Publication Database

Human stem cell-derived neurospheres to explore the consequences of Listeria infection on brain development (2024)

Art

Poster

Autoren

Fischer, Annika (WE 14)

Lemke, Christine (WE 7)

van Vorst, Kira (WE 7)

Haiber, L. M.

Fulde, Marcus (WE 7)

Seeger, B.

Bröer, S. (WE 14)

Kongress

Berlin Neuroscience Meeting 2024

Berlin, 10. – 11.10.2024

Quelle

Sprache

Englisch

Verweise

URL (Volltext): https://www.ecn-berlin.de/files/img/08_events/Q3_2024/BNM2024_Poster-Index.pdf

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Zoonotic infections during pregnancy can lead to central nervous system (CNS) infections in surviving offspring, often resulting in neurodevelopmental disorders as long-term consequences of pre- and perinatal CNS infections. Despite these known associations, the underlying mechanisms remain poorly understood.
To investigate these mechanisms we employ human induced pluripotent stem cell (iPSC)-derived neurospheres as a model to mimic the developing human brain. The neurospheres contain neural stem cells (NSCs) that differentiate into various neural and glial subtypes, including mature neuronal cell populations. Our objective is to assess the suitability of these neurospheres for studying the consequences of fetal CNS infections.
Our initial studies focus on infection with Listeria monocytogenes, a bacterial pathogen known to cause enduring neurological impairments in affected offspring. Pregnant women typically transmit the bacteria to the fetus via bloodstream following the ingestion of contaminated food.
We hypothesize that infection induces primary NSC depletion and accelerates the maturation of surviving NSCs, which in turn impairs differentiation, alters migratory behavior, and disrupts network activity.
In the initial phase of the project, we successfully generated NSCs from a human iPSC line (IMR90, WiCell®) that can be stably cryopreserved to produce neurospheres from the same batch for reproducible follow-up infection experiments. Neurosphere differentiation protocols were established and the resulting spheres are currently being characterized via RT-qPCR and immunocytochemistry. Furthermore, we implemented cryopreservation protocols using two distinct media enabling the direct transfer of neurospheres to biosafety level facilities for subsequent infection experiments. The two media differ from one another in their use of either fetal calf serum (FCS) or Knockout Serum Replacement (KSR) in order to achieve greater standardization of the freezing medium. Preliminary results show no significant difference in recovery post-cryopreservation between the two media; however, long-term cell survival rates
did not meet our requirements for further experiments, necessitating optimization of the cryopreservation protocol. Our next steps include conducting initial infection experiments to establish an appropriate infection dose. The results from previously conducted growth curve 16 analyses suggest that the optimal time for infection is 9 hours after the initiation of Listeria cultivation. Additionally, based on the results of performed dilution series, an optical density
(OD) measurement of 0.5 corresponds to a bacterial concentration of 108 colony-forming units (CFU).