Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

BackgroundTranscriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS).MethodsTo address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes.ResultsThe average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30.ConclusionIn this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.Electronic supplementary materialThe online version of this article (10.1186/s12575-017-0065-2) contains supplementary material, which is available to authorized users.

Introduction: This real-world study investigated glycaemic control and quality of life (QoL) in insulin-experienced Japanese patients with type 2 diabetes (T2D) who switched to insulin degludec/insulin aspart (IDegAsp). Methods: This was a prospective, non-interventional, open-label, single-arm study. Eligible patients were adults (aged C 20 years) with T2D, previously treated with insulin glargine 100 or 300 units/mL (glargine U100/U300) with or without prandial insulin, who switched to IDegAsp as part of routine practice. Change from baseline to end of study (EOS; 26 weeks after initiation or IDegAsp discontinuation) in the following endpoints was assessed by adjusted mixed models for repeated measures: glycated haemoglobin (HbA1c; primary endpoint), fasting plasma glucose (FPG), insulin dose and total Diabetes Therapy-Related Quality of Life (DTR-QoL) score. Non-severe hypoglycaemia was assessed in the 4-week period prior to initiating IDegAsp and in the 4-week period before EOS or discontinuation using negative binomial regression. Results: The full analysis set included 236 patients from 29 centres in Japan with mean (± SD) age 63.2 years (± 12.3), HbA1c 7.7% (± 1.0) and diabetes duration 14.9 (± 9.3) years. After 26 weeks with IDegAsp, HbA1c (estimated change -0.1% [-0.2; 0.0] 95% confidence interval (CI) , p = 0.3036) and FPG (-7.5 mg/dL [-23.5; 8.5] 95% CI , p = 0.3477) were maintained; there were significant reductions in basal and total insulin dose: estimated change of -3.4 units/day [-3.8; -3.0] 95% CI and -1.0 units/day [-1.9; -0.1] 95% CI , respectively (both p \ 0.05). Non-severe hypoglycaemia rates were similar in the periods before and after initiating IDegAsp, while there was a significant improvement in total DTR-QoL score after 26 weeks with IDegAsp (p = 0.0012). Conclusion: These real-world data suggest that switching to IDegAsp from glargine U100 or U300 was well tolerated in a Japanese population with T2D, with no new safety or tolerability signals, and associated with maintenance of glycaemic control and improved QoL.