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Volume 137, Issue 6 , Pages 2063-2073.e4, December 2009

Expansion and Differentiation of Neural Progenitors Derived From the Human Adult Enteric Nervous System

Marco Metzger
- Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany
Petra M. Bareiss
- Institute of Anatomy, Center for Regenerative Biology and Medicine, University of Tuebingen, Tuebingen, Germany
Timm Danker
- Department of Electrophysiology, Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany
Silvia Wagner
- Clinic for General, Visceral and Transplantation Surgery, University Hospital of Tuebingen, Tuebingen, Germany
Joerg Hennenlotter
- Department of Urology, University of Tuebingen, Tuebingen, Germany
Elke Guenther
- Department of Electrophysiology, Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany
Florian Obermayr
- Department of Pediatric Surgery, University Children′s Hospital Tuebingen, University of Tuebingen, Tuebingen, Germany
Arnulf Stenzl
- Department of Urology, University of Tuebingen, Tuebingen, Germany
Alfred Koenigsrainer
- Clinic for General, Visceral and Transplantation Surgery, University Hospital of Tuebingen, Tuebingen, Germany
Thomas Skutella
- Institute of Anatomy, Center for Regenerative Biology and Medicine, University of Tuebingen, Tuebingen, Germany
Lothar Just
- Institute of Anatomy, Center for Regenerative Biology and Medicine, University of Tuebingen, Tuebingen, Germany
- Reprint requests Address requests for reprints to: Lothar Just, PhD, Institute of Anatomy, Center for Regenerative Biology and Medicine, Eberhardt-Karls-University Tuebingen, Oesterbergstrasse 3, 72074 Tuebingen, Germany. fax: (49) 7071-29-5124

Received 13 February 2008; accepted 10 June 2009. published online 22 June 2009.

Background & Aims

Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract.

Methods

Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies.

Results

The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment.

Conclusions

It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Abbreviations used in this paper: BrdU, 5-bromo-2-deoxyuridine, DAPI, 4′-6-diamidino-2-phenylindole, ENS, enteric nervous system, RT-PCR, reverse-transcription polymerase chain reaction