GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS BY PROTEIN TRANSDUCTION.

Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2 and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in frame to the GST tag for affinity purification and to the TAT-NLS polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-hour intervals, and cultured on mitomycin C treated MEF (mouse embryonic fibroblast) cells in complete embryonic stem cell medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to embryonic stem (ESC) cells and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by RT-PCR. The protein iPSCs (piPSCs) formed EBs and subsequently differentiated towards all three germ layer lineages. Importantly the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming mouse embryonic fibroblasts to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESC cells: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation

Generation of Mucopolysaccharidosis type II (MPS II) human induced pluripotent stem cell (iPSC) line from a 3-year-old male with pathogenic IDS mutation

AbstractPeripheral blood was collected from a 3-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies

Establishment of a rabbit induced pluripotent stem cell (RbiPSC) line using lentiviral delivery of human pluripotency factors

Rabbit Embryonic Fibroblast (RbEF) cells (from Hycole hybrid rabbit foetus) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the newly generated RbiPSC was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the spontaneous differentiation potential of the iPSC was also tested in vivo by teratoma assay. The iPSC line showed normal karyotype. The advantages of using RbiPSC are the easy access to primary material and the possibility to study the efficacy and safety of the iPSC-based therapies on a non-rodent animal model

Screening of Bioactive Peptides Using an Embryonic Stem Cell-Based Neurodifferentiation Assay

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons

A májfunkció romlásának rizikófaktorai sikeres vesetranszplantációt követően = Risk factors for deterioration of liver functions after successful kidney transplantation

Absztrakt: Bevezetés: A vesetranszplantáció utáni májfunkcióromlás az egyik leggyakoribb szövődmény, melynek oka a hepatitis C-vírus (HCV)-fertőzés mellett az alkalmazott immunszuppresszív terápia és hyperlipidaemia. Módszer: A beválasztási kritériumokat követően (n = 59) vizsgáltuk az alkalmazott immunszuppresszív terápiát, a betegek alapadatait, további a HCV és a májfunkció romlása közötti összefüggést. A betegeknél éhomi laboratóriumi vizsgálat történt, melynek során néztük a szérumionokat, húgysav-, albuminszintet. Az immunszuppresszív terápia lipidekre (TG, TC, HDL, LDL), valamint májenzimekre (GOT, GPT, ALP, GGT) gyakorolt hatását néztük. Vizsgálatunk részét képezte a lipidek és a májenzimek közötti kapcsolat elemzése is. Eredmények: A betegek alapadatait vizsgálva a takrolimuszt (n = 50) és a ciklosporint (n = 9) szedők körében szignifikáns különbséget nem találtunk. A laboratóriumi eredmények tekintetében a Mg-szint szignifikánsan eltért a két csoport között (p = 0,044). A HCV-fertőzés májenzimekre gyakorolt hatását nézve a GGT (p = 0,008) szignifikánsan különbözött. A lipideket vizsgálva a takrolimusz- és a ciklosporinalapú immunszuppresszióban részesülő betegek között az összkoleszterin (p = 0,005), valamint a májenzimek közül a GOT (p = 0,05) szignifikánsan eltért a két betegcsoport között. A hyperlipidaemia a takrolimuszalapú immunszuppressziót szedők körében 26%-ban, míg a ciklosporint szedőknél 89%-ban fordult elő, melyek között a különbség szignifikáns volt (p = 0,002). A hyperlipidaemia májenzimekre gyakorolt hatását nézve az ALP (p = 0,006) és a GGT (p = 0,0001) szignifikánsan magasabb volt. Következtetés: A májenzimek, az ALT és a GGT emelkedése utal a májsejtek sérülésére. A májfunkció romlásának a legfőbb rizikófaktora a HCV talaján kialakult hepatitis mellett az alkalmazott immunszuppresszív terápia és a hyperlipidaemia, mely az allograftfunkció romlásához és hosszú távon graftvesztéshez vezet. Orv Hetil. 2019; 160(5): 186–190. | Abstract: Introduction: Increase of liver function is one of the most common complications after kidney transplantation due to the use of immunosuppressive therapy and hyperlipidemia in addition to hepatitis C virus (HCV) infection. Method: Following the selection criteria (n = 59), the study is based on applied immunosuppressive therapy, baseline data of patients, further correlation between HCV and liver function deterioration. Patients were subjected to fasting laboratory examination to monitor serum electrolytes, uric acid and albumin levels. We looked at the effects of immunosuppressive therapy on the lipids (TG, TC, HDL, LDL) and liver enzymes (GOT, GPT, ALP, GGT). The analysis of the relationship between lipids and liver enzymes was also included in our study. Results: The data basics were not significantly different between the tacrolimus and the cyclosporine groups. In the laboratory results, Mg levels were significantly different between the two groups (p = 0.044). The impact of HCV on the liver function was significantly different on GGT (p = 0.008). We examed the lipids and liver function level between the tacrolimus and the patients receiving cyclosporine-based immunosuppression and the total cholesterol (p = 0.005) and GOT (p = 0.05) were significantly different between the two groups. Hyperlipidemia was associated with 26% of patients taking tacrolimus-based immunosuppression, and 89% of those receiving cyclosporine; the difference was significant (p = 0.002). Regarding the effect of hyperlipidemia on liver enzymes, ALP (p = 0.006) and GGT (p = 0.0001) were significantly higher. Conclusion: Increases in hepatic enzymes, ALT and GGT indicate the damage to hepatocytes. Beside the increase of liver function, which is the main risk factor in hepatitis on HCV soil, the applied immunosuppressive therapy and hyperlipidemia lead to degradation of allograft function and long-term graft loss. Orv hetil. 2019; 160(5): 186–190

Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes

To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel. Rescuing the folding defect or down-regulating the E1 ubiquitin (Ub)-activating enzyme stabilized the mutant CFTR without interfering with its internalization. These observations with the preferential association of mutant CFTRs with Hrs, STAM-2, TSG101, hVps25, and hVps32, components of the Ub-dependent endosomal sorting machinery, establish a functional link between Ub modification and lysosomal degradation of misfolded CFTR from the cell surface. Our data provide evidence for a novel cellular mechanism of CF pathogenesis and suggest a paradigm for the quality control of plasma membrane proteins involving the coordinated function of ubiquitination and the Ub-dependent endosomal sorting machinery

Neurosphere based differentiation of human iPSC improves astrocyte differentiation

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes