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1.
Stem Cell Res ; 72: 103197, 2023 10.
Article in English | MEDLINE | ID: mdl-37689041

ABSTRACT

Mitochondrial membrane Protein-Associated Neurodegeneration (MPAN) is a lethal neurodegenerative disorder caused by mutations in the human gene C19orf12. The molecular mechanisms underlying the disorder are still unclear, and no established therapy is available. Here, we describe the generation and characterization of two human induced pluripotent stem cell (iPSC) lines derived from skin fibroblasts of two MPAN patients carrying homozygous recessive mutations in C19orf12. These iPSC lines represent a useful resource for future investigations on the pathology of MPAN, as well as for the development of successful treatments.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Mitochondrial Proteins/genetics , Mutation/genetics , Membrane Proteins/genetics , Fibroblasts
2.
Nucleic Acids Res ; 51(3): 1297-1316, 2023 02 22.
Article in English | MEDLINE | ID: mdl-36651277

ABSTRACT

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.


Subject(s)
RNA-Binding Proteins , Transcription Factors , Humans , DNA-Binding Proteins/genetics , Epilepsy , Processing Bodies , RNA, Messenger/metabolism , Transcription Factors/metabolism
3.
Stem Cell Res ; 61: 102773, 2022 05.
Article in English | MEDLINE | ID: mdl-35397396

ABSTRACT

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.


Subject(s)
Cardiomyopathy, Dilated , Induced Pluripotent Stem Cells , Coenzyme A , Fibroblasts , Humans , Mutation/genetics
4.
CRISPR J ; 4(2): 178-190, 2021 04.
Article in English | MEDLINE | ID: mdl-33876960

ABSTRACT

STAT3-hyper IgE syndrome (STAT3-HIES) is a primary immunodeficiency presenting with destructive lung disease along with other symptoms. CRISPR-Cas9-mediated adenine base editors (ABEs) have the potential to correct one of the most common STAT3-HIES causing heterozygous STAT3 mutations (c.1144C>T/p.R382W). As a proof-of-concept, we successfully applied ABEs to correct STAT3 p.R382W in patient fibroblasts and induced pluripotent stem cells (iPSCs). Treated primary STAT3-HIES patient fibroblasts showed a correction efficiency of 29% ± 7% without detectable off-target effects evaluated through whole-genome and high-throughput sequencing. Compared with untreated patient fibroblasts, corrected single-cell clones showed functional rescue of STAT3 signaling with significantly increased STAT3 DNA-binding activity and target gene expression of CCL2 and SOCS3. Patient-derived iPSCs were corrected with an efficiency of 30% ± 6% and differentiated to alveolar organoids showing preserved plasticity in treated cells. In conclusion, our results are supportive for ABE-based gene correction as a potential causative treatment of STAT3-HIES.


Subject(s)
Gene Editing/methods , Job Syndrome/genetics , Job Syndrome/therapy , STAT3 Transcription Factor/genetics , Adenine , CRISPR-Cas Systems , Cell Differentiation , Clustered Regularly Interspaced Short Palindromic Repeats , Fibroblasts , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Immunoglobulin E/genetics , Induced Pluripotent Stem Cells , Mutation , Whole Genome Sequencing
5.
BMC Biol ; 18(1): 42, 2020 04 22.
Article in English | MEDLINE | ID: mdl-32321486

ABSTRACT

BACKGROUND: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. RESULTS: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. CONCLUSIONS: Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.


Subject(s)
Cell Differentiation , Pluripotent Stem Cells/physiology , RNA, Long Noncoding/metabolism , Cell Nucleus/genetics , Cell Nucleus/physiology , DNA/genetics , DNA/physiology , Humans
6.
Stem Cell Reports ; 12(5): 861-868, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31006630

ABSTRACT

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.


Subject(s)
Cell Differentiation/genetics , Craniosynostoses/genetics , Gene Expression Profiling/methods , Intellectual Disability/genetics , Mutation , Neural Crest/metabolism , Pluripotent Stem Cells/metabolism , Repressor Proteins/genetics , Animals , Cell Line , Cells, Cultured , Chick Embryo , Craniosynostoses/metabolism , Craniosynostoses/pathology , Gene Regulatory Networks , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intellectual Disability/metabolism , Intellectual Disability/pathology , Neural Crest/cytology , Pluripotent Stem Cells/cytology , Repressor Proteins/metabolism , Transplantation, Heterologous
7.
Mol Biotechnol ; 56(8): 697-713, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24677035

ABSTRACT

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Animals , Biotechnology , Cell Differentiation , Cells, Cultured , Cellular Reprogramming/genetics , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , Female , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/genetics , Induced Pluripotent Stem Cells/transplantation , Integrases/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Transcriptome
8.
PLoS One ; 8(1): e55170, 2013.
Article in English | MEDLINE | ID: mdl-23383095

ABSTRACT

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.


Subject(s)
Cell Dedifferentiation/genetics , Genetic Vectors/genetics , Induced Pluripotent Stem Cells/cytology , Animals , Blotting, Southern , Cell Culture Techniques , Cell Line , Embryoid Bodies/physiology , Genes, myc/genetics , Immunohistochemistry , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Octamer Transcription Factor-3/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription Factors/genetics , Sequence Analysis, DNA , Transfection/methods
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