A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Phenotype microarray analysis of the drug efflux systems in Salmonella enterica serovar Typhimurium.

A large number of drug efflux transporters have been identified in Salmonella enterica serovar Typhimurium, and increased expression of these transporters confers drug resistance in this organism. Here we compared the respiration activities of the wild-type strain and a mutant with nine deleted transporters by phenotype microarray analysis. The mutant was susceptible to 66 structurally unrelated compounds including many antibiotics, dyes, detergents, antihistamine agents, plant alkaloids, antidepressants, antipsychotic drugs, and antiprotozoal drugs. To investigate the effect of each transporter on the susceptibilities to these drugs, we used the single transporter mutants, several multiple deletion mutants, and the transporter overexpressor strains to determine minimum inhibitory concentrations of ampicillin, erythromycin, minocycline, ciprofloxacin, orphenadrine, amitriptyline, thioridazine, and chlorpromazine. The data indicate that the increased susceptibilities of the mutant lacking nine transporter genes are mainly dependent on the absence of the acrAB efflux genes as well as the tolC gene. In addition to the AcrAB-TolC efflux system, the results from the overexpressor strains show that AcrEF confers resistance to these compounds as well as AcrAB of Escherichia coli, MexAB-OprM and MexXY-OprM of Pseudomonas aeruginosa. The results highlight the importance of the efflux systems not only for resistance to antibiotics but also for resistance to antihistamine agents, plant alkaloids, antidepressants, antipsychotic drugs, and antiprotozoal drugs.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa.

Transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into retinal disease animal models has yielded promising results, and several clinical trials on iPSC-derived retinal pigment epithelial cell transplantation have confirmed its safety. In this study, we performed allogeneic iPSC-derived retinal organoid sheet transplantation in two subjects with advanced retinitis pigmentosa (jRCTa050200027). The primary endpoint was the survival and safety of the transplanted retinal organoid sheets in the first year post-transplantation. The secondary endpoints were the safety of the transplantation procedure and visual function evaluation. The grafts survived in a stable condition for 2 years, and the retinal thickness increased at the transplant site without serious adverse events in both subjects. Changes in visual function were less progressive than those of the untreated eye during the follow-up. Allogeneic iPSC-derived retinal organoid sheet transplantation is a potential therapeutic approach, and the treatment's safety and efficacy for visual function should be investigated further.

Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture.

BACKGROUND: The BMP signaling pathway plays a key role in growth, differentiation and patterning during neural development. Recent work on the generation of a self-organization of three-dimensional retinal organoid (3D-retina) from human pluripotent stem cells (hPSCs) revealed that addition of recombinant human BMP4 (rhBMP4) promotes retinal differentiation in the early neural differentiation stage. For clinical application, efficient differentiation from hPSCs to retinal cells with minimal numbers of off-target non-retinal cells is desirable. We therefore aimed to further improve an efficient retinal differentiation method for future up-scaling of cell production. METHODS: hPSCs were differentiated into 3D-retina using a modified SFEBq method. The effect of rhBMP4 with or without Checkpoint kinase 1 (Chk1) inhibitor (PD407824), a modulator of BMP signaling pathway, at day 3 was compared by characterizing the differentiating 3D-retina by the use of the hPSCs and immunohistochemical analysis. RESULTS: The Chk1 inhibitor treatment promoted retinal differentiation from hPSCs, in combination with low-concentration rhBMP4. Addition of a Chk1 inhibitor generated a unique type of organoid with neural retina (NR) encapsulated in retinal pigment epithelium (RPE), possibly by promoting phosphorylation of SMAD1/5/9 in the cells inside the early aggregates. We confirmed that the Chk1-inhibitor-treated hPSC-3D-retina differentiated into rod and cone photoreceptor precursors and other types of retinal neurons, in long-term culture. CONCLUSIONS: In this study, we found that combined use of rhBMP4 and a Chk1 inhibitor cooperatively promoted retinal differentiation from hPSCs. Our new retinal differentiation method is a promising option for the stable supply and up-scaling of production of 3D-retina for future cell therapy.

A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation.

Pluripotent stem cell (PSC)-derived retinal sheet transplanted in vivo can form structured photoreceptor layers, contact with host bipolar cells, and transmit light signals to host retinas. However, a major concern is the presence of graft bipolar cells that may impede host-graft interaction. In this study, we used human ESC-retinas with the deletion of Islet-1 (ISL1) gene to achieve the reduced graft ON-bipolar cells after xenotransplantation into end-stage retinal degeneration model rats. Compared with wild-type graft, ISL1 -/- hESC-retinas showed better host-graft contact, with indication of host-graft synapse formation and significant restoration of light responsiveness in host ganglion cells. We further analyzed to find out that improved functional integration of ISL1 -/- hESC-retinas seemed attributed by a better host-graft contact and a better preservation of host inner retina. ISL1 -/- hESC-retinas are promising for the efficient reconstruction of a degenerated retinal network in future clinical application.

Competency of iPSC-derived retinas in MHC-mismatched transplantation in non-human primates.

Transplantation of embryonic/induced pluripotent stem cell-derived retina (ESC/iPSC-retina) restores host retinal ganglion cell light responses in end-stage retinal degeneration models with host-graft synapse formation. We studied the immunological features of iPSC-retina transplantation using major histocompatibility complex (MHC)-homozygote monkey iPSC-retinas in monkeys with laser-induced retinal degeneration in MHC-matched and -mismatched transplantation. MHC-mismatched transplantation without immune suppression showed no evident clinical signs of rejection and histologically showed graft maturation without lymphocytic infiltration, although immunological tests using peripheral blood monocytes suggested subclinical rejection in three of four MHC-mismatched monkeys. Although extensive photoreceptor rosette formation was observed on histology, evaluation of functional integration using mouse models such as mouse ESC-retina (C57BL/6) transplanted into rd1(C3H/HeJ, MHC-mismatched model) elicited light responses in the host retinal ganglion cells after transplantation but with less responsiveness than that in rd1-2J mice (C57BL/6, MHC-matched model). These results suggest the reasonable use of ESC/iPSC-retina in MHC-mismatched transplantation, albeit with caution.

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor ß signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.

Publisher Correction: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Crystal structure of the multidrug resistance regulator RamR complexed with bile acids.

During infection, Salmonella senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for Salmonella to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the ramRA locus, which is composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π-π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6 G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of ramA and acrAB-tolC. Our results reveal that Salmonella senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of acrAB-tolC expression with resulting tolerance to bile-rich environments.

Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.

Medium- to long-term survival and functional examination of human iPSC-derived retinas in rat and primate models of retinal degeneration.

BACKGROUND: We have previously reported that xeno-transplanted human ESC-derived retinas are able to mature in the immunodeficient retinal degeneration rodent models, similar to allo-transplantations using mouse iPSC-derived retina. The photoreceptors in the latter developed outer segments and formed synapses with host bipolar cells, driving light responses of host retinal ganglion cells. In view of clinical application, here we further confirmed the competency of human iPSC-derived retina (hiPSC-retina) to mature in the degenerated retinas of rat and monkey models. METHODS: Human iPSC-retinas were transplanted in rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats and two monkeys with laser-induced photoreceptor degeneration. Graft maturation was studied by immunohistochemistry and its function was examined by multi-electrode array (MEA) recording in rat retinas and visually-guided saccade (VGS) in a monkey. FINDINGS: A substantial amount of mature photoreceptors in hiPSC-retina graft survived well in the host retinas for at least 5 months (rat) to over 2 years (monkey). In 4 of 7 transplanted rat retinas, RGC light responses were detected at the grafted area. A mild recovery of light perception was also suggested by the VGS performance 1.5 years after transplantation in that monkey. INTERPRETATION: Our results support the competency of hiPSC-derived retinas to be clinically applied for transplantation therapy in retinal degeneration, although the light responses observed in the present models were not conclusively distinguishable from residual functions of degenerating host retinas. The functional analysis may be further elaborated using other models with more advanced retinal degeneration.

Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation.

Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets.

The crystal structure of multidrug-resistance regulator RamR with multiple drugs.

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.

Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses.

BACKGROUND: Many Gram-positive and Gram-negative bacteria produce large quantities of indole as an intercellular signal in microbial communities. Indole demonstrated to affect gene expression in Escherichia coli as an intra-species signaling molecule. In contrast to E. coli, Salmonella does not produce indole because it does not harbor tnaA, which encodes the enzyme responsible for tryptophan metabolism. Our previous study demonstrated that E. coli-conditioned medium and indole induce expression of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium for inter-species communication; however, the global effect of indole on genes in Salmonella remains unknown. RESULTS: To understand the complete picture of genes regulated by indole, we performed DNA microarray analysis of genes in the S. enterica serovar Typhimurium strain ATCC 14028s affected by indole. Predicted Salmonella phenotypes affected by indole based on the microarray data were also examined in this study. Indole induced expression of genes related to efflux-mediated multidrug resistance, including ramA and acrAB, and repressed those related to host cell invasion encoded in the Salmonella pathogenicity island 1, and flagella production. Reduction of invasive activity and motility of Salmonella by indole was also observed phenotypically. CONCLUSION: Our results suggest that indole is an important signaling molecule for inter-species communication to control drug resistance and virulence of S. enterica.