Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation.

The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46-/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46-/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.

Establishment of keratinocyte cell lines from human hair follicles.

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells.

Aging is accompanied by loss of subcutaneous adipose tissue. This may be due to reduced differentiation capacity or deficiency in DNA damage repair (DDR) factors. Here we investigated the role of SNEVhPrp19/hPso4, which was implicated in DDR and senescence evasion, in adipogenic differentiation of human adipose stromal cells (hASCs). We showed that SNEV is induced during adipogenesis and localized both in the nucleus and in the cytoplasm. Knockdown of SNEV perturbed adipogenic differentiation and led to accumulation of DNA damage in hASCs upon oxidative stress. In addition, we demonstrated that SNEV is required for fat deposition in Caenorhabditis elegans. Consequently, we tested other DDR factors and found that WRN is also required for adipogenesis in both models. These results demonstrate that SNEV regulates adipogenesis in hASCs and indicate that DDR capacity in general might be a pre-requisite for this process.

Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells.

Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor.

Osteogenesis from Dental Pulp Derived Stem Cells: A Novel Conditioned Medium Including Melatonin within a Mixture of Hyaluronic, Butyric, and Retinoic Acids.

Human dental pulp stem cells (hDPSCs) have shown relevant potential for cell therapy in the orthopedic and odontoiatric fields. The optimization of their osteogenic potential is currently a major challenge. Vascular endothelial growth factor A (VEGF A) has been recently reported to act as a major conductor of osteogenesis in vitro and in vivo. Here, we attempted to prime endogenous VEGF A expression without the need for viral vector mediated gene transfer technologies. We show that hDPSCs exposure to a mixture of hyaluronic, butyric, and retinoic acids (HA + BU + RA) induced the transcription of a gene program of osteogenesis and the acquirement of an osteogenic lineage. Such response was also elicited by cell exposure to melatonin, a pleiotropic agent that recently emerged as a remarkable osteogenic inducer. Interestingly, the commitment to the osteogenic fate was synergistically enhanced by the combinatorial exposure to a conditioned medium containing both melatonin and HA + BU + RA. These in vitro results suggest that in vivo osteogenesis might be improved and further studies are needed.

Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.

Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC.

SNEV(Prp19/PSO4) deficiency increases PUVA-induced senescence in mouse skin.

Senescent cells accumulate during ageing in various tissues and contribute to organismal ageing. However, factors that are involved in the induction of senescence in vivo are still not well understood. SNEV(P) (rp19/) (PSO) (4) is a multifaceted protein, known to be involved in DNA damage repair and senescence, albeit only in vitro. In this study, we used heterozygous SNEV(+/-) mice (SNEV-knockout results in early embryonic lethality) and wild-type littermate controls as a model to elucidate the role of SNEV(P) (rp19/) (PSO) (4) in DNA damage repair and senescence in vivo. We performed PUVA treatment as model system for potently inducing cellular senescence, consisting of 8-methoxypsoralen in combination with UVA on mouse skin to induce DNA damage and premature skin ageing. We show that SNEV(P) (rp19/) (PSO) (4) expression decreases during organismal ageing, while p16, a marker of ageing in vivo, increases. In response to PUVA treatment, we observed in the skin of both SNEV(P) (rp19/) (PSO) (4) and wild-type mice an increase in γ-H2AX levels, a DNA damage marker. In old SNEV(P) (rp19/) (PSO) (4) mice, this increase is accompanied by reduced epidermis thickening and increase in p16 and collagenase levels. Thus, the DNA damage response occurring in the mouse skin upon PUVA treatment is dependent on SNEV(P) (rp19/) (PSO) (4) expression and lower levels of SNEV(P) (rp19/) (PSO) (4) , as in old SNEV(+/-) mice, result in increase in cellular senescence and acceleration of premature skin ageing.

Urine is a novel source of autologous mesenchymal stem cells for patients with epidermolysis bullosa.

BACKGROUND: Regenerative medicine is strictly dependent on stem cells as a source for a high diversity of somatic cells. However, the isolation of such from individuals suffering from severe genetic skin blistering diseases like epidermolysis bullosa (EB) is often associated with further organ damage. METHODS: Stem cells were isolated from 112 urine samples from 21 different healthy donors, as well as from 33 urine samples from 25 donors with EB. The cultivation of these cells was optimized by testing different media formulations and pre-coating of culture vessels with collagen. The identity of cells was confirmed by testing marker expression, differentiation potential and immune-modulatory properties. RESULTS: We provide here an optimized protocol for the reproducible isolation of mesenchymal stem cells from urine, even from small volumes as obtained from patients with EB. Furthermore, we offer a basic characterization of those urine-derived stem cells (USCs) from healthy donors, as well as from patients with EB, and demonstrate their potential to differentiate into chondrocytes, osteoblasts and adipocytes, as well as their immune-modulatory properties. CONCLUSIONS: Thus, USCs provide a novel and non-invasive source of stem cells, which might be applied for gene-therapeutic approaches to improve medical conditions of patients with EB.

Differentially circulating miRNAs after recent osteoporotic fractures can influence osteogenic differentiation.

Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level and are known to take part in the control of bone formation and bone resorption. In addition, it is known that miRNAs are secreted by many cell types and can transfer "messages" to recipient cells. Thus, circulating miRNAs might not only be useful as surrogate biomarkers for the diagnosis or prognosis of pathological conditions, but could be actively modulating tissue physiology. Therefore, the aim of this study was to test whether circulating miRNAs that exhibit changes in recent osteoporotic fracture patients could be causally related to bone metabolism. In the first step we performed an explorative analysis of 175 miRNAs in serum samples obtained from 7 female patients with recent osteoporotic fractures at the femoral neck, and 7 age-matched female controls. Unsupervised cluster analysis revealed a high discriminatory power of the top 10 circulating miRNAs for patients with recent osteoporotic fractures. In total 6 miRNAs, miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p exhibited significantly different serum levels in response to fracture (adjusted p-value<0.05). These miRNAs were subsequently analyzed in a validation cohort of 23 patients (11 control, 12 fracture), which confirmed significant regulation for miR-22-3p, miR-328-3p, and let-7g-5p. A set of these and of other miRNAs known to change in the context of osteoporotic fractures were subsequently tested for their effects on osteogenic differentiation of human mesenchymal stem cells (MSCs) in vitro. The results show that 5 out of 7 tested miRNAs can modulate osteogenic differentiation of MSCs in vitro. Overall, these data suggest that levels of specific circulating miRNAs change in the context of recent osteoporotic fractures and that such perturbations of "normal" levels might affect bone metabolism or bone healing processes.

Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines.

Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, especially in early drug discovery and development phases of biopharmaceuticals. The need for fast production of sufficient recombinant protein for initial tests has dramatically increased with increase in the identification of potential novel pharmaceutical targets. One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol. Thereby, we show the loss of PCN during a typical batch process of HEK293 cells after transfection from 606,000 to 4560 copies per cell within 5 days. Finally two novel human kidney cell lines, RS and RPTEC/TERT1 were compared to HEK293 and proved competitive in terms of PCN and specific productivity. In conclusion, since trafficking and degradation of plasmid DNA is not fully understood yet, improved methods for analysis of PCN may contribute to design specific and more stable plasmids for high yield transient gene expression systems.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan.

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

The role of microRNAs in cellular senescence and age-related conditions of cartilage and bone.

BACKGROUND AND PURPOSE: We reviewed the current state of research on microRNAs in age-related diseases in cartilage and bone. METHODS: PubMed searches were conducted using separate terms to retrieve articles on (1) the role of microRNAs on aging and tissue degeneration, (2) specific microRNAs that influence cellular and organism senescence, (3) microRNAs in age-related musculoskeletal conditions, and (4) the diagnostic and therapeutic potential of microRNAs in age-related musculoskeletal conditions. RESULTS: An increasing number of studies have identified microRNAs associated with cellular aging and tissue degeneration. Specifically in regard to frailty, microRNAs have been found to influence the onset and course of age-related musculoskeletal conditions such as osteoporosis, osteoarthritis, and posttraumatic arthritis. Both intracellular and extracellular microRNAs may be suitable to function as diagnostic biomarkers. INTERPRETATION: The research data currently available suggest that microRNAs play an important role in orchestrating age-related processes and conditions of the musculoskeletal system. Further research may help to improve our understanding of the complexity of these processes at the cellular and extracellular level. The option to develop microRNA biomarkers and novel therapeutic agents for the degenerating diseases of bone and cartilage appears to be promising.

Human cell lines for the production of recombinant proteins: on the horizon.

The market of recombinant proteins as human pharmaceuticals has surpassed annual revenues of more than 150 billion dollars. The marketed proteins are often complex in terms of post-translational modifications and conventional hosts have shown weaknesses in terms of quality of these recombinant proteins. Especially the non-human glycopatterns leading to immunogenicity or shortened in vivo half-life have gained attention over the past decade. Therefore, production cell lines with better or novel characteristics are required and human cell lines seem to be the most genuine and logical choice. Thus, several human cell lines have been used to generate biopharmaceuticals. We here present an overview of such examples and highlight their promise for biopharmaceutical production processes of the future.

Characterization of a novel cell penetrating peptide derived from human Oct4.

BACKGROUND: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells. RESULTS: A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide. CONCLUSIONS: Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs. Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.

MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro.

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.