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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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Characterization of multipotent neural progenitors isolated from human adult gut.(A) Bright-field view of representative adult enteric neurosphere-like bodies (NLBs) after 14 days in vitro. Free-floating NLBs could be observed with an estimated average number of 35 NLBs per preparation and a sphere diameter of 71.6 μm. Different NLB phenotypes include round- (star, comparable to B), ellipsoid- (white arrow, comparable to C) or irregular-shaped (black arrow, comparable to D) NLBs. (B–D) Depending to the individual NLB phenotype, different cellular compositions can be derived as shown for the ENS marker gene p75 in immunocytochemistry. (E–G) Proliferative NLB cells also incorporated BrdU and expressed previously described progenitor markers including Sox2 and Nestin. (H–J) After 2 weeks of differentiation NLBs built a monolayer composed of neuronal (TuJ), glial (S100β) and smooth muscle-like cells (SMA). (K–M) Molecular cytogenetic FISH analyses revealed no chromosomal alterations as representatively shown for three chromosomal interphase nuclei. (N) A schematic illustration of a chromosome is shown. In total, 15 different probe sets recognizing 41 different unique subtelomeric repeats were applied. (O) Efficacy and cellular distribution of NLBs strongly depends to the individual preparation, relying on age, amount and quality of tissue, as demonstrated by qPCR analysis for three representative marker genes: p75, Ret and tropomyosin (TPM). Scale bars represent in (A) 100 μm, (B–G) 25 μm, (H–J) 50 μm.
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pone-0093605-g001: Characterization of multipotent neural progenitors isolated from human adult gut.(A) Bright-field view of representative adult enteric neurosphere-like bodies (NLBs) after 14 days in vitro. Free-floating NLBs could be observed with an estimated average number of 35 NLBs per preparation and a sphere diameter of 71.6 μm. Different NLB phenotypes include round- (star, comparable to B), ellipsoid- (white arrow, comparable to C) or irregular-shaped (black arrow, comparable to D) NLBs. (B–D) Depending to the individual NLB phenotype, different cellular compositions can be derived as shown for the ENS marker gene p75 in immunocytochemistry. (E–G) Proliferative NLB cells also incorporated BrdU and expressed previously described progenitor markers including Sox2 and Nestin. (H–J) After 2 weeks of differentiation NLBs built a monolayer composed of neuronal (TuJ), glial (S100β) and smooth muscle-like cells (SMA). (K–M) Molecular cytogenetic FISH analyses revealed no chromosomal alterations as representatively shown for three chromosomal interphase nuclei. (N) A schematic illustration of a chromosome is shown. In total, 15 different probe sets recognizing 41 different unique subtelomeric repeats were applied. (O) Efficacy and cellular distribution of NLBs strongly depends to the individual preparation, relying on age, amount and quality of tissue, as demonstrated by qPCR analysis for three representative marker genes: p75, Ret and tropomyosin (TPM). Scale bars represent in (A) 100 μm, (B–G) 25 μm, (H–J) 50 μm.

Mentions: Genomic alterations of human enteric progenitors were evaluated by DNA fluorescence in situ hybridization (FISH) as described recently [22]. Briefly, after preparation of cell metaphases and interphases, cells were denatured, dehydrated and hybridized with multi-colour DNA FISH probe mixtures for 12–16 h at 37°C (ToTelVysionTM, Abbott, Wiesbaden, Germany). These probes bind to the sensitive subtelomeric chromosomal regions which are immediately adjacent to the long (TTAGGG)n repeats and also contain repetitive stretches of DNA (Figure 1I). After washing, slides were air-dried for 20 min in the dark, incubated with 4′-6-Diamidino-2-phenylindole (DAPI) solution (125 μg/ml; Abbott, Wiesbaden, Germany) for 10 min at room temperature and coverslipped with Kaiser’s gelatine (Merck, Darmstadt, Germany).


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)

Characterization of multipotent neural progenitors isolated from human adult gut.(A) Bright-field view of representative adult enteric neurosphere-like bodies (NLBs) after 14 days in vitro. Free-floating NLBs could be observed with an estimated average number of 35 NLBs per preparation and a sphere diameter of 71.6 μm. Different NLB phenotypes include round- (star, comparable to B), ellipsoid- (white arrow, comparable to C) or irregular-shaped (black arrow, comparable to D) NLBs. (B–D) Depending to the individual NLB phenotype, different cellular compositions can be derived as shown for the ENS marker gene p75 in immunocytochemistry. (E–G) Proliferative NLB cells also incorporated BrdU and expressed previously described progenitor markers including Sox2 and Nestin. (H–J) After 2 weeks of differentiation NLBs built a monolayer composed of neuronal (TuJ), glial (S100β) and smooth muscle-like cells (SMA). (K–M) Molecular cytogenetic FISH analyses revealed no chromosomal alterations as representatively shown for three chromosomal interphase nuclei. (N) A schematic illustration of a chromosome is shown. In total, 15 different probe sets recognizing 41 different unique subtelomeric repeats were applied. (O) Efficacy and cellular distribution of NLBs strongly depends to the individual preparation, relying on age, amount and quality of tissue, as demonstrated by qPCR analysis for three representative marker genes: p75, Ret and tropomyosin (TPM). Scale bars represent in (A) 100 μm, (B–G) 25 μm, (H–J) 50 μm.
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Related In: Results  -  Collection

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pone-0093605-g001: Characterization of multipotent neural progenitors isolated from human adult gut.(A) Bright-field view of representative adult enteric neurosphere-like bodies (NLBs) after 14 days in vitro. Free-floating NLBs could be observed with an estimated average number of 35 NLBs per preparation and a sphere diameter of 71.6 μm. Different NLB phenotypes include round- (star, comparable to B), ellipsoid- (white arrow, comparable to C) or irregular-shaped (black arrow, comparable to D) NLBs. (B–D) Depending to the individual NLB phenotype, different cellular compositions can be derived as shown for the ENS marker gene p75 in immunocytochemistry. (E–G) Proliferative NLB cells also incorporated BrdU and expressed previously described progenitor markers including Sox2 and Nestin. (H–J) After 2 weeks of differentiation NLBs built a monolayer composed of neuronal (TuJ), glial (S100β) and smooth muscle-like cells (SMA). (K–M) Molecular cytogenetic FISH analyses revealed no chromosomal alterations as representatively shown for three chromosomal interphase nuclei. (N) A schematic illustration of a chromosome is shown. In total, 15 different probe sets recognizing 41 different unique subtelomeric repeats were applied. (O) Efficacy and cellular distribution of NLBs strongly depends to the individual preparation, relying on age, amount and quality of tissue, as demonstrated by qPCR analysis for three representative marker genes: p75, Ret and tropomyosin (TPM). Scale bars represent in (A) 100 μm, (B–G) 25 μm, (H–J) 50 μm.
Mentions: Genomic alterations of human enteric progenitors were evaluated by DNA fluorescence in situ hybridization (FISH) as described recently [22]. Briefly, after preparation of cell metaphases and interphases, cells were denatured, dehydrated and hybridized with multi-colour DNA FISH probe mixtures for 12–16 h at 37°C (ToTelVysionTM, Abbott, Wiesbaden, Germany). These probes bind to the sensitive subtelomeric chromosomal regions which are immediately adjacent to the long (TTAGGG)n repeats and also contain repetitive stretches of DNA (Figure 1I). After washing, slides were air-dried for 20 min in the dark, incubated with 4′-6-Diamidino-2-phenylindole (DAPI) solution (125 μg/ml; Abbott, Wiesbaden, Germany) for 10 min at room temperature and coverslipped with Kaiser’s gelatine (Merck, Darmstadt, Germany).

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