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

Hetz S, Acikgoez A, Voss U, Nieber K, Holland H, Hegewald C, Till H, Metzger R, Metzger M - PLoS ONE (2014)

Bottom Line: In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature.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.

View Article: PubMed Central - PubMed

Affiliation: Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Clinic-oriented Therapy Assessment Unit, Leipzig, Germany.

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.

Show MeSH

Related in: MedlinePlus

Macroscopic evaluation and organ bath analysis of transplanted gut tissues.(A, B) Concentration-dependent severity of gut/ENS damage after BAC-treatment is visible in resected gut tissues. Higher concentrations resulted in severe obstruction proximal to the treated gut segments indicated by condensed feces and tissue dilatations (arrows) leading to death of the majority of animals within few days. Lower BAC-concentrations instead led to a moderate damage of the gut tissue with good animal survival. (C) Segments treated with 0.01% BAC (black bar, with and without cell transplantation) and control gut tissues (i.e. normal gut) were placed in the organ bath between two electrodes (star) for further analysis of EFS-induced gut contractility. (D) Quantification of EFS-induced isometric contractility relative to the Acetylcholine (ACh)-induced control contraction is shown in box-whisker plots. The tissue damage caused by BAC (§P = 0.06) could be partly abolished after cell transplantation (P = 0.43) compared to control tissues. (E) Representative plots showing the ACh control peaks at the beginning and end of each measurement. After EFS application, contractions were particularly diminished in the BAC-only treatment group, which could be partly restored after cell transplantation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3974735&req=5

pone-0093605-g005: Macroscopic evaluation and organ bath analysis of transplanted gut tissues.(A, B) Concentration-dependent severity of gut/ENS damage after BAC-treatment is visible in resected gut tissues. Higher concentrations resulted in severe obstruction proximal to the treated gut segments indicated by condensed feces and tissue dilatations (arrows) leading to death of the majority of animals within few days. Lower BAC-concentrations instead led to a moderate damage of the gut tissue with good animal survival. (C) Segments treated with 0.01% BAC (black bar, with and without cell transplantation) and control gut tissues (i.e. normal gut) were placed in the organ bath between two electrodes (star) for further analysis of EFS-induced gut contractility. (D) Quantification of EFS-induced isometric contractility relative to the Acetylcholine (ACh)-induced control contraction is shown in box-whisker plots. The tissue damage caused by BAC (§P = 0.06) could be partly abolished after cell transplantation (P = 0.43) compared to control tissues. (E) Representative plots showing the ACh control peaks at the beginning and end of each measurement. After EFS application, contractions were particularly diminished in the BAC-only treatment group, which could be partly restored after cell transplantation.

Mentions: According to our ISH results, there was the possibility that we might able to detect beneficial effects of human cell transplantation on a functional level. Therefore, we first demonstrated that BAC could cause detrimental effects on gut contractility after the 4 weeks follow-up as a consequence of the ENS damage seen in histology. Resected gut segments (Figure 5A–C) were placed into an organ bath filled with physiological Krebs solution and electrical field stimulations (EFS) was performed to assess neurally mediated contractions of the smooth musculature. Acetycholine (ACh)-induced contractions were included before and after EFS and served as reference control (Figure 5E). For unknown reasons, 5 of 24 analyzed guts (2×control, 1×BAC, 2×BAC +cells) showed no reaction, neither to ACh nor to EFS, probably due to technical problems and therefore could not be included in the analysis. In the remaining guts, BAC-treatment caused a notable ∼50% inhibition of contractile response after EFS (Figure 5D; n = 7, P = 0.06 vs. controls), although the variability of results was relatively high, which resulted in a moderate statistical P-value. In contrast, this inhibitory effects on contractility was reduced in the transplantation group (Figure 5D; n = 6, P = 0.43 vs. controls), which was even more distinct if only the younger donors were considered. The functional data therefore suggest that NLB transplantation indeed may have physiological effects, even if not clearly reflected on a histological level.


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

Hetz S, Acikgoez A, Voss U, Nieber K, Holland H, Hegewald C, Till H, Metzger R, Metzger M - PLoS ONE (2014)

Macroscopic evaluation and organ bath analysis of transplanted gut tissues.(A, B) Concentration-dependent severity of gut/ENS damage after BAC-treatment is visible in resected gut tissues. Higher concentrations resulted in severe obstruction proximal to the treated gut segments indicated by condensed feces and tissue dilatations (arrows) leading to death of the majority of animals within few days. Lower BAC-concentrations instead led to a moderate damage of the gut tissue with good animal survival. (C) Segments treated with 0.01% BAC (black bar, with and without cell transplantation) and control gut tissues (i.e. normal gut) were placed in the organ bath between two electrodes (star) for further analysis of EFS-induced gut contractility. (D) Quantification of EFS-induced isometric contractility relative to the Acetylcholine (ACh)-induced control contraction is shown in box-whisker plots. The tissue damage caused by BAC (§P = 0.06) could be partly abolished after cell transplantation (P = 0.43) compared to control tissues. (E) Representative plots showing the ACh control peaks at the beginning and end of each measurement. After EFS application, contractions were particularly diminished in the BAC-only treatment group, which could be partly restored after cell transplantation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974735&req=5

pone-0093605-g005: Macroscopic evaluation and organ bath analysis of transplanted gut tissues.(A, B) Concentration-dependent severity of gut/ENS damage after BAC-treatment is visible in resected gut tissues. Higher concentrations resulted in severe obstruction proximal to the treated gut segments indicated by condensed feces and tissue dilatations (arrows) leading to death of the majority of animals within few days. Lower BAC-concentrations instead led to a moderate damage of the gut tissue with good animal survival. (C) Segments treated with 0.01% BAC (black bar, with and without cell transplantation) and control gut tissues (i.e. normal gut) were placed in the organ bath between two electrodes (star) for further analysis of EFS-induced gut contractility. (D) Quantification of EFS-induced isometric contractility relative to the Acetylcholine (ACh)-induced control contraction is shown in box-whisker plots. The tissue damage caused by BAC (§P = 0.06) could be partly abolished after cell transplantation (P = 0.43) compared to control tissues. (E) Representative plots showing the ACh control peaks at the beginning and end of each measurement. After EFS application, contractions were particularly diminished in the BAC-only treatment group, which could be partly restored after cell transplantation.
Mentions: According to our ISH results, there was the possibility that we might able to detect beneficial effects of human cell transplantation on a functional level. Therefore, we first demonstrated that BAC could cause detrimental effects on gut contractility after the 4 weeks follow-up as a consequence of the ENS damage seen in histology. Resected gut segments (Figure 5A–C) were placed into an organ bath filled with physiological Krebs solution and electrical field stimulations (EFS) was performed to assess neurally mediated contractions of the smooth musculature. Acetycholine (ACh)-induced contractions were included before and after EFS and served as reference control (Figure 5E). For unknown reasons, 5 of 24 analyzed guts (2×control, 1×BAC, 2×BAC +cells) showed no reaction, neither to ACh nor to EFS, probably due to technical problems and therefore could not be included in the analysis. In the remaining guts, BAC-treatment caused a notable ∼50% inhibition of contractile response after EFS (Figure 5D; n = 7, P = 0.06 vs. controls), although the variability of results was relatively high, which resulted in a moderate statistical P-value. In contrast, this inhibitory effects on contractility was reduced in the transplantation group (Figure 5D; n = 6, P = 0.43 vs. controls), which was even more distinct if only the younger donors were considered. The functional data therefore suggest that NLB transplantation indeed may have physiological effects, even if not clearly reflected on a histological level.

Bottom Line: In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature.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.

View Article: PubMed Central - PubMed

Affiliation: Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Clinic-oriented Therapy Assessment Unit, Leipzig, Germany.

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.

Show MeSH
Related in: MedlinePlus