Generation of human induced pluripotent stem cell lines from five patients with Myofibrillar myopathy carrying different heterozygous mutations in the DES gene

Myofibrillar myopathy (MFM) is a rare genetic disorder characterized by muscular dystrophy that is often associated with cardiac disease. This disease is caused by mutations in several genes, among them DES (encoding desmin) is the most frequently affected. Peripheral blood mononuclear cells from 5 different MFM patients with different DES mutations were reprogrammed into induced pluripotent stem cells (IPSC


Resource utility
These IPSC lines, generated from five different patients affected by MFM associated with dilated cardiomyopathy (DCM) with five different DES mutations, can be used to study cellular and molecular mechanisms of the MFM.They also can be used as a cell model to develop new therapeutic approaches, especially based on drug-screening.

Resource details
Desmin is the specific intermediate filament of muscle cells, within which it forms a three-dimensional network connecting the various cellular components to each other, including organelles and contractile apparatus (Brodehl et al., 2018).Mutations in the gene encoding this protein is known to cause desmin-related myofibrillar myopathy (MFM1: OMIM:601419), a heterogeneous disease affecting primarily skeletal muscles (weakness) (Joanne et al., 2013).However, approximately 75 % of patients also present signs of cardiac disease (van Spaendonck-Zwarts et al., 2010) that mainly evolve toward dilated cardiomyopathy and heart failure.At the cell level, mutations of desmin cause several disorganizations of the muscle cell that lead to contractile and metabolic dysfunctions (Hovhannisyan et al., 2023;Winter et al., 2016).
Here we described the generation of human induced pluripotent stem cells from the peripheral blood mononuclear cells (PBMC) of patients affected by MFM associated with a cardiac dysfunction.These five patients have different mutations in DES even if they present similar cardiac symptoms (see Table 1.).These mutations (DES D214-E245Del , DES S46Y , DES P419H , DES E439K , DES E245D ) have been previously described (Wahbi et al., 2012) and are linked to the development of dilated cardiomyopathy.Five different clones (INSRMi012-C from the patient PC173T19 with the mutation DES D214-E245Del , INSRMi013-A from the patient PC179c1 with the mutation DES S46Y , INSRMi019-A from the patient PC129k8 with the mutation DES P419H , INSRMi020-A from the patient PC130k2c with the mutation DES E439K and INSRMi021-A from the patient PC177 3c14 with the mutation DES E245D ) were derived and are described here (Table 1).In details, PBMC of these patients were reprogrammed using non-integrative strategy (Phenocell SAS, France) and clones for each patient were manually isolated and amplified until passage 20 where they were frozen in liquid nitrogen with CTS PSC Cryomedium (Thermo Fisher Scientific, France).After thawing on Matrigel, these clones were passaged at least one time before analysis.Colonies of all clones demonstrate normal morphology in culture with a high nucleus/cytoplasm ratio (Fig. 1A, scale bar = 100 µm) and express pluripotency markers as shown by immunocytochemistry (Fig. 1B, scale bar = 100 µm) and cytometry (Fig. 1C and Supplemental Data A).The absence of residual reprogramming plasmids was confirmed by PCR.Genomic integrity, gender and identity were confirmed for each clone by G-banding karyotyping (Fig. 1E) and by SNP analysis through the comparison of the iPSCs genotype with that of parental PBMCs to confirm their origin (see Table 1 and Supplemental Data C and D).Moreover, each clone was tested for its capacity to differentiate into the three germ layer lineages using the STEMdiff™ Trilineage Differentiation Kit (Stem Cell Technologies, France).As shown in Fig. 1D, each clone expresses endodermic, mesodermic and ectodermic markers after specific differentiation (expresses as fold change compared to undifferentiated condition) further confirming their pluripotency.The presence of the specific mutations (Table 1) was assessed by sequencing the genomic DNA of each clone and reveal that the expected mutation was correctly observed (Supplemental Data B).Finally, absence of mycoplasma was confirmed using the Mycoplasma check service of Eurofins Genomics.

Immunocytochemistry
Cells cultured on coverslip coated with Matrigel were fixed with 4 % paraformaldehyde for 5 min and permeabilization were performed with cold methanol (-20 • C) for 5 min.After incubation with 5 % bovine serum albumin for one hour cells were incubated with primary antibodies (see Table 2) overnight at 4 • C.After 45 min incubation with secondary antibodies (see Table 2), nuclei were counterstained with Hoescht 33,342 (Life Technologies).Then, slides were mounted with homemade medium containing Mowiol.Images were taken on an P. Joanne et al. inverted epifluorescence microscope (DMi8, Leica).

Karyotyping
At least 2 passages after thawing cells were karyotyped by Cell Guidance systems (UK) and at least 10 metaphases were counted.

Differentiation assay
STEMdiff Trilineage Differentiation Kit (Cat.# 05230, StemCell Technologies) was used according to the manufacturer's instructions, at a density of 400,000 cells/well for the ectoderm and endoderm conditions and 100,000 cells/well for the mesoderm condition.Differentiated cells were then collected for RNA isolation using TRIzol.RevertAid First Strand cDNA Synthesis Kit was used to generate cDNA using random hexamer primers and expression of lineages markers (Table 2) was performed in SYBR Green Master Mix (Roche) on a LightCycler480 (Roche).

Genomic DNA extraction
Genomic DNA was extracted using the Genelute Mammalian

Table 1
Characterization and validation.
P.Joanne et al.