 was emerged at E16 and gradually enhanced during prenatal development. Scale bar: E12/E14=100 µm, E16/E19/E21=200 µm.
during postnatal development. The expression of CK15 was strongly detectable in both the corneal superficial squamous epithelial cells and LECs at P7 and specifically detectable in LECs during P14-P70. (B) was the high magnification image of (A). The cornea in (B) was corresponds to the green frame in (A), the limbus in (B) was corresponds to the white frame in (A), the
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conjunctiva in (B) was corresponds to the yellow frame in (A). (A) scale bar: 500 µm; (B) scale
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Fig. 8 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
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Fig. 9 Schematic of the IHC staining of LSCs putative biomarkers dynamic expression in mouse
bar: 20 µm.
during P7-P14. The expression of CK15 was gradually weakened in CECs (P7-P13) until undetectable (P14), while it was persistently strongly expressed in LECs. (B) was the high magnification image of (A). The cornea in (B) was corresponds to the green frame in (A), the limbus in (B) was corresponds to the white frame in (A), the conjunctiva in (B) was corresponds to the yellow frame in (A). (A) scale bar: 500 µm; (B) scale bar: 20 µm.
limbal epithelial basal cells (A) and corneal epithelial basal cells (B) during pre- and post-natal development. The expression of LSCs putative biomarkers in limbal epithelial basal cells (A) were emerged in chronological order as follows: Vim=p63>CK14>CK15. The expression of LSCs putative biomarkers in corneal epithelial basal cells (B) were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. The degree of colors represents the expression levels of LSCs putative biomarkers.
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Table 1. The antibodies used for IHC staining Antibody source Antibody (clone)
Antibody dilution
CK12 (EPR17882)
1:100
Abcam (ab185627)
CK13 (AE8)
1:100
Abcam (ab16112)
CK14 (EPR17350)
1:200
Abcam (ab181595)
CK15 (EPR1614Y)
1:100
Abcam (ab52816)
CK19 (EP1580Y)
1:50
Abcam (ab52625)
Vim (EPR3776)
1:100
Abcam (ab92547)
p63 (EPR5701)
1:200
Abcam (ab124762)
ABCG2 (BXP-53)
1:50
Abcam (ab24115)
Fzd7
1:100
Millipore (06-1063)
Actn1
1:50
Proteintech (11313-2-AP)
1:200
Abcam (ab150113)
1:200
Abcam (ab150080)
1:200
Abcam (ab150157)
(Product Code)
Goat anti-Mouse IgG H&L (Alexa Fluro 488) Goat anti-Rabbit IgG H&L (Alexa Fluro 594) Goat anti-Rat IgG H&L (Alexa Fluro 488)
506
507 508 509
Fig. 1
510 511
Fig. 2
512 513 514
Fig. 3
515
516 517
Fig. 4
518 519
Fig. 5
520 521
Fig. 6
522 523
Fig. 7A
524 525
Fig. 7B
526 527
Fig. 8A
528 529
Fig. 8B
530 531 532
Fig. 9
S0014-4835(19)30318-5
DOI:
https://doi.org/10.1016/j.exer.2020.107915
Reference:
YEXER 107915
To appear in:
Experimental Eye Research
Received Date: 5 May 2019 Revised Date:
20 December 2019
Accepted Date: 2 January 2020
Please cite this article as: Guo, Z.H., Zeng, Y.M., Lin, J.S., Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development, Experimental Eye Research (2020), doi: https://doi.org/10.1016/j.exer.2020.107915. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Research article
2 4
Dynamic Spatiotemporal Expression Pattern of Limbal Stem Cell Putative Biomarkers During Mouse Development
5
Zhi Hou Guo1，2, Yi Ming Zeng3, Jun Sheng Lin1*
3
6 7 8 9 10 11 12 13 14 15
，
1. School
of
Medicine,
Huaqiao
University,
Quanzhou
362021,
Fujian,
China;
[email protected] (Z.H.G.); [email protected] (J.S.L); 2. Stem cell laboratory, The second affiliated Hospital of Fujian Medical University, Quanzhou 362000, China; [email protected] (Z.H.G.); 3. The second affiliated Hospital of Fujian Medical University, China; [email protected] (Z.Y.M); Corresponding to: Jun Sheng Lin* Email: [email protected]; Tel.: +86-595-2269-0889
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Abstract: Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to
39
Keywords: Limbal stem cells; biomarkers; niche; spatiotemporal expression; stemness
the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim=p63>CK14>CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.
40 41
Highlight:
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1. The differentiation of CECs is related to eye opening in mice. 2. Dynamic spatial expression pattern reveals a temporal restriction of LSCs putative biomarkers. 3. The order of Vim>p63>CK15>CK14 might represent the stemness degree in ocular surface during mouse development.
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1. Introduction Limbal stem cells (LSCs) are the basal cells of limbal epithelium with self-renewal, high-proliferation and differentiation potential, etc[1]. LSCs undergo a symmetric and/or asymmetric mitosis fashion to divide into the transient amplifying cells (TACs), which have limited stemness with the capable of detach from the basement membrane and centripetal migration. Subsequently, TACs anteriorly centripetally migrate and continuously divide into the terminally differentiated cells (TDCs) to maintain the homeostasis of corneal epithelium and to heal
up
the
wounded
area
during
physiological
and
pathophysiological
processes,
respectively[2-3]. The stemness of LSCs relies on the microenvironment of the LSCs niche, composed of the cells, extracellular matrix, and functional mediators, such as cytokines, growth factors, and exosomes, etc[4-6]. The isolation and identification of LSCs are mainly depended on the LSCs biomarkers. To date, several LSCs putative biomarkers, such as CK14[7], CK15[8], CK19[9], p63[10], ABCG2[9], etc. were reported. However, their specificity was still controversial. Lack of identification of specific LSCs biomarker is the bottleneck of basic research of the LSCs. Many reports have demonstrated that the microenvironment of stem cells niche was age-related. Zheng et al. found that the human periodontal ligament stem cells from young donors showed a stronger proliferation and differentiation capacity compared to those from aged donors, which were rejuvenated by exposure to the young extrinsic environment[11]. Okaley et al. reported that the functions of mouse hematopoietic stem cells, such as proliferation and differentiation, were influenced by the age-related hematopoietic stem cells niche cells[12]. Notara et al. comparatively analyzed the functions and morphology of the human corneas derived from the different age groups, they found that the surface area, degrees of arc occupied by limbal niche structures, and the colony forming efficiency of limbal epithelial cells (LECs) were reduced with aging. These indicated that the LSCs niche and proliferation were age-related[13]. The LSCs niche and the capacity of proliferation and differentiation were closely related to the stemness of LSCs[14-15]. Hence, the expression of LSCs biomarkers might be dynamic during development. As one of the common animal models, mice have controllable advantages in the study of LSCs biomarkers, such as the source of samples, age grouping and many other influencing factors compared with sampling of human limbus. The correlation between the mouse cornea development and both the LSCs niche morphology and the spatiotemporal expression patterns of LSCs biomarkers have not as far been investigated. By this study, we provide an insight into the spatiotemporal expression patterns of LSCs putative biomarkers during mouse development. The pre- and post-natal mouse corneas were stained
with
Haematoxylin-eosin
(HE),
4',6-diamidino-2-phenylindole
(DAPI),
and
immunohistochemistry (IHC). By combining the obtained data, the development process of mouse corneal and limbal epithelium was analyzed and the unique LSCs niche in mice that supports the stemness of LSCs was discussed. In addition, the time spectrum of the expression of putative biomarkers of mouse LSCs was revealed. These findings may help us understand the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness. Our data could be useful to determine a strategy for identifying LSCs at different physiological ages in the absence of a known lifelong LSCs-specific biomarker.
90
2. Material and methods
91
2.1 Ethics statement
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The postnatal 0d, 7d, 8d, 9d, 10d, 11d, 12d,13d, 14d, 28d, 56d, 70d and the pregnant ICR mice were housed，respectively, in the medical research facility at Huaqiao University. All experimental protocols and surgery followed the animal welfare of ethical regulations. The Institutional Animal Care and Use Committee (IACUC) of the Huaqiao University approved all animal protocols (A2018006). 2.2 Sample preparation Prenatal, obtained by caesarean section, and postnatal eyes were fixed with a pH 7.4 4% paraformaldehyde (AR1069, BOSTER) at 4℃ overnight. The tissues were dehydrated using an ethanol gradient (30 min each of 70%, 80%, 90%, 100% ethanol) and embedded in paraffin (39601095, Leica Biosystems). The paraffin-embedded tissue blocks were cut into serial 5 µm thickness using microtome (RM2235, Leica). These were further processed for HE, DAPI, and IHC staining. Three more pre- and post-natal mice were assayed at each timepoint. 2.3 HE staining Paraffin sections were deparaffinized using xylene for 10 min and rehydrated using an ethanol gradient (5 min each of 100%, 90%, 80%, 70% ethanol). Then washed with ddH2O and stained with hematoxylin (G1080, Solarbio) for 10 min, and washed with ddH2O following differentiation with 1% acid alcohol (G1861, Solarbio) for 5 s, washed with ddH2O for 10 min, and then counterstained with Eosin Y solution (G1140, Solarbio) for 2 min, washed with ddH2O for 5 min, dehydrated with 90%, 100% alcohols and cleared with xylene, and then mounted with polyvinylpyrrolidone mounting medium (C0185, Beyotime). 2.4 Immunohistochemistry staining Deparaffinization and rehydration were done with xylene and ethanol gradient (100%, 90%, 80%, 70% ethanol). Followed antigen retrieval with pH 6.0 sodium citrate buffer by heating (110℃, 5 min). Slides were blocked with 5% bovine serum albumin (E661003, Sangon Biotech) for 30min at room temperature (RT) and incubated with primary antibodies (Table 1) at 4℃ overnight, washed thrice with phosphate-buffered saline containing 0.05% Tween (PBST) for 5 min. They were then incubated with secondary antibodies (Table 1) for 1 h at RT, washed thrice with PBST for 5 min. DAPI (AR1177, BOSTER) was used for counterstaining. The slides were then washed and mounted with coverslips using the antifade mounting medium (P0126, Beyotime). The primary antibodies （Table 1） were detected by western blotting. The normal rabbit and mouse serum were used as primary antibodies negative controls for IHC staining.
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3. Results
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3.1 Morphological features of mouse limbus
125 126
The HE staining was conducted for mouse limbus histological analysis. As our results (Fig. 1), the limbal epithelium was the thinnest. The epithelial cell layer was gradually increased from
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limbus to central cornea. The stroma showed about 3-4 times thickness of epithelium and the
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3.2 Spatial expression of LSCs putative biomarkers in mouse ocular surface
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Bowman's layer was not observed. Palisade of Vogt (POV) is a structure in the limbal epithelium which provided the microenvironment of LSCs niche to protect LSCs from the physical, chemical, and UV damaged so as to maintain the stemness of LSCs in human[16]. The limbal epithelium in our studied mice showed smoothly without detectable POV.
In order to investigate the expression pattern of putative biomarkers of LSCs, CK14, CK15, CK19, Vim, p63, and ABCG2 were used for IHC study. To accurately identify the LSCs, the corneal epithelial cells (CECs) biomarker (CK12) and conjunctival epithelial biomarker (CK13) were also used to exclude the influence of the CECs and conjunctival epithelial cells. As our 4-week aged mice corneas IHC staining results (Fig. 2), CK12 was specifically expressed in CECs whereas CK13 was in both the conjunctival epithelial cells and the superficial cells of the limbal epithelium. CK14 and CK15 were strongly expressed in both LECs and conjunctival epithelial cells. CK14 was also weakly expressed in basal cells of the corneal epithelium. Double IHC staining of CK13 and CK19 showed that the expression pattern of CK19 was highly similar to the CK13, which expressed in superficial cells of limbal epithelium and conjunctival epithelium. The expression of Vim was detected in the stroma of mouse ocular surface. However, it was not detectable in the epithelium. ABCG2 and p63 are mostly reported as stem cell biomarkers. We found that both of them were not only expressed in limbal basal epithelial cells but also in corneal basal epithelial cells (Fig. 3). 3.3 The development of mouse cornea We further isolated the corneas from the mice with different ages to investigate the histological characteristics of corneal development by HE and DAPI staining (Fig. 4, Fig. 5, Fig. A2.1, and Fig. A2.2). During E12-E16, the corneal epithelium was composed of the monolayer squamous cells (Fig. A2.1A-C and Fig. 4A-I). At E19, the corneal epithelium began to consist of the squamous and cuboidal epithelial cells (Fig. A2.1D and Fig. 4J-M). Underneath of epithelium, the stroma showed more cell layers, which were increased during E12-E19 (Fig. 4 and Fig. A2.1). The monolayer corneal endothelial cells emerged at E16 were gradually tight (Fig. 4G). Furthermore, the eyelids were first observable at E14, then underwent centripetal growth until closure during E14-E19 (Fig. 4 and Fig. A2.1). During P0-P7, the central corneal epithelium was similar to the limbal epithelium. However, the stromal cells at the central corneal region and limbal region showed tightly and loosely, respectively (Fig. A2.2A-B and Fig. 5A-F). At P14, the eyelids began to open, then the cell layers of central corneal epithelium were quickly increased. Whereas the limbal epithelium kept no obviously change (Fig. A2.2C and Fig. 5G-I). From P28 to P70, the squamous epithelial cells in superficial increased up to 3-4 layers, the cuboidal epithelial cells in suprabasal increased to 1-2 layers and the cell layers of the columnar epithelial cells in basal didn't increase (Fig. A2.2D-F and Fig. 5J-R). 3.4 Spatial expression of LSCs putative biomarkers during development To explore the spatial expression patterns of LSCs putative biomarkers during mouse cornea
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development, the mice corneas from designated ages were collected and the putative biomarkers
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4. Discussions
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(CK14, CK15, CK19, Vim, p63, Fzd7 and Actn1) were used for IHC staining. The primary antibodies showed a high specificity by western blotting (Fig. A1.1). The normal rabbit and mouse serum used as the primary antibodies for IHC staining showed good negative controls (Fig. A1.2). The expression of CK15 was emerged at E16. From E16 to E21, the expression of CK15 was detectable in epithelial cells overall the ocular surface and gradually enhanced (Fig. 6). At P7, CK15 was strongly expressed in both the superficial squamous epithelial cells and LECs. However, during P14-P70, CK15 was specifically detectable in LECs (Fig. 7A-B). It was found that the expression of CK15 was gradually weakened in CECs (P7-P13) until undetectable (P14), while it was persistently strongly expressed in LECs (Fig. 8A-B). The expression of CK14 was emerged at E12 (Fig. A2.3). From E12 to P7, CK14 was expressed in both the CECs and LECs, the expression was gradually enhanced (Fig. A2.3, A2.4). During P14-P70, the CK14 was weakly expressed in corneal epithelial basal cells and strongly expressed in LECs (Fig. A2.4). Additionally, the mice corneas on the time points of P8, P9, P10, P11, P12 and P13 were also collected for IHC staining to clarify the expression changes of CK14 in the CECs. The results (Fig. A2.5) showed that the expression of CK14 was gradually weakened in CECs from P7 to P14, while it was persistently strongly expressed in LECs. The expression of CK19 was emerged in superficial epithelial cells at E16 and gradually enhanced during prenatal. At P7, CK19 was strongly expressed in both the superficial squamous epithelial cells and LECs. During P14-P70, CK19 was strongly specifically detectable in the limbal superficial epithelial cells. We also found that the expression of CK19 was gradually weakened in corneal superficial epithelial cells (P7-P13) until undetectable (P14), while it was persistently strongly expressed in limbal superficial epithelial cells (Fig. A2.6-8). The expression of p63 was strongly detected in both CECs and LECs at E12, then gradually weakened from E12 to P70 (Fig. A2.9-11). Moreover, the expression of p63 showed stronger in corneal basal epithelial cells compared to the limbal basal epithelial cells (Fig. A2.10-11). At E12, Vim was strongly expressed in both CECs and LECs, then gradually weakened until undetectable at E19. During prenatal, Vim was strongly expressed in both corneal and limbal stroma tissues. During postnatal, the expression of Vim was gradually weakened in the corneal stroma, while it was persistently strongly expressed in the limbal stroma (Fig. A2.12-14). From E12 to P70, the expression of Fzd7 (Fig. A2.15-17) and Actn1(Fig. A2.18-20) were both strongly detectable in the epithelial cells overall the ocular surface. To determine the boundary of the cornea, limbus, and conjunctiva, CECs biomarker (CK12) and conjunctival epithelial cells biomarker (CK13) were also be used for IHC staining. The expression of CK12 was emerged in CECs at E19, then gradually enhanced from E19 to P70. During pre- to post-natal, CK12 was always specifically expressed in CECs (Fig. A2.21-23). The expression of CK13 was emerged in superficial epithelial cells at E19. The spatial expression patterns of CK13 was similar to CK19 during prenatal to postnatal (Fig. A2.24-26).
LSCs are the basal cells of limbal epithelium resided in the POV in human[17]. The limbal epithelium and stroma have the most cell layers and thickness in the human ocular surface[9].
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However, in our 4-week aged mouse model, the limbal epithelium and stroma have the least cell layers, which was consistent with the recent reports[18-19]. Lin et al.[20] and Yang et al.[21] reported that the morphology and thickness of human corneal and limbal epithelium were varied with the phases of development and growth. In our results, the limbal basal epithelium in 4 weeks old mouse was smooth and without POV structure (Fig. 1). To analyze whether the morphological differences were related to age, the mouse corneas of different ages were isolated for HE and DAPI staining (Fig. 4, Fig. 5, Fig. A2.1, and Fig. A2.2). The results showed that the mouse limbal epithelium and stroma were always the thinnest in ocular surface and no POV was observed in prenatal and postnatal. These were different from the well-known fact that the limbus in ocular surface with the thickest corrugated epithelium, known as the limbal POV, and without a distinctive Bowman's layer in human[9, 16]. As reported, the deep stromal location of limbal POV[16] and the distinctive Bowman's layer[22-23] provided the environment of protection from the potential light damage. Moreover, Grieve et al. demonstrated that the three-dimensional (3D) architecture of LSCs niche in the species of human, pig, and mouse appears associated with eye exposure to light[24]. Furthermore, the mouse was a nocturnal activity animal which adapted to live in low-light environments. Hence, the mouse limbus morphological feature observed in this study might be related to the adaptation of low-light environment. The cornea is composed of the epithelium, endothelium and stroma[25]. During the prenatal and neonatal (Fig. 4, Fig. 5, Fig. A2.1, and Fig. 2.2), the mouse cornea was composed of the monolayer of epithelial cells. At this stage, the development of mouse cornea was dominated by the stroma. The stroma was thickened with increasing of the cell layer, and the stromal cells were tight, providing the structural basis for the passage of light. As reported, the liquid-air interface promoted the differentiation of epithelial cells[26-27]. In our results, the eyelids of the mice were opened at P14, then the development of cornea was dominated by corneal epithelium (Fig. 5 and Fig. A2.2). It was found that after the eyelids opening, the CECs quickly go stratification in response to the liquid-air interface, suggesting that the rate of the differentiation of mouse CECs was increased. The corneal epithelium was thickened with the increasing of CECs layers. The thickening corneal epithelium and the tightening stroma, leading the ocular surface to be parallel with the posterior stroma from P14. This increased the transparency of mouse cornea, decreased the scattering of light, and promoted light transmission. All in all, our results supported that the liquid-air interface promotes the differentiation of CECs. To date, the LSCs specific biomarkers are still remained elusive[28-29]. The main method for the identification of LSCs is based on the co-expression of LSCs putative biomarkers and negative biomarkers[30]. In this study, the most promising LSCs putative biomarkers, CK14[7], CK15[31], CK19[9], Vim[32], p63[33], and ABCG2[34], expressed in human limbal basal cells were used for the spatial expression analysis of mouse LSCs. To exclude the CECs and conjunctival epithelial cells, the CECs differentiated biomarker (CK12) [9] and conjunctival epithelial cells biomarker (CK13) [35] were also used. In our 4 weeks old mice, the expression of these putative biomarkers was detectable not only in the LECs, but also in both the corneal basal cells (CK14, p63, ABCG2) and conjunctival epithelial cells (CK14, CK15, CK19, p63, ABCG2) (Fig. 2 and Fig. 3), indicating a unique spatial expression of mouse LSCs. Recently, Lee et al. reported the microenvironment, generated by bone marrow stem cells, for the regulation of normal
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hematopoietic and leukemia stem cells showed age-related. They found that the self-renewal of leukemia stem cells was higher in adult bone marrow than in neonatal bone marrow[36]. Sagga et al. comparatively analyzed the cell-cycle time of the limbal and corneal epithelia in young adult and aging mouse eyes, found that the cell cycle was significant faster in central corneal epithelium of aging eyes compared to young adult mice[37]. These indicated that age was an important factor influence of the stemness of LSCs. The unique spatial expression of mouse LSCs in our results might be age-related. To explore the spatial expression pattern of the putative biomarkers of LSCs during mouse development, the prenatal and postnatal of mice corneas were further collected for IHC staining under the same exposure time (Fig. 6-8 and Fig. A2.3-26). In current study, we found that the expression of p63 were detected throughout the ocular surface at all ageing groups, in which the highest expressed in cornea, the second in limbus, and the lowest in conjunctiva. This expression pattern of p63 was consistent with Hsueh et al. reported in rat[38] and Sonam et al. reported in Xenopus frogs[39], whereas contrary to Davies et al. reported in human[40]. The expression of CK15 were initially observed across the ocular surface in early mouse development, eventually confined to the limbal and conjunctival epithelium. It was contrast to the finding by Davies et al. reported in the developing human fetal and adult cornea[40]. CK14 was known as a LSCs putative biomarker specifically expressed in limbal epithelial basal cells in human[41]. However, in our result, the expression of CK14 were detectable across the ocular surface and then progressively weakened in CECs, which were consistence with Richardson et al. reported[42]. In our IHC results, the presents of Vimentin expressing cells were observed in stroma during postnatal stage, which supported previous reported in a variety of vertebrates, including chick[43], cat[44], Xenopus frogs[39], and rabbit[45]. However, we found the expression of Vimentin were observed in CECs and LECs during prenatal stage. The expression of CK19 was observed in corneal superficial epithelial cells only during prenatal and
early postnatal stage, whereas persistently in limbal superficial epithelial cells. This expression pattern of CK19 was contrast to the Sonam et al. reported in larval versus adult Xenopus frogs[39]. Moreover, we further analyzed the dynamic expression of the examined LSCs putative biomarkers in different timepoints during mouse development to understand the dynamic stemness of LSCs and the temporal restriction of LSCs putative biomarkers. Before E19, the expression of LSCs' putative
biomarkers (CK14, CK15, CK19, p63, Fzd7, Actn1 and Vim) were detectable in both CECs and LECs, while the differentiation biomarker, CK12, had not yet begun to express, suggesting that CECs still had some degree of stemness. At E19, the expression of CK12 was detectable in CECs, indicating that CECs began to loss stemness. Just at the same time point, the expression of Vim was progressively weakened and eventually disappeared in CECs while others examined LSCs putative biomarkers (CK14, CK15, CK19, p63, Fzd7, and Actn1) remained detectable. This finding suggested that Vim might represent higher potency of stemness than the others examined LSCs putative biomarkers. During E19-P14, the expression of CK14 and CK15 in CECs were enhanced in E19-P7 and weakened in P7-P14, whereas the expression of p63 was progressively weakened, meaning that p63 represented weaker stemness degree than Vim, but higher than CK14 and CK15. At P14, the mouse eyelids were opened, CK14 was weakly expressed in corneal basal epithelial cells, whereas the expression of CK15 was not detectable in CECs, which suggested that the expression of CK15 represents weaker stemness degree than p63, but higher than CK14. As
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reported, CK19 was used as human LSCs putative biomarker[9]. However, Donisi et al.
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5. Conclusion
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demonstrated that CK19 was a specific biomarker of conjunctival epithelial cells for the diagnosis of limbal stem cells deficiency (LSCD)[46]. In our results, the spatial expression patterns of CK19 was similar to the CK13 during mouse development (Fig. A2.6-8 and Fig. A2.24-26), indicating that the CK19 was more suitable served as a conjunctival epithelial cell biomarker than LSCs biomarker. Fzd7[47] and Actn1[48] have been reported to be the LSCs putative biomarkers expressed in the limbal epithelial basal cells. However, in this study, the expression of Fzd7 and Actn1 were always detectable in both CECs and LECs during mouse development (Fig. A2.15-20), therefore, whether they could serve as mouse LSCs biomarkers need to be confirmed by more experimental data. Above all, our results suggest that the expression of the examined LSCs putative biomarkers in limbal epithelial basal cells (Fig. 9A) were emerged in chronological order as follows: Vim=p63>CK14>CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells (Fig. 9B) were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development (Fig. 9) also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others LSCs interested gene expression products, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mice and revealed the time spectrum of the expression of LSCs in mice, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.
Stemness of the LSCs in mice could be maintained without the structure of POV. The differentiation of CECs is related to eye opening in mice. Furthermore, our data demonstrated that the dynamic spatial expression patterns of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs putative biomarkers in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness. Appendix A. Supplementary data The following is the Supplementary data to this article. Funding
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No.2016YFE0101700), Huaqiao University Grant (No. 13Y0391), and the fourth round of joint
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Declaration of Interest
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This research was funded by the National Key R&D Program of China (Grant key research project jointly with health and education in Fujian province (No. WKJ2016-2-13).
None.
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Figure captions.
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Fig. 1 Morphological features of the cornea, limbus and conjunctiva in the 4 weeks old ICR mouse.
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Fig. 2 Double IHC staining of LSCs putative biomarkers on the 4 weeks old ICR mouse ocular
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Fig. 3 Double IHC staining of p63 and ABCG2 on the 4 weeks old ICR mouse ocular surface. The
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Fig. 4 DAPI staining of mouse ocular surface during the prenatal development. The development
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Fig. 5 DAPI staining of mouse ocular surface during the postnatal development. The development
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Fig. 6 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
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Fig. 7 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
(A) and (B) showed the boundaries of the cornea, limbus and conjunctiva in the mouse eye; (C) exhibited the thickest epithelium in mouse central cornea and the thinnest epithelium in limbus, which with no visible POV. Scale bar: 100 µm.
surface. The CK12 and CK13 were used for the identification of CECs and conjunctival epithelial cells. The detectable gene products of CK14, CK15, CK19, Vim, and p63 were not only limited to the limbal epithelial basal cells but also corneal epithelial basal cells and conjunctival epithelial cells. L: limbus, C: cornea, Cj: conjunctiva. Scale bar: 100 µm.
expression of LSCs putative biomarkers p63 and ABCG2 were detectable not only in limbal epithelial basal cells but also in corneal epithelial basal cells and conjunctival epithelial basal cells. L: limbus, C: cornea, Cj: conjunctiva. Scale bar: 100 µm.
was dominated by the stroma, the corneal and limbal epithelium were composed of the monolayer epithelial cells. The white arrow and dotted line indicated the emerged monolayer corneal endothelial cells at E16 (G). The left panel represents the magnified area of the green block within central cornea, and the right panel represents the magnified area of the red block surround limbus, respectively. Scale bar: middle panel, E12/E14=100 µm, E16/E19=200 µm; left and right panels, 20 µm.
was dominated by the stroma during neonatal. At P14, the eyelids were opened responding to the stimulation of the liquid-air interface. The development of cornea was then dominated by the epithelium, the speed of the differentiation of mouse CECs was increased and the epithelium was thickened with the increasing of CECs layers. The white dotted frame indicated the Eyelid (H and I). The left panel represents the magnified area of the green block within central cornea, and the right panel represents the magnified area of the red block surround limbus, respectively. Scale bar: middle panel, 500 µm; left and right panel, 100 µm.
during prenatal development. The expression of CK15 was emerged at E16 and gradually enhanced during prenatal development. Scale bar: E12/E14=100 µm, E16/E19/E21=200 µm.
during postnatal development. The expression of CK15 was strongly detectable in both the corneal superficial squamous epithelial cells and LECs at P7 and specifically detectable in LECs during P14-P70. (B) was the high magnification image of (A). The cornea in (B) was corresponds to the green frame in (A), the limbus in (B) was corresponds to the white frame in (A), the
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conjunctiva in (B) was corresponds to the yellow frame in (A). (A) scale bar: 500 µm; (B) scale
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Fig. 8 Dynamic expression of CK15 in mouse corneal, limbal and conjunctival epithelial cells
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Fig. 9 Schematic of the IHC staining of LSCs putative biomarkers dynamic expression in mouse
bar: 20 µm.
during P7-P14. The expression of CK15 was gradually weakened in CECs (P7-P13) until undetectable (P14), while it was persistently strongly expressed in LECs. (B) was the high magnification image of (A). The cornea in (B) was corresponds to the green frame in (A), the limbus in (B) was corresponds to the white frame in (A), the conjunctiva in (B) was corresponds to the yellow frame in (A). (A) scale bar: 500 µm; (B) scale bar: 20 µm.
limbal epithelial basal cells (A) and corneal epithelial basal cells (B) during pre- and post-natal development. The expression of LSCs putative biomarkers in limbal epithelial basal cells (A) were emerged in chronological order as follows: Vim=p63>CK14>CK15. The expression of LSCs putative biomarkers in corneal epithelial basal cells (B) were weakened in chronological order as follows: Vim>p63>CK15>CK14, which might also represent the stemness degree. The degree of colors represents the expression levels of LSCs putative biomarkers.
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Table 1. The antibodies used for IHC staining Antibody source Antibody (clone)
Antibody dilution
CK12 (EPR17882)
1:100
Abcam (ab185627)
CK13 (AE8)
1:100
Abcam (ab16112)
CK14 (EPR17350)
1:200
Abcam (ab181595)
CK15 (EPR1614Y)
1:100
Abcam (ab52816)
CK19 (EP1580Y)
1:50
Abcam (ab52625)
Vim (EPR3776)
1:100
Abcam (ab92547)
p63 (EPR5701)
1:200
Abcam (ab124762)
ABCG2 (BXP-53)
1:50
Abcam (ab24115)
Fzd7
1:100
Millipore (06-1063)
Actn1
1:50
Proteintech (11313-2-AP)
1:200
Abcam (ab150113)
1:200
Abcam (ab150080)
1:200
Abcam (ab150157)
(Product Code)
Goat anti-Mouse IgG H&L (Alexa Fluro 488) Goat anti-Rabbit IgG H&L (Alexa Fluro 594) Goat anti-Rat IgG H&L (Alexa Fluro 488)
506
507 508 509
Fig. 1
510 511
Fig. 2
512 513 514
Fig. 3
515
516 517
Fig. 4
518 519
Fig. 5
520 521
Fig. 6
522 523
Fig. 7A
524 525
Fig. 7B
526 527
Fig. 8A
528 529
Fig. 8B
530 531 532
Fig. 9