Establishment of human pluripotent stem cell-derived cortical neurosphere model to study pathomechanisms and chemical toxicity in Kleefstra syndrome

Sci Rep. 2024 Sep 29;14(1):22572. doi: 10.1038/s41598-024-72791-4.

Abstract

In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies.

Keywords: In vitro toxicology; EHMT1; Human induced pluripotent stem cells; Kleefstra syndrome; Neurospheroid.

MeSH terms

  • Brain-Derived Neurotrophic Factor / metabolism
  • Cell Differentiation* / drug effects
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Cerebral Cortex / drug effects
  • Cerebral Cortex / metabolism
  • Cerebral Cortex / pathology
  • Chromosome Deletion*
  • Chromosomes, Human, Pair 9* / genetics
  • Craniofacial Abnormalities / metabolism
  • Craniofacial Abnormalities / pathology
  • Heart Defects, Congenital
  • Humans
  • Induced Pluripotent Stem Cells* / cytology
  • Induced Pluripotent Stem Cells* / drug effects
  • Induced Pluripotent Stem Cells* / metabolism
  • Intellectual Disability / metabolism
  • Nerve Tissue Proteins
  • Neurons / drug effects
  • Neurons / metabolism
  • Neurons / pathology
  • Pluripotent Stem Cells / drug effects
  • Pluripotent Stem Cells / metabolism
  • Receptors, N-Methyl-D-Aspartate / metabolism
  • Rotenone / toxicity
  • Spheroids, Cellular* / drug effects
  • Spheroids, Cellular* / metabolism

Substances

  • Receptors, N-Methyl-D-Aspartate
  • GRIN1 protein, human
  • Brain-Derived Neurotrophic Factor
  • Rotenone
  • Nerve Tissue Proteins

Supplementary concepts

  • Kleefstra Syndrome