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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.

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Alterations of the ENS four weeks post-operation.(A, B) Low-magnification bright-field view of NADPH-diaphorase staining demonstrates a visible and relative reduction (i.e. hypoganglionosis) of neuronal cells (shown as %NOS+ of Fast Red+ myenteric plexus cells, dotted lines) as well as neuronal fibers compared to untreated gut control tissues. Note that the musculature is thickened due to hypertrophic effects of BAC as indicated by the white bar. Nuclei are stained red with Nuclear Fast Red dye. (C, D) Compared to the control, hypertrophic effects are also detectable in the ENS as shown by immunohistochemistry for PGP9.5 (arrowheads). Nuclei were stained with DAPI (blue). (E) Quantification of NOS-positive cells in the myenteric plexus, (F) mean ganglia size indicated by PGP9.5 cytoplasmatic immunostaining, and (G) muscle thickness. As seen in box-whisker plots, damage of the ENS and muscle was still quantifiable four weeks after BAC-treatment, however in this analysis, no statistical significant effects were seen by additional cell transplantation compared to the BAC-only treated group. (A–D) Scale bars represent 100 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle. (E–G) *P<0.05 and 0.05< §P<0.15 as compared to the untreated controls.
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pone-0093605-g003: Alterations of the ENS four weeks post-operation.(A, B) Low-magnification bright-field view of NADPH-diaphorase staining demonstrates a visible and relative reduction (i.e. hypoganglionosis) of neuronal cells (shown as %NOS+ of Fast Red+ myenteric plexus cells, dotted lines) as well as neuronal fibers compared to untreated gut control tissues. Note that the musculature is thickened due to hypertrophic effects of BAC as indicated by the white bar. Nuclei are stained red with Nuclear Fast Red dye. (C, D) Compared to the control, hypertrophic effects are also detectable in the ENS as shown by immunohistochemistry for PGP9.5 (arrowheads). Nuclei were stained with DAPI (blue). (E) Quantification of NOS-positive cells in the myenteric plexus, (F) mean ganglia size indicated by PGP9.5 cytoplasmatic immunostaining, and (G) muscle thickness. As seen in box-whisker plots, damage of the ENS and muscle was still quantifiable four weeks after BAC-treatment, however in this analysis, no statistical significant effects were seen by additional cell transplantation compared to the BAC-only treated group. (A–D) Scale bars represent 100 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle. (E–G) *P<0.05 and 0.05< §P<0.15 as compared to the untreated controls.

Mentions: To constitute a small colonic segment with disturbed ENS function (i.e. hypoganglionosis), we applied the cationic surfactant benzalkonium chloride (BAC) (Figure 2). Dependent to the concentration and time of incubation, treatment with BAC has been shown to lead to transient enteric plexus ablation (i.e. hypoganglionosis) and hypertrophy as well as hypertrophy of the musculature. We have tested 2 different concentrations of BAC (0.01% and 0.05%) aiming to generate a moderate damage with minimal animal suffering and good survival of the recipient mice. The higher BAC concentration resulted in an already macroscopically visible damage of the treated gut segment (e.g. damaged vasculature) after 20 minutes of application (data not shown). Consequently, approximately 90% of all treated animals (n = 24) developed a severe intestinal obstruction and either died or were killed within the first 5 days to shorten animal suffering. After gut dissection, we noticed the constricted segments accompanied by extremely distended proximal colon/caecum as well as small intestine filled with abnormal amounts of condensed feces still visible in the surviving animals 4 weeks following operation. In contrast, 80% of animals (n = 28) treated with the lower BAC concentration survived the 4 weeks follow-up phase without any visible signs of suffering, weight-loss or macroscopically alterations of gut integrity. On a microscopic level, however we could still identify and quantify hypertrophic effects in the regenerating plexus structures and smooth musculature (Figure 3A–D, Figure 3F, G). In average, the mean size of ganglia, measured via the PGP9.5+ area, significantly increased from 180 μm2 (control) to 294 and 389 μm2 (BAC-treated groups without and with cells, respectively); 30–62 ganglia per animal were analyzed. In contrast, the mean cell number per ganglion, determined by Fast Red-positive cell number, was only moderately, but not significantly decreased after BAC-treatment. Between 250–450 total Fast Red+ cells per animal, organized as densely-packed ganglion structures, were counted. Interestingly, a relative decrease of NOS-positive neurons (in % of Fast Red-positive myenteric plexus cells), which represent the majority of plexus cells, and neuronal fibers was detectable in the BAC-treated animals (Figure 3B, E).


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)

Alterations of the ENS four weeks post-operation.(A, B) Low-magnification bright-field view of NADPH-diaphorase staining demonstrates a visible and relative reduction (i.e. hypoganglionosis) of neuronal cells (shown as %NOS+ of Fast Red+ myenteric plexus cells, dotted lines) as well as neuronal fibers compared to untreated gut control tissues. Note that the musculature is thickened due to hypertrophic effects of BAC as indicated by the white bar. Nuclei are stained red with Nuclear Fast Red dye. (C, D) Compared to the control, hypertrophic effects are also detectable in the ENS as shown by immunohistochemistry for PGP9.5 (arrowheads). Nuclei were stained with DAPI (blue). (E) Quantification of NOS-positive cells in the myenteric plexus, (F) mean ganglia size indicated by PGP9.5 cytoplasmatic immunostaining, and (G) muscle thickness. As seen in box-whisker plots, damage of the ENS and muscle was still quantifiable four weeks after BAC-treatment, however in this analysis, no statistical significant effects were seen by additional cell transplantation compared to the BAC-only treated group. (A–D) Scale bars represent 100 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle. (E–G) *P<0.05 and 0.05< §P<0.15 as compared to the untreated controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0093605-g003: Alterations of the ENS four weeks post-operation.(A, B) Low-magnification bright-field view of NADPH-diaphorase staining demonstrates a visible and relative reduction (i.e. hypoganglionosis) of neuronal cells (shown as %NOS+ of Fast Red+ myenteric plexus cells, dotted lines) as well as neuronal fibers compared to untreated gut control tissues. Note that the musculature is thickened due to hypertrophic effects of BAC as indicated by the white bar. Nuclei are stained red with Nuclear Fast Red dye. (C, D) Compared to the control, hypertrophic effects are also detectable in the ENS as shown by immunohistochemistry for PGP9.5 (arrowheads). Nuclei were stained with DAPI (blue). (E) Quantification of NOS-positive cells in the myenteric plexus, (F) mean ganglia size indicated by PGP9.5 cytoplasmatic immunostaining, and (G) muscle thickness. As seen in box-whisker plots, damage of the ENS and muscle was still quantifiable four weeks after BAC-treatment, however in this analysis, no statistical significant effects were seen by additional cell transplantation compared to the BAC-only treated group. (A–D) Scale bars represent 100 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle. (E–G) *P<0.05 and 0.05< §P<0.15 as compared to the untreated controls.
Mentions: To constitute a small colonic segment with disturbed ENS function (i.e. hypoganglionosis), we applied the cationic surfactant benzalkonium chloride (BAC) (Figure 2). Dependent to the concentration and time of incubation, treatment with BAC has been shown to lead to transient enteric plexus ablation (i.e. hypoganglionosis) and hypertrophy as well as hypertrophy of the musculature. We have tested 2 different concentrations of BAC (0.01% and 0.05%) aiming to generate a moderate damage with minimal animal suffering and good survival of the recipient mice. The higher BAC concentration resulted in an already macroscopically visible damage of the treated gut segment (e.g. damaged vasculature) after 20 minutes of application (data not shown). Consequently, approximately 90% of all treated animals (n = 24) developed a severe intestinal obstruction and either died or were killed within the first 5 days to shorten animal suffering. After gut dissection, we noticed the constricted segments accompanied by extremely distended proximal colon/caecum as well as small intestine filled with abnormal amounts of condensed feces still visible in the surviving animals 4 weeks following operation. In contrast, 80% of animals (n = 28) treated with the lower BAC concentration survived the 4 weeks follow-up phase without any visible signs of suffering, weight-loss or macroscopically alterations of gut integrity. On a microscopic level, however we could still identify and quantify hypertrophic effects in the regenerating plexus structures and smooth musculature (Figure 3A–D, Figure 3F, G). In average, the mean size of ganglia, measured via the PGP9.5+ area, significantly increased from 180 μm2 (control) to 294 and 389 μm2 (BAC-treated groups without and with cells, respectively); 30–62 ganglia per animal were analyzed. In contrast, the mean cell number per ganglion, determined by Fast Red-positive cell number, was only moderately, but not significantly decreased after BAC-treatment. Between 250–450 total Fast Red+ cells per animal, organized as densely-packed ganglion structures, were counted. Interestingly, a relative decrease of NOS-positive neurons (in % of Fast Red-positive myenteric plexus cells), which represent the majority of plexus cells, and neuronal fibers was detectable in the BAC-treated animals (Figure 3B, E).

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