Enhancing the Functionality of Immunoisolated Human SC-ßeta Cell Clusters through Prior Resizing.

The transplantation of immunoisolated stem cell derived beta cell clusters (SC-ß) has the potential to restore physiological glycemic control in patients with type I diabetes. This strategy is attractive as it uses a renewable ß-cell source without the need for systemic immune suppression. SC-ß cells have been shown to reverse diabetes in immune compromised mice when transplanted as ≈300 µm diameter clusters into sites where they can become revascularized. However, immunoisolated SC-ß clusters are not directly revascularized and rely on slower diffusion of nutrients through a membrane. It is hypothesized that smaller SC-ß cell clusters (≈150 µm diameter), more similar to islets, will perform better within immunoisolation devices due to enhanced mass transport. To test this, SC-ß cells are resized into small clusters, encapsulated in alginate spheres, and coated with a biocompatible A10 polycation coating that resists fibrosis. After transplantation into diabetic immune competent C57BL/6 mice, the "resized" SC-ß cells plus the A10 biocompatible polycation coating induced long-term euglycemia in the mice (6 months). After retrieval, the resized A10 SC-ß cells exhibited the least amount of fibrosis and enhanced markers of ß-cell maturation. The utilization of small SC-ß cell clusters within immunoprotection devices may improve clinical translation in the future.

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

Interplay between mitochondrial reactive oxygen species, oxidative stress and hypoxic adaptation in facioscapulohumeral muscular dystrophy: Metabolic stress as potential therapeutic target.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

The Satellite Cell at 60: The Foundation Years.

The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, together with generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, and also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the supportive role of non-satellite cell types in muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided further foundations for the dramatic escalation of work on muscle stem cells in the 21st century.

Employing core regulatory circuits to define cell identity.

The interplay between extrinsic signaling and downstream gene networks controls the establishment of cell identity during development and its maintenance in adult life. Advances in next-generation sequencing and single-cell technologies have revealed additional layers of complexity in cell identity. Here, we review our current understanding of transcription factor (TF) networks as key determinants of cell identity. We discuss the concept of the core regulatory circuit as a set of TFs and interacting factors that together define the gene expression profile of the cell. We propose the core regulatory circuit as a comprehensive conceptual framework for defining cellular identity and discuss its connections to cell function in different contexts.

Live Cell Monitoring and Enrichment of Stem Cell-Derived ß Cells Using Intracellular Zinc Content as a Population Marker.

Our laboratory and others have developed protocols to generate glucose-responsive stem cell-derived ß cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell-based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell-derived ß cells and gene-editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell-derived ß cells. This purification of SC-ß cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors. Basic Protocol: Fluorescent labeling and isolation of stem cell-derived ß cells.

Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development.

In vitro differentiation of pluripotent cells into ß cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.

Charting cellular identity during human in vitro ß-cell differentiation.

In vitro differentiation of human stem cells can produce pancreatic ß-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro ß-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to ß-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo ß-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the ß-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro ß-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine.

Alginate-microencapsulation of human stem cell-derived ß cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic immunosuppression.

Pancreatic ß-cell replacement by islet transplantation for the treatment of type 1 diabetes (T1D) is currently limited by donor tissue scarcity and the requirement for lifelong immunosuppression. The advent of in vitro differentiation protocols for generating functional ß-like cells from human pluripotent stem cells, also referred to as SC-ß cells, could eliminate these obstacles. To avoid the need for immunosuppression, alginate-microencapsulation is widely investigated as a safe path to ß-cell replacement. Nonetheless, inflammatory foreign body responses leading to pericapsular fibrotic overgrowth often causes microencapsulated islet-cell death and graft failure. Here we used a novel approach to evade the pericapsular fibrotic response to alginate-microencapsulated SC-ß cells; an immunomodulatory chemokine, CXCL12, was incorporated into clinical grade sodium alginate to microencapsulate SC-ß cells. CXCL12 enhanced glucose-stimulated insulin secretion activity of SC-ß cells and induced expression of genes associated with ß-cell function in vitro. SC-ß cells co-encapsulated with CXCL12 showed enhanced insulin secretion in diabetic mice and accelerated the normalization of hyperglycemia. Additionally, SC-ß cells co-encapsulated with CXCL12 evaded the pericapsular fibrotic response, resulting in long-term functional competence and glycemic correction (>150 days) without systemic immunosuppression in immunocompetent C57BL/6 mice. These findings lay the groundwork for further preclinical translation of this approach into large animal models of T1D.

A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation.

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.