DMD and Human Induced Pluripotent Stem Cells: History
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The development of somatic cell reprograming technology has enabled generation of human induced pluripotent stem cells (hiPSCs) which can be differentiated into different cell types. This technology provides a potentially endless pool of human cells for research. Furthermore, hiPSCs can be generated from patients, thus providing patient-specific cells and enabling research tailored to different mutations.

  • Duchenne muscular dystrophy
  • DMD
  • dystrophin gene

1. Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene with an estimated prevalence between 1.3 and 2.1 per 10,000 live male births [1,2,3]. Dystrophin is the longest gene in the human DNA spanning 2.4 Mbp, with its 14 kb transcript consisting of 79 exons; it encodes the 427 kDa dystrophin protein [4,5]. Dystrophin is a major structural protein which is also involved in important metabolic processes [6,7,8]. In skeletal and cardiac muscle, dystrophin provides mechanical stability essential for contracting myocytes, and anchors the cellular cytoskeleton to the extracellular matrix (ECM) via the transmembrane dystrophin–glycoprotein complex (DGC) which links directly to extracellular laminin-2 [9,10,11]. Dystrophin can be grossly divided into three major domains: (1) the N-terminus and actin-binding domain (ABD) (exons 1–8), (2) the central rod segment consisting of 24 spectrin repeats (exons 9–63), and (3) the DGC-binding domain and C-terminus (exons 64–79) [12,13]. The majority of DMD mutations are deletions of one or more exons (60–65%), while duplications make up to 5–10% of the cases. The remaining (25–35%) are single-nucleotide variants, small deletions or insertions in the coding sequence, or splice site variants [14,15].
DMD symptoms start at an early age, usually around 2–3 years, when proximal muscles of the lower extremities begin to weaken. Gradually, the weakness progresses to the distal muscles and the upper limbs. With age, symptoms become more prominent, and by the early teens, patients are usually wheelchair-dependent. The cardiac involvement of DMD includes dilated cardiomyopathy (DCM) which is present in virtually all patients by their late teen years, along with conduction abnormalities, various arrhythmias and extensive fibrosis. Eventually, patients die by their late 20s or early 30s due to respiratory and cardiac failure [16,17,18].
The current gold-standard treatment of DMD includes glucocorticoids (GCs), usually from the age of 4 years, aimed at improving motor and pulmonary function, while also potentially delaying the onset of DCM [16,19,20]. Beyond known side-effects including weight gain, hirsutism and other Cushing’s syndrome symptoms, GCs do not change the disease outcome, but rather can only slow its course [19,21]. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) are administered from around the age of 10 years to reduce mechanical stress on the heart [16,17]. Novel therapies include Eteplirsen, an exon 51 skipping drug, and Ataluren (PTC124), which promotes ribosomal readthrough of nonsense mutations [22,23]. However, these treatments are not intended for all DMD mutations, and there is a need for additional research and therapies to be developed.
The primary animal model used for DMD research is the mdx mouse, which carries a nonsense point mutation in exon 23 of the dystrophin gene [24,25]. Although mdx mice exhibit chronic degeneration of myofibers, they do not manifest some prominent symptoms of DMD. mdx mice display a slower disease progression compared to human DMD patients, and their relative lifespan is significantly longer. The slow progression of muscle pathology does not lead to extensive fibrosis as in humans, and the mice retain their mobility. Cardiac involvement follows a different course than in humans, as mdx mice initially develop hypertrophic cardiomyopathy (HCM), while human DMD patients suffer from contractile dysfunction and DCM [26,27,28]. It has been previously found that mdx mice heart mitochondria display an increase in Ca2+ uptake rate via activation of Ca2+ transport, possibly compensating for a defective sarcoplasmic reticulum (SR) [29,30,31]. This adaptiveness of mdx mice may be a key feature differentiating this model from human patients [32]. Indeed, these challenges led to the development of the D2.mdx model which exhibits a significantly more prominent disease phenotype [33]. An additional limitation of the mouse model lies in gender differences between female and male mdx mice. These differences include a more prominent cardiac involvement and skeletal muscle degeneration in female compared to male mice [34,35], contrary to slower disease progression in human female carriers [36].
Another important animal model was developed in rats by means of TALENs targeting DMD exon 23 [37]. mdx rats manifest progressive muscle degeneration accompanied by a reduction in muscle force, as well as dilated cardiomyopathy. Importantly, mdx rats display significant fibrosis in skeletal and cardiac muscle, similar to human patients but contrary to mdx mice [37,38]. However, some differences remain between mdx rats and human patients, as well as cells derived from human patients, including the lack of muscle calcifications [37], normal L-type Ca2+ current (ICa,L) [39,40,41], and unimpaired β-adrenergic cascade [39,42,43,44] in mdx rats.

2. Human Induced Pluripotent Stem Cells

In 2006 Takahashi and Yamanaka published their successful attempt to reprogram differentiated rat somatic cells into induced pluripotent stem cells (iPSCs) by means of the induction of four factors: Oct3/4, SOX2, c-Myc, and KLF4 [45]; in 2007, these breakthroughs were repeated in human somatic cells [46]. Human iPSCs (hiPSCs) present classic embryonic stem-cell (ESC) characteristics including trilineage differentiation capability [47,48]. Thus, provided proper culture and media conditions, hiPSCs can be differentiated into various cell types. Like ESCs, hiPSCs and hiPSC-derived cells can be used for disease modeling and drug testing [49,50,51,52,53]. Furthermore, hiPSCs can generate a potentially endless pool of differentiated cells from a minute biopsy of a single living human donor, whereas ESC generation requires the sacrifices of embryos [54]. This enables previously unmatched research capabilities of various human diseases without the limitations of different animal models. Indeed, in the past years, numerous papers utilized the reprogramming technique for disease modeling and regenerative medicine. Patient-specific hiPSCs served as a means for many discoveries and advancements in research of different diseases [51,52,55,56,57].
Due to the multitude of different mutations causing DMD [58], patient-specific hiPSCs provide a valuable approach to investigate the precise disease mechanisms resulting from these mutations. Furthermore, hiPSC research enables the potential development of new drugs and therapeutic approaches targeting specific mutations with higher efficacy than previous generic treatments. Importantly, investigating human-derived cells is preferable to using animal models which display different disease course and characteristics, compared to human patients.

This entry is adapted from the peer-reviewed paper 10.3390/ijms24108657

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