BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0738-4) contains supplementary material, which is available to authorized users.

SummaryHuman induced pluripotent stem cells (hiPSCs) offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF) in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

PurposeThe impact of visual acuity (VA) on Health-Related Quality of Life (HRQoL) and the cross-sectional and longitudinal differences in HRQoL during the 11-year follow-up were investigated. The aim was to examine the impact declining vision has on HRQoL and to provide comparable data to facilitate the allocation of health-care resources.MethodsWe utilized nationwide health examination surveys carried out by the National Institute for Health and Welfare in 2000 and 2011, providing a representative sampling of the Finnish adult population aged 30 and older. VA was assessed through Snellen E test, and HRQoL scores were evaluated using EQ-5D and 15D questionnaires. Multiple imputations with Markov chain Monte Carlo method was used to utilize the data more effectively. Regression analyses were conducted to assess the impact of declining VA on HRQoL, adjusted for incident comorbidities.ResultsLower VA status was associated with significantly lower HRQoL at both time points, most clearly observable below the VA level of 0.5. Declining VA resulted in statistically significant decline in HRQoL during the follow-up, greater with distance than near VA. 15D impairment associated with decline in the distance VA was also clinically meaningful and greater than that associated with any of the examined comorbidities.ConclusionsHRQoL was significantly and meaningfully impaired even before the threshold of severe vision loss or blindness was reached. The results encourage the improvement of available treatment options aiming to postpone the onset of visual impairment or declining VA, to maintain better quality of life among the population.Electronic supplementary materialThe online version of this article (10.1007/s11136-019-02260-3) contains supplementary material, which is available to authorized users.

BackgroundAdvances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed. MethodsA total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry.ResultsWe discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins < 20% RSD), compared to iTRAQ (43% of proteins < 20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers.ConclusionsOverall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points.Trial RegisterationThe study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).Electronic supplementary materialThe online version of this article (10.1186/s12014-018-9201-5) contains supplementary material, which is available to authorized users.