Hansjoerg Keller

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

We describe the synthesis and characterization of 3-alkoxy-pyrrolo[1,2-b]pyrazolines as novel selective androgen receptor (AR) modulators that possess excellent physicochemical properties for transdermal administration. Compound 26 bound to human AR with an IC₅₀ of 0.7 nM with great selectivity over other nuclear hormone receptors and potently activated AR in a C2C12 muscle cell reporter gene assay with an EC₅₀ of 0.5 nM. It showed high aqueous solubility of 1.3 g/L at pH 7.4, and an in silico model as well as a customized parallel artificial membrane permeability assay indicated good skin permeation. Indeed, when measuring skin permeation through excised human skin, an excellent flux of 2 μg/(cm²·h) was determined without any permeation enhancers. In a 2 week Hershberger model using castrated rats, the compound showed dose-dependent effects fully restoring skeletal muscle weight at 0.3 mg/kg/day after subcutaneous administration with high selectivity over prostate stimulation.

Expression of the osteocyte‐derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH. Introduction: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52‐kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness. Materials and Methods: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast‐like UMR‐106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody‐induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT‐PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA‐mediated RNA knockdown. Results: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co‐localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co‐expression of a dominant negative MEF2C mutant. Finally, siRNA‐mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR‐106 osteoblast‐like cells. Conclusions: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.