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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
A resource is provided investigating temporal short and long RNA dynamics in induced pluripotent stem cell‐derived sensory neuron differentiation, with a special focus on miRNA::mRNA interactions regulating synapse development, neurite outgrowth, and ion channels. The containerized docker tool NOCICEPTRA allows for exploration of temporal trajectories, Kyoto Encyclopedia of Genes and Genomes pathway onsets, and more.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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-β 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-β-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-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-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-β 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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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.
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.
•Different immunosuppressive agents lead to different effects on the renal cellular aging process.•Cyclosporine A induces cellular senescence in renal epithelial cells but not Tacrolimus and Sirolimus.•This effect is induced by Hydrogenperoxide over the CDKN1A/p21 pathway.•These results may have a substantial impact in the decision of immunosuppressive therapy especially in renal transplantation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
L-type Ca²⁺ currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca²⁺ signal, which regulates the contraction force. The auxiliary Ca²⁺ channel subunits ...α₂δ-1 and β₂ are important regulators of membrane expression and current properties of the cardiac Ca²⁺ channel (CaV1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the α₂δ-1 subunit in a muscle expression system with simulation of APs and Ca²⁺ transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with CaV1.2 (GFP-α₁C) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the α₂δ-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-α₁C subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, α₂δ-1 depletion shifted the voltage dependence of Ca²⁺ 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 Ca²⁺ concentration during each contraction. Thus, the Ca²⁺ channel α₂δ-1 subunit is not essential for normal Ca²⁺ channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of α₂δ-1 function is predicted to severely perturb normal heart function.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
We present an ab initio molecular dynamics study on hydrogen adsorption in potassium-intercalated graphite of second stage. The simulation utilizes the ultrasoft pseudopotenals plane wave method ...under local density functional approximation. The optimized lattice structures and the calculated H2 adsorption energy are in excellent agreement with experiments. The simulation also well reproduces the previously observed lattice expansion due to H2 uptake. The dynamics investigations reveal that not only the adsorbed hydrogen molecules but also the intercalated potassium atoms are highly mobile and assume a variety of two-dimensional configurations. The hydrogen dynamics is essentially chaotic within a layer of about 1.4 Å thickness centered between the graphite sheets, with a closest C−H distance of about 2.2 Å.
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IJS, KILJ, NUK, PNG, UL, UM
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