PhD Scientific Days 2023

Budapest, 22-23 June 2023

Molecular Sciences - Posters B

Generation and Differentiation of Neurospheres from Avian Enteric Nervous System

Ádám Soós1, Csongor-László Pethő1, Emőke Szőcs1, Viktória Halasy1, Nándor Nagy1

1 Semmelweis University, Faculty of Medicine, Department of Anatomy, Histology and Embryology, Budapest, Hungary

Text of the abstract

Introduction:
The enteric nervous system (ENS) develops from neural crest cells (NCC) that migrate along the intestine, and differentiate into neurons and glia within the gut wall. Congenital abnormalities of the ENS cause severe intestinal disorders, such as Hirschsprung disease, which is characterized by the absence of enteric ganglia along a variable length of colon. Recently the enteric neurosphere technique has been used to isolate and culture enteric nervous system derived stem cells (ENCDCs) for cell transplantation to treat congenital neurointestinal diseases: however, the optimal method of generating neurospheres is not well defined.
ENS development is highly dependent on reciprocal interactions between NCCs and their stromal environment that is conserved between mammals and avian species. The avian embryo offers a unique opportunity to study ENS development, where ENCDCs and their interaction with the gut mesenchyme-derived growth factors can be followed, and to leverage our understanding to bioengineer neurospheres, to enhance the migration of transplanted ENSCs.
Aims and Methods:
The objective of this study was to compare the quantity and neurogenic potential of ENCDCs isolated from embryonic chicken large intestine and to develop efficient methodologies for their neurosphere cultivation. Unsorted gut cells were grown in Neurocult Basal Medium. To optimize stem cell growth and proliferation potential we supplemented the culture media with glial-cell-line-derived neurotrophic factor (GDNF) and Noggin growth factors that have been described to influence the ontogeny of ENCDCs. Neurospheres were cultured for 3 and 7 days, co-cultured with embryonic hindgut for 24 hours, recombinants were transplanted to CAM membrane of chicken embryo in ovo for another 7 days and processed for immunostaining to characterize the cells and their microenvironment. Exposure to GDNF+Noggin promoted the expansion of ENCDCs and resulted in a robust increase in neurosphere volume. Chicken neurospheres transplanted to aneural hindgut migrate normally and differentiate into appropriate neural crest-derived cell types.
Results and Conclusions:
Our data show that the ability of avian ENCDCs to generate enteric neurospheres can be enhanced by exposure to appropriate growth factors. Using this technique we can better understand interactions of different cell types and the microenvironmental factors that increase proliferation potential and guide differentiation into specific ENS cell types.
Funding:
-SUPPORTED BY THE ÚNKP-22-3-I NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR CULTURE AND INNOVATION FROM THE SOURCE OF THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND.
- OTKA, Hungarian Scientific Research Fund K-138664
- SE250+ Excellence PhD Funding