Induction of Pluripotent Stem Cells from a Manifesting Carrier of Duchenne Muscular Dystrophy and Characterization of Their X-Inactivation Status

Abstract

Three to eight percent of female carriers of Duchenne muscular dystrophy (DMD) develop dystrophic symptoms ranging from mild muscle weakness to a rapidly progressive DMD-like muscular dystrophy due to skewed inactivation of X chromosomes during early development. Here, we generated human induced pluripotent stem cells (hiPSCs) from a manifesting female carrier using retroviral or Sendai viral (SeV) vectors and determined their X-inactivation status. Although manifesting carrier-derived iPS cells showed normal expression of human embryonic stem cell markers and formed well-differentiated teratomas in vivo, many hiPS clones showed bi-allelic expression of the androgen receptor (AR) gene and loss of X-inactivation-specific transcript and trimethyl-histone H3 (Lys27) signals on X chromosomes, suggesting that both X chromosomes of the hiPS cells are in an active state. Importantly, normal dystrophin was expressed in multinucleated myotubes differentiated from a manifesting carrier of DMD-hiPS cells with XaXa pattern. AR transcripts were also equally transcribed from both alleles in induced myotubes. Our results indicated that the inactivated X chromosome in the patient's fibroblasts was activated during reprogramming, and XCI occurred randomly during differentiation.

Electrophysiological abnormalities in induced pluripotent stem cell‐derived cardiomyocytes generated from Duchenne muscular dystrophy patients

“…Genetic analysis demonstrates the DMD female iPSC‐CM population mixed expression of WT dystrophin allele alongside the mutated allele (Figure ). During reprogramming, X chromosome status is destined to be one of the following three options: (1) conserved inactivation pattern of the originating somatic cell (XaXi); (2) reactivation of the inactive X chromosome (XaXa); (3) erosion of XCI (XaXe) . Considering XIST expression does not sufficiently indicate X chromosome status, and based on the NGS dystrophin expression analyses, a likely possibility is that a mixed population was established during reprogramming and each differentiation .…”

“…During reprogramming, X chromosome status is destined to be one of the following three options: (1) conserved inactivation pattern of the originating somatic cell (XaXi); (2) reactivation of the inactive X chromosome (XaXa); (3) erosion of XCI (XaXe). [12][13][14][15][16][17][18] Considering XIST expression does not sufficiently indicate X chromosome status, and based on the NGS dystrophin expression analyses, a likely possibility is that a mixed population was established during reprogramming and each differentiation. 17,18 In this scenario, DMD female iPSC-CMs can be divided to two sub-populations: (1) cells expressing the WT allele, and (2) cells expressing the mutated allele.…”

“…Previous studies investigating the effect of reprogramming on X chromosome status suggest three possible outcomes: (1) X chromosome inactivation (XCI) status is maintained throughout the reprogramming process; (2) inactivated X chromosome is reactivated; (3) inactivated X chromosome is eroded. [11][12][13][14][15][16][17][18] To investigate the electrophysiological abnormalities caused by dystrophin mutations in cardiomyocytes, we generated iPSCs from two DMD patients: a male and a female heterozygous manifesting carrier. X inactivation status and dystrophin gene expression were analysed in iPSCs and iPSC-CMs generated from healthy controls and patients of both sexes with regard to genetic, molecular and functional abnormalities.…”

Recent advances in lineage differentiation from stem cells: hurdles and opportunities?

“…For example, in the case of Duchenne muscular dystrophy, female carriers of a dystrophin gene mutation rarely manifest with muscular dystrophy because the random nature of X-chromosome inactivation leads to a mixture of cells expressing either the wild-type or the disease gene. In this case, in vitro X-chromosome skewing could misrepresent the in vivo situation 16 . Together with the fact that primed cells are not lineage-neutral, this may be a major contributing factor to the variability observed within and between different cell lines 17 .…”

Duchenne and Becker Muscular Dystrophies: A Review of Animal Models, Clinical End Points, and Biomarker Quantification

“…Typically, BMD manifests later in life with a mean onset of 12 years, loss of ambulation is delayed until the third decade of life, onset of cardiac involvement is variable, and the overall life expectancy is longer compared with DMD patients (Bushby et al , 1993, Finsterer and Stollberger, 2008, Flanigan, 2014, Nigro and Piluso, 2015). Though the disease is X-linked, 3–8% of female carriers can present with symptoms ranging from mild (symptomatic carriers) to rapidly-progressive DMD-like muscular dystrophy due to skewed inactivation of X chromosomes (Guiraud et al , 2015, Miyagoe-Suzuki et al , 2017). …”