Abstract
BackgroundStem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Pluripotent embryonic stem (ES) cells represent an attractive cell source; however, little or no information is currently available on how ES cells will respond to the gut environment. In this study, we investigated the ability of ES cells to respond to environmental cues derived from the ENS and related tissues, both in vitro and in vivo.MethodsNeurospheres were generated from mouse ES cells (ES-NS) and co-cultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LM-MP).ResultsLM-MP co-culture led to a significant increase in the expression of pan-neuronal markers (βIII-tubulin, PGP 9.5) as well as more specialized markers (peripherin, nNOS) in ES-NS, both at the transcriptional and protein level. The increased expression was not associated with increased proliferation, thus confirming a true neurogenic effect. LM-MP preparations exerted also a myogenic effect on ES-NS, although to a lesser extent. After transplantation in vivo into the mouse pylorus, grafted ES-NS failed to acquire a distinct phenotype al least 1 week following transplantation.ConclusionsThis is the first study reporting that the gut explants can induce neuronal differentiation of ES cells in vitro and induce the expression of nNOS, a key molecule in gastrointestinal motility regulation. The inability of ES-NS to adopt a neuronal phenotype after transplantation in the gastrointestinal tract is suggestive of the presence of local inhibitory influences that prevent ES-NS differentiation in vivo.
Highlights
Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS)
Murine ES cells grown in feeder-free conditions formed colonies with the typical round and compacted morphology (Figure 1A) and with a high expression of alkaline phosphatase, a marker expressed by embryonic stem cells but not by their differentiated derivatives (Figure 1B) [27]
Under these conditions ES cells grow as floating multicellular aggregates, which morphologically resemble the neurospheres that are formed by neural stem cells (NSC) isolated from fetal and adult brain (Figure 1C)
Summary
Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Several enteric neuronal disorders, such as achalasia, Hirschprung disease, congenital pyloric stenosis and pseudo-obstruction, are characterized by the loss or malfunction of critical neuronal subpopulations of the enteric nervous system (ENS) [1,2]. These conditions result in a severe impairment of digestive functions, for which current surgical and pharmacological therapeutic approaches are not very satisfactory, with the exception of pyloromyotomy for pyloric stenosis [3]. Recent evidence has shown that enteric neuronal progenitor cells (ENPC) can be derived by autologous biopsy and can be grafted in organotypic gut preparations in vitro [9]. The consistency of this approach and the functional recovery after ENPC grafting need further investigation
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