Ramon-Azcon, J. | Search bioRxiv

bioRxiv2026

Patient-derived 3D engineered human muscle model recapitulates CLCN1 mis-splicing and myotonia in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) lacks human in vitro models that directly link RNA toxicity to mature skeletal muscle function, particularly myotonia. Here, we engineer contractile 3D human skeletal muscle tissues from immortalized myoblasts derived from three DM1 patients representing juvenile, adult, and late-onset subtypes. These tissues reproduce key molecular features of DM1, including nuclear RNA foci, MBNL1 sequestration, and widespread mis-splicing. Functionally, DM1 tissues exhibit impaired calcium handling, subtype-dependent weakness, rapid fatigue, and a fiber-type distribution characterized by increased slow type I fibers and pathological MyHC-I/IIx hybrids. Notably, the 3D environment enables expression and complete pathogenic mis-splicing of CLCN1--undetectable in matched 2D cultures--accompanied by myotonia-like delayed relaxation. Using this model, we assessed therapeutic responses of candidate small-molecule modulators. Phenylbutazone reduced RNA foci and MBNL1 sequestration but failed to rescue spliceopathy or function. In contrast, calcitriol induced coordinated transcriptomic remodeling and robustly rescued myotonia-like relaxation despite persistent CLCN1 mis-splicing. These findings establish a functionally mature human DM1 muscle model and highlight compensatory network activation as a strategy to improve muscle function in DM1.

bioengineering↗

bioRxiv2021

Direct reprogramming of human fibroblasts into insulin-producing cells by transcription factors

Direct lineage reprogramming of one somatic cell into another bypassing an intermediate pluripotent state has emerged as an alternative to embryonic or induced pluripotent stem cell differentiation to generate clinically relevant cell types. One cell type of clinical interest is the pancreatic {beta} cell that secretes insulin and whose loss and/or dysfunction leads to diabetes. Generation of functional {beta}-like cells from developmentally related somatic cell types (pancreas, liver, gut) has been achieved via enforced expression of defined sets of transcription factors. However, clinical applicability of these findings is challenging because the starting cell types are not easily obtainable. Skin fibroblasts are accessible and easily manipulated cells that could be a better option, but available studies indicate that their competence to give rise to {beta} cells through similar direct reprogramming approaches is limited. Here, using human skin fibroblasts and a protocol that ensures high and consistent expression of adenovirus-encoded reprogramming factors, we show that the transcription factor cocktail consisting of Pdx1, Ngn3, MafA, Pax4 and Nkx2-2 activates key {beta} cell genes and down-regulates the fibroblast transcriptional program. The converted cells produce insulin and exhibit intracellular calcium responses to glucose and/or membrane depolarization. Furthermore, they secrete insulin in response to glucose in vitro and after transplantation in vivo. These findings demonstrate that transcription factor-mediated direct reprogramming of human fibroblasts is a feasible strategy to generate insulin-producing cells.

developmental biology↗