NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC-Derived Nociceptor Maturation.

Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human induced pluripotent stem cell-derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co-sequencing, immunofluorescence staining, and qPCR validations, reveal expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerge as regulatory hubs for neurite outgrowth, synapse development, and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis reveals distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors.

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

Differentiation of human iPSCs into functional podocytes.

Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.

Effect of cyclosporine, tacrolimus and sirolimus on cellular senescence in renal epithelial cells.

INTRODUCTION: In transplantation medicine calcineurin inhibitors (CNI) still represent the backbone of immunosuppressive therapy. The nephrotoxic potential of the CNI Cyclosporine A (CsA) and Tacrolimus (FK506) is well recognized and CNI not only have been linked with toxicity, but also with cellular senescence which hinders parenchymal tissue regeneration and thus may prime kidneys for subsequent insults. To minimize pathological effects on kidney grafts, alternative immunosuppressive agents like mTOR inhibitors or the T-cell co-stimulation blocker Belatacept have been introduced. METHODS: We compared the effects of CsA, FK506 and Sirolimus on the process of cellular senescence in different human renal tubule cell types (HK2, RPTEC). Telomere length (by real time PCR), DNA synthesis (by BrdU incorporation), cell viability (by Resazurin conversion), gene expression (by RT-PCR), protein (by western blotting), Immuncytochemistry and H2O2 production (by Amplex Red® conversion) were evaluated. RESULTS: DNA synthesis was significantly reduced when cells were treated with cyclosporine but not with tacrolimus and sirolimus. Resazurin conversion was not altered by all three immunosuppressive agents. The gene expression as well as protein production of the cell cycle inhibitor p21 (CDKN1A) but not p16 (CDKN2A) was significantly induced by cyclosporine compared to the other two immunosuppressive agents when determined by western blotting an immuncytochemistry. Relative telomere length was reduced and hydrogen peroxide production increased after treatment with CsA but not with FK506 or sirolimus. CONCLUSION: In summary, renal tubule cells exposed to CsA show clear signs of cellular senescence where on the contrary the second calcineurin inhibitor FK506 and the mTOR inhibitor sirolimus are not involved in such mechanisms. Chronic renal allograft dysfunction could be in part triggered by cellular senescence induced by immunosuppressive medication and the choice of drug could therefore influence long term outcome. Tacrolimus and Sirolimus are equally effective in avoiding cellular senescence compared to cyclosporine at least in parts due to a lack of induction of reactive oxygen species.

Towards optimisation of induced pluripotent cell culture: Extracellular acidification results in growth arrest of iPSC prior to nutrient exhaustion.

Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose, lactate, extracellular pH and cell cycle phases were quantified. Without daily feeding, FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast, RGA cultured cells grew to confluence over 3days. Surprisingly, glucose was not exhausted under any condition. However, extracellular pH reached 6.8 after 72h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle, while adding an additional 10mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification, a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification, while still maintaining pluripotency and allowing longer feeding cycles.

Interleukin-19 as a translational indicator of renal injury.

Accurate detection and prediction of renal injury are central not only to improving renal disease management but also for the development of new strategies to assess drug safety in pre-clinical and clinical testing. In this study, we utilised the well-characterised and differentiated human renal proximal tubule cell line, RPTEC/TERT1 in an attempt to identify markers of renal injury, independent of the mechanism of toxicity. We chose zoledronate as a representative nephrotoxic agent to examine global transcriptomic alterations using a daily repeat bolus protocol over 14 days, reflective of sub-acute or chronic injury. We identified alterations in targets of the cholesterol and mevalonate biosynthetic pathways reflective of zoledronate specific effects. We also identified interleukin-19 (IL-19) among other inflammatory signals such as SERPINA3 and DEFB4 utilising microarray analysis. Release of IL-19 protein was highly induced by an additional four nephrotoxic agents, at magnitudes greater than the characterised marker of renal injury, lipocalin-2. We also demonstrate a large increase in levels of IL-19 in urine of patients with chronic kidney disease, which significantly correlated with estimated glomerular filtration rate levels. We suggest IL-19 as a potential new translational marker of renal injury.

Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-ß is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-ß-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-ß as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-ß receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-ß receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-ß1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-ß1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-ß is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

Effect of different immunosuppressive drugs on immune cells from young and old healthy persons.

BACKGROUND: Life expectancy, as well as the average age of patients undergoing solid organ transplantation, increases constantly. Consequently, immunosuppressive therapy is no longer limited to young organ recipients. OBJECTIVE: Here, we investigate how different types of immunosuppressive therapy, namely the calcineurin inhibitors cyclosporin A and tacrolimus, as well as the mTOR inhibitor rapamycin, affect the function of immune cells in young and elderly persons. METHODS: Proliferation, cell viability, cytokine production (IL-2, IFN-γ), H2O2 production and telomere length of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) of young (n = 13; median age 27 years) and old (n = 19; median age 71 years) healthy donors were analyzed. RESULTS: The inhibition of proliferation was dampened in PBMCs from elderly donors, especially after incubation with rapamycin. All three immunosuppressive drugs inhibited the production of IL-2 equally well, whereas the production of IFN-γ was less well inhibited by rapamycin. Both calcineurin inhibitors increased H2O2 concentrations after stimulation with PHA and led to a shortening of telomeres in PBMCs from young and old individuals. Rapamycin had only minor effects on H2O2 production and telomere length. CONCLUSION: Our results demonstrate that the effects of immunosuppressive drugs on PBMCs differ between young and elderly persons. Calcineurin inhibitors compared to rapamycin have a more pronounced prosenescence effect. These data indicate that specific treatment regimens for the elderly might therefore be considered.

Biomarkers of aging with prognostic and predictive value in non-oncological diseases.

In this review we discuss the current knowledge on Biomarkers of Aging (BoAs) in the context of human diseases and their value as predictive or prognostic markers. The vast majority of studies using BoAs in a clinical context have been undertaken by determining telomere length in peripheral blood mononuclear cells (PBMCs), whereas the expression of cell cycle inhibitors and an increase in advanced glycation end products (AGEs) have rarely been used. Here we summarize the impact of BoAs on non-oncological, hematological, cardiovascular, metabolic, renal and neurological diseases, as well as on overall survival. The specific methodologies utilized are described and evaluated for their high-throughput potential.

SRP-27 is a novel component of the supramolecular signalling complex involved in skeletal muscle excitation-contraction coupling.

SRP-27 (sarcoplasmic reticulum protein of 27 kDa) is a newly identified integral membrane protein constituent of the skeletal muscle SR (sarcoplasmic reticulum). We identified its primary structure from cDNA clones isolated from a mouse skeletal muscle cDNA library. ESTs (expressed sequence tags) of SRP-27 were found mainly in cDNA libraries from excitable tissues of mouse. Western blot analysis confirmed the expression of SRP-27 in skeletal muscle and, to a lower extent, in heart and brain. Mild trypsin proteolysis combined with primary-structure prediction analysis suggested that SRP-27 has four transmembrane-spanning alpha helices and its C-terminal domain faces the cytoplasmic side of the endo(sarco)plasmic reticulum. The expression of SRP-27 is higher in fast twitch skeletal muscles compared to slow twitch muscles and peaks during the first month of post-natal development. High-resolution immunohistochemistry and Western blot analysis of subcellular fractions indicated that SRP-27 is distributed in both longitudinal tubules and terminal cisternae of the SR, as well as in the perinuclear membrane systems and the nuclear envelope of myotubes and adult fibres. SRP-27 co-sediments with the RyR (ryanodine receptor) macromolecular complex in high-salt sucrose-gradient centrifugation, and is pulled-down by anti-RyR as well as by maurocalcin, a well characterized RyR modulator. Our results indicate that SRP-27 is part of a SR supramolecular complex, suggesting the involvement of SRP-27 in the structural organization or function of the molecular machinery underlying excitation-contraction coupling.

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

Positive and negative spatial priming in schizophrenia.

Priming tasks are used for investigating the deficits of selective attention in schizophrenia, which are thought to be related to the psychotic symptoms. Priming was assessed in acutely psychotic unmedicated (n = 22) and medicated (n = 36) schizophrenia patients and in control subjects (n = 42). The subjects had to indicate the location of a target stimulus in two consecutive stimulus displays (prime and probe). Each stimulus appeared together with a distractor on a screen. Negative Priming is characterized by an increase in reaction time, whenever a probe target is presented at a prime distractor location. Positive Priming is characterized by a decrease in reaction time, when the probe target is presented at the prime target location. Schizophrenia patients altogether showed no abnormalities in priming effects, neither in the acute phase of illness (medicated and unmedicated) nor in partial remission (one month later, medicated). In unmedicated patients however Negative Priming was inversely correlated with the severity of positive symptoms. This indicates a priming deficit in a small subgroup of severely ill patients. The data support the notion that automatic (implicit) mechanisms of learning are unimpaired in schizophrenia patients unless symptoms exceed a certain critical level.

The role of auxiliary dihydropyridine receptor subunits in muscle.

The skeletal muscle dihydropyridine receptor is a slowly-activating calcium channel that functions as the voltage sensor in excitation-contraction coupling. In addition to the pore-forming alpha(1S) subunit it contains the transmembrane alpha(2)delta-1 and gamma(1) subunits and the cytoplasmic beta(1a) subunit. Although the roles of the auxiliary subunits in calcium channel function have been intensively studied in heterologous expression systems, their functions in excitation-contraction coupling has only recently been elucidated in muscle cells of various null-mutant animal models. In this article we will briefly outline the current state of these investigations.