3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Compartmentation of cGMP Signaling in Induced Pluripotent Stem Cell Derived Cardiomyocytes during Prolonged Culture.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and ß3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-ß-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the ß3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

Stable methylation at promoters distinguishes epiblast stem cells from embryonic stem cells and the in vivo epiblasts.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.