Biology subjects

Graffmann, N.

Publications and source records attributed to Graffmann, N..

6 recordsLinked to original sources

bioRxiv2026

SAHA increases chaperone expression and reduces Z-alpha-1-antitrypsin polymers in a patient specific iPSC-based liver model for alpha-1-antitrypsin deficiency

The most severe phenotype of alpha-1-antitrypsin deficiency (AATD) is caused by the Z-mutation within the SERPINA1 gene. The Glu342Lys substitution causes misfolding and polymerisation of the alpha-1-antitrypsin (AAT) protein, its accumulation in the ER and increases the susceptibility of hepatocytes towards ER-stress. Here, we present an induced pluripotent stem cell (iPSC)-based hepatic model to study AATD. We demonstrate that iPSCs from AATD patients differentiate equally well to hepatocyte-like cells (HLCs) as control iPSCs. We detected ZAAT polymers in patient-derived HLCs which could be reduced by SAHA or CBZ treatment. Transcriptome analyses revealed major differences in metabolism and signalling between control and AATD HLCs and indicated increased stress levels affecting intracellular organelles. Importantly, the transcriptomes of control and patient-derived cells separated into distinct clusters with respect to the expression of Heat-shock protein (HSP) encoding genes. SAHA treatment increased expression of various HSPs which might contribute towards reduced ZAAT polymers.

cell biology↗

bioRxiv2025

Human CSB-deficient iPSCs exhibit impaired DNA damage repair and stress responses following BPDE exposure in an early developmental model.

Maintenance of genome integrity is essential for normal human development, particularly during pre-gastrulation stages when rapid proliferation and intense transcriptional activity increase susceptibility to DNA damage. Environmental genotoxins such as benzo[a]pyrene (BaP), a widespread polycyclic aromatic hydrocarbon, and its reactive metabolite benzo[a]pyrene diol epoxide (BPDE) form bulky DNA adducts that interfere with replication and transcription, thereby posing significant risks to embryonic genome stability. To examine how genetic defects in DNA repair influence these effects, we assessed human induced pluripotent stem cells (iPSCs) carrying pathogenic mutations in ERCC6 (encoding the Cockayne syndrome B, CSB, protein), a key component of transcription-coupled nucleotide excision repair. Pathogenic ERCC6 mutations result in Cockayne syndrome- a severe neurodevelopmental disorder characterized by growth failure, premature aging, and multisystemic degeneration, thus underscoring the essential developmental functions of CSB. Exposure of healthy and CSB-deficient patient derived iPSCs to BPDE revealed impaired proliferation, persistent accumulation of DNA damage and defective checkpoint activation in CSB-deficient lines. Although the levels of key pluripotency-regulating proteins such as OCT4 and NANOG remained unaltered, we observed altered levels of SOX2 and p-SMAD1/5 signaling thus implying that unrepaired DNA damage can perturb developmental-associated signaling pathways and biological processes. Transcriptomic profiling revealed broad suppression of DNA repair and cell-cycle pathways together with activation of p53-, TNF-, and MAPK/JNK-mediated stress responses in CSB-deficient lines. Failure to induce anti-oxidant defenses, including SOD2 and IDO, further contributed to oxidative imbalance and incomplete apoptotic clearance. These findings demonstrate that CSB function is essential for coupling DNA repair with transcriptional recovery and redox homeostasis in pluripotent cells. Loss of CSB destabilizes the genome stability under genotoxic stress, providing a mechanistic basis for developmental toxicity of environmental polycyclic aromatic hydrocarbons and underscoring the importance of considering genetic susceptibility in developmental toxicology risk assessment.

developmental biology↗

bioRxiv2024

Investigating BPDE-induced embryonic toxicity employing hiPSC-based models

Benzo[a]pyrene diol epoxide (BPDE) is a metabolite of the environmental contaminant Benzo[a]pyrene- a byproduct of incomplete combustion of organic matter. BPDE reacts with DNA to form BPDE-DNA bulky adducts which if not removed can lead to mutations due to DNA base-pair substitutions. While the effects of BPDE on somatic cells are fairly well described, its effects on early human development are currently unknown. In this study, we investigated for the first time the effect of BPDE on human induced pluripotent stem cells (hiPSCs) and their differentiated neuroprogenitor cells (NPCs) as a model for early embryonic development. Furthermore, we compared hiPSCs and NPCs derived from cells of patients suffering from Nijmegen Breakage Syndrome (NBS), which is a chromosomal instability disorder characterized by defective DNA repair and increased risk of malignancies. Transcriptome analysis, coupled with protein content analysis employing immunostaining and Western blots, revealed that hiPSCs are more sensitive to BPDE exposure when compared to NPCs with an enhanced expression of several genes associated with p53-mediated DNA damage response, including DNA repair by lesion bypass, cell cycle checkpoints and extrinsic apoptosis. We also identified that cells from NBS patients showed less apoptotic response and a distinct p53 response than their healthy counterparts. This iPSC-based study enhances our meagre knowledge of the effects of BPDE on early human development in both healthy individuals and NBS patients. Furthermore, our model conforms with the 3Rs principle.

pharmacology and toxicology↗

bioRxiv2024

Inhibiting DPP4 activity protects hiPSC-derived steatotic HLCs by supporting fatty acid and purine metabolism and dampening inflammation

BackgroundMetabolic dysfunction-associated steatotic liver disease (MAFLD) has a high prevalence and high co-morbidity for other diseases. Due to the complexity of this multifactorial disease, therapy options are still rather limited. We employed an in vitro pluripotent stem cell-based model to decipher potential disease-associated molecular pathways and to study the mode of action of prospective drugs. Dipeptidyl peptidase 4 (DPP4) or Cluster of differentiation 26 (CD26) is involved in inflammation, infections, immune disorders, type 2 diabetes, kidney disease and cancer. MethodsWe induced the steatosis phenotype in human induced pluripotent stem cell (iPSC) derived hepatocyte-like cells (HLCs) by oleic acid (OA)-feeding and confirmed regulation of clinically relevant pathways by NGS-based global transcriptomic analyses. Analysis of the secretome of steatotic HLCs revealed DPP4 as a potential key mediator of the disease. To further elucidate its role in the development of MAFLD, we inhibited DPP4 activity with Vildagliptin (VILDA) and analyzed the global transcriptome changes as well as specific gene and protein expression of steatosis-associated genes with and without DPP4 inhibition. ResultsMAFLD-associated pathways such as PPAR- and TNF signaling were differentially regulated in hiPSC-derived steatotic HLCs. We found increased hepatic DPP4 activity and secretion upon OA. Gene expression of fatty acid and purine metabolism and inflammation-associated pathways were regulated upon DPP4 inhibition. ConclusionsOur HLC-model confirmed association of DPP4 with metabolism and inflammation which foster the development of MAFLD. Inhibiting DPP4 with VILDA partially relieved the steatotic phenotype on a global transcriptomic level. Impact and implicationsGiven the difficulties of identifying suitable anti-MAFLD drugs, novel model systems are urgently needed. Our in vitro HLC-model reproduced DPP4-dependent aspects of the disease and responded positively to Vildagliptin treatment. Further elucidation of the role of DPP4 in the etiology of MAFLD and other diseases is warranted.

cell biology↗

bioRxiv2021

Investigation of Thyroid Hormone Associated Gene-Regulatory Networks during Hepatogenesis using an Induced Pluripotent Stem Cell based Model

Currently, the only treatment of end-stage liver diseases is liver transplantation. The worldwide shortage of donor organs and the high number of patients suffering from end-stage liver diseases, require alternative treatment approaches. Therefore, the generation of hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) is a promising treatment option. HLCs are suitable for patient-specific drug screening, disease modeling and regenerative medicine. So far, they are immature and resemble the fetal state. In this study, we employed the combined thyroid hormones triiodo-L-thyronine (T3) and L-thyroxine (T4) to drive HLCs towards maturation. HLCs expressed the maturation markers ALBUMIN (ALB), ALPHA-1 ANTITRYPSIN (A1AT), CYTOCHROME P450 3A4 (CYP3A4) and TRANSTHYRETIN (TTR). Remarkably, stimulation with T3 and T4 slightly reduced the expression of the fetal marker CYTOCHROME P450 3A7 (CYP3A7) and had an even more pronounced effect on ALPHA-FETOPROTEIN (AFP) levels. Comparative transcriptome and associated pathways in unstimulated and T3 and T4 stimulated HLCs revealed regulated expression of numerous genes within for example the peroxisome proliferator-activated receptor (PPAR), transforming growth factor beta (TGF-{beta}), mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways and thyroid hormone synthesis. We propose the inclusion of combined T3 andT4 in HLC differentiation protocols to enhance maturation and therefore provide additional improvement in their applications in drug screening and disease modeling.

developmental biology↗

bioRxiv2019

A Comprehensive Molecular Portrait of Human Urine-derived Renal Progenitor Cells

BackgroundHuman urine is now recognised as a non-invasive source of stem cells with regeneration potential. These cells are mesenchymal stem cells but their detailed molecular and cellular identities are poorly defined. Furthermore, unlike the mouse, the gene regulatory network driving self-renewal and differentiation into functional renal cells in vitro remain unresolved.\n\nMethodsWe isolated urine stem cells from 10 individuals from both genders and distinct ages, characterized them as renal progenitor cells and explored the gene regulatory network sustaining self-renewal.\n\nResultsThese cells express pluripotency-associated proteins-TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133. Expression of pluripotency-associated proteins enabled rapid reprogramming into iPSCs using episomal-based plasmids without pathway perturbations. Transcriptome analysis revealed expression of a plethora of nephrogenesis-related genes such as SIX2, OSR1, CITED1, NPHS2, NPHS1, PAX2, SALL1, AQP2, EYA1, SLC12A1 and UMOD. As expected, the cells transport Albumin by endocytosis. Based on this, we refer to these cells as urine derived renal progenitor cells-UdRPCs. Associated GO-term analysis of UdRPCs and UdRPC-iPSCs underlined their renal identity and functionality. Upon differentiation by WNT activation using the GSK3{beta}-inhibitor (CHIR99021), transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative regulator of self-renewal and differentiation in UdRPCs. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGF{beta}-SMAD2/3 pathway.\n\nConclusionThis in vitro model and the data presented should lay the foundation for studying nephrogenesis in man.\n\nSignificance StatementHuman urine is a non-invasive source of stem cells with regeneration potential. Here, we investigated the cellular and molecular identities, and the gene regulation driving self-renewal and differentiation of these cells in vitro. These cells express pluripotency-associated markers enabling easy reprogramming. Based on the expression of renal associated genes, proteins and functionality, we refer to these cells as urine derived renal progenitor cells-UdRPCs. CHIR99021-induced differentiation of UdRPCs activated WNT-related genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN as a putative regulator of differentiation whereas self-renewal is maintained by FGF2-driven TGF{beta}-SMAD2/3. Our data will enhance understanding of the molecular identities of UdRPCs, and enable the generation of renal disease models in vitro and eventually kidney-associated regenerative therapies.

developmental biology↗