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We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.
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Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.
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Células Madre Pluripotentes Inducidas , Enfermedades Renales , Riñón , Organoides , Humanos , Organoides/patología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/patología , Riñón/patología , Animales , Sistemas CRISPR-Cas/genéticaRESUMEN
Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them. Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN. Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.
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Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.
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Anticuerpos Monoclonales Humanizados , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Ratones Endogámicos C57BL , Linfocitos B , Ratones Transgénicos , Antígeno HLA-A2/genética , Antígenos HLA/metabolismo , Inmunoglobulina G/metabolismo , Células Madre Mesenquimatosas/metabolismoRESUMEN
BACKGROUND: Multiple risk factors are involved in new-onset diabetes mellitus (DM) after organ transplantation; however, their ability to predict clinical prognosis remains unclear. Therefore, we investigated whether patient-specific induced pluripotent stem cells (iPSCs) could help predict DM development before performing kidney transplantation (KT). METHODS: We first performed whole transcriptome and functional enrichment analyses of KT patient-derived iPSCs. Our results revealed that insulin resistance, type 2 DM, and transforming growth factor beta signaling pathways are associated between the groups of DM and non-DM. We next determined whether the genetic background was associated with development of iPSCs into pancreatic progenitor (PP) cells. RESULTS: The levels of differentiation-related key markers of PP cells were significantly lower in the DM group than in the non-DM group. Moreover, the results of tacrolimus toxicity screening showed a significant decrease in the number of PP cells of the DM group compared with the non-DM group, suggesting that these cells are more susceptible to tacrolimus toxicity. CONCLUSION: Taken together, these results indicate that PP cells of the DM group showed low developmental potency accompanied by a significantly different genetic background compared with the non-DM group. Thus, genetic analysis can be used to predict the risk of DM before KT.
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Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/-), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.
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Células Madre Pluripotentes Inducidas , Nefritis Intersticial , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Riñón/metabolismo , Endonucleasas , Organoides/metabolismo , Enzimas MultifuncionalesRESUMEN
Dent disease, an X-linked tubular disorder, is a rare condition that leads to low-molecular-weight proteinuria, hypercalciuria, kidney stones, and chronic kidney disease. Here, we successfully established a human induced pluripotent stem cells (hiPSC) line from peripheral blood mononuclear cells of 10-year-old male with Dent disease 1 caused by the mutation of Chloride Voltage-Gated Channel 5 gene. This hiPSCs displayed features similar to human embryonic stem cells, including pluripotency-associated markers expression, normal karyotype, and the ability to differentiate into cells representing all three germ layers. The implications of this research extend to the potential development of novel treatments for Dent disease.
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Enfermedad de Dent , Células Madre Pluripotentes Inducidas , Masculino , Humanos , Niño , Enfermedad de Dent/complicaciones , Enfermedad de Dent/genética , Leucocitos Mononucleares , Mutación , Proteinuria/genética , Proteinuria/orinaRESUMEN
BACKGROUND/AIMS: Although the conversion from tacrolimus (TAC) to cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) is effective in reducing TAC-induced nephrotoxicity, it remains unclear whether CTLA4-Ig has a direct effect on TAC-induced renal injury. In this study, we evaluated the effects of CTLA4-Ig on TAC-induced renal injury in terms of oxidative stress. METHODS: In vitro study was performed to assess the effect of CTLA4-Ig on TAC-induced cell death, reactive oxygen species (ROS), apoptosis, and the protein kinase B (AKT)/forkhead transcription factor (FOXO) 3 pathway in human kidney 2 cells. In the in vivo study, the effect of CTLA4-Ig on TAC-induced renal injury was evaluated using renal function, histopathology, markers of oxidative stress (8-hydroxy-2'-deoxyguanosine) and metabolites (4-hydroxy-2-hexenal, catalase, glutathione S-transferase, and glutathione reductase), and activation of the AKT/FOXO3 pathway with insulin-like growth factor 1 (IGF-1). RESULTS: CTLA4-Ig significantly decreased cell death, ROS, and apoptosis caused by TAC. TAC treatment increased apoptotic cell death and apoptosis-related proteins (increased Bcl-2-associated X protein and caspase-3 and decreased Bcl-2), but it was reversed by CTLA4-Ig treatment. The activation of p-AKT and p-FOXO3 by TAC decreased with CTLA4-Ig treatment. TAC-induced renal dysfunction and oxidative marker levels were significantly improved by CTLA4-Ig in vivo. Concomitant IGF-1 treatment abolished the effects of CTLA4-Ig. CONCLUSION: CTLA4-Ig has a direct protective effect on TAC-induced renal injury via the inhibition of AKT/FOXO3 pathway.
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Insuficiencia Renal , Tacrolimus , Ratas , Humanos , Animales , Tacrolimus/farmacología , Abatacept/farmacología , Abatacept/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Proto-Oncogénicas c-akt , Especies Reactivas de Oxígeno , Ratas Sprague-Dawley , Transducción de Señal , Estrés Oxidativo , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Forkhead Box O3/metabolismo , Antígeno CTLA-4RESUMEN
In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of -13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.
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Lípidos , Nanopartículas , ARN Interferente Pequeño/genéticaRESUMEN
OBJECTIVES: To explore the possibility of kidney organoids generated using patient derived human induced pluripotent stem cells (hiPSC) for modeling of Fabry disease nephropathy (FDN). METHODS: First, we generated hiPSC line using peripheral blood mononuclear cells (PBMCs) from two male FD-patients with different types of GLA mutation: a classic type mutation (CMC-Fb-001) and a non-classic type (CMC-Fb-003) mutation. Second, we generated kidney organoids using wild-type (WT) hiPSC (WTC-11) and mutant hiPSCs (CMC-Fb-001 and CMC-Fb-003). We then compared alpha-galactosidase A (α-GalA) activity, deposition of globotriaosylceremide (Gb-3), and zebra body formation under electromicroscopy (EM). RESULTS: Both FD patients derived hiPSCs had the same mutations as those detected in PBMCs of patients, showing typical pluripotency markers, normal karyotyping, and successful tri-lineage differentiation. Kidney organoids generated using WT-hiPSC and both FD patients derived hiPSCs expressed typical nephron markers without structural deformity. Activity of α-GalA was decreased and deposition of Gb-3 was increased in FD patients derived hiPSCs and kidney organoids in comparison with WT, with such changes being far more significant in CMC-Fb-001 than in CMC-Fb-003. In EM finding, multi-lammelated inclusion body was detected in both CMC-Fb-001 and CMC-Fb-003 kidney organoids, but not in WT. CONCLUSIONS: Kidney organoids generated using hiPSCs from male FD patients might recapitulate the disease phenotype and represent the severity of FD according to the GLA mutation type.
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Enfermedad de Fabry , Células Madre Pluripotentes Inducidas , Enfermedades Renales , Humanos , Masculino , Enfermedad de Fabry/genética , Leucocitos Mononucleares , Riñón , Diferenciación Celular , OrganoidesRESUMEN
The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.
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Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas , Humanos , Sistemas CRISPR-Cas/genética , Miembro 3 de la Familia de Transportadores de Soluto 12 , Mutación , Riñón , Fenotipo , OrganoidesRESUMEN
The objective of this study was to investigate whether CRISPR/Cas9-mediated suppression of A4GALT could rescue phenotype of Fabry disease nephropathy (FDN) using human induced pluripotent stem cells (hiPSCs) derived kidney organoid system. We generated FDN patient-derived hiPSC (CMC-Fb-002) and FD-specific hiPSCs (GLA-KO) by knock-out (KO) of GLA in wild-type (WT) hiPSCs using CRISPR/Cas9. We then performed A4GALT KO in both CMC-Fb-002 and GLA-KO to make Fb-002-A4GALT-KO and GLA/A4GALT-KO, respectively. Using these hiPSCs, we generated kidney organoids and compared alpha-galactosidase-A enzyme (α-GalA) activity, globotriaosylceramide (Gb-3) deposition, and zebra body formation under electron microscopy (EM). We also compared mRNA expression levels using RNA-seq and qPCR. Generated hiPSCs showed typical pluripotency markers without chromosomal disruption. Expression levels of GLA in CMC-Fb-002 and GLA-KO and expression levels of A4GALT in Fb-002-A4GALT-KO and GLA/A4GALT-KO were successfully decreased compared to those in WT-hiPSCs, respectively. Generated kidney organoids using these hiPSCs expressed typical nephron markers. In CMC-Fb-002 and GLA-KO organoids, α-GalA activity was significantly decreased along with increased deposition of Gb-3 in comparison with WT organoids. Intralysosomal inclusion body was also detected under EM. However, these disease phenotypes were rescued by KO of A4GALT in both GLA/A4GALT-KO and Fb-002-A4GALT-KO kidney organoids. RNA-seq showed increased expression levels of genes related to FDN progression in both GLA-mutant organoids compared to those in WT. Such increases were rescued in GLA/A4GALT-KO or Fb-002-A4GALT-KO organoids. CRISPR/Cas9 mediated suppression of A4GALT could rescue FDN phenotype. Hence, it can be proposed as a therapeutic approach to treat FDN.
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Enfermedad de Fabry , Células Madre Pluripotentes Inducidas , Enfermedades Renales , Humanos , Enfermedad de Fabry/genética , Enfermedad de Fabry/metabolismo , Sistemas CRISPR-Cas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/metabolismo , Enfermedades Renales/genética , Fenotipo , OrganoidesRESUMEN
BACKGROUND/AIMS: We previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system. METHODS: We engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model. RESULTS: We confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells. CONCLUSION: These findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.
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Rechazo de Injerto , Interleucina-10 , Aloinjertos , Animales , Rechazo de Injerto/prevención & control , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores CXCR3/genética , Linfocitos T ReguladoresRESUMEN
Sodium/glucose co-transporter-2 inhibitor (SGLT2i) or dipeptidyl peptidase IV inhibitor (DPP4i) is a newer anti-diabetic drug in type II diabetes mellitus (DM), but their use in tacrolimus (TAC)-induced DM is still undetermined. We performed this study to evaluate the effect of these two drugs in TAC-induced DM and nephrotoxicity in ex vivo and in vivo. In the experimental Sprague Dawley rat model of TAC-induced DM and nephrotoxicity, dual inhibition of DPP4 and SGLT2 significantly decreased blood glucose level, HbA1C and increased plasma insulin levels and pancreatic islet size compared with each drug. In the kidney, dual inhibition improved renal function decreased interstitial fibrosis and profibrotic cytokines compared with DPP4i and SGLT2i alone. Increased oxidative stress by TAC was remarkably decreased with DPP4i or SGLT2i in serum, pancreatic and renal tissues and this decrease was much more significant in the combination group. In in vitro study, TAC decreased the cell viability of human kidney-2(HK-2) cells and insulin-secreting beta-cell-derived line(INS-1) cells. SGLT2i protected TAC-induced cell death in HK-2 cells, but not in INS-1 cells. The addition of DPP4i to SGLT2i compensated for a lack of protective effect of SGLT2i on INS-1 cells. This finding provides the rationale for the combined treatment of SGLG2i and DPP4i in TAC-induced DM and nephrotoxicity.
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Diabetes Mellitus Tipo 2 , Inhibidores de la Dipeptidil-Peptidasa IV , Insulinas , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Hipoglucemiantes/farmacología , Insulinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , TacrolimusRESUMEN
BACKGROUND/AIMS: Renal ischemia followed by reperfusion (I/R) is a leading cause of acute kidney injury (AKI), which is closely associated with high morbidity and mortality. Studies have shown that induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) exert powerful therapeutic effects in renal ischemia. However, the efficacy of iMSC-derived exosomes (iExo) on I/R injuries remains largely unknown. METHODS: Human iPSCs were differentiated into iMSCs using a modified one-step method. Ultrafiltration, combined with purification, was used to isolate iExo from iMSCs. iExo was administered following I/R injury in a mouse model. The effect of iExo on I/R injury was assessed through changes in renal function, histology, and expression of oxidative stress, inflammation, and apoptosis markers. Further, we evaluated its association with the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. RESULTS: Mice subjected to I/R injury exhibited typical AKI patterns; serum creatinine level, tubular necrosis, apoptosis, inflammatory cytokine production, and oxidative stress were markedly increased compared to sham mice. However, treatment with iExo attenuated these changes, significantly improving renal function and tissue damage, similar to the renoprotective effects of iMSCs on I/R injury. Significant induction of activated ERK 1/2 signaling molecules was observed in mice treated with iExo compared to those in the I/R injury group. CONCLUSION: The present study demonstrates that iExo administration ameliorated renal damage following I/R, suggesting that iMSC-derived exosomes may provide a novel therapeutic approach for AKI treatment.
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Lesión Renal Aguda , Exosomas , Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Daño por Reperfusión , Lesión Renal Aguda/terapia , Animales , Apoptosis , Exosomas/metabolismo , Exosomas/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Isquemia/patología , Riñón/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
BACKGROUND: Endothelial cell (EC) dysfunction is a frequent feature in patients with end-stage renal disease (ESRD). The aim of this study was to generate human induced pluripotent stem cells, differentiate ECs (hiPSC-ECs) from patients with ESRD, and appraise the usefulness of hiPSC-ECs as a model to investigate EC dysfunction. METHODS: We generated hiPSCs using peripheral blood mononuclear cells (PBMCs) isolated from three patients with ESRD and three healthy controls (HCs). Next, we differentiated hiPSC-ECs using the generated hiPSCs and assessed the expression of endothelial markers by immunofluorescence. The differentiation efficacy, EC dysfunction, and molecular signatures of EC-related genes based on microarray analysis were compared between the ESRD and HC groups. RESULTS: In both groups, hiPSCs and hiPSC-ECs were successfully obtained based on induced pluripotent stem cell or EC marker expression in immunofluorescence and flow cytometry. However, the efficiency of differentiation of ECs from hiPSCs was lower in the ESRD-hiPSCs than in the HC-hiPSCs. In addition, unlike HC-hiPSC-ECs, ESRD-hiPSC-ECs failed to form interconnecting branching point networks in an in vitro tube formation assay. During microarray analysis, transcripts associated with oxidative stress and inflammation were upregulated and transcripts associated with vascular development and basement membrane extracellular matrix components were downregulated in ESRD-hiPSC-ECs relative to in HC-hiPSC-ECs. CONCLUSION: ESRD-hiPSC-ECs showed a greater level of EC dysfunction than HC-hiPSC-ECs did based on functional assay results and molecular profiles. hiPSC-ECs may be used as a disease model to investigate the pathophysiology of EC dysfunction in ESRD.
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The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using a murine model of chronic tacrolimus neurotoxicity, we evaluated the effects of tacrolimus on cognitive function, synaptic balance, its regulating protein (Klotho), and oxidative stress in the hippocampus. Compared to vehicle-treated mice, tacrolimus-treated mice showed significantly decreased hippocampal-dependent spatial learning and memory function. Furthermore, tacrolimus caused synaptic imbalance, as demonstrated by decreased excitatory synapses and increased inhibitory synapses, and downregulated Klotho in a dose-dependent manner; the downregulation of Klotho was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with activation of the PI3K/AKT pathway in the hippocampus. These results indicate that tacrolimus impairs cognitive function via synaptic imbalance, and that these processes are associated with Klotho downregulation at synapses through tacrolimus-induced oxidative stress in the hippocampus.
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Trastornos del Conocimiento/inducido químicamente , Hipocampo/fisiopatología , Inmunosupresores/toxicidad , Proteínas Klotho/fisiología , Proteínas del Tejido Nervioso/fisiología , Sinapsis/efectos de los fármacos , Tacrolimus/toxicidad , Animales , Trastornos del Conocimiento/metabolismo , Dendritas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Hipocampo/patología , Inmunosupresores/farmacología , Proteínas Klotho/biosíntesis , Proteínas Klotho/genética , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Prueba de Campo Abierto , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Distribución Aleatoria , Transducción de Señal , Aprendizaje Espacial , Memoria Espacial , Sinapsis/fisiología , Tacrolimus/farmacologíaRESUMEN
Subcontractors depend heavily on their prime contractor and thus find it very risky to enter a new business on their own. This study proposes a framework for these subcontractors to develop blue ocean technologies related to their prime contractor. First, the primary technologies predicted to be promising are extracted from the business reports of the prime contractor. Sub-technologies are then selected through a patent-based search using keywords and International Patent Classification codes of the primary technologies. From them, blue ocean technologies are proposed by optimizing the weighted mean of the min-max normalized market value, degree of competition in the technology market, and subcontractors' potential technological capabilities for each sub-technology. This study shows that subcontractors can enhance their technology competitiveness by finding a low-risk blue ocean technology. Our empirical research on the subcontractors of a semiconductor firm identified technological patent fields for them to pursue. From our framework, subcontractors can identify blue ocean technologies by considering their prime contractor's future industrial areas and technologies of interest as well as their own technological capabilities. Furthermore, the prime contractors can gain the synergy effect of technology expansion through cooperation.
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Comercio/normas , Competencia Económica/tendencias , Industrias/normas , Invenciones/tendencias , Patentes como Asunto , Comercio/métodos , Humanos , Industrias/métodos , Industrias/estadística & datos numéricosRESUMEN
Human-induced pluripotent stem cell lines (hiPSCs) derived from the peripheral blood mononuclear cells (PBMCs) of a woman (CMCi007-A) and her son (CMCi006-A) diagnosed with Fabry disease (FD) caused by the frameshift deletion mutation c.969delC in the alpha-galactosidase A (GLA) gene were generated. These hiPSCs showed typical human embryonic stem cell-like morphology and expressed pluripotency-associated markers, and directly differentiated into all three germ-layers. Karyotyping showed normal 46, XY (CMCi006-A) and 46, XX (CMCi007-A). In summary, we generated novel patient-specific hiPSC lines from both a female and male containing the same mutation, which may provide additional insight into the pathophysiology of FD.
Asunto(s)
Enfermedad de Fabry , Células Madre Pluripotentes Inducidas , Enfermedad de Fabry/genética , Femenino , Humanos , Leucocitos Mononucleares , Masculino , Mutación , Eliminación de Secuencia , alfa-Galactosidasa/genéticaRESUMEN
The human-induced pluripotent stem cell lines (hiPSCs) (CMCi009), derived from peripheral blood mononuclear cells (PBMCs) of a 42-year-old woman who were diagnosed as Birt-Hogg-Dubé syndrome (BHD) caused by the frameshift deletion mutation c.1285delC in FCLN gene, was generated using synthetic mRNA. Generated hiPSCs showed a typical human embryonic stem cell like morphology and expressed all pluripotency-associated markers, and directly differentiated into all three germ layers. Karyotyping of generated iPSCs showed normal 46, XY (CMCi009-A) respectively. In summary, we generated a novel patient-specific hiPSCs line containing the same mutation of FLCN gene and it can be used to provide additional insights for BHD pathophysiology.