MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

Abstract

PURPOSE. To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy.METHODS. The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen).RESULTS. Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44 À SP from SPs with CD44 þþ~C D44 þþþ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. CONCLUSIONS. The specified cultured SPs sharing the CD44À surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44 þþþ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.Keywords: heterogeneity of cultured corneal endothelial cells, CD44, miR, gutatta, epithelium-mesenchymal transition (EMT) A lthough human corneal endothelial cells (HCECs) have the ability to proliferate in vitro, 1 culturing HCECs for an extended period of time is known to be extremely difficult.2 To date, most researchers have conceptualized cultured HCECs (cHCECs) only from the aspect that they are derived from corneal endothelium tissue, and disregarded details pertaining to the refinement of the biochemical features. In fact, cHCECs are known to be heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster-ofdifferentiation (CD) antigens.3-5 Studies, either directly or indirectly, and including those from Joyce et al., [6][7][8][9][10][11][12][13] have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al.3 of the presence of frequent chromosomal aneuploidy in cHCECs, directly indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.Cultured HCECs have an inclination toward cell-state transition (CST) into a senescence phenotype, epitheliummesenchymal transition (EMT), and fibroblastic cell morphology. In addition, the plasticity of metabolic profiles of cH...

Corneal Endothelial Cells Over the Past Decade: Are We Missing the Mark(er)?

“…31 Furthermore, miR34a detection in culture medium was linked to a lack of CD44 expression and thus to the absence of aneuploidy. 32 Although miRNA analysis shows some potential for use in the validation of cell therapy products, we are concerned about the concomitant isolation of miRNAs from the fetal bovine serum and mesenchymal stem cell–conditioned medium that are used as additives in their cell culture medium. 33,34…”

“…18,28–30 The unique phenotypic markers that have reportedly been found to be linked to healthy HCEnCs in these studies are CD166, CD200, GPC4, HLA-ABC, and PD-L1. 21,32,40,41 In addition to markers that positively identify HCEnCs, attention should also be paid to the absence of certain gene or protein expressions, in particular α-SMA, CD9, CD24, CD26, CD44, CD73, CD90, CD105, and CD133 for phenotypic studies, and Snail, ZEB1, and vimentin, which have been shown to be negative in HCEnCs. 19,32–35 Nevertheless, these markers are challenged (i.e., CD166 20 ) and have not been widely adopted by other groups to date.…”

Production of Homogeneous Cultured Human Corneal Endothelial Cells Indispensable for Innovative Cell Therapy

“…It is noteworthy that the only miR capable of discriminating CD44 À effector cells from CD44 þþ -CD44 þþþ SPs was identified as miR34a. 35 Consequently, it is conceivable that in the previous experiment of Koizumi et al, 40 either CD44 þþþ or CD44 þþ SPs with downregulated miR34a used the RhoA/MMP2 pathway activated in the undifferentiated SPs. 33 If this assumption is correct, FIGURE 2.…”

“…In a previous study, we confirmed the upregulation of miR29 in parallel with that of CD44 expression among distinct SPs, as our findings showed that miR29 was most downregulated in effector cells with a CD44-phenotype among cHCEC SPs. 35 Interestingly, miR29 reportedly has an EMT-promoting effect. 36 Reportedly, TGF-b can initiate and maintain the EMT in various biological and pathologic systems.…”

“…In a series of recent studies, [17][18][19][20][21][22][23] we reported the existence of diverse SPs in cHCECs and identified a cluster of differentiation (CD)166…”