Small fibre neuropathy in Fabry disease: a human-derived neuronal <i>in vitro</i> disease model and pilot data

Klein, Thomas; Grüner, Julia; Breyer, Maximilian; Schlegel, Jan; Schottmann, Nicole Michelle; Hofmann, Lukas; Gauss, Kevin; Mease, Rebecca; Erbacher, Christoph; Finke, Laura; Klein, Alexandra; Klug, Katharina; Karl-Schöller, Franziska; Vignolo, Bettina; Reinhard, Sebastian; Schneider, Tamara; Günther, Katharina; Fink, Julian; Dudek, Jan; Maack, Christoph; Klopocki, Eva; Seibel, Jürgen; Edenhofer, Frank; Wischmeyer, Erhard; Sauer, Markus; Üçeyler, Nurcan

doi:10.1093/braincomms/fcae095

Cited by 6 publications

(1 citation statement)

References 85 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…iSN were differentiated from a healthy control iPSC line as described recently (Klein et al, 2024). In brief, 1.125*10 6 iPSC were seeded into Growth Factor Reduced Matrigel-coated wells (bMg, Corning, Corning, NY, USA) in a six-well plate in 2 ml StemMACS iPS-Brew (Miltenyi Biotec, Bergisch Gladbach, Germany).…”

Section: Methodsmentioning

confidence: 99%

Human sensory-like neuron cultivation – an optimized protocol

Schottmann,

Grüner,

Bär

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

IntroductionReprogramming of human induced pluripotent stem cells (iPSC) and their differentiation into specific cell types, such as induced sensory-like neurons (iSN), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving survival and quality of iSN.MethodsiSN were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-β-D-arabinofuranosyl-cytosin-hydrochlorid (AraC) treatment, Magnetic-Activated Cell Sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively.ResultsPassaging of cells during differentiation did not increase the iSN-to-total-cell-count ratio, and MACS of immature iSN led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10 μM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSN shortly after seeding also helped to reduce non-iSN cells.ConclusionIn direct comparison to other methods, treatment with 10 μM FdU for 24 h after differentiation represents a promising approach to improve iSN culture purity, offering a potential benefit for downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of this method and enhance its reproducibility and applicability. Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC-based research.

show abstract

Section: Methodsmentioning

confidence: 99%

Human sensory-like neuron cultivation – an optimized protocol

Schottmann,

Grüner,

Bär

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Genetic variants of unknown significance in alpha‐galactosidase A: Cellular delineation from Fabry disease

Klein,

Klug,

Breyer

et al. 2024

J of Inher Metab Disea

Self Cite

View full text Add to dashboard Cite

Fabry disease (FD) is an X‐linked multiorgan disorder caused by variants in the alpha‐galactosidase A gene (GLA). Depending on the variant, disease phenotypes range from benign to life‐threatening. More than 1000 GLA variants are known, but a link between genotype and phenotype in FD has not yet been established for all. p.A143T, p.D313Y, and p.S126G are frequent examples of variants of unknown significance (VUS). We have investigated the potential pathogenicity of these VUS combining clinical data with data obtained in human cellular in vitro systems. We have analyzed four different male subject‐derived cell types for alpha‐galactosidase A enzyme (GLA) activity and intracellular Gb3 load. Additionally, Gb3 load in skin tissue as well as clinical data were studied for correlates of disease manifestations. A reduction of GLA activity was observed in cells carrying p.A143T compared with controls (p < 0.05). In cells carrying the p.D313Y variant, a reduced GLA activity was found only in endothelial cells (p < 0.01) compared with controls. No pathological changes were observed in cells carrying the p.S126G variant. None of the VUS investigated caused intracellular Gb3 accumulation in any cell type. Our data of aberrant GLA activity in cells of p.A143T hemizygotes and overall normal cellular phenotypes in cells of p.D313Y and p.S126G hemizygotes contribute a basic science perspective to the clinically highly relevant discussion on VUS in GLA.

show abstract

Human in vitro models for Fabry disease: new paths for unravelling disease mechanisms and therapies

Borisch,

Thum,

Bär

et al. 2024

J Transl Med

View full text Add to dashboard Cite

Fabry disease is a multi-organ disease, caused by mutations in the GLA gene and leading to a progressive accumulation of glycosphingolipids due to enzymatic absence or malfunction of the encoded alpha-galactosidase A. Since pathomechanisms are not yet fully understood and available treatments are not efficient for all mutation types and tissues, further research is highly needed. This research involves many different model types, with significant effort towards the establishment of an in vivo model. However, these models did not replicate the variety of symptoms observed in patients. As an alternative strategy, patient-derived somatic cells as well as patient-independent cell lines were used to model specific aspects of the disease in vitro. Fabry disease patients present different phenotypes according to the mutation and the level of residual enzyme activity, pointing to the necessity of personalized disease modeling. With the advent of induced pluripotent stem cells, the derivation of a multitude of disease-affected cell types became possible, even in a patient-specific and mutation-specific manner. Only recently, three-dimensional Fabry disease models were established that even more closely resemble the native tissue of investigated organs and will bring research closer to the in vivo situation. This review provides an overview of human in vitro models and their achievements in unravelling the Fabry disease pathomechanism as well as in elucidating current and future treatment strategies.

show abstract

Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data

Cited by 6 publications

References 85 publications

Human sensory-like neuron cultivation – an optimized protocol

Human sensory-like neuron cultivation – an optimized protocol

Genetic variants of unknown significance in alpha‐galactosidase A: Cellular delineation from Fabry disease

Human in vitro models for Fabry disease: new paths for unravelling disease mechanisms and therapies

Contact Info

Product

Resources

About