Jumelle C.,University of Sfax |
Mauclair C.,CNRS Hubert Curien Laboratory |
Houzet J.,CNRS Hubert Curien Laboratory |
Bernard A.,University of Sfax |
And 11 more authors.
PLoS ONE | Year: 2015
Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future cell or tissue therapy. © 2015 Jumelle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source
Ha Thi B.M.,Jean Monnet University |
Campolmi N.,Jean Monnet University |
He Z.,Jean Monnet University |
Pipparelli A.,Jean Monnet University |
And 9 more authors.
PLoS ONE | Year: 2014
Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts. Copyright: © 2014 Ha Thi et al. Source
All animal studies were performed with the approval of the Institutional Animal Care Committees of Sun Yat-sen University, the University of California San Diego, West China Hospital, and the University of Texas Southwestern Medical Center. The eyeball was enucleated from a one-month-old New Zealand white rabbit and washed with PBS (containing antibiotics) three times. After the cornea and iris were removed, a small cut was made in the posterior capsule of the lens; the capsule with attached epithelium was removed and cut into 1 × 1 mm2 pieces. The pieces of epithelium were cultured in minimum essential media supplemented with 20% FBS, NEAA, and 50 μg ml−1 gentamicin. A 17-week-old human fetal eyeball was purchased from Advanced Bioscience Resources, Inc. (San Francisco, California). Post-mortem human eyes were obtained from San Diego Eye Bank. The human LECs were cultured according to the same methods as above. For in vitro differentiation, LECs were cultured on Matrigel-coated six-well plates or eight-well chambers. Lentoid body was formed after 21 days in minimum essential media supplemented with NEAA, 1% FBS, 100 ng ml−1 FGF2, and 5 μg ml−1 insulin. Images of lentoid tissue were obtained using a Leica M205FA stereo microscope. Membrane-tomato/membrane-green (mTmG)-targeted ROSAmTmG mice were purchased from the Jackson Laboratory (Bar Harbour, ME; stock no. 7576) and maintained as homozygotes. P0-3.9-GFPcre mice expressing an eGFP–Cre recombinase fusion protein under the control of the Pax6 lens ectoderm enhancer and the Pax6 P0 promoter26 were maintained in a FVB/N background. Lineage-tracing experiments were performed by crossing the homozygous ROSAmTmG reporter mouse strain with the P0-3.9-GFPcre deleter strain. Eyes were dissected at P1, P14, and P30 and fixed overnight in 4% formaldehyde. Tissues were then incubated in 10% sucrose and embedded in OCT for cryo-sectioning. Frozen sections were washed in PBS and imaged on a Zeiss Axio Imager fluorescence microscope. Bmi1fl/fl mice were generated as previously described27. Nestin-cre mice28 were obtained from the Jackson Laboratory. For BrdU pulses, mice were injected with 100 mg kg−1 BrdU (Sigma) dissolved in PBS, then maintained on drinking water that contained 1 mg ml−1 BrdU until sacrifice. For gene expression studies, lenses of Pax6P0-3.9-GFPcre mice were dissected under a dissecting microscope. Lens capsular bag was opened from the posterior surface by making three crisscross incisions. The capsular bag was opens and lens material extruded. GFP-positive LECs in the mid-anterior capsular area were separated mechanically from GFP-negative LECs in the remaining capsular areas under a fluorescence microscope. RNA was isolated using RNeasy Mini Kit (Qiagen). To image cataracts, mice were anaesthetized with Avertin, and one drop of 1% Mydriacyl (Alcon) was administered per eye. Eyes were immediately visualized in vivo using a light microscope. For histology, mice were perfused with heparinized saline followed by 4% paraformaldehyde (PFA) in PBS. Dissected eyes were fixed in 4% PFA overnight, embedded in paraffin, and sectioned by the UT Southwestern Molecular Pathology core facility. For BrdU staining, slides were deparaffinized, and subjected to heat-mediated antigen retrieval (in 10 mM sodium citrate, pH 6.0). Slides were stained with primary mouse anti-BrdU (Caltag, MD5000, 1:200) overnight at 4 °C. Slides were subsequently stained with Alexa Fluor 555-conjugated goat anti-mouse IgG1 secondary antibody (Life Technologies, 1:500) and 1 mg ml−1 DAPI (1:500) for 1 h at room temperature. The number of BrdU-labelled cells was divided by the total number of DAPI+ cells in a single layer of LECs. Lentiviral shRNA targeting the human BMI1 gene (NCBI Reference Sequence: NM_005180.8) was purchased from Origene (TL314462), ShRNA targeting sequences were as follow: 5′-AATGCCATATTGGTATATGACATAACAGG-3′ and 5′-GTAAGAATCAGATGGCATTATGCTTGTTG-3′. Two shRNAs were used separately, and a non-effective 29-mer scrambled shRNA was used as a control. Lentiviral shRNA particles were prepared using shRNA lentiviral packaging kit (Origene, TR30022). Viruses were harvested at 48 h and 72 h post-transfection. LECs were cultured on Matrigel-coated 3.5-mm dishes with lentoid formation medium for 30 days. Cells were washed twice with ice-cold PBS, and lysed in RIPA lysis buffer with PMSF. Protein concentration was determined by BCA protein assay kit. Thirty micrograms of total protein lysate was loaded onto 10% SDS–PAGE gel and then transferred to a PVDF membrane (Millipore) at 70 V for 2 h. The membrane was probed with the following primary antibody at 4 °C overnight: anti-αA-crystallin (sc-22389, Santa Cruz), anti-β-crystallin (sc-48335, Santa Cruz), anti-γ-crystallin (sc-22415, Santa Cruz) and anti-β-actin (sc-47778, Santa Cruz), and then incubated with HRP-conjugated anti-rabbit, anti-mouse, or anti-goat secondary antibody for 1 h at room temperature. The immunodetection was visualized using a blot imaging system (Fluor Chem Q, Protein Simple) with ECL buffer (Millipore). New Zealand white rabbits (n = 29, four rabbits died from systemic infections unrelated to surgery. The remaining 25 rabbits were used to assess regeneration), and long-tailed macaques (Macaca fascicularis) monkeys (n = 6) underwent minimally invasive capsulorhexis surgery. Only the left eye of each animal was used for experiments. Slit-lamp biomicroscopy and photography were performed at different time points to monitor lens regeneration. Rabbits were euthanized at day 1, day 7, and one month after surgery, and the treated eyes were enucleated. The lenses were harvested for histologic analysis using haematoxylin and eosin staining. For the macaques, enucleation of the treated eye was performed 4 months post-surgery and the lenses were harvested for the same histologic examinations. The eyes were fixed, paraffin-embedded, and sectioned at 5 μm through the cornea, pupil, and optic nerve with the lens in situ. RNA was isolated from rabbit LECs, mature lens fibre cells and LECs in P0-3.9-GFPcre mice using an RNeasy Mini Kit (Qiagen) and subjected to on-column DNase digestion. cDNA was synthesized using a Superscript III reverse transcriptase kit according to the manufacturer’s instructions (Invitrogen). Quantitative PCR was performed via 40 cycle amplification using gene-specific primers (Supplementary Table 1) and Power SYBR Green PCR Master Mix on a 7500 Real-Time PCR System (Applied Biosystems). Measurements were performed in triplicate and normalized to endogenous GAPDH levels. The relative fold change in expression was calculated using the ΔΔC method (C values <30). Rabbit LECs were fixed in 4% PFA for 20 min, then permeabilized with 0.3% Triton X-100-PBS for 10 min and blocked in PBS solution containing 5% BSA, followed by an overnight incubation in primary antibodies at 4 °C. After three washes in PBS, cells were incubated with secondary antibody for 1 h in room temperature. Cell nuclei were counterstained with DAPI. The following antibodies were used: goat anti-Sox2 polyclonal antibody (Santa Cruz), rabbit anti-PAX6 polyclonal antibody (PRB-278P, Covance), mouse anti-Bmi1 antibody (ab14389, Abcam), and mouse anti-Ki67 monoclonal antibody (550609, BD Sciences). The secondary antibodies, Alexa Fluor 488- or 568-conjugated anti-mouse or anti-rabbit IgG (Invitrogen), were used at a dilution of 1:500. Images were obtained using an Olympus FV1000 confocal microscope. We used BrdU labelling to identify and quantify proliferating LECs from human cadaver eyes. Whole-mount human lens capsules were pulsed with BrdU and then stained with an antibody against BrdU to determine the distribution and density of proliferating LECs. In brief, within 12–24 h after death, lenses from post-mortem donor eyes were obtained from the Eye Bank of Zhongshan Ophthalmic Center in Guangzhou, China. Twelve lenses in total from six donors were used for the experiment. A small puncture injury was made on the anterior surface of a post-mortem human lens using a 30-gauge needle. The lenses were cultured at 37 °C in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS in a humidified incubator with 5% CO . The contralateral lens from the same donor was treated under the same conditions but did not receive a puncture injury and was used as a control. To label the proliferating LECs, both groups of lenses were incubated in 100 μg ml−1 BrdU (Sigma-Aldrich) 24 h after the puncture injury. The lens was then removed from the capsular bag, and the lens capsules were fixed in 4% formaldehyde and subjected to BrdU staining using a standard immunohistochemistry protocol according to the manufacturer’s instructions (CST, Boston, Massachusetts). Images were taken using a Carl Zeiss microscope (Jena, Germany). This study was approved by the institutional review board of the Zhongshan Ophthalmic Center (ZOC). Informed written consent was obtained from the parents or guardians of the infants before enrolment, and the tenets of the Declaration of Helsinki were followed throughout the study. The study was conducted in accordance with an international guideline and protocol for visual function measurements in paediatric cataract surgery and a protocol of the Childhood Cataract Program of the Chinese Ministry of Health (CCPMOH) and had an independent data and safety monitoring board of ZOC-CCPMOH. The current standard-of-care treatment for paediatric cataract involves removal of the cataractous lens through a relatively large opening using anterior continuous curvilinear capsulorhexis (ACCC, about 6 mm in diameter, Extended Data Fig. 1), followed by cataract extraction and artificial lens implantation or placement of postoperative aphakic eyeglasses/contact lens in paediatric cataract patients younger than two years. Some patients underwent additional posterior continuous curvilinear capsulorhexis (PCCC) and anterior vitrectomy. We established a new capsulorhexis surgery method to facilitate lens regeneration (Fig. 3a). First, we decreased the size of the capsulorhexis opening to 1.0–1.5 mm in diameter. This results in a minimal wound of about 1.2 mm2 in area, which is only about 4.3% the size of the wound created by the current method. Second, we moved the location of the capsulorhexis to the peripheral area of the lens instead of the central area. A 0.9 mm phacoemulsification probe was used to remove the lens contents and/or cortical opacities. These changes provide significant advantages. First, it considerably reduces the size of the injury, which resulted in a lower incidence of inflammation and much faster healing. Second, it moves the wound scar away from the central visual axis to the periphery, leading to improved visual axis transparency. Third, it preserves a nearly intact transparent lens capsule and layer of LECs, which have regenerative potential and are critically required for the regeneration of a natural lens. The clinical trial is an open label, randomized controlled trial in a study population of paediatric cataract patients (age: 0–2 years). Except the trial participants, all other parties (care providers, outcome assessors) were blinded to treatment allocation. A clinical trial consort flowchart is listed in the Extended Data Fig. 8a. Paediatric patients were enrolled accordingly inclusion and exclusion criteria below (ClinicalTrials.gov identifier: NCT01844258). Inclusion criteria were the following: infants were ≤24 months old, and diagnosed with bilateral uncomplicated congenital cataract with an intact non-fibrotic capsular bag. Exclusion criteria included preoperative intraocular pressure (IOP) >21 mm Hg, premature birth, family history of ocular disease, ocular trauma, or other abnormalities, such as microcornea, persistent hyperplastic primary vitreous, rubella, or Lowe syndrome. In total, twelve paediatric cataract patients (24 eyes) received the new minimally invasive lens surgery (Table 1). Twenty-five paediatric cataract patients (50 eyes in total) were enrolled as the control group to receive the current standard surgical treatment (Extended Data Fig. 8a). Bilateral eye surgeries of the same patient were conducted during the same operation session. We defined the incidence of corneal oedema as a >5% increase in central corneal thickness one week post-surgery, and the incidence of severe anterior chamber inflammation as a Flare value >10 evaluated by Pentacam system (OCULUS, Germany) and Laser flare meter (KOWA FM-600, Japan). Early-onset ocular hypertension was identified as IOP >21 mm Hg by Tonopen (Reichert, Seefeld, Germany) within one month after surgery. Macular oedema was identified by fundus OCT (iVue, Optovue, Germany) as an increase in central macular thickness >10% one week post-surgery. When indicated, VAO, defined by visual decline and the degree to which the fundus was obscured, was treated with YAG laser capsulotomy at follow-up. Compared to infants operated on using our new surgical technique, infants who received the traditional technique had a higher incidence of anterior chamber inflammation one week after surgery, early-onset ocular hypertension, and increased VAO (Table 1). However, in the group treated with our new method, a transparent regenerated biconvex lens was found in 100% of eyes three months after surgery, while no regenerated biconvex lenses formed in the group treated with the standard technique. In addition, 100% of the capsular openings healed within one month after surgery in the experimental group, but no capsular openings healed in the control group. Testing equipment included a set of Teller Acuity Cards (Vistech Consultants, Dayton, Ohio). The set of cards consists of 15 cards with gratings ranging in spatial frequency from 0.32 to 38 cycles per cm, in half-octave steps, and one blank grey card. A 4-mm peephole in each card allows the tester to view the child’s face through the card during testing. Test distance was kept constant by use of an aid to measure the distance from the child’s eyes to the card throughout testing. For 38 cm, the aid was the distance measured from the tester’s elbow to a specific knuckle on the tester’s hand, and for 55 cm, the aid was the length (55 cm) of the Teller Acuity Card. Testers were instructed to hold the cards without wrapping their fingers around the front side of the card, as this may attract the child’s attention. Testers presented the cards directly in front of the child and observed the child either over the top of the card or through the peephole in the card. During each acuity test, a masked visual acuity examiner was aware that the gratings were arranged in order from lower to higher spatial frequencies in half-octave steps, but were masked to the absolute spatial frequency of the grating on each card. The subset of spatial frequencies used for each test was selected according to a pseudorandom order from among three possible subsets of spatial frequencies for the subject’s age group. All three subsets for each age group included spatial frequencies known to be well above the threshold for that age group. To keep the visual acuity examiner masked to the absolute spatial frequency, the visual acuity examiner was not permitted to look at the front of the card to confirm the location of the grating. Instead, the visual acuity examiner asked an assistant to confirm the location of the grating on the card, after the visual acuity examiner had shown a card to the subject enough times to assess whether or not the subject could detect the grating. A clinical examiner was masked to the acuity results and the assigned patient group. Acuity was scored as the spatial frequency of the finest grating and was converted to log values before data analysis. We used a handheld auto-refractometer (PlusoptiX A09, OptiMed, Sydney, Australia) to evaluate the function of the regenerated lenses according to the manufacturer’s methods. Descriptive statistics was provided for the primary and secondary endpoints measured by intervention groups at each time point. Mean and standard deviation was reported for continuous variables and count and percentage is reported for categorical variables. To assess whether the primary outcome, decimal acuity, was significantly improved within each group, we performed the pre-post comparison between decimal acuity measured at baseline and study endpoint using paired t-tests. Normality of the data was checked and non-parametric alternatives, Wilcoxon signed-rank test is considered if the assumption was severely violated. To evaluate whether the mean response profiles in two groups were similar, we used the linear mixed-effect model taking account for the within-subjects correlation. The baseline decimal acuity was not adjusted by the model due to the homogeneity of this measurement as shown in the summary statistics. As the standard-of-care approach requires laser surgery at 3 months while the novel treatment does not, we fit two models using before and after laser surgery data, separately, to demonstrate the superiority of the novel approach. In each model, the outcome is the decimal acuity measured at four time points: baseline, 1 week, 3 months (before or after laser surgery) and 6 months; time (baseline as the reference level), treatment assignment and their interaction are the fixed effects; and patient is the random effect. Significant associations are identified using likelihood ratio test (LRT) by comparing models with and without a fixed effect. A linear mixed-effect model is fit again by dropping out the insignificant fixed effect until the final model is selected. A contrast test is performed when necessary. For the secondary aim, we compared the proportions of each condition of complications between two groups. We assumed the occurrence of complications for eyes from the same patient were independent. The mean difference and its 95% confidence interval was reported. A two-proportion z-test was used with the nonparametric χ2 test as alternative if the normality assumption was violated. All tests were two-sided and a P value less than 0.05 is considered to be statistically significant. Accommodative response was measured by an open-field autorefractor (SRW-5001K; Shin-Nippon, Tokyo, Japan), which allows targets to be viewed at any distance. The paediatric patients were positioned for autorefractor measurement with assistance from their parents. The patients were guided to fixate binocularly at a near target (33 cm, 5 × 5 array of smiley faces of N10 size) and a far target (3 m, 5 × 5 array of smiley faces of N10 size) by a trained and certified investigator or study coordinator. The measurements from non-cycloplegic autorefraction were performed three times at each target distance by the same trained and certified investigator throughout the study, in order to maintain accuracy and consistency throughout the trial. Measurements were taken in the same quiet environment with consistent room illumination to diminish the influence of distracting factors and to maintain subjects’ concentration. The spherical equivalent refractive value (SER) was recorded for each measurement and the mean value was calculated for evaluation of an accommodative response. The value of accommodative response was the difference between SER values for the near and the far target. We also used dynamic retinoscopy to measure the infants’ accommodation29, 30, 31. In brief, we recorded a lens dioptre value using retinoscopy when a patient was guided to fixate on a target 3 m away. Then another lens dioptre value was recorded when the target was moved closer, at a distance of 33 cm from the eyes. The difference between these two measurements was used to evaluate lens accommodative power.
News Article | August 23, 2016
Getting screened for Alzheimer’s disease could soon mean taking a trip to the eye doctor. Decreased retinal thickness, the presence of abnormal proteins, and changes in how the retinal blood vessels respond to light all appear to be signs of neurodegenerative disease, according to researchers who spoke at the recent Alzheimer’s Association International Conference (AAIC 2016) in Toronto. All of these could be detected with non-invasive eye exams, which would represent a huge leap forward for patients and Alzheimer’s researchers alike. Alzheimer’s is the most common cause of dementia, and it’s irreversible. It affects an estimated 5 million Americans, and the numbers are growing. But right now, there’s no perfect way to diagnose it: Doctors perform memory tests on their patients, or take a detailed family history, which means the disease sometimes isn’t caught until it’s progressed. A definitive diagnosis generally can’t be done until after the patient’s death, when clusters of abnormal proteins called amyloid plaques (a hallmark of the disease) can be found in brain tissue samples. Earlier detection would mean that patients and their families could plan ahead, and that researchers could better study the disease. Improved screening methods would enable doctors to identify who’s at risk, maybe even before their symptoms start to show. Read More: Can Learning to Code Delay Alzheimer's? The eyes are attracting attention as a portal to what’s happening in the brain. At a session at the AAIC 2016, researchers focused on the retina, which sits in the back of the eye and is made up of nerve tissue. The eyes are like windows into the brain, said Melanie Campbell, professor of optometry and vision science at the University of Waterloo. She told Motherboard in an interview that amyloid plaques can appear in the back of the eyes on the retina. It’s possible amyloids leak into the vitreous fluid of the eye from the cerebrospinal fluid, Campbell said. Researchers also hypothesize that amyloid proteins are synthesized by neural cells within the eye, a similar process to what happens in the brains of Alzheimer’s patients, appearing in both the retina and the vitreous fluid. Right now, in the lab, amyloids can be detected on retinas using rather complicated and expensive eye-imaging techniques. But Campbell and colleagues developed a prototype device that does the job more easily and cheaply. This new technology, called polarimetry, uses polarized light. “It turns out amyloids show up very clearly under polarized light,” she said. She presented results of a series of proof-of-concept scans done on human and canine retinas. The scans were conducted on a series of cadaver retinas from the Eye Bank of Canada (20 from people who had Alzheimer’s, and 22 controls), as well on living and postmortem canine retinas. The researchers found that amyloid deposits were not only easy to detect with this new technology, but it was relatively easy to count them, and to measure their size—something other imaging techniques can’t do. The next step will be testing the device clinically on patients with Alzheimer’s disease, Campbell said. However, the presence of amyloids isn’t a guaranteed way to diagnose it; they show risk so this would be for screening. Another clue of the disease is thin retinal nerve fiber layers (RNFL). In fact, the thinner RNFLs are, the poorer the cognition levels of subjects, according to Fang Ko, clinical associate professor of ophthalmology, Florida State University and Moorfields Eye Hospital in the UK, who also spoke at the conference. Here, researchers used data from the UK Biobank, which included medical and health details of 500,000 volunteers aged between 40 to 69 years from across England. Of these, 67,000 underwent eye exams, which included retinal imaging. Many were ultimately excluded (including those with diabetes or other conditions that affect the retina), leaving about 32,000 subjects. They completed four different cognitive tests. Of those, a total of 1,251 participants went on to repeat the cognitive tests after three years. Researchers found that people with thinner RNFLs performed worse on each of the cognitive tests than those whose RNFLs were thicker. And those who started the study with thinner RNFL had greater cognitive decline at the three year follow-up than those who had thicker ones. It may be possible to use thin RNFL as a predictor of cognitive decline, she said, but it isn’t a surefire method: diseases like glaucoma can also affect its thickness, so once again, this could be a useful tool for screening rather than diagnosis. A third technique, using a flickering light exam of the retinal blood vessels, could also help screen for Alzheimer’s, according to Konstantin Kotliar, a biomedical engineer at the Aachen University of Applied Sciences in Germany. In healthy eyes, a flickering light shone on the retina causes immediate dilation of both retinal arteries and veins. “In people with Alzheimer’s disease, retinal arteries and veins have a delayed reaction to a flickering light test,” he said. But, they undergo greater dilation than in people without the disease. (Diminished and sometimes delayed dilation is also seen in eye diseases like glaucoma, he said.) At the conference, Kotliar presented a study (unpublished as of yet) measuring and comparing retinal vessel reactions to flickering light in patients aged 60 to 79. Fifteen had mild-to-moderate dementia due to Alzheimer’s; 24 had mild cognitive impairment, also from Alzheimer’s, and 15 were healthy controls with no cognitive impairment. Retinal artery and vein reactions to 20-second-long flicker stimulation were measured. Both arteries and veins dilated more in people with mild to moderate Alzheimer’s than in controls. Also, the start of dilation in the retinal arteries took longer in people with Alzheimer’s than in controls—though the delay wasn’t as pronounced in the veins. How the retinal vessels behaved in Alzheimer’s patients was a surprise, and this might contribute to another screening test, he said. Finding new ways to screen for Alzheimer’s has never been more important: with the number of patients expected to balloon in years to come, so finding new ways to detect it will crucial.
No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. The human iPS cell lines 201B7, 253G1, and 454E2 were obtained from the RIKEN Bio Resource Center (Tsukuba, Japan)21, 22. The 1231A3 and 1383D2 human iPS cells were provided by the Center for iPS Cell Research and Application, Kyoto University23. All cells were cultured in StemFit medium (Ajinomoto, Tokyo, Japan) on LN511E8-coated (0.5 μg cm−2) dishes23, 24. LN511E8, produced using cGMP-banked CHO-S cells (Life Technologies, Carlsbad, CA), was obtained from Nippi (Tokyo, Japan). In part, LN511E8 was produced using human 293-F cells as previously described12. The 201B7 and 454E2 human iPS cell lines were used in the in vitro experiments, while 201B7 and 1383D2 cells were used in the animal experiments; 253G1 and 1231A3 cells were used in the supplementary experiments, the results of which are reported in Extended Data Fig. 7. All of the experiments using recombinant DNA were approved by the Recombinant DNA Committees of Osaka University and were performed according to our institutional guidelines. The differentiation culture for human iPS cells was performed as indicated in Fig. 3a. First, human iPS cells were seeded on LN511E8-coated dishes at 350–700 cells cm−2, after which they were cultivated in StemFit medium for 8–12 days. The culture medium was then changed to DM (differentiation medium; GMEM (Life Technologies) supplemented with 10% knockout serum replacement (KSR; Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM non-essential amino acids (Life Technologies), 2 mM l-glutamine (Life Technologies), 1% penicillin-streptomycin solution (Life Technologies) and 55 μM 2-mercaptoethanol (Life Technologies) or monothioglycerol (Wako, Osaka, Japan))25. In some experiments, as indicated in the Results section, Noggin (R&D systems, Minneapolis, MN), LDN-193189 (Wako) or SB-431542 (Wako) were added for the first four days. BMP4 (R&D systems) was used in some early experiments at concentrations up to 0.125 nM. This had no discernible effect on SEAM formation, however, so its use was discontinued. After four weeks of culture in DM, the medium was changed to corneal differentiation medium (CDM; DM and Cnt-20 or Cnt-PR (w/o; EGF and FGF2) (1:1, CELLnTEC Advanced Cell Systems, Bern, Switzerland) containing 5 ng ml−1 FGF2 (Wako), 20 ng ml−1 KGF (Wako) 10 μM Y-27632 (Wako) and 1% penicillin-streptomycin solution). FGF2 in CDM was not essential for corneal epithelial induction. During CDM culture (around six to eight weeks of differentiation), non-epithelial cells were removed by manual pipetting under microscopy (Extended Data Fig. 2a, b). After pipetting, the medium was changed to fresh CDM. After four weeks of culture in CDM, the medium was changed to corneal epithelium maintenance medium (CEM; DMEM/F12 (2:1), Life Technologies) containing 2% B27 supplement (Life Technologies), 1% penicillin-streptomycin solution, 20 ng ml−1 KGF and 10 μM Y-27632 for two to seven weeks. To achieve retinal differentiation (Fig. 2c) after four weeks of differentiation the medium was directly changed to CEM. Isolated RPE cell colonies were cultivated in CEM on separate dishes coated with LN511E8. Phase-contrast microscopic observations were performed with an Axio-observer.Z1, D1 (Carl Zeiss, Jena, Germany) and an EVOS FL Auto (Life Technologies). Differentiated human iPS cells in CEM were dissociated using Accutase (Life Technologies), and resuspended in ice-cold KCM medium (DMEM without glutamine and Nutrient Mixture F-12 Ham (3:1, Life Technologies) supplemented with 5% FBS (Japan Bio Serum, Hiroshima, Japan), 0.4 μg ml−1 hydrocortisone succinate (Wako), 2 nM 3,3′,5-Triiodo-l-thyronine sodium salt (MP biomedicals, Santa Ana, CA), 1 nM cholera toxin (List Biological Laboratory, Campbell, CA), 2.25 μg ml−1 bovine transferrin HOLO form (Life Technologies), 2 mM l-glutamine, 0.5% insulin transferrin selenium solution (Life Technologies) and 1% penicillin-streptomycin solution). The harvested cells were filtered with a cell strainer (40 μm, BD Biosciences, San Diego, CA) and then stained with anti-SSEA-4 (MC813-70, Biolegend, San Diego, CA), TRA-1-60 (TRA-1-60-R, Biolegend) and CD104 (ITGB4; 58XB4, Biolegend) antibodies for 1 h on ice. After being washed twice with PBS, stained cells underwent cell sorting with a FACSAria II instrument (BD Biosciences). For intracellular protein staining, a BD Cytofix/Cytoperm (BD Biosciences) kit was used. In all of the experiments, cells were stained with non-specific isotype IgG or IgM as controls (Biolegend). The data were analysed using the BD FACSDiva Software (BD Biosciences) and the FlowJo software program (TreeStar, San Carlos, CA). Sorted human iPS cell-derived epithelial cells obtained from zone 3 of the SEAM (human iCECs) were seeded on LN511E8 coated (0.5 μg cm−2) cell culture inserts or temperature-responsive dishes (UpCell, CellSeed, Tokyo, Japan) without cell passaging, and were cultured in CEM until confluence26. To promote maturation, the epithelial cells were cultivated in CMM (corneal epithelium maturation medium; KCM medium containing 20 ng ml−1 KGF and 10 μM Y-27632) for an additional 3–14 days after CEM culture. The human iCECs cultivated on temperature-responsive dishes were released from their substrate by reducing the temperature to 20 °C. Total RNA was obtained from differentiated human iPS cells after specific culture periods, from human epidermal keratinocytes (EKs (foreskin), Life Technologies and TaKaRa Bio, Otsu, Japan), and from human corneal limbal epithelial cells (CECs) using the RNeasy total RNA kit or the QIAzol reagent (Qiagen, Valencia, CA). Reverse transcription was performed using the SuperScript III First-Strand Synthesis System for qRT–PCR (Life Technologies) according to the manufacturer’s protocol, and cDNA was used as a template for PCR. qRT–PCR was performed using the ABI Prism 7500 Fast Sequence Detection System (Life Technologies) in accordance with the manufacturer’s instructions. The TaqMan MGB used in the present study are shown in Supplementary Table 2. The thermocycling program was performed with an initial cycle at 95 °C for 20 s, followed by 45 cycles at 95 °C for 3 s and 60 °C for 30 s. Research grade human skin tissue sections were obtained from US Biomax Inc. (MD, USA) and human oral mucosal tissue was obtained from Science Care (Phoenix, AZ). The cells were fixed in 4% paraformaldehyde (PFA) or cold methanol, washed with Tris-buffered saline (TBS, TaKaRa Bio) three times for 10 min and incubated with TBS containing 5% donkey serum and 0.3% Triton X-100 for 1 h to block non-specific reactions. They were then incubated with the antibodies shown in Supplementary Table 3 at 4 °C overnight or at room temperature for 3 h. The cells were again washed twice with TBS for 10 min, and were incubated with a 1:200 dilution of Alexa Fluor 488-, 568-, 647-conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. Counterstaining was performed with Hoechst 33342 (Molecular Probes) before fluorescence microscopy (Axio Observer.D1, Carl Zeiss). Fabricated human iCEC sheets were fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque, Kyoto, Japan). After washing with distilled water, the human iCEC sheets were embedded in paraffin from which 3-μm-thick sections were cut. These were stained with haematoxylin and eosin following deparaffinization and hydration. The sections were observed with a NanoZoomer-XR C12000 (Hamamatsu Photonics, Hamamatsu, Japan), BZ-9000 (KEYENCE, Osaka, Japan) and an Axio Observer.D1. Differentiated human iPS cells (more than 12 weeks of differentiation) were fixed with 10% formaldehyde neutral buffer solution, after which PAS staining was performed with a PAS staining kit (MERCK KGaA, Darmstadt Germany) according to the manufacturer’s protocol. The sections were observed with an Axio Observer.D1. Epithelial cells were seeded onto MMC-treated NIH-3T3 feeder layers at a density of 3,000–20,000 cells per well. These were cultivated in CMM for 7–14 days. The colonies were fixed with 10% formaldehyde neutral buffer solution and then stained with rhodamine B (Wako). Colony formation was then assessed using a dissecting microscope and the colony-forming efficiency (CFE) was calculated. For the holoclone analysis, a single human iCEC colony derived from the SEAM was cultivated on 3T3-J2 (provided by H. Green, Harvard Medical School, Boston, MA) in CMM for 7–11 days was picked up under a dissecting microscope and dissociated by TrypLE Select (Life Technologies). The dissociated human iCECs were again seeded on a MMC-treated 3T3-J2 feeder layer and cultivated in CMM for 10–13 days. The colonies were scored under a microscope and classified as holoclones, paraclones or meloclones based on previously reported methods27. Human CECs were harvested from corneoscleral rims (Northwest Lions Eye Bank, Seattle, WA) as reported previously28. Human CECs and human oral keratinocytes (OKs; ScienCell, Carlsbad, CA) along with SEAM-derived human iCECs were cultivated on LN511E8 coated cell culture inserts in CEM until confluent. They were then cultivated in CMM. Human dermal fibroblasts (DFs; ScienCell) were cultivated in DMEM/F12 (2:1) containing 10% FBS. Total RNA was obtained from human iPS cells, iCECs, CECs, OKs, DFs, and six-week differentiated iPS cells (that is, OSE) using the QIAzol reagent. A microarray analysis using Sure Print G3 human 8x60K slides (Agilent technologies, Palo Alto, CA) was performed at Takara Bio. The data were analysed using the GeneSpring GX software program (Agilent technologies). Microarray data used in this study are deposited in Gene Expression Omnibus under accession number GSE73971. The cultivated epithelial cell sheets were fixed in 2.5% glutaraldehyde (Nacalai Tesque) at 4 °C overnight. Subsequently, the sheets were washed in buffer, dehydrated with ethanol and tert-butyl alcohol (Wako), and critical point dried (JFD-320, JEOL, Tokyo, Japan). After sputter-coating with platinum in an auto fine coater (JFCL-1600, JEOL), the samples were observed by scanning electron microscopy (JSM-6510LA, JEOL) at 5 kV. FACS-isolated human iCECs were cultivated on MMC-treated NIH-3T3 feeder layers in CMM up to 70–80% confluence. The human iCECs were harvested using TrypLE Select following the removal of feeder cells by manual pipetting. The total cell numbers were counted, after which the cells were passaged at a 1:8 ratio onto newly prepared feeder layers. These were cultivated in CMM until sub-confluence was reached again. The G-band karyotype analysis for human iCECs was performed at Nihon Gene Research Laboratories (Sendai, Japan). All animal experimentation was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the animal ethics committees of Osaka University. To examine embryonic mouse eyes, pregnant females (C57/BL6, E9.5–18.5) were acquired from SLC Japan (Shizuoka, Japan). For the transplantation experiments, Female New Zealand white rabbits (2.5–3.0 kg (approximately 12–14 weeks)) were obtained from Kitayama Labes (Nagano, Japan). Harvested human iCEC sheets were grafted onto rabbit corneas, in which a total epithelial limbal stem-cell deficiency had been created following a corneal and limbal lamellar keratectomy (Extended Data Fig. 8c–j). After surgery, 0.3% ofloxacin ointment (Santen Pharmaceutical, Osaka, Japan), 0.1% betamethasone phosphate eye drops (Shionogi Pharmaceutical, Osaka, Japan) and 0.1% sodium hyaluronate eye drops (Santen Pharmaceutical) were applied three to four times per day. Triamcinolone acetonide (8 mg; Bristol Myers Squibb, Tokyo, Japan) was also administered by subconjunctival injection. Tacrolimus (0.05 mg kg−1 per day, Astellas Pharma, Tokyo, Japan) and Mizoribine (4.0 mg kg−1 per day Sawai Pharmaceutical, Osaka, Japan) were systemically administered using an osmotic pump (DURECT, Cupertino, CA). The corneal barrier function following surgery was assessed by 0.5% fluorescein eye drop instillation at day 7 and day 14 after surgery and the fluorescein negative area was calculated using the AxioVision software program (Carl Zeiss). Throughout the healing period, the cornea was observed with a digital slit-lamp camera (SL-7F, TOPCON, Tokyo, Japan) and 3D OCT1000 MARK II (TOPCON) or CASIA SS-1000 (TOMEY, Nagoya, Japan) machines. If an infection was found or if unexpected weight loss occurred, animals were excluded from the analysis. The rabbits were euthanized by the intravenous administration of sodium pentobarbitone 14 days after transplantation, after which the eyes were immediately enucleated for the histological analyses. No blinding or randomization was conducted to allocate animals to each group. The data are expressed as means ± standard deviation (s.d.). The statistical analyses were performed using the Mann–Whitney rank sum test or Steel’s test. Bonferroni’s correction was applied to the data in animal experiments. All of the statistical analyses were performed using the JMP software program (SAS institute Inc., Cary, NC). No statistical methods were used to predetermine sample size. Comprehensive technical details can be found in Protocols Exchange, http://dx.doi.org/10.1038/protex.2016.009.