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A 69-year-old woman developed severe right suprabulbar pain with blurred right-sided vision. There were no haloes around lights, photophobia, nausea or vomiting. Investigations in the emergency department excluded a posterior communicating/internal carotid artery aneurysm. However, she did not have an ophthalmological assessment and the initial diagnosis was of sinusitis-related headache. An urgent ear, nose and throat assessment found no abnormality, but a local ophthalmologist subsequently diagnosed and managed the patient's acute angle closure crisis. Periocular pain always deserves detailed assessment with an accurate history, visual acuity assessment and intraocular pressure measurement., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor., Methods: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions., Results: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 10 6 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 10 6 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process., Conclusion: A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

In this study, 50 tri-substituted imidazoles (TIs), which are analogs of the small molecules TA-01 and SB203580, were synthesized and screened for cardiomyogenic activities. Several TIs displayed cardiomyogenic activities when applied during the differentiation from days 3-5. The TIs did not affect the Wnt/β-catenin pathway during cardiomyogenesis and the likely mechanism of action is through the inhibition of ALK5 of the TGFβ pathway. Interestingly, these TIs promoted the neural differentiation of human pluripotent stem cells (hPSCs) with a similar potency to that of the dual SMAD inhibitors SB431542/LDN-193189 when dosed from days 1 to 9. The neural induction activities of the TIs correlated with their ALK5 inhibitory activities. This study reports the discovery of small molecule inhibitors of ALK5, which can promote the differentiation of hPSCs into cardiomyocytes or neural cells depending on the time of dosing, showing potential for the production of clinical-grade cardiac/neural cells for regenerative therapy. Stem Cells Translational Medicine 2018;7:709-720., (© 2018 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3β inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3β inhibitors to induce cardiac differentiation. GSK3β inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Differentiation of human pluripotent stem cells as embryoid bodies (EBs) has been achieved previously with p38alfa MAPK inhibitors such as SB203580 with moderate efficiency of 10-15%. We synthesized and screened 42 compounds that are 2,4,5-trisubstituted azole analogues of SB203580 for efficient cardiomyocyte differentiation. Our screen identified novel compounds that have similar cardiac differentiation activity as SB203580. However, the cardiac differentiation did not correlate with p38alfa MAPK inhibition, indicating an alternative mechanism in cardiac differentiation. Upon profiling several 2,4,5-trisubstituted azole compounds against a panel of 97 kinases we identified several off targets, among them casein kinases 1 (CK1). The cardiomyogenic activities of SB203580 and its analogues showed a correlation with post mesoderm Wnt/beta-catenin pathway inhibition of CK1 epsilon and delta. These findings united the mechanism of 2,4,5-trisubstituted azole with the current theory of Wnt/beta-catenin regulated pathway of cardiac differentiation. Consequently an efficient cardiomyocyte protocol was developed with Wnt activator CHIR99021 and 2,4,5-trisubstituted azoles to give high yields of 50-70% cardiomyocytes and a 2-fold increase in growth., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10⁶ cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10⁶ cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.

Objective: To report a retrospective case series of 29 Australian and New Zealand patients with orbital and adnexal Wegener's granulomatosis (WG)., Design: Retrospective case series., Participants: Twenty-nine cases of orbital and adnexal WG were identified., Methods: A number of oculoplastic surgeons and other clinicians in Australia and New Zealand was asked about their experience with orbital and adnexal WG. Clinical data regarding these cases were conveyed by means of a questionnaire. Cases of ophthalmic WG without features of orbital or adnexal disease were excluded., Main Outcome Measures: Data obtained from the questionnaire includes age, gender, limited or generalized disease, antineutrophil cytoplasmic antibody (ANCA) status, symptoms and signs: nasolacrimal obstruction, sinusitis, fistula/orbital bone erosion, orbital mass/proptosis, extraocular muscle/diplopia, visual acuity reduction caused by optic nerve compression, orbital pain, lid edema/erythema, biopsy status, and treatment status., Results: Twenty-nine patients with orbital and adnexal WG were identified and described. Symptoms included awareness of an orbital mass, epiphora, orbital pain and diplopia. Signs included an orbital mass or proptosis (69%), nasolacrimal duct obstruction (52%), limited ocular rotations (52%), lid erythema and edema (31%), bony destruction (21%), and reduced visual acuity (17%). Two patients had a persistent nasolacrimocanthal fistula. Cytoplasmic pattern antineutrophil cytoplasmic antibodies (c-ANCA) were present in 52% of patients, and in 9 of 10 patients with generalized disease. However, c-ANCA was positive in only 32% (6 of 19) of patients with limited WG. Perinuclear pattern antineutrophil cytoplasmic antibodies (p-ANCA) was positive in 10% of cases., Conclusions: To diagnose and treat ophthalmic WG effectively, the clinician must be aware of its protean orbital and adnexal manifestations. WG may occur with or without systemic involvement, and c-ANCA was negative in approximately half our cases. Our cases also demonstrated two orbital fistulae, an observation previously believed to be rare.