Maxi Hofrichter’s research while affiliated with Leibniz Research Institute for Environmental Medicine and other places

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Publications (5)


Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction
  • Article
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October 2020

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130 Reads

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15 Citations

STEM CELLS TRANSLATIONAL MEDICINE

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Maxi Hofrichter

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The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at -80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level.

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Table 1 Calculated EC 50 values for MeHgCl treatment on migration distance and viability.
Fig. 3. Comparative functional analyses of hiPSC-neurospheres and primary human neurospheres using the 'Neurosphere Assay'. (A): The 'Neurosphere Assay' is a method to determine basic processes of neurodevelopment (proliferation, migration, differentiation and viability) in vitro. (B): Proliferation was measured for 14 days in neural proliferation medium (NPM) with (solid line) or without growth factors (GF; dotted line). Values represent mean ± SD, n = 3. Scale bars = 200 μm. (C): Migration distance of migrating cells 3 days after plating of neurospheres on Poly-D-Lysin (PDL)/Laminin coated plates in NDM. Values represent mean + SEM, n = 3. Scale bars = 500 μm. (D): Immunocytochemical stainings and quantification of at least five representative immunocytochemical images per neural induction for the neuronal marker βIII-Tubulin in 7 days differentiated NPCs of different origins plated on PDL/Laminin coated plates in NDM. Nuclei were stained with Hoechst 33258 (blue). Each n represents an independent neural induction; Scale bars in large images = 100 μm; scale bar in insertions = 20 μm.
Fig. 4. Comparative immunofluorecent stainings of 28-days differentiated neurospheres. Primary hNPCs (A/C) and hiPSC-NPCs (B/D) were stained for βIII-Tubulin (neurons, red, A-D) and GFAP (astrocytes/NPCs, green, A/B) after 28 days of differentiation. hiPSC-NPCs show co-localization of βIII-Tubulin (red) with the synapse markers Synapsin1 (green, (E)) and PSD-95 (green, (F)) and three representative images of results from three independent neural inductions were quantified (G). Nuclei are counter-stained with Hoechst 33,258. Scale bars A-F = 100 μm.
Fig. 5. Electrophysiological activity of hiPSC-derived neuronal networks grown on MEAs. (A): Representative spike raster plot of hiPSC-NPCs differentiated for 85 days on a MEA. Each cross denotes a spike representing an action potential. (B): Spike train of hiPSC-NPCs after 82 days of differentiation. (C): Detected spikes after 85 days of differentiation in a spike overlay. Depicted is one representative out of 7 electrically active chips. (D): After treatment with 1 μM TTX no action potentials (APs) were detected in a hiPSC-NPCs culture differentiated on MEAs for 84 days. After removal of TTX the networks recovered after 48 h resulting in APs with higher frequency and higher amplitude.
Human induced pluripotent stem cell-derived neurospheres develop into electrically active neuronal networks which suit as an alternative method to study neurotoxicity in vitro

October 2018

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335 Reads

Toxicology Letters



Fig. 1. Neural Induction Protocols to differentiate hiPSCs into neurospheres. hiPSCs were differentiated into neurospheres resembling primary human fetal neurospheres (A) using different protocols. (B): For the noggin protocol hiPSCs colonies were treated with 500 ng/μL noggin for 14 days. Afterwards they were cut into pieces and cultured as suspens culture in neural proliferation medium (NPM) containing basic fibroblast growth factor (bFGF; Denham and Dottori, 2011). (C): For the NIM protocol hiPSC colonies were cut i pieces and directly cultured as suspension culture in NIM. After 7 days, bFGF was added to the culture for additional 14 days. Finally, hiPSC-derived neurospheres were culture NPM containing bFGF for at least 28 days (modified from Hibaoui et al., 2014). Scale bars = 500 μm. (D): hiPS-derived neurospheres generated with the Noggin protocol (B) and NIM protocol (C) were analyzed for their expression of the neural stem/progenitor markers Nestin and SOX2 via flow cytometry analyses and compared to primary hum neurospheres. Number of analyzed cells = 2000. 
Table 1 Calculated EC 50 values for MeHgCl treatment on migration distance and viability.
Fig. 3. Comparative functional analyses of hiPSC-neurospheres and primary human neurospheres using the 'Neurosphere Assay'. (A): The 'Neurosphere Assay' is a method to det basic processes of neurodevelopment (proliferation, migration, differentiation and viability) in vitro. (B): Proliferation was measured for 14 days in neural proliferation medium with (solid line) or without growth factors (GF; dotted line). Values represent mean ± SD, n = 3. Scale bars = 200 m. (C): Migration distance of migrating cells 3 days after μ of neurospheres on Poly-D-Lysin (PDL)/Laminin coated plates in NDM. Values represent mean + SEM, n = 3. Scale bars = 500 μm. (D): Immunocytochemical stainin quantifi fi cation of at least ve representative immunocytochemical images per neural induction for the neuronal marker βIII-Tubulin in 7 days differentiated NPCs of different plated on PDL/Laminin coated plates in NDM. Nuclei were stained with Hoechst 33258 (blue). Each n represents an independent neural induction; Scale bars in large images μm; scale bar in insertions = 20 μm. 
Fig. 4. Comparative immunofluorecent stainings of 28-days differentiated neurospheres. Primary hNPCs (A/C) and hiPSC-NPCs (B/D) were stained for βIII-Tubulin (neurons, red, A-D) GFAP (astrocytes/NPCs, green, A/B) after 28 days of differentiation. hiPSC-NPCs show co-localization of III-Tubulin (red) with the synapse markers Synapsin1 (green, (E)) and PSD β (green, (F)) and three representative images of results from three independent neural inductions were quantified (G). Nuclei are counter-stained with Hoechst 33,258. Scale bars = 100 μm.
Fig. 5. Electrophysiological activity of hiPSC-derived neuronal networks grown on MEAs. (A): Representative spike raster plot of hiPSC-NPCs differentiated for 85 days on a MEA. Eac denotes a spike representing an action potential. (B): Spike train of hiPSC-NPCs after 82 days of differentiation. (C): Detected spikes after 85 days of differentiation in a spike o Depicted is one representative out of 7 electrically active chips. (D): After treatment with 1 μM TTX no action potentials (APs) were detected in a hiPSC-NPCs culture differentia MEAs for 84 days. After removal of TTX the networks recovered after 48 h resulting in APs with higher frequency and higher amplitude. 
Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro

October 2017

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679 Reads

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70 Citations

Stem Cell Research

Developmental neurotoxicity (DNT) testing performed in rats is resource-intensive (costs, time, animals) and bears the issue of species extrapolation. Thus, reliable alternative human-based approaches are needed for predicting neurodevelopmental toxicity. Human induced pluripotent stem cells (hiPSCs) represent a basis for an alternative method possibly being part of an alternative DNT testing strategy. Here, we compared two hiPSC neural induction protocols resulting in 3D neurospheres: one using noggin and one cultivating cells in neural induction medium (NIM protocol). Performance of Nestin⁺/SOX2⁺ hiPSC-derived neural progenitor cells (NPCs) was compared to primary human NPCs. Generally, primary hNPCs first differentiate into Nestin⁺ and/or GFAP⁺ radial glia-like cells, while the hiPSC-derived NPCs (hiPSC-NPC) first differentiate into βIII-Tubulin⁺ neurons suggesting an earlier developmental stage of hiPSC-NPC. In the ‘Neurosphere Assay’, NIM generated hiPSC-NPC produced neurons with higher performance than with the noggin protocol. After long-term differentiation, hiPSC-NPC form neuronal networks, which become electrically active on microelectrode arrays after 85 days. Finally, methylmercury chloride inhibits hiPSC-NPC and hNPC migration with similar potencies. hiPSC-NPCs-derived neurospheres seem to be useful for DNT evaluation representing early neural development in vitro. More system characterization by compound testing is needed to gain higher confidence in this method.


Citations (2)


... On NBA, FGR rabbits have increased levels of motoric and sensorial impairment at early assessment, develop behavior that suggests increased anxiety 29 , and show alterations in short-term memory and attention up until pre-adolescence, as previously published in the same model at PND 70 23 . At neuropathological assessment, the multiregional white matter reduction in oligodendrocyte populations confirms the MRI microstructural changes and, moreover, the hampered ability of neural progenitor cells to differentiate into oligodendrocytes precursors reported in this model at an earlier time point 23,24,30 . Future interventions for FGR may also aim at ameliorating these long-lasting neurocognitive sequelae 22,30 . ...

Reference:

Long-term pulmonary and neurodevelopmental impairment in a fetal growth restriction rabbit model
Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction

STEM CELLS TRANSLATIONAL MEDICINE

... The developing brain is more susceptible to chemical exposure compared to the adult brain because the protective blood-brain barrier is not fully functional in embryo, fetus, and newborn [2][3][4][5][6][7]. The brain development process including differentiation, proliferation, migration, and cell-cell communication must take place in a controlled time frame, and any disturbance in this process by a trace amount of chemical exposure could lead to permanent damage to the developing brain [8]. Despite the potential vulnerability of the developing brain to compounds, only a few hundred compounds among tens of thousands of chemicals have been tested for DNT. ...

Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro

Stem Cell Research