Dehesa, M. | bioRxiv Science

bioRxiv2026

Effect of the functional environment on the cardiac differentiation of iPSC

Pristine carbon nanotubes (CNTs) have proven to be excellent supports for cardiac cell growth, survival and maturation, as well to improve cellular function, enhance spontaneous beating activity and benefit their cellular structure. Due to the large quantity of cardiomyocytes that have to be replaced for myocardial regeneration, iPSCs are the most promising candidates for robust generation of cardiomyocytes in vivo/vitro. Herein, iPSCs are cultured and differentiated into cardiomyocytes on functionalized carbon nanotubes (fCNTs). For this purpose, a first optimization of the type of plate and the number of iPSCs suitable for the passaging is performed. Thus, 5{middle dot}105 cells per cm2 are cultured in 24-well and 8-well plates. After 19-days of differentiation and maturation, calcium imaging was done to analyze the spontaneous beating behavior by means of beat frequency and amplitude, immunofluorescence was done to observe evaluate the degree of maturation by staining the sarcomere and the cell nucleus. A set of diverse functionalized CNTs were also tested: pristine CNT, ox-CNT, CNT-COOH, CNT-NH2, CNT-NO2 and CNT-SO3. Calcium analysis showed that all but the nitro-functionalization were beating, with acid-and oxygen derivative CNTs producing an increase in frequency with respect to control, while amino-functional groups decrease it. This suggests that the beating and contractile behavior of cardiomyocyte can be modulated according to the cardiac issue to be faced. In addition, CNT-SO3 produces a striated and elongated sarcomere, proper of the real tissue.

bioengineering↗

bioRxiv2025

Enhanced muscle uptake of chemically optimized miR-23b antisense oligonucleotides as lead compounds for Myotonic Dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by CTG repeat expansions in DMPK. Mutant transcripts containing expanded CUG repeats form ribonuclear foci that sequester muscleblind-like splicing regulator (MBNL) proteins, key regulators of RNA splicing and metabolism. This functional depletion leads to widespread mis-splicing and persistence of fetal transcript profiles, which underlie muscle weakness, myotonia, and muscle atrophy. In addition, miR-23b is upregulated in DM1 muscle and further represses MBNL1 translation, amplifying molecular defects. We developed chemically optimized miRNA-targeting antisense oligonucleotides (antimiRs) to inhibit miR-23b and restore functional MBNL1 levels. Using a multi-step screening process, we evaluated antimiRs with varying sequences, lengths, chemical modifications, and lipid conjugations. A key optimization was a 3-oleic acid conjugation combined with specific chemical modifications, which enhanced muscle uptake and efficacy. Lead candidates showed strong activity in preclinical models (HSALR and DMSXL mice, and human myoblasts), increasing MBNL1 levels, correcting mis-splicing, improving muscle strength, and reducing myotonia. They also exhibited efficient biodistribution to skeletal muscle, a critical DM1-affected tissue. In vitro toxicology indicated a favorable safety profile with minimal immune or renal toxicity. The antimiR mechanism was conserved in rat and pig fibroblasts. Overall, two lead antimiRs emerged as promising therapeutic candidates for DM1, with improved pharmacokinetics, tissue targeting, and safety, supporting the potential of microRNA-based approaches to correct key molecular defects in this disorder.

genetics↗