Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Weiand, Matthias; Ballmaier, Paula; Niemietz, Christoph; Schmidt, Hartmut; Zibert, Andree

doi:10.1016/j.bbrep.2022.101308

Cited by 3 publications

(6 citation statements)

References 27 publications

(41 reference statements)

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“…Two patient-specific cellular platforms, representing urinary epithelial cells and iPSC-derived cells [18], were used throughout the study to model the molecular consequences of a R236C [6]. Urine cells (UCs) derived from an ADPLD patient encoding R236C were subjected to retroviral gene transfer for immortalization (UCi) or reprogrammed to iPSCs followed by differentiation to cholangiocyte progenitor-like cells (CPLC) (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Human embryonic kidney HEK293T cells were maintained in DMEM/F12 (Gibco) with 10 % FCS (Gibco) and 1 % PenStrep. For generation of retroviral supernatants, HEK293T cells were transfected with plasmid pLXSN16E6E7 encoding the human papilloma virus HPVE6E7 gene (Addgene #52394) as described [18]. Urine cells following retroviral transduction were cultivated in UC medium (DMEM/F12, 10 % FCS, 1 % L-Glu, 1 % PenStrep).…”

Section: Retroviral Transductionmentioning

confidence: 99%

“…Establishment of iPSCs was performed as reported before [18]. 0.5 x 10 6 primary urine cells were reprogrammed using the Amaxa Basic Nucleofector Kit (Lonza) according to the protocol of the manufacturer.…”

Section: Induced Pluripotent Stem Cellsmentioning

confidence: 99%

“…We have previously reported that primary cells derived from urine (UCs), representing a non-invasive cell source, can be immortalized via retroviral expression or reprogrammed to induced pluripotent stem cells (iPSCs) followed by liver-specific differentiation [18]. In the present study, basic molecular functions of SEC61A1 mutation R236C were studied using this methodology of different cellular platforms.…”

Section: Introductionmentioning

confidence: 99%

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Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Weiand,

Sandfort,

Nadzemova

et al. 2023

Preprint

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Background & objectivesSEC61A1 encodes a central protein of the mammalian translocon machinery residing in the ER. Mutations ofSEC61A1are implicated to result in various severe diseases. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD), which originates from defects of cholangiocytes in the liver. The molecular phenotype of R236C was assessed.MethodsTwo cellular platforms were established from the same cell source of an ADPLD patient by different methodology. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to several analyses of molecular pathways that were previously associated with development of polycystic disease.ResultsUCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis.ConclusionAlthough the grade of aberrant cellular functions differed between the two platforms, overall the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways that cause ADPLD. Our approach may be valuable to decipher the molecular pathways of other rare genetic diseases using patient-specific cell models.

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Section: Resultsmentioning

confidence: 99%

Section: Retroviral Transductionmentioning

confidence: 99%

Section: Induced Pluripotent Stem Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Weiand,

Sandfort,

Nadzemova

et al. 2023

Preprint

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“…Spontaneously voided urine was collected and processed as described 33 . One well of a 12‐well cell culture plate (Greiner Bio‐One) was precoated with 0.1% gelatin (Millipore).…”

Section: Methodsmentioning

confidence: 99%

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease

Schlevogt

Schlieper

Krader

et al. 2022

Liver International

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Background and Aims Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. Methods Customized next‐generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. Results In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin‐2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. Conclusions Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

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Comparative analysis of SEC61A1 mutant R236C in two patient-derived cellular platforms

Weiand,

Sandfort,

Nadzemova

et al. 2024

Sci Rep

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SEC61A1 encodes a central protein of the mammalian translocon and dysfunction results in severe disease. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD). The molecular phenotype of R236C was assessed in two cellular platforms. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to analyses of molecular pathways that were associated with development of disease. UCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis. Although the grade of aberrant cellular functions differed between the two platforms, the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways.

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Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Cited by 3 publications

References 27 publications

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease

Comparative analysis of SEC61A1 mutant R236C in two patient-derived cellular platforms

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