RESUMEN
Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.
Asunto(s)
Canal de Potasio ERG1/genética , Células Madre Pluripotentes Inducidas/fisiología , Síndrome de QT Prolongado/genética , Mutación/genética , Miocitos Cardíacos/fisiología , Transporte de Proteínas/genética , Potenciales de Acción/genética , Adolescente , Adulto , Membrana Celular/genética , Femenino , Edición Génica/métodos , Humanos , Leucocitos Mononucleares/fisiología , Masculino , Fenotipo , Adulto JovenRESUMEN
The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.
Asunto(s)
Adenosina Trifosfato/deficiencia , Micropartículas Derivadas de Células/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Línea Celular , Proliferación Celular , Citoprotección , Células Epiteliales/metabolismo , Células Epiteliales/patología , Ontología de Genes , Humanos , Receptores de Hialuranos/metabolismo , Integrina beta1/metabolismo , MicroARNs/genética , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: Renal tubular cells are the main target of ischemic insult associated with acute renal injury. Low oxygen and nutrient supplies result in ATP depletion, leading to cell death and loss of renal function. A possible mechanism by which bone marrow-derived cells support renal tissue regeneration relies on the capacity of mononuclear cells (BMMC), particularly mesenchymal stem cells (MSC), to secrete paracrine factors that mediate support for kidney regeneration. METHODS: BMMC/MSC and renal cells (LLC-PK(1) from pig and IRPTC from rat) were co-cultured under stressful conditions (ATP depletion and/or serum free starvation), physically separated by a microporous membrane (0.4 µm), was used to determine whether bone marrow-derived cells can interact with renal cells in a paracrine manner. RESULTS: This interaction resulted in stimulation of renal cell proliferation and the arrest of cell death. MSC elicit effective responses in renal cells in terms of stimulating proliferation and protection. Such effects are observed in renal cells co-cultured with rat BMMC/MSC, an indication that paracrine mechanisms are not entirely species-specific. CONCLUSION: The paracrine action of BMMC/MSC was influenced by a renal cell stimulus released during stress, indicating that cross-talk with injured cells is required for renal regeneration supported by bone marrow-derived cells.
