Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells.

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.

Wnt/ß-catenin signaling stimulates the self-renewal of conjunctival stem cells and promotes corneal conjunctivalization.

Limbal stem cell deficiency causes conjunctivalization characterized by the covering of the corneal surface with conjunctival epithelium. However, the driving force for the encroachment of these conjunctival cells is unclear. Conjunctival stem cells are bipotent stem cells that can proliferate and differentiate into conjunctival epithelial cells and goblet cells to maintain regeneration of the conjunctival epithelium. Here, we show a robust proliferative response of conjunctival stem cells and upregulation of Wnt2b and Wnt3a gene expression in the conjunctivae of mice with induced limbal stem cell deficiency. Topical application of the Wnt/ß-catenin signaling activator CHIR resulted in increased proliferation of ΔNp63α-positive stem cells in the basal layers of the bulbar and forniceal conjunctivae and enhanced invasion of conjunctival epithelial and goblet cells into the corneal surface. We also found that in cultures of stem cells isolated from the human conjunctiva, Wnt/ß-catenin pathway activation improved the expansion of the ΔNp63α/ABCG2 double-positive cell population by promoting the proliferation and preventing the differentiation of these cells. These expanded stem cells formed a stratified epithelium containing goblet cells under airlift culture conditions. Our data reveal that Wnt/ß-catenin signaling contributes to the pathological process of limbal stem cell deficiency by promoting the self-renewal of conjunctival stem cells and suggest that these cells are a driving force in corneal conjunctivalization.

Sliding or Nonsliding Arthroscopic Knots for Shoulder Surgery: A Systematic Review.

BACKGROUND: Knot tying is a crucial component of successful arthroscopic shoulder surgery. It is currently unknown whether sliding or nonsliding techniques result in superior clinical outcomes. PURPOSE: To assess the clinical outcomes of arthroscopic sliding knot (SK)- versus nonsliding knot (NSK)-tying techniques during arthroscopic shoulder surgery, including rotator cuff repair, Bankart repair, and superior labral anterior-posterior (SLAP) repair. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: A systematic search of the PubMed, Embase, and Cochrane Library databases was performed using PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guidelines. All English-language literature published between 2000 and 2018 reporting clinical outcomes utilizing SK- or NSK-tying techniques during rotator cuff repair, Bankart repair, and SLAP repair with a minimum 24-month follow-up was reviewed by 2 independent reviewers. Information on type of surgery, knot used, failure rate, patient satisfaction, and patient-reported outcomes was collected. Patient-reported outcome measures included the Constant-Murley score, Rowe score, and visual analog scale for pain. Study quality was evaluated using the modified Coleman Methodology Score. RESULTS: Overall, 9 studies (6 level 3 and 3 level 4) with a total of 671 patients (mean age, 52.8 years [range, 16-86 years]; 65.7% male; 206 SK and 465 NSK) were included. There were 4 studies that reported on Bankart repair in 148 patients (63 SK and 85 NSK), 3 on SLAP repair in 59 patients (59 SK), and 2 on rotator cuff repair in 464 patients (84 SK and 380 NSK). Also, 6 studies compared knot-tying with knotless techniques (3 Bankart repair studies and 3 SLAP repair studies), while the studies reporting the outcomes of SLAP repair evaluated SK-tying techniques only. The failure rate for Bankart repair was 3.2% (2/63) for SKs and 4.7% (4/85) for NSKs. The failure rate for rotator cuff repair was 2.4% (2/84) for SKs and 6.3% (24/380) for NSKs. The failure rate for SLAP repair was 11.9% (7/59). Because of inconsistencies in outcomes and procedures, no quantitative analysis was possible. The mean modified Coleman Methodology Score for all studies was 65.1 ± 8.77, indicating adequate methodology. CONCLUSION: The literature on clinical outcomes using SKs or NSKs for shoulder procedures is limited to level 4 evidence. Future studies should be prospective and focus on comparing the use of SKs and NSKs for shoulder procedures to elucidate which arthroscopic knot results in superior clinical outcomes.