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

Integration of transplanted human NLBs in vivo.(A) Example of in situ hybridization (ISH) for human-specific Alu sequence with good cell integration within the gut musculature. Note that ganglia-like structures can be detectedat the expected location between the circular and longitudinal muscle layer (arrowhead). (B) Representative example for poor cell integration i.e. only a single cell was detected in this microscopic view (arrowhead). (C) Alu-ISH on human control gut tissue demonstrates the specifity of Alu probe for human cells. (D) ISH with Alu probe on mouse tissue served as negative control. Note, that there is some unspecific background staining in the cytoplasm and extracellular matrix, which does not co-localize with Dapi+ nuclei (stars). (E) Alu-ISH was combined with immunohistochemistry (IHC) to demonstrate in vivo differentiation state of neuronal cells between the longitudinal and circular muscle layers (Alu, black nuclei and PGP9.5, brown cytoplasmic staining). (F–H) Fluorescence ISH for Alu combined with IHC in a higher magnification (Alu-ISH, red nuclei and IHC, green cytoplasma) (F) A PGP9.5/Alu co-stained human cell has been integrated in a small ganglion structure (arrowhead). (G) Differentiated human glial cells within a ganglion as shown by Alu/GFAP-co-staining (arrowheads). (H) Most of the transplanted cells (arrowheads) were found around ganglion structures (stars) in the smooth muscle layers as shown by Alu/SMA-co-staining. (I) Variability of human cell integration was quantified and demonstrated as box-whisker plots. Most cells were found within the longitudinal muscle layer. Scale bars in (A–D) 100 μm, (E) 50 μm, (F–H) 200 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0093605-g004: Integration of transplanted human NLBs in vivo.(A) Example of in situ hybridization (ISH) for human-specific Alu sequence with good cell integration within the gut musculature. Note that ganglia-like structures can be detectedat the expected location between the circular and longitudinal muscle layer (arrowhead). (B) Representative example for poor cell integration i.e. only a single cell was detected in this microscopic view (arrowhead). (C) Alu-ISH on human control gut tissue demonstrates the specifity of Alu probe for human cells. (D) ISH with Alu probe on mouse tissue served as negative control. Note, that there is some unspecific background staining in the cytoplasm and extracellular matrix, which does not co-localize with Dapi+ nuclei (stars). (E) Alu-ISH was combined with immunohistochemistry (IHC) to demonstrate in vivo differentiation state of neuronal cells between the longitudinal and circular muscle layers (Alu, black nuclei and PGP9.5, brown cytoplasmic staining). (F–H) Fluorescence ISH for Alu combined with IHC in a higher magnification (Alu-ISH, red nuclei and IHC, green cytoplasma) (F) A PGP9.5/Alu co-stained human cell has been integrated in a small ganglion structure (arrowhead). (G) Differentiated human glial cells within a ganglion as shown by Alu/GFAP-co-staining (arrowheads). (H) Most of the transplanted cells (arrowheads) were found around ganglion structures (stars) in the smooth muscle layers as shown by Alu/SMA-co-staining. (I) Variability of human cell integration was quantified and demonstrated as box-whisker plots. Most cells were found within the longitudinal muscle layer. Scale bars in (A–D) 100 μm, (E) 50 μm, (F–H) 200 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle.

Mentions: To clarify the transplanted cell fate i.e. whether and where the transplanted human progenitors have been integrated within the host tissue, we performed DNA in situ hybridization (ISH) using a probe for the highly repetitive Alu sequence that specifically detects human cell nuclei as proved in human and mouse control tissues (Figure 4C, D). In our experiments, we detected Alu-positive human cells in 8 of 10 transplanted animals, although with high variability (Figure 4A, B, I; n = 10 animals ranging from 0 to 176 Alu+ cells/slice, mean: 35.2+–53.7 cells/slice). 5 of the 8 animals revealed relative high numbers of integrated human cells (∼20–180 cells/slice), although the estimated total number of surviving cells in the treated gut segment is far less than the initial transplanted cell number suggesting relatively moderate cell survival and/or integration. Noteworthy, there seem to be slight age-dependent effects of donors with respect to an increased extent of cell integration; however, the number of analyzed patients was too low to apply any robust statistics. Most of the human cells were found within the longitudinal muscle (79%), the remaining cells in the inner circular muscle region (21%). Interestingly, we also discovered cells clustering in the known plexus region suggesting either neo-ganglia formation or integration of transplanted neural cells into existing/regenerating plexus structures (Figure 4A, arrowhead). This observation could also be verified by co-immunostaining with characteristic ENS differentiation markers, such as PGP9.5 to identify neurons (Figure 4E, F) and GFAP for glial cells (Figure 4G) indicating the presence and survival of neural cells in vivo. However, the majority of cells were not found within ganglia structures, but rather as single cells located in the smooth muscle layers or in close contact to ganglia. Co-staining for SMA verified the presence of non-neural phenotypes such as smooth muscle-like cells (Figure 4H). Unfortunately, due to technical reasons a quantification of double-stained human cells was not applicable since all used antibodies, established for in situ hybridization and immunohistochemistry, are cytoplasmic markers, and therefore not suitable for a precise allocation.


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)

Integration of transplanted human NLBs in vivo.(A) Example of in situ hybridization (ISH) for human-specific Alu sequence with good cell integration within the gut musculature. Note that ganglia-like structures can be detectedat the expected location between the circular and longitudinal muscle layer (arrowhead). (B) Representative example for poor cell integration i.e. only a single cell was detected in this microscopic view (arrowhead). (C) Alu-ISH on human control gut tissue demonstrates the specifity of Alu probe for human cells. (D) ISH with Alu probe on mouse tissue served as negative control. Note, that there is some unspecific background staining in the cytoplasm and extracellular matrix, which does not co-localize with Dapi+ nuclei (stars). (E) Alu-ISH was combined with immunohistochemistry (IHC) to demonstrate in vivo differentiation state of neuronal cells between the longitudinal and circular muscle layers (Alu, black nuclei and PGP9.5, brown cytoplasmic staining). (F–H) Fluorescence ISH for Alu combined with IHC in a higher magnification (Alu-ISH, red nuclei and IHC, green cytoplasma) (F) A PGP9.5/Alu co-stained human cell has been integrated in a small ganglion structure (arrowhead). (G) Differentiated human glial cells within a ganglion as shown by Alu/GFAP-co-staining (arrowheads). (H) Most of the transplanted cells (arrowheads) were found around ganglion structures (stars) in the smooth muscle layers as shown by Alu/SMA-co-staining. (I) Variability of human cell integration was quantified and demonstrated as box-whisker plots. Most cells were found within the longitudinal muscle layer. Scale bars in (A–D) 100 μm, (E) 50 μm, (F–H) 200 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0093605-g004: Integration of transplanted human NLBs in vivo.(A) Example of in situ hybridization (ISH) for human-specific Alu sequence with good cell integration within the gut musculature. Note that ganglia-like structures can be detectedat the expected location between the circular and longitudinal muscle layer (arrowhead). (B) Representative example for poor cell integration i.e. only a single cell was detected in this microscopic view (arrowhead). (C) Alu-ISH on human control gut tissue demonstrates the specifity of Alu probe for human cells. (D) ISH with Alu probe on mouse tissue served as negative control. Note, that there is some unspecific background staining in the cytoplasm and extracellular matrix, which does not co-localize with Dapi+ nuclei (stars). (E) Alu-ISH was combined with immunohistochemistry (IHC) to demonstrate in vivo differentiation state of neuronal cells between the longitudinal and circular muscle layers (Alu, black nuclei and PGP9.5, brown cytoplasmic staining). (F–H) Fluorescence ISH for Alu combined with IHC in a higher magnification (Alu-ISH, red nuclei and IHC, green cytoplasma) (F) A PGP9.5/Alu co-stained human cell has been integrated in a small ganglion structure (arrowhead). (G) Differentiated human glial cells within a ganglion as shown by Alu/GFAP-co-staining (arrowheads). (H) Most of the transplanted cells (arrowheads) were found around ganglion structures (stars) in the smooth muscle layers as shown by Alu/SMA-co-staining. (I) Variability of human cell integration was quantified and demonstrated as box-whisker plots. Most cells were found within the longitudinal muscle layer. Scale bars in (A–D) 100 μm, (E) 50 μm, (F–H) 200 μm. M = mucosa, CM = circular muscle, LM = longitudinal muscle.
Mentions: To clarify the transplanted cell fate i.e. whether and where the transplanted human progenitors have been integrated within the host tissue, we performed DNA in situ hybridization (ISH) using a probe for the highly repetitive Alu sequence that specifically detects human cell nuclei as proved in human and mouse control tissues (Figure 4C, D). In our experiments, we detected Alu-positive human cells in 8 of 10 transplanted animals, although with high variability (Figure 4A, B, I; n = 10 animals ranging from 0 to 176 Alu+ cells/slice, mean: 35.2+–53.7 cells/slice). 5 of the 8 animals revealed relative high numbers of integrated human cells (∼20–180 cells/slice), although the estimated total number of surviving cells in the treated gut segment is far less than the initial transplanted cell number suggesting relatively moderate cell survival and/or integration. Noteworthy, there seem to be slight age-dependent effects of donors with respect to an increased extent of cell integration; however, the number of analyzed patients was too low to apply any robust statistics. Most of the human cells were found within the longitudinal muscle (79%), the remaining cells in the inner circular muscle region (21%). Interestingly, we also discovered cells clustering in the known plexus region suggesting either neo-ganglia formation or integration of transplanted neural cells into existing/regenerating plexus structures (Figure 4A, arrowhead). This observation could also be verified by co-immunostaining with characteristic ENS differentiation markers, such as PGP9.5 to identify neurons (Figure 4E, F) and GFAP for glial cells (Figure 4G) indicating the presence and survival of neural cells in vivo. However, the majority of cells were not found within ganglia structures, but rather as single cells located in the smooth muscle layers or in close contact to ganglia. Co-staining for SMA verified the presence of non-neural phenotypes such as smooth muscle-like cells (Figure 4H). Unfortunately, due to technical reasons a quantification of double-stained human cells was not applicable since all used antibodies, established for in situ hybridization and immunohistochemistry, are cytoplasmic markers, and therefore not suitable for a precise allocation.

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