Wadhwa Himanshu, Ismail Salim, McGhee Jennifer J, Van der Werf Bert, Sherwin Trevor
Department of Ophthalmology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1023, New Zealand.
Department of Epidemiology and Biostatistics, School of Population Health, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1023, New Zealand.
World J Stem Cells. 2020 Jan 26;12(1):35-54. doi: 10.4252/wjsc.v12.i1.35.
Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which have shown the potential to repopulate the normal peripheral corneal stroma upon sphere implantation but have not been previously studied in keratoconic tissue.
To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.
Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells. These spheres were implanted into incisions created in full thickness and onto the surface of 10 µm thin sections of keratoconic and normal stromal tissues . Tissue sections were used to maximise use of limited keratoconic tissue available for research. Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d (D14) from implantation. Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.
Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10 µm thin tissue sections. No observable differences were seen in sphere migration, proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14. Spheres stained positively with Calcein-AM up to D14. Cell migration increased from day 0 to D14, occurring radially in three dimensions from the sphere and in alignment with tissue edges. Cell proliferation marker, EdU, was detected at day 10. Implanted spheres stained positively for putative stem cell markers ∆Np63α and ABCB5, while ABCG2, ABCB5, ∆Np63 and p63α were detectable by droplet digital PCR up to D14. Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin (myofibroblast marker) in some migrated cells. Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers, indicating an appropriate response to repopulating diseased tissue.
Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.
圆锥角膜是一种退行性角膜疾病，其特征是细胞行为异常和基质丢失，可导致视力丧失。从周边角膜提取的细胞可形成富含干细胞的球体，这些球体已显示出在球体植入后有重新填充正常周边角膜基质的潜力，但此前尚未在圆锥角膜组织中进行研究。
研究从人周边角膜提取的细胞形成的富含干细胞的球体引入圆锥角膜组织后的治疗潜力。
从正常尸体人周边角膜细胞提取物中形成富含干细胞的球体。将这些球体植入圆锥角膜和正常基质组织10 µm薄片表面及全层切口中。使用组织切片以最大限度利用有限的可用于研究的圆锥角膜组织。活细胞用钙黄绿素-AM染色，并用立体显微镜和荧光显微镜观察，以评估植入后第0天至第14天（D14）之间的存活情况和行为。使用免疫组织化学和液滴数字PCR对植入组织中的球体细胞进行干细胞和分化标志物鉴定，以评估这些特征在圆锥角膜治疗中使用球体的潜在意义。
球体成功植入全层中央角膜组织及10 µm薄片组织表面。在第0天至D14期间，与正常组织相比，圆锥角膜组织中球体的迁移、增殖或分化未见明显差异。直到D14，球体用钙黄绿素-AM染色均呈阳性。细胞迁移从第0天到D14增加，从球体向三维径向发生，并与组织边缘对齐。在第10天检测到细胞增殖标志物EdU。植入的球体对假定的干细胞标志物∆Np63α和ABCB5染色呈阳性，而直到D14，通过液滴数字PCR可检测到ABCG2、ABCB5、∆Np63和p63α。双重免疫标记显示迁移细胞中无ABCB5染色，但一些迁移细胞中α平滑肌肌动蛋白（肌成纤维细胞标志物）染色呈阳性。液滴数字PCR显示分化标志物的表达模式相似，但正常组织和圆锥角膜组织之间干细胞标志物减少，基质细胞标志物增加，上皮细胞标志物减少，表明对填充病变组织有适当反应。
植入的富含干细胞的球体中的细胞可以定向填充圆锥角膜基质表面，并表现出迁移性基质细胞表型。
