Introduction: Although DM animal models have been developed to investigate pathophysiologic mechanisms and evaluate the efficacy of therapeutic approaches, cellular models remain needed for prior compounds evaluation or screenings. Primary muscle cell cultures derived from biopsies of DM patients represent a valuable model since the C/CTG expansions are expressed within its natural genomic context. However several concern such as accessibility and availability of DM biopsies, limited proliferative capacity of human myoblasts support the development of immortalized human DM muscle cell lines in which replicative senescence is bypassed but that display robust and reliable disease-associated features. Methods: Immortalized DM1 (2600CTG) and DM2 (4000CCTG) human muscle cell lines displaying nuclear RNA foci were generated by transduction of lentiviral vectors expressing the catalytic subunit of the human telomerase (hTERT) and the natural p16 ligand, Cdk4. Results: Selected clones of DM immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of DM1 myoblasts when compared to control and DM2 cells. Alternative splicing defects were measured in differentiated DM1 but not in DM2 muscle cell lines. Splicing alterations did not result from differentiation delay because similar changes were fond in immortalized DM1 transdifferentiated fibroblasts in which the myogenic differentiation has been forced by MyoD overexpression. As a proof-of-concept, we showed that antisense approaches alleviate disease-associated defects and a RNA-seq analysis confirmed that the vast majority of misspliced events in immortalized DM1 muscle cells were modulated by antisense treatment. Discussion: We generated new immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA-aggregates and splicing defects that can be used as robust readouts for the screening of therapeutic compounds.