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Calcium phosphates (CaP) are widely used synthetic bone graft substitutes, having bioactivity that is regulated by a set of intertwined physico-chemical and structural properties. While some CaPs have shown to be as effective in regenerating large bone defects as autologous bone, there is still the need to understand the role of individual material properties in CaP performance. Here, we aimed to decouple the effects of chemical composition and surface-microstructure of a beta-tricalcium phosphate (TCP) ceramic, with proven osteoinductive potential, on human mesenchymal stromal cells (hMSCs) differentiation. To this end, we replicated the surface structure of the TCP ceramic into polylactic acid without inorganic additives, or containing the chemical constituents of the ceramic, i.e., a calcium salt, a phosphate salt, or TCP powder. The microstructure of the different materials was characterized by confocal laser profilometry. hMSCs were cultured on the materials, and the expression of a set of osteogenic genes was determined. The cell culture medium was collected and the levels of calcium and phosphate ions were quantified by inductively-coupled plasma mass spectrometry. The results revealed that none of the tested combinations of properties in polymer/composite replicas was as potent in supporting the osteogenic differentiation of hMSCs as the original ceramic. Nevertheless, we observed some effects of the surface structure in the absence of inorganics, as well as combined effects of surface structure and the added salts, in particular calcium, on osteogenic differentiation. The approach presented here can be used to study the role of independent properties in other CaP-based biomaterials.

Drug induced liver injury (DILI) is one of the major reasons of drug withdrawal during the different phases of drug development. The later in the drug development a drug is discovered to be toxic, the higher the economical as well as the ethical impact will be. In vitro models for early detection of drug liver toxicity are under constant development, however to date a superior model of the liver is still lacking. Ideally, a highly reliable model should be established to maintain the different hepatic cell functionalities to the greatest extent possible, during a period of time long enough to allow for tracking of the toxicity of compounds. In the case of DILI, toxicity can appear even after months of exposure. To reach this goal, an in vitro model should be developed that mimics the in vivo liver environment, function and response to external stimuli. The different approaches for the development of liver models currently used in the field of tissue engineering will be described in this review. Combining different technologies, leading to optimal materials, cells and 3D-constructs will ultimately lead to an ideal superior model that fully recapitulates the liver.

Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships. ispartof: NATURE GENETICS vol:55 issue:5 ispartof: location:United States status: Published online

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100β, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

Mutations in LRRK2 increase its kinase activity and cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab proteins which allows for their binding to RILPL1. The phospho-Rab/RILPL1 interaction causes deficits in ciliogenesis and interferes with the cohesion of duplicated centrosomes. We show here that centrosomal deficits mediated by pathogenic LRRK2 can also be observed in patient-derived iPS cells, and we have used transiently transfected cell lines to identify the underlying mechanism. The LRRK2-mediated centrosomal cohesion deficits are dependent on both the GTP conformation and phosphorylation status of the Rab proteins. Pathogenic LRRK2 does not displace proteinaceous linker proteins which hold duplicated centrosomes together, but causes the centrosomal displacement of CDK5RAP2, a protein critical for centrosome cohesion. The LRRK2-mediated centrosomal displacement of CDK5RAP2 requires RILPL1 and phospho-Rab proteins, which stably associate with centrosomes. These data provide fundamental information as to how pathogenic LRRK2 alters the normal physiology of a cell. We are grateful to Erich Nigg and Francis Barr for providing a variety of constructs and antibodies, and to Dario Alessi for providing various A549 cell lines and MEF cells. We thank LauraMontosa for excellent technical assistance with confocal microscopy. This work was supported by The Michael J. Fox Foundation for Parkinson's research (to S.H.), intramural funding from Rutgers University (to S.H.), the Spanish Ministry of Economy and Competitiveness (SAF2017-89402-R to S.H.), the BBVA Foundation (to S.H., S.A.C., and R.W. M.), the Spanish Ministry of Education, Culture and Sport (FPU12/04367 to J.M. P., FPU15/05233 to A.J. L.O.), and the Spanish Ministry of Science, Innovation and Universities (EST18/00412 to A.J.L. O.).

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies. ispartof: CELLS vol:12 issue:8 ispartof: location:Switzerland status: published

The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography (OPT) enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify the density of villi throughout centimetre-long segments of intestine. Furthermore, we exhibit the age and microbiota dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D imaging with reverse-OPT and high-resolution 2D imaging allows accurate localisation of ROIs and adds value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment., A workflow for 3D characterization of the mouse small intestine with optical projection tomography allows the identification of sparsely-distributed regions of interest in large volumes while retaining compatibility with high-resolution microscopy modalities.

Recent years have seen growing scholarly interest in female physiology in general. Moreover, particular attention has been devoted to how concentrations of female sex hormones vary during the menstrual cycle and menopausal transition and how hormonal contraception and hormonal therapy influence skeletal muscle tissue. While much effort has been paid to macro outcomes, such as muscle function or mass, rather less attention has been paid to mechanistic work that may help explain the underlying mechanism through which sex hormones regulate skeletal muscle tissue. Evidence from animal studies shows a strong relationship between the female sex hormone estrogen and satellite cells (SCs), a population of muscle stem cells involved in skeletal muscle regulation. A few human studies investigating this relationship have been published only recently. Thus, the purpose of this study was to bring an updated review on female sex hormones and their role in SC regulation. First, we describe how SCs regulate skeletal muscle maintenance and repair and introduce sex hormone signaling within the muscle. Second, we present evidence from animal studies elucidating how estrogen deficiency and supplementation influence SCs. Third, we present results from investigations from human trials including women whose concentrations of female hormones differ due to menopause, hormone therapy, hormonal contraceptives, and the menstrual cycle. Finally, we discuss research and methodological recommendations for future studies aiming at elucidating the link between female sex hormones and SCs with respect to aging and training.

Aging is the largest risk factor for Achilles tendon associated disorders and rupture. Although Achilles tendon macroscale elastic properties are suggested to decline with aging, less is known about the effect of maturity and aging on multiscale viscoelastic properties and their effect on tendon cell behavior. Here, we show dose dependent changes in native multiscale tendon mechanical and structural properties and uncover several nanoindentation properties predicted by tensile mechanics and echogenicity. Alginate hydrogel systems designed to mimic juvenile tendon microscale mechanics revealed that stiffness and viscoelasticity affected Achilles tendon cell aspect ratio and proliferation during aging. This knowledge provides further evidence for the negative impact of maturity and aging on tendon and begins to elucidate how viscoelasticity can control tendon derived cell morphology and expansion., Statement of significance, Aging is the largest risk factor for Achilles tendon associated disorders and rupture. Although Achilles tendon macroscale elastic properties are suggested to decline with aging, less is known about the effect of maturity and aging on multiscale viscoelastic properties and their effect on tendon cell behavior. Here, we show dose dependent changes in native multiscale tendon mechanical and structural properties and uncover several nanoindentation properties predicted by tensile mechanics and echogenicity. Alginate hydrogel systems designed to mimic juvenile tendon microscale mechanics revealed that stiffness and viscoelasticity affected Achilles tendon cell spreading and proliferation during aging. This knowledge provides further evidence for the negative impact of maturity and aging on tendon and begins to elucidate how viscoelasticity can control tendon derived cell morphology and expansion. (c) 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Organoids are increasingly used as model systems in precision medicine, but they are delicate, making it difficult to get accurate spatial chemical information. This protocol describes how to prepare organoid samples for mass spectrometry imaging.Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics.

Funder: Core funding from the Wellcome Sanger Institute (Wellcome Trust, No. 206194)., Funder: Wellcome PhD Studentship (Wellcome Trust, No. 206194), Funder: Wellcome Clinical PhD fellowship (Wellcome Trust, No. 206194), Funder: Pathological Society of Great Britain and Ireland; doi: https://doi.org/10.13039/501100000672, Funder: Jean Shank/Pathological Society of Great Britain and Ireland Intermediate Clinical Fellowship(JSPS IF 2019 01), Funder: Finnish Center of Excellence Program 2018–2025, No. 312041, Funder: NIHR Cambridge Biomedical Research Centre, Funder: Core funding from The Pharsalia Trust., APOBEC mutational signatures SBS2 and SBS13 are common in many human cancer types. However, there is an incomplete understanding of its stimulus, when it occurs in the progression from normal to cancer cell and the APOBEC enzymes responsible. Here we whole-genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals and found that SBS2/SBS13 mutations were present in 17% of crypts, more frequent than most other normal tissues. Crypts with SBS2/SBS13 often had immediate crypt neighbors without SBS2/SBS13, suggesting that the underlying cause of SBS2/SBS13 is cell-intrinsic. APOBEC mutagenesis occurred in an episodic manner throughout the human lifespan, including in young children. APOBEC1 mRNA levels were very high in the small intestine epithelium, but low in the large intestine epithelium and other tissues. The results suggest that the high levels of SBS2/SBS13 in the small intestine are collateral damage from APOBEC1 fulfilling its physiological function of editing APOB mRNA.

Growing evidence suggests the importance of the small intestinal bacteria in the diet-host-microbiota dialogue in various facets of health and disease. Yet, this body site is still poorly explored and its ecology and mechanisms of interaction with the host are just starting to be unraveled. In this review, we describe the current knowledge on the small intestinal ecology, its composition and diversity, and how the intestinal bacteria in homeostatic conditions participate in nutrient digestion and absorption. We illustrate the importance of a controlled bacterial density and of the preservation of absorptive surface for the host's nutritional status. In particular, we discuss these aspects of the small intestinal environment in the framework of two disease conditions, namely small intestinal bacterial overgrowth (SIBO) and short bowel syndrome (SBS). We also detail in vivo, ex vivo and in vitro models developed to simulate the small intestinal environment, some applied for (diet-)host-bacteria interaction studies. Lastly, we highlight recent technological, medical and scientific advances applicable to investigate this complex and yet understudied body environment to broaden our knowledge in support of further progress in the medical practice, and to proceed towards the integration of the (small)intestinal bacteria in personalized therapeutic approaches.

Mutations in the non-coding snoRNA component of mitochondrial RNA processing endoribonuclease (RMRP) are the cause of cartilage-hair hypoplasia (CHH). CHH is a rare form of metaphyseal chondrodysplasia characterized by disproportionate short stature and abnormal growth plate development. The process of chondrogenic differentiation within growth plates of long bones is vital for longitudinal bone growth. However, molecular mechanisms behind impaired skeletal development in CHH patients remain unclear. We employed a transdifferentiation model (FDC) combined with whole transcriptome analysis to investigate the chondrogenic transdifferentiation capacity of CHH fibroblasts and to examine pathway regulation in CHH cells during chondrogenic differentiation. We established that the FDC transdifferentiation model is a relevant in vitro model of chondrogenic differentiation, with an emphasis on the terminal differentiation phase, which is crucial for longitudinal bone growth. We demonstrated that CHH fibroblasts are capable of transdifferentiating into chondrocyte-like cells, and show a reduced commitment to terminal differentiation. We also found a number of key factors of BMP, FGF, and IGF-1 signalling axes to be significantly upregulated in CHH cells during the chondrogenic transdifferentiation. Our results support postulated conclusions that RMRP has pleiotropic functions and profoundly affects multiple aspects of cell fate and signalling. Our findings shed light on the consequences of pathological CHH mutations in snoRNA RMRP during chondrogenic differentiation and the relevance and roles of non-coding RNAs in genetic diseases in general., Graphical abstract Image 1

Providing a cost-efficient feeding strategy for cell expansion processes remains a challenging task due to, among other factors, donor variability. The current method to use a fixed medium replacement strategy for all cell batches results often in either over- or underfeeding these cells. In order to take into account the individual needs of the cells, a model predictive controller was developed in this work. Reference experiments were performed by expanding human periosteum derived progenitor cells (hPDCs) in tissue flasks to acquire reference data. With these data, a time-variant prediction model was identified to describe the relation between the accumulated medium replaced as the control input and the accumulated lactate produced as the process output. Several forecast methods to predict the cell growth process were designed using multiple collected datasets by applying transfer function models or machine learning. The first controller experiment was performed using the accumulated lactate values from the reference experiment as a static target function over time, resulting in over- or underfeeding the cells. The second controller experiment used a time-adaptive target function by combining reference data as well as current measured real-time data, without over- or underfeeding the cells. ispartof: PROCESSES vol:11 issue:1 status: published

The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic–embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood. This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics. ispartof: FRONTIERS IN ENDOCRINOLOGY vol:12 ispartof: location:Switzerland status: published

Although gene therapy has its conceptual origins in the treatment of Mendelian disorders, it has potential applications in regenerative medicine, including bone healing. Research into the use of gene therapy for bone healing began in the 1990s. Prior to this period, the highly osteogenic proteins bone morphogenetic protein (BMP)-2 and -7 were cloned, produced in their recombinant forms and approved for clinical use. Despite their promising osteogenic properties, the clinical usefulness of recombinant BMPs is hindered by delivery problems that necessitate their application in vastly supraphysiological amounts. This generates adverse side effects, some of them severe, and raises costs; moreover, the clinical efficacy of the recombinant proteins is modest. Gene delivery offers a potential strategy for overcoming these limitations. Our research has focused on delivering a cDNA encoding human BMP-2, because the recombinant protein is Food and Drug Administration approved and there is a large body of data on its effects in people with broken bones. However, there is also a sizeable literature describing experimental results obtained with other transgenes that may directly or indirectly promote bone formation. Data from experiments in small animal models confirm that intralesional delivery of BMP-2 cDNA is able to heal defects efficiently and safely while generating transient, local BMP-2 concentrations 2-3 log orders less than those needed by recombinant BMP-2. The next challenge is to translate this information into a clinically expedient technology for bone healing. Our present research focuses on the use of genetically modified, allografted cells and chemically modified messenger RNA.