Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells

角膜上皮位于角膜最表层，对维持角膜的透明性发挥非常重要的作用。角膜上皮细胞处于不断更新之中，它们更新的源泉在于角膜缘处的上皮干细胞，即角膜缘干细胞。眼表面化学伤、热烧伤以及部分先天性遗传疾病可以引起角膜缘干细胞缺乏，或者使干细胞丧失向正常角膜上皮细胞分化的能力，导致经久不愈的角膜上皮缺损、角膜新生血管、角膜上皮结膜化、角膜溃疡等病理改变，严重影响患者视力，甚至导致失明。由于眼表面的严重破坏，这类患者的治疗具有很大的挑战性，目前比较理想的治疗方法是进行组织工程角膜上皮移植。 厦门大学眼科研究所和中国科学院动物研究所干细胞与生殖生物学国家重点实验室合作研究采用中科院动物研究所研发的我国第一个临床级胚胎干细胞系Q-CTS-hESC-1，体外诱导分化为角膜上皮祖细胞，在此基础上构建组织工程角膜上皮，移植于兔角膜缘干细胞缺乏动物模型，成功重建眼表面。 厦门大学眼科研究所博士研究生贺佳和欧尚坤为该论文的共同第一作者，厦门大学眼科研究所李炜教授、刘祖国教授以及中科院动物所郝捷副研究员为共同通讯作者。【Abstract】Purpose: To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation.Methods: A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, realtime quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. Results: The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch,Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. Conclusion: The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.This study was supported in part by the National Key R&D Program of China (2018YFA0107301 [to WL], 2018YFA0107304 [to ZL], 2013CB967003 [to WL]), the National Natural Science Foundation of China (NSFC, No.81770894, No.81470602 [to WL], and No.81330022 [to ZL]). 该论文获得了国家重点研发计划项目和国家自然科学基金项目的资助

Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction

Unique expression pattern and functional role of periostin in human limbal stem cells.

Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC

Transcription Factor TCF4 Maintains the Properties of Human Corneal Epithelial Stem Cells

TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of beta-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells. STEM CELLS 2012; 30:753761DOD CDMRP [FY06 PR064719]; NIH [EY11915]; National Natural Science Foundation of China [30901634]; Fight for Sight; Research to Prevent Blindness, Oshman Foundatio

Human primer sequences used for RT-PCR.

<p>Human primer sequences used for RT-PCR.</p

Wnt/β-catenin signaling pathway activation in conjunctival epithelium of airlift explant cultures.

<p>β-catenin (column A) and phosphor-β-catenin (Ser-552, column B) staining were performed at different time points after airlifting culture. β-catenin mostly anchored to the cell membrane and showed very low expression levels in cytoplasm of normal conjunctival epithelium (D0). Phosphor-β-catenin was only found in the cell membrane of basal conjunctival epithelium. Cytoplasmic accumulation of β-catenin and phosphor β-catenin in conjunctival epithelium in airlift explants started at day 2, and then gradually decreased after day 6. Nuclear translocation of β-catenin and phosphor β-catenin was seen in the basal and suprabasal layers of conjunctival epithlial cells from day 2 to day 6 (see inserts). At days 12 and 14, β-catenin and the majority of phosphor-β-catenin has translocated to the cell membrane. Inserts are high power magnification of specific locations. Bar represents 100 µm.</p

Air Exposure Induced Characteristics of Dry Eye in Conjunctival Tissue Culture

<div><p>There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.</p></div

Conjunctival epithelial changes in explant cultures.

<p>Hematoxylin & Eosin staining showed increases in conjunctival epithelial cell layers in airlift culture. Their number particularly increased from day 8 to day 14, resulting in an undulated basal epithelial plane and digital invasion. In contrast, the cell layers of submerged culture did not show significant increase. Bar represents 200 µm.</p

Representative immuno-fluorescent staining profiles for corneal epithelial phenotype.

<p>Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.</p