Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells.

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

Fate choice of post-natal mesoderm progenitors: skeletal versus cardiac muscle plasticity.

Regenerative medicine for skeletal and cardiac muscles still constitutes a fascinating and ambitious frontier. In this perspective, understanding the possibilities of intrinsic cell plasticity, present in post-natal muscles, is vital to define and improve novel therapeutic strategies for acute and chronic diseases. In addition, many somatic stem cells are now crossing the boundaries of basic/translational research to enter the first clinical trials. However, it is still an open question whether a lineage switch between skeletal and cardiac adult myogenesis is possible. Therefore, this review focuses on resident somatic stem cells of post-natal skeletal and cardiac muscles and their plastic potential toward the two lineages. Furthermore, examples of myogenic lineage switch in adult stem cells are also reported and discussed.

The synthetic purine reversine selectively induces cell death of cancer cells.

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.

Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles.

Mesoangioblasts (MABs) are mesoderm-derived stem cells, associated with small vessels and originally described in the mouse embryonic dorsal aorta. Similar though not identical cells have been later identified and characterized from postnatal small vessels of skeletal muscle and heart. They have in common the expression of pericyte markers, the anatomical location, the ability to self-renew in culture, and to differentiate into various types of mesodermal lineages upon proper culture conditions. Currently, the developmental origin of MABs and the relationship with other muscle stem cells are not understood in detail and are the subject of active research. This chapter provides an outline of the latest techniques for isolation and characterization of adult MABs from human and mouse skeletal muscles.

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a, and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC-derived cardiac differentiation.

Intrinsic cell memory reinforces myogenic commitment of pericyte-derived iPSCs.

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.6 antigen. Furthermore, chimeric mice from mdx-iPSCs (DYS-HAC) cells showed tissue-specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions. Here we show that iPSCs generated from prospectively sorted MABs (MAB-iPSCs) are pluripotent as fibroblast-derived iPSCs (f-iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB-iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB-iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB-iPSCs than in f-iPSCs. A larger amount of CD56(+) progenitors can be sorted from the MAB-iPSCs differentiating pool and, after transplantation into αsg-KO mice, can efficiently participate to skeletal muscle regeneration and restore αsg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom-made cell protocols for muscular dystrophies.

NEU3 sialidase strictly modulates GM3 levels in skeletal myoblasts C2C12 thus favoring their differentiation and protecting them from apoptosis.

Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase," has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.