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Hippo signaling pathway is involved in many biological processes including the fate decision of embryonic stem cells (ESCs). Yes-associated protein (Yap) function as a key effector of Hippo pathway, but its role in ESCs is still controversial. So far, only two isoforms of Yap have been identified and they have both overlapping and distinct functions. Here, we identify six novel isoforms of mouse Yap, bringing the total number of isoforms to eight. According to the differences in the first exon, they are divided into two subtypes (a and b). Isoform-a and isoform-b exhibit different subcellular localizations. Moreover, isoform-a can fully reverse the impaired self-renewal phenotype induced by Yap knockout (KO). Upon overexpression, isoform-a moderately promotes mESCs self-renewal and markedly delays differentiation. On the contrary, no significant pro-self-renewal phenotype is observed when isoform-b overexpressed in wildtype (WT) mESCs or re-expressed in Yap KO cell lines. These finding not only help to clarify the role of Yap in mESCs, but also lay the foundation for advancing functional researches of Yap in other processes.

Background Human umbilical cord mesenchymal stem cells (UCMSCs) transplantation is a promising therapy for the treatment of retinitis pigmentosa (RP). However, intravenously infused cells may be blocked in the lung, increasing the risk of vascular obstruction, which needs to be optimized to further improve safety and efficacy. Methods We derived small UCMSCs (S-UCMSCs) from filtering UCMSCs with a 10-μm filter, and compared with UCMSCs by flow cytometry, directional differentiation culture and transcriptome sequencing. Then the S-UCMSCs and UCMSCs were intravenously infused in the Royal College Surgeons (RCS) rats to evaluate the safety and the efficacy. Results The diameter of S-UCMSCs ranged from 5.568 to 17.231 μm, with an average diameter of 8.636 ± 2.256 μm, which was significantly smaller than that of UCMSCs. Flow cytometry, immunofluorescence and transcriptome sequencing demonstrated that the S-UCMSCs and UCMSCs were the same kind of MSCs, and the S-UCMSCs were more proliferative. After the S-UCMSCs and UCMSCs were intravenously infused into the Royal College of Surgeons (RCS) rats at a dose of 1 × 106 cells/rat, the S-UCMSCs blocked in the lungs were significantly fewer and disappeared more quickly than UCMSCs. The b wave of the flash electroretinogram was improved at 7 d, and the retinal outer nuclear layer thickness was thicker at 7 d and 14 d. The expression level of inflammation was inhibited, and the expression level of neurotrophic factors was upregulated in the retina and serum after transplantation. Conclusions S-UCMSCs intravenous infusion was safer than UCMSCs and could delay retinal degeneration and protect visual function in RCS rats, which may be a preferable therapeutic approach for RP.

PURPOSE To compare changes of chorioretinal blood perfusion between Bietti crystalline dystrophy (BCD) and typical retinitis pigmentosa and perform a staging and a longitudinal analysis of chorioretinal perfusion in BCD. METHODS Twenty-eight patients with BCD (56 eyes), 28 patients with typical retinitis pigmentosa (56 eyes), and 28 healthy subjects (56 eyes) were enrolled. Macular structural parameters and subfoveal choroidal thickness were measured using optical coherence tomography. Retinal vessel and perfusion densities were calculated using optical coherence tomography angiography. Choroidal blood perfusion was assessed through indocyanine green angiography. The results of the BCD group were compared with those of the retinitis pigmentosa and control groups and followed by a staging and a longitudinal analysis of BCD. RESULTS Macular structural and perfusion parameters were decreased less in the BCD group than those in the retinitis pigmentosa group. Subfoveal choroidal thickness was significantly thinner in the BCD group, with a remarkable choroidal perfusion deficit using indocyanine green angiography. The staging analysis revealed damage of both retinal and choroidal perfusion in BCD; however, the longitudinal analysis showed the impairment of choroidal perfusion outweighed retinal. CONCLUSION Both retinal and choroidal blood perfusion are impaired in BCD, but choroidal perfusion deficit caused by CYP4V2 mutations may play a more vital pathologic role.

Regeneration/repair after injury can be endowed by adult stem cells (ASCs) or lineage restricted and even terminally differentiated cells. In corneal epithelium, regeneration after a large wound depends on ASCs (limbal epithelial stem cells, LESCs), whereas repair after a small wound is LESCs-independent. Here, using rat corneal epithelial wounds with different sizes, we show that YAP activation promotes the activation and expansion of LESCs after a large wound, as well as the reprogramming of local epithelial cells (repairing epithelial cells) after a small wound, which contributes to LESCs-dependent and -independent wound healing, respectively. Mechanically, we highlight that the reciprocal regulation of YAP activity and the assembly of cell junction and cortical F-actin cytoskeleton accelerates corneal epithelial healing with different-sized wounds. Together, the common YAP activation and the underlying regulatory mechanism are harnessed by LESCs and lineage-restricted epithelial cells to cope with corneal epithelial wounds with different sizes.

BackgroundHuman embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of hESC-RPE cells is a great challenge.MethodsHere, we report hESC-RPE cells that are embedded with polyelectrolytes gelatin and alginate by layer-by-layer (LbL) self-assembly technique, based on the opposite charge of alternate layers. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo.ResultsThis strategy obviously decreased the immunogenicity of hESC-RPE cells without affecting its activity. LbL-RPE cell transplants into the subretinal space of Royal College of Surgeons (RCS) rats optimized cell engraftment and decreased immunogenicity compared to untreated RPE cell transplants (immunosuppression was not used during the 21-week study). Visual-functional assay with electroretinogram recordings (ERGs) also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants.ConclusionsWe demonstrate that transplanted LbL-RPE cells have better viability and grafting efficiency, optimized immunogenicity, and visual function. Therefore, LbL engineering is a promising method to increase the efficacy of hESC-RPE cell transplantation.

Embryonic stem cells (ESCs) provide an ideal model for investigating developmental processes and are great sources for developing regenerative medicine. Harnessing apoptosis facilitates accurate recapitulation of signalling events during embryogenesis and allows efficient expansion of the ESCs during differentiation. Bcl2, a key regulator of intrinsic anti-apoptotic pathway, encodes two splicing isoforms. However, the identification and functional comparison of Bcl2 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we provide the evidence that both Bcl2 splicing variants are expressed in mESCs. Despite the structural difference, they have similar subcellular localisation. Both Bcl2α and Bcl2β enhance differentiation efficiency of the ESCs and effectively improve the survival and growth of ESCs under serum-free conditions. However, the functional effect of Bcl2α was more potent than that of Bcl2β. Moreover, only Bcl2α could maintain the long-term expansion and pluripotency of ESCs cultured in serum-free medium. Taken together, our results demonstrate previously unknown functional differences in Bcl2 alternative splicing isoforms in ESCs, and lay the foundation for future efforts to engineer ESCs for regenerative medicine.

Retinal pigment epithelial (RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of RPE cells is a great challenge. Here, we established human embryonic stem cells-derived RPE cells, then used layer-by-layer (LbL) self-assembly to coat RPE cells. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo. LbL-RPE cell cultures, IFN-γ ELISIA, a mixed lymphocyte reaction assay, and a T-cell rejection assay indicated that a LbL coating optimized survival, function, and immunogenicity of RPE cells. LbL-RPE cell transplants into Royal Collage of Surgeons (RCS) rats showed in vivo self-assembly to optimize cells engraftment, and decreased immunogenicity as judged by the lack of a robust invasion of the transplant area by Iba1+ cells, CD3+ T cells, or MHC class II+ cells compared to untreated RPE cell transplants (immunosuppression was not used during the 5-wk study). Electroretinogram recordings also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants. We demonstrate transplanted LbL in vivo self-assembly RPE cells enhance cell survival and engraftment optimizing immunogenicity and visual function. Thus, LbL in vivo self-assembly may be suite for treating retinal degenerative disease.

Alternative splicing (AS) is a key process to expand the diversity of mRNA and protein from the genome and it is crucial for fate determination of embryonic stem cells (ESCs) by encoding isoforms with different functions to regulate the balance between pluripotency maintenance and differentiation. Since the role of the Hippo pathway in ESCs is controversial, there may be novel isoforms of Taz, a key effector of the Hippo pathway, previously unknown to us. Here, we identified three variants of Taz in mESCs. Apart from the canonical Taz1185, there were also two novel variants, Taz402 and Taz1086. We found their structure and subcellular localization to be different, while they could all interact with TEAD2 with similar binding affinities and activate transcription. Under the LIFlow condition, overexpression of them all induced apoptosis and differentiation of mESCs, among which the phenotype of Taz1086 was the most dramatic. Taken together, we discovered novel variants of Taz and compared their structure and functional differences in mESC pluripotency maintenance. These findings will help us to understand the Taz gene and clarify its role in mESC.

Purpose. To investigate the relationship between transient pupillary light reflex (PLR) and visual function in patients with retinitis pigmentosa (RP). Methods. A retrospective study was performed with 137 eyes of 73 patients with RP. Transient pupillary light reflex was measured by the vision monitor system (MonColor; Metrovision, France). Dark-adapted transient PLRs were elicited by four specific levels of stimulus luminance (−5, −3, −1, and 0 log cd/m2, blue or white light). Best-corrected visual acuity (BCVA) was recorded based on Early Treatment Diabetic Retinopathy Study (ETDRS) acuity charts. Fixation stability and retinal sensitivity of radial 10° areas were measured with microperimetry. The retinal sensitivity (RS) was divided into central RS (fovea and radial 1° areas) and peripheral RS (radial 3° and 5° areas from the fovea). The patients were further classified into 2 groups (P1 > 75% and P1 < 75%) according to fixation stability. Spearman’s correlation was performed to identify significant associations between BCVA, fixation stability, RS, and PLR. Results. Under the stimuli of the same color light, relative pupillary constriction (RPC), latency, or velocity of constriction in the same patients was statistically different in multiple luminance, respectively. Under the same luminance, blue light induced greater RPC and velocity (except for −3 log cd/m2) than white light. Most patients showed varying degrees of threshold elevation and visual function deficiency. Besides, there was a statistically significant difference in the distribution of BCVA, MRS, or fixation stability under different thresholds. The correlation between pupillary constrictive area (PCA) and retinal sensitivity was mainly determined by the peripheral region. Moreover, patients with stable fixation showed a greater correlation between PCA and RS. Conclusion. PLR induced by specific colors and luminance may serve as a promising clinical approach for assessing and monitoring rod function in advanced RP patients.