In vivo transplantation of neurosphere-like bodies derived from the human postnatal and adult enteric nervous system: a pilot study.

Susan Hetz,Susan Hetz,Karen Nieber,Ali Acikgoez,Heidrun Holland,Marco Metzger,Marco Metzger,Marco Metzger,Holger Till,Cindy Hegewald,Ulrike Voss,Roman Metzger

doi:10.1371/journal.pone.0093605

Abstract

Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via ‘bystander’ mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.

Highlights

The Enteric Nervous System (ENS) poses the largest and probably most complex division of the autonomic nervous system and mainly controls the principal activities of the gut including motility and secretion
Cells were initially seeded onto plastic dishes coated with fibronectin, ornithin and laminin (2 mg/cm2; Sigma). 50% of medium was supplemented with conditioned medium obtained from fast growing human fetal ENS in vitro cell cultures
neurosphere-like bodies (NLBs) generation efficiency tend to be higher from younger donors compared to older donors considering same biopsy size and cultivation duration

Summary

Introduction

The Enteric Nervous System (ENS) poses the largest and probably most complex division of the autonomic nervous system and mainly controls the principal activities of the gut including motility and secretion. Multipotent neural stem and progenitor cells of the ENS have been identified as a promising cell source for the treatment of at least some ENS disorders, which may involve replacing or restoring neural components or, alternatively, stimulating endogenous regenerative response [3,4,5,6,7,8,9,10,11]. In vitro protocols have been developed which describe the propagation of mammalian neural stem and progenitor cells, mainly as neurosphere-like bodies (NLBs). These cells are capable of colonizing intestinal tissue and differentiating into neural subtypes demonstrated mostly ex vivo in organotypic tissue cultures [5,8,12]. These studies were restricted to fetal or neonatal cell sources and often without proof of functional effects in vivo [13,14,15,16,17]

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