Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation.

Introduction: The retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. Methods: A retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. Results: Gelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. Conclusion: Gelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.

Inducing human retinal pigment epithelium-like cells from somatic tissue.

Regenerative medicine relies on basic research outcomes that are only practical when cost effective. The human eyeball requires the retinal pigment epithelium (RPE) to interface the neural retina and the choroid at large. Millions of people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among RPE degradation pathologies. Recently, autologous pluripotent stem-cell-derived RPE cells were prohibitively expensive due to time; therefore, we developed a faster reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts (Fibs) by conditional overexpression of both broad plasticity and lineage-specific transcription factors (TFs). iRPE cells displayed critical RPE benchmarks and significant in vivo integration in transplanted retinas. Herein, we detail the iRPE system with comprehensive single-cell RNA sequencing (scRNA-seq) profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for basic research and affordable autologous human RPE tissue for regenerative cell therapy.

A Case of Vogt-Koyanagi-Harada Disease-Like Uveitis Induced by Nivolumab and Ipilimumab Combination Therapy.

Nivolumab and ipilimumab are widely used immune checkpoint inhibitors (ICPIs) for the treatment of metastatic melanoma. ICPIs cause an array of side effects called immune-related adverse events (IRAEs) due to activation of an immune response. ICPI-uveitis can cause irreversible vision loss if untreated. There are few reports of recurrent Vogt-Koyanagi-Harada (VKH) disease-like uveitis induced by nivolumab and ipilimumab. We report a case of VKH disease-like uveitis recurrence after resuming ICPIs. A 73-year-old man with advanced melanoma was referred to our clinic with visual loss 25 days after starting nivolumab/ipilimumab. His corrected visual acuity was 0.5 in the right eye and 0.02 in the left eye. Enhanced-depth imaging optical coherence tomography (EDI-OCT) showed marked choroid thickening. The patient was diagnosed with VKH disease-like uveitis due to IRAEs. Subtenon injection of triamcinolone acetonide was performed, and nivolumab/ipilimumab was suspended, but serous retinal detachment (SRD) markedly worsened and choroidal detachment appeared. With 2 courses of steroid pulse therapy and oral steroids, SRD disappeared, and corrected visual acuity recovered in both eyes. Five months after the first injection, exacerbation of melanoma was observed, and nivolumab and oral steroids were restarted. Six weeks later, an increase in choroidal thickness was observed with EDI-OCT and diagnosed as a recurrence of VKH disease-like uveitis. Monitoring for the recurrence of VKH disease-like uveitis during the administration of ICPIs, even after uveitis is treated, is essential. Assessment of choroidal thickness with EDI-OCT may be useful for detecting early signs of VKH disease-like uveitis.

HuR regulates alternative splicing of the TRA2ß gene in human colon cancer cells under oxidative stress.

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2ß gene encodes splicing factor transformer 2ß (Tra2ß) and generates 5 mRNA isoforms (TRA2ß1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38(MAPK))-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2ß exon 2, generating a TRA2ß4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38(MAPK) double knockdown inhibited the arsenite-stimulated production of TRA2ß4 and increased Tra2ß protein, facilitating Tra2ß-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2ß minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2ß4 interaction and TRA2ß4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2ß4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.

Oxidative stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 production in human colon cancer cells.

Serine/arginine-rich splicing factor 3 (SRSF3) is a member of the SR protein family and plays wide-ranging roles in gene expression. The human SRSF3 gene generates two alternative splice transcripts, a major mRNA isoform (SRSF3-FL) encoding functional full-length protein and a premature termination codon (PTC)-containing isoform (SRSF3-PTC). The latter is degraded through nonsense-mediated mRNA decay (NMD). Treatment of a human colon cancer cell line (HCT116) with 100 µM sodium arsenite increased SRSF3-PTC mRNA levels without changing SRSF3-FL mRNA levels. A chemiluminescence-based NMD reporter assay system demonstrated that arsenite treatment inhibited NMD activity and increased SRSF3-PTC mRNA levels in the cytoplasm, facilitating translation of a truncated SRSF3 protein (SRSF3-TR) from SRSF3-PTC mRNA. SRSF3-TR lacked two-thirds of the Arg/Ser-rich (RS) domain whose phosphorylation state is known to be crucial for subcellular distribution. SRSF3-FL was localized in the nucleus, while overexpressed SRSF3-TR was diffusely distributed in the cytoplasm and the nucleus. A part of SRSF3-TR was also associated with stress granules in the cytoplasm. Interestingly, treatment of HCT116 cells with a small interference RNA specifically targeting SRSF3-PTC mRNA significantly attenuated arsenite-stimulated induction of c-JUN protein, its binding activity to the AP-1 binding site (-126 to 120 bp) in the interleukin (IL)-8 gene promoter, and AP-1 promoter activity, resulting in significant reduction of arsenite-stimulated IL-8 production. Our results suggest that SRSF3-TR may function as a positive regulator of oxidative stress-initiated inflammatory responses in colon cancer cells.

Truncated serine/arginine-rich splicing factor 3 accelerates cell growth through up-regulating c-Jun expression.

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the SRSF family, plays a wide-ranging role in gene expression. The human SRSF3 gene generates a major mRNA isoform encoding a functional, full-length protein and a PTC-containing isoform (SRSF3-PTC). The latter is expected to be degraded through the nonsense-mediated mRNA decay system. However, it was reported that SRSF3-PTC mRNA was produced under stressful conditions and translated into a truncated SRSF3 protein (SRSF3-TR). To disclose unknown functions of SRSF3-TR, we established Flp-In-293 cells stably expressing SRSF3-TR. The SRSF3-TR-expressing cells increased mRNA and protein levels of positive regulators for G1 to S phase transition (cyclin D1, cyclin D3, CDC25A, and E2F1) and accelerated their growth. c-Jun is required for progression through the G1 phase, the mechanism by which involves transcriptional control of the cyclin D1 gene. We also found that the JUN promoter activity was significantly increased in the Flp-In-293 cells stably expressing SRSF3-TR, compared with mock-transfected control cells. The SRSF3-TR-expressing cells increased c-Jun and Sp-1 levels, which are important for the positive autoregulation and basal transcription of JUN, respectively. Our results suggest that stress-inducible SRSF3-TR may participate in the acceleration of cell growth through facilitating c-Jun-mediated G1 progression under stressful conditions.

Ets1 and heat shock factor 1 regulate transcription of the Transformer 2ß gene in human colon cancer cells.

BACKGROUND: Transformer (Tra) 2ß is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2ß expression, while the regulatory mechanism of Tra2ß expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2ß in human colon cancer cells. METHODS: We cloned 959 bp-upstream of the human TRA2ß 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2ß promoter. Tra2ß expression in the human colon and colon cancer tissues was examined by immunohistochemistry. RESULTS: In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2ß1 mRNA encoding a functional, full-length Tra2ß with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2ß promoters and Chip assay demonstrated that an Ets1-binding site present at -64 to -55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at -145 to -99 bp mediated the oxidant-induced transactivation of TRA2ß. Tra2ß knockdown caused apoptosis of HCT116 cells. Tra2ß were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. CONCLUSIONS: Our results suggest that oxidative stress-responsive Tra2ß may play an important role in colon cancer growth.

MicroRNAs miR-144/144* and miR-16 in peripheral blood are potential biomarkers for naturalistic stress in healthy Japanese medical students.

Non-coding microRNAs (miRNAs) are suggested to serve fundamental roles in cellular stress responses and in coping with sudden environmental changes in experimental animals. We examined whether naturalistic stressor-responsive miRNAs were detectable in whole blood. Blood and saliva were collected between 16:00 and 17:00 from 10 healthy medical students (5 males and 5 females; aged 22.4±0.8 years, mean±SD) 7 weeks before, one day before, immediately after, and one week after a nationally administered examination for academic promotion. Samples obtained one week after the examination were used as baseline controls. State anxiety and salivary cortisol levels reached maximum levels the day before the examination. Eleven candidate miRNAs (miR-144, -144*, -16, -15a, -19a, -19b, -26b, -30b, -106b, -126, and -142-3p) were extracted using a human miRNA microarray, and quantitative real-time reverse transcription PCR confirmed significant elevation of miR-144/144* and miR-16 levels immediately after finishing the examination. miR-16 levels in individual students were positively correlated with those of serum tumor necrosis factor (TNF)-α measured immediately after the examination. Percentage changes in miR-144* and miR-16 levels from immediately after to one week after the examination were significantly correlated with percentage changes in circulating interferon-γ and/or TNF-α levels over the same time points. Our results suggest that miR-144/144* and miR-16 may constitute a part of an integrated response to naturalistic stressors in healthy young adults.

Skipping of an alternative intron in the srsf1 3' untranslated region increases transcript stability.

The srsf1 gene encodes serine/arginine-rich splicing factor 1 (SRSF1) that participates in both constitutive and alternative splicing reactions. This gene possesses two ultraconserved elements in the 3' untranslated region (UTR). Skipping of an alternative intron between the two elements has no effect on the protein-coding sequence, but it generates a premature stop codon (PTC)-containing mRNA isoform, whose degradation is considered to depend on nonsense-mediated mRNA decay (NMD). However, several cell lines (HCT116, RKO, HeLa, and WI38 cells) constitutively expressed significant amounts of the srsf1 PTC variant. HCT116 cells expressed the PTC variant nearly equivalent to the major isoform that includes the alternative intron in the 3' UTR. Inhibition of NMD by silencing a key effecter UPF1 or by treatment with cycloheximide failed to increase amounts of the PTC variant in HCT116 cells, and the PTC variant was rather more stable than the major isoform in the presence of actinomycin D. Our results suggest that the original stop codon may escape from the NMD surveillance even in skipping of the alternative intron. The srsf1 gene may produce an alternative splice variant having truncated 3' UTR to relief the microRNA- and/or RNA-binding protein-mediated control of translation or degradation.

Brief naturalistic stress induces an alternative splice variant of SMG-1 lacking exon 63 in peripheral leukocytes.

Alternative splicing (AS) not only regulates the gene expression program in response to surrounding environment, but also produces protein isoforms with unique properties under stressful conditions. However, acute psychological stress-initiated AS events have not been documented in human studies. After assessments of changes in salivary cortisol levels and anxiety among 28 fourth-grade medical students 7 weeks prior to, 1 day before, immediately after, and 1 week after an examination for promotion, we selected 5 male students, who showed a typical stress response, and screened AS events in their circulating leukocytes using the GeneChip human exon 1.0 ST array. AS events of 27 genes with splicing indices >1.0 could be detected between immediately after and either 7 weeks before, 1 day before, or 1 week after the examination. The examination stress preferentially caused skipping rather than inclusion: 21 out of the 27 pre-mRNAs underwent skipping of exons, and skipping in 3'UTR was observed in 8 genes. Among the candidate genes, real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG-1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p53-dependent pathways and nonsense-mediated mRNA decay. Our results indicate a significant impact of brief naturalistic stressors on AS-mediated regulation of gene expression in peripheral leukocytes, and suggest the SMG-1 splice variant as a potential biomarker for acute psychological stress.