Urine-derived Cells Research Articles

Detection of Canine Urothelial Carcinoma Cells in Urine Using 5-Aminolevulinic Acid

Simple Summary5-aminolevulinic acid (5-ALA) is a natural amino acid that is metabolized in the mitochondria and used in the synthesis of heme. In human medicine, the fluorescence property of 5-ALA has been used for photodynamic tumor diagnosis. Nevertheless, research on 5-ALA in veterinary medicine is very limited. In this study, we aimed to evaluate the efficacy of 5-ALA to detect canine urothelial carcinoma and to clarify its diagnostic accuracy. The addition of 5-ALA to tumor cells from the urine of patients with urothelial carcinoma and canine urothelial carcinoma cell lines caused red fluorescence, and the amount of fluorescence was significantly higher than that in healthy dogs. Additionally, cases with high fluorescence intensity had more tumor invasion and metastasis. This study showed that 5-ALA can be used to detect canine urothelial carcinoma cells in urine with relatively high diagnostic accuracy.This study aimed to establish a method to detect canine urothelial carcinoma cells in urine using 5-aminolevulinic acid (5-ALA) and to evaluate its diagnostic accuracy. Urine samples were collected from 21 dogs diagnosed with urothelial carcinoma and three urothelial carcinoma cell lines were used. Urine samples obtained from seven healthy dogs were used as controls. Cells in the urine sediment, or urothelial carcinoma cell lines, were cultured with 5-ALA and then observed under a fluorescence microscope. Moreover, we examined the relationship between fluorescence intensity and the presence of metastasis as well as tumor invasion into the bladder wall in cases of urothelial carcinoma. Urine-derived cells from urothelial carcinoma and urothelial carcinoma cell lines showed clearer red fluorescence with the addition of 5-ALA compared to that exhibited by the cells from healthy dogs. The sensitivity and specificity of the diagnosis of urothelial carcinoma were 90% and 86%, respectively. Significant associations were found between fluorescence intensity and tumor metastasis and bladder wall invasion. This study showed that 5-ALA can be used to detect urothelial carcinoma cells in dogs with relatively high diagnostic accuracy. Further, the fluorescence intensity of tumor cells caused by 5-ALA correlated with the clinical condition of urothelial carcinoma cases, which suggested that 5-ALA could be used as a prognostic marker for canine urothelial carcinoma.

Induction of Salivary Gland-Like Tissue by Induced Pluripotent Stem Cells In Vitro.

To investigate the in vitro induction of salivary gland-like tissue by ips cells in an interferon regulatory factor 6 (IRF6) overexpression and parotid conditioned medium environment. Urine-derived ips cells were isolated, identified, transfected with IRF6 and cultured in parotid conditioned medium to induce ips cells into salivary gland differentiation, morphological changes of ips cells were observed, CCK-8 was used to determine the cell proliferation efficiency and transcriptome sequencing was used to detect the expression of genes related to parotid gland formation. Immunofluorescence staining showed that the isolated ips cells were positive for NANOG, SSEA4 and OCT4 and had embryonic-like stem cell characteristics; CCK-8 showed that there was no statistical difference in the proliferation efficiency between the IRF6+ induced group and the simple induced group after induction of ips cells into salivary glands. The results of transcriptome sequencing showed that there were a total of 643 differentially expressed genes, including 365 up-regulated genes and 278 down-regulated genes in the IRF6+ induced group compared to the blank control group, and the salivary gland related genes HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM were all highly expressed in the IRF6+ induced group. IRF6 promotes salivary gland differentiation in urine-derived iPSCs, and its mechanism of promoting differentiation may be that IRF6 upregulates the expression of HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM to promote epithelial differentiation.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.

Generation of a non-integrated induced pluripotent stem cell line (SMBCi009-A) from urine-derived cells of a Chinese Familial hypercholesterolemia patient

Familial hypercholesterolemia (FH; OMIM: # 143890) is a common inherited autosomal dominant disease, characterized by high-level low-density lipoprotein cholesterol (LDL-C) in plasma. Elevated LDL-C levels is closely related with atherosclerotic plaques and premature cardiovascular disease if not treated in time. Here we generated an induced pluripotent stem cell (iPSC) line using urine cells (UCs) derived from an 8-year-old male FH patient who carrying two coding and pathogenic mutations of low-density lipoprotein receptor (LDLR) gene (exon12:c.C1747T and exon13: c. 1948 del G). This induced pluripotent stem cell line named SMBCi009-A can be used to understand more cellular details about FH.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

A New Genetic Insight for Orphan Renal Disorder, Fabry: A Review

Fabry is the rare X-linked genetic disorder caused due to mutation in Alpha –Galactosidase encoding GLA gene mutation in chromosome number 22. It has wide diversification in prevalence due to clinical heterozygosity. There are some potential biomarkers for the evaluation of normal or altered genes responsible for Fabry. Advances in the research of biomarkers over the years have made significant development for several clinical indicators, viz. urine-derived cells, oxidative stress, DNA methylation, etc. At present days the recommended therapies for the disease are Enzyme Replacement Therapy (ERT), Chaperone therapy (CT), and mRNA-based therapy, besides, some second-generation therapies which are still under clinical trials.

Generation of Induced Nephron Progenitor-like Cells from Human Urine-Derived Cells.

Background: Regenerative medicine strategies employing nephron progenitor cells (NPCs) are a viable approach that is worthy of substantial consideration as a promising cell source for kidney diseases. However, the generation of induced nephron progenitor-like cells (iNPCs) from human somatic cells remains a major challenge. Here, we describe a novel method for generating NPCs from human urine-derived cells (UCs) that can undergo long-term expansion in a serum-free condition. Results: Here, we generated iNPCs from human urine-derived cells by forced expression of the transcription factors OCT4, SOX2, KLF4, c-MYC, and SLUG, followed by exposure to a cocktail of defined small molecules. These iNPCs resembled human embryonic stem cell-derived NPCs in terms of their morphology, biological characteristics, differentiation potential, and global gene expression and underwent a long-term expansion in serum-free conditions. Conclusion: This study demonstrates that human iNPCs can be readily generated and expanded, which will facilitate their broad applicability in a rapid, efficient, and patient-specific manner, particularly holding the potential as a transplantable cell source for patients with kidney disease.

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq.

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end‐stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co‐expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA‐seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine‐derived renal progenitor cells and human kidney‐derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single‐cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co‐expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.

Generation of Urine-Derived Induced Pluripotent Stem Cell Line from Patients with Acute Kidney Injury.

Acute kidney injury (AKI) is mainly characterized by rapid decline of renal function. Currently, the strategy of stem cells might be a therapy to treat AKI. The objective of this study was to obtain human urine-derived cells (HUCs) from patients with AKI, followed by establishing induced pluripotent stem (iPS) cell line. We isolated urine cells from patients with AKI and found that the cells could survive long term with epithelioid morphology and maintain a normal karyotype. The cell line had expression of renal-specific markers and renal development-related genes. After induction, the urine cells cotransfecting with TET-ON vectors were converted into iPS cells. The HUC-derived iPS (HUC-iPS) was positive for alkaline phosphatase staining, and had expression of pluripotency markers, consistent with human embryonic fibroblast-derived iPS cell. Notably, HUC-iPS could be induced to undergo directional kidney precursor cells (KPCs) differentiation under defined conditions, and transplantation of KPCs resulted in reducing kidney damage from ischemia-reperfusion injury in mice. Therefore, we successfully established HUC-iPS cell from patients with AKI and provided a novel stem cell resource for cell therapy in AKI.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80kDa) translated from full-length OCRL, whereas only one protein (80kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50%enzyme activity, whereas the Lowe syndrome variants retained <20%activity. We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Donor HLA genotyping of ex vivo expanded urine cells from kidney transplant recipients.

Antibody-mediated rejection (AMR) induced by donor-specific anti-HLA antibodies (DSA) remains a major cause of long-term graft loss after kidney transplantation. Currently, the presence of DSA cannot always be determined at a specific allele level, because existing donor HLA typing is low resolution and often incomplete, lacking HLA-DP, and occasionally HLA-C and HLA-DQ information and historical donor DNA samples are not available for HLA retyping. Here we present a novel, non-invasive technique for obtaining donor DNA from selectively expanded donor cells from urine of renal transplant recipients. Urine-derived cells were successfully expanded ex vivo from 31 of 32 enrolled renal transplant recipients, and with DNA obtained from these cells, donor HLA typing was unambiguously determined for HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 loci by next-generation sequencing. Our results showed 100% concordance of HLA typing data between donor peripheral blood and recipient urine-derived cells. In comparison, HLA typing showed that DNA derived from urine sediments mainly contained recipient-derived DNA. We also present the successful application of our novel technique in a clinical case of AMR in a renal transplant recipient. Urine-derived donor cells can be isolated from kidney transplant recipients and serve as a suitable source of donor material for reliable high-resolution HLA genotyping. Thus, this approach can aid the assessment of DSA specificity to support the diagnosis of AMR as well as the evaluation of treatment efficacy in kidney transplant recipients when complete donor HLA information and donor DNA are unavailable.

Epigenetic Alterations in Podocytes in Diabetic Nephropathy.

Recently, epigenetic alterations have been shown to be involved in the pathogenesis of diabetes and its complications. Kidney podocytes, which are glomerular epithelial cells, are important cells that form a slit membrane—a barrier for proteinuria. Podocytes are terminally differentiated cells without cell division or replenishment abilities. Therefore, podocyte damage is suggested to be one of the key factors determining renal prognosis. Recent studies, including ours, suggest that epigenetic changes in podocytes are associated with chronic kidney disease, including diabetic nephropathy. Furthermore, the association between DNA damage repair and epigenetic changes in diabetic podocytes has been demonstrated. Detection of podocyte DNA damage and epigenetic changes using human samples, such as kidney biopsy and urine-derived cells, may be a promising strategy for estimating kidney damage and renal prognoses in patients with diabetes. Targeting epigenetic podocyte changes and associated DNA damage may become a novel therapeutic strategy for preventing progression to end-stage renal disease (ESRD) and provide a possible prognostic marker in diabetic nephropathy. This review summarizes recent advances regarding epigenetic changes, especially DNA methylation, in podocytes in diabetic nephropathy and addresses detection of these alterations in human samples. Additionally, we focused on DNA damage, which is increased under high-glucose conditions and associated with the generation of epigenetic changes in podocytes. Furthermore, epigenetic memory in diabetes is discussed. Understanding the role of epigenetic changes in podocytes in diabetic nephropathy may be of great importance considering the increasing diabetic nephropathy patient population in an aging society.

Urine Cells-derived iPSCs: An Upcoming Frontier in Regenerative Medicine.

There is a momentous surge in the development of stem cell technology, such as therapeutic and diagnostic tools. Stem cell-derived cells are currently used in various clinical trials. However, key issues and challenges faced involve the low differentiation efficiency, integration and functioning of transplanted stem cells-derived cells. Extraction of bone marrow, adipose or other mesenchymal stem cells (MSCs) involves invasive methods, specialized skills and expensive technologies. Urine-derived cells, on the other hand, are obtained by non-invasive methods; samples can be obtained repeatedly from patients of any age. Urine-derived cells are used to generate reprogrammed or induced pluripotent stem cells (iPSCs) which can be cultured and differentiated into various types of cell lineages for biomedical investigations and drug testing in vitro or in vivo using model animals of human diseases. Urine cells-derived iPSCs (UiPSCs) have emerged as a major area of research having immense therapeutic significance. Given that preliminary preclinical studies are successful in terms of safety and as a regenerative tool, the UiPSCs will pave the way to the development of various types of autologous stem cell therapies.

Genetic compensation for cilia defects in cep290 mutants by upregulation of cilia-associated small GTPases.

ABSTRACTMutations in CEP290 (also known as NPHP6), a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish, the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes, whereas deficiencies in a CRISPR/Cas9 genetic mutant were restricted to photoreceptor defects. Here, we show that milder phenotypes in genetic mutants were associated with the upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human Joubert syndrome CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies.This article has an associated First Person interview with the first author of the paper.

Induced pluripotent stem cells from urine of Duchenne muscular dystrophy patients.

The most common muscular dystrophy, Duchenne muscular dystrophy (DMD), is a lethal, X-linked disorder with no widespread cure. Worldwide, invitro studies involving new, mutation-specific cures and regenerative therapies are employing disease-specific patient-specific cells. However, these may not be completely relevant for Pakistani children because of the human genome diversities and geographic variation in mutation type and frequency. Therefore, this study aimed to generate DMD induced pluripotent stem cells (iPSCs) from the urine of Pakistani children with DMD, to serve as a precious source of differentiated cells, such as Pakistani DMD-cardiomyocytes, for future disease-modelling, drug testing, and gene therapy. Urine-derived cells (UDCs) isolated from mid-stream urine underwent molecular characterization and cellular reprogramming towards iPSCs using the episomal vector system followed by molecular profiling of the iPSCs. Colonies of elongated and spindle-shaped or rounded rice-grain like UDCs were spotted 4-7days after plating and expanded rapidly with a second passage at 2-3weeks. Multicolor flow cytometry confirmed the expression of mesenchymal stem-cell markers. The reprogramed iPSCs consisted of colonies of round, tightly-packed cells with large nuclei that were positively fluorescent for the pluripotency markers octamer binding transcription factor-4 (OCT-4), tumour resistance antigen 1-60 (TRA-1-60), and stage specific embryonic 4 antigen (SSEA-4), but not for the negative pluripotency marker SSEA-1. To the best of our knowledge, this was the first time DMD-iPSCs have been generated for Pakistani children. This integration-free, feeder-free, efficient, and reproducible reprogramming method employed UDCs. Urine is a low-cost, non-invasive, painless, and repeatable source of rapidly expandable cells from children and morbid individuals for obtaining autologous cells for drug-assays and disease-modelling, suitable for DMD and other debilitating diseases.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases.

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

FC 008INTERDEPENDENT REGULATION OF POLYCYSTIN EXPRESSION INFLUENCES STARVATION-INDUCED AUTOPHAGY AND CELL DEATH

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in either PKD1 (ca. 78%) or PKD2 (ca. 15%), encoding for the proteins polycystin-1 (PC1) or polycystin-2 (PC2), respectively. Mutations in PKD1 generally lead to a more severe disease progression compared to PKD2 patients. The exact function of the polycystins in cyst formation remains unclear, but it is clear that the levels of PC1 and PC2 are inversely correlated to cyst formation. Moreover, renal stress has been proposed to enhance cystogenesis. We therefore aimed to investigate the cellular response towards nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 or PC2 (PC1KO or PC2KO), and unique human urine-derived proximal tubular epithelial cells (PTECs) of early-stage ADPKD patients with truncating PKD1 mutations versus healthy individuals, with a focus on cell survival (autophagy) and cell death. Method Cell death was assessed with Cytotox green-based live cell imaging in the Incucyte, by trypane blue exclusion and by analyzing the levels of cleaved Caspase 3. Autophagy was measured by LC3 immunoblotting and by counting GFP-LC3 punctae. Autophagy was blocked with Bafilomycin A1. To modulate the levels of PC1 and PC2, transient overexpression of human PC1 or siRNA-mediated knockdown of PC2 was performed. Results During chronic starvation, cell death was reduced and autophagy was increased in mouse PC1- and PC2-deficient mIMCDs. This was validated in human cells from early-stage ADPKD patients. Autophagy inhibition restored cell death resistance in KO cells, implying that decrease in cell death was caused by autophagy upregulation in PC1- and PC2KO cells. Interestingly, PC2 expression was increased in PC1KO cells, while PC2KO displayed a downregulation of PC1. Although PC2 is known to regulate autophagy, PC2 knockdown did not reduce autophagy in PC1KO cells, while the effect in PC2KO could be reversed by overexpression of PC1. Conclusion These findings indicate that PC1 levels determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress. Moreover, PC1 also indirectly influences this process by regulating PC1 levels during starvation. Our findings imply that in early stage ADPKD, cells with the lowest polycystin levels (which are most prone to form cysts) are more resistant to stress by autophagy upregulation. This is important, as renal stress is inherent to the cystic environment and has been proposed as an additional trigger in cystogenesis.

ISOLATION AND REPROGRAMMING OF URINARY PROGENITOR CELLS FROM A PATIENT WITH MUTATION IN GENE CYFIP2 THAT CAUSES INTELLECTUAL DISABILITY

<h3>Background</h3> West syndrome (WS) is an infantile neural disorder that causes a delay in development and infantile spasms. Mutations in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene were recently described as possible causes of WS. CYFIP2 has a role in actin polymerization through WAVE regulatory complex (WRC), especially on dendritic spines and synaptosomes. To study the molecular mechanism of mutation in CYFIP2 and to propose a possible therapy, this study aimed to establish a cell model based on induced pluripotent stem cells (iPSC), through a non-invasive cell sample collected from the patient and its subsequent reprogramming. <h3>Methods</h3> The urine sample was collected for a 2 years old Brazilian female patient characterized with the R87C variant of CYFIP2, with the parent's consent. For isolation, the sample was washed, centrifuged, and the cellular pellet was cultivated until confluence. For reprogramming, the episomal vectors expressing hSK, hOCT4, shp53, hUL, and EBNA1 were electroporated in cells by Nucleofector. Cells were cultivated until reprogrammed cell colonies appeared. Colonies were labeled with anti-TRA-1-60 and anti-TRA-1-81 antibodies on days 15 and 24 to follow up. <h3>Results</h3> Isolation and expansion of urine-derived cells lasted 21 days and based on its morphology and growth rate, cells were classified as type II urinary progenitor cells (UPC), according to the literature. After 15 days of reprogramming, pluripotent stem cells-like colonies can be visualized in culture. The TRA-1-60 and TRA-1-81 markers were even more evident in colonies on day 24, compared to day 15. On day 26 after reprogramming, colonies were manually harvested, and the cells were expanded until confluence. <h3>Conclusions</h3> The isolation and reprogramming of UPC collected from a patient with the CYFIP2 R87C variant were possible, even with the possibility of CYFIP2 mutation impaired cell growth or reprogramming. Also, the reprogrammed cells expressed two of the pluripotent markers, TRA-1-60 and TRA-1-81. The next step is the characterization of the reprogrammed cells, through expression analysis for different pluripotent markers, differentiation in the three germ layers, and karyotype. After the establishment of the iPSC as a study model for CYFIP2, cells will be differentiated into neural cells, making possible the study of the molecular mechanisms and drug screening.

Highly sensitive screening of antisense sequences for different types of DMD mutations in patients' urine-derived cells

Exon skipping using short antisense oligonucleotides (AONs) is a promising treatment for Duchenne muscular dystrophy (DMD). Several exon-skipping drugs, including viltolarsen (NS-065/NCNP-01), have been approved worldwide. Immortalized human skeletal muscle cell lines, such as rhabdomyosarcoma cells, are frequently used to screen efficient oligonucleotide sequences. However, rhabdomyosarcoma cells do not recapitulate DMD pathophysiology as they express endogenous dystrophin. To overcome this limitation, we recently established a direct human somatic cell reprogramming technology and successfully developed a cellular skeletal muscle DMD model by using myogenic differentiation 1 (MYOD1)-transduced urine-derived cells (MYOD1-UDCs). Here, we compared in vitro drug screening systems in MYOD1-UDCs and rhabdomyosarcoma cells. We collected UDCs from patients with DMD amenable to exon 51 skipping, and obtained MYOD1-UDCs. We then compared the efficiency of exon 51 skipping induced by various morpholino-based AONs, including eteplirsen in differentiated MYOD1-UDCs (UDC-myotubes) and rhabdomyosarcoma cells. Exon skipping was induced more efficiently in UDC-myotubes than in rhabdomyosarcoma cells even at a low AON concentration (1 μM). Furthermore, exon 51 skipping efficiency was higher in UDC-myotubes with a deletion of exons 49–50 than in those with a deletion of exons 48–50, suggesting that the skipping efficiency may vary depending on the DMD mutation pattern. An essential finding of this study is that the sequence of eteplirsen consistently leads to much lower efficiency than other sequences. These findings underscore the importance of AON sequence optimization by our cellular system, which enables highly sensitive screening of exon skipping drugs that target different types of DMD mutations.

Expanding the Spectrum of FAT1 Nephropathies by Novel Mutations That Affect Hippo Signaling

IntroductionDisease-causing mutations in the protocadherin FAT1 have been recently described both in patients with a glomerulotubular nephropathy and in patients with a syndromic nephropathy.MethodsWe identified 4 patients with FAT1-associated disease, performed clinical and genetic characterization, and compared our findings to the previously published patients. Patient-derived primary urinary epithelial cells were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblotting to identify possible alterations in Hippo signaling.ResultsHere we expand the spectrum of FAT1-associated disease with the identification of novel FAT1 mutations in 4 patients from 3 families (homozygous truncating variants in 3, compound heterozygous missense variants in 1 patient). All patients show an ophthalmologic phenotype together with heterogeneous renal phenotypes ranging from normal renal function to early-onset end-stage kidney failure. Molecular analysis of primary urine-derived urinary renal epithelial cells revealed alterations in the Hippo signaling cascade with a decreased phosphorylation of both the core kinase MST and the downstream effector YAP. Consistently, we found a transcriptional upregulation of bona fide YAP target genes.ConclusionA comprehensive review of the here identified patients and those previously published indicates a highly diverse phenotype in patients with missense mutations but a more uniform and better recognizable phenotype in the patients with truncating mutations. Altered Hippo signaling and de-repressed YAP activity might be novel contributing factors to the pathomechanism in FAT1-associated renal disease.