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Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

Lehrberg J, Gardiner DM - PLoS ONE (2015)

Bottom Line: Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo.Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves.Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

No MeSH data available.


Related in: MedlinePlus

Organotypic blastema slices survive and proliferate in vitro.(A) Hematoxylin and eosin stained section of a blastema slice that had been cultured for 10 days in the presence of 5% FBS. Regions of pre-cartilage condensations are evident (arrows). The blastema epithelium has begun to migrate over the proximal cut end. (B) EdU labeling of a blastema slice after 10 days in vitro. EdU positive nuclei (green) are present in the mesenchyme but absent from the epithelium. (C) A bright field image of a blastema slice (upper right) and DRG (lower left) after 10 days of co-culture. (D) Immunofluorescence of the blastema slice/DRG co-culture illustrated in (C). Proliferating cells in the slices (green) are restricted to the mesenchyme, nuclei are stained with DAPI (blue), and neurofilaments are labeled with RT97 (red). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.
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pone.0123186.g002: Organotypic blastema slices survive and proliferate in vitro.(A) Hematoxylin and eosin stained section of a blastema slice that had been cultured for 10 days in the presence of 5% FBS. Regions of pre-cartilage condensations are evident (arrows). The blastema epithelium has begun to migrate over the proximal cut end. (B) EdU labeling of a blastema slice after 10 days in vitro. EdU positive nuclei (green) are present in the mesenchyme but absent from the epithelium. (C) A bright field image of a blastema slice (upper right) and DRG (lower left) after 10 days of co-culture. (D) Immunofluorescence of the blastema slice/DRG co-culture illustrated in (C). Proliferating cells in the slices (green) are restricted to the mesenchyme, nuclei are stained with DAPI (blue), and neurofilaments are labeled with RT97 (red). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.

Mentions: Blastema slices appeared healthy and the cells continued to proliferate in vitro over an extended period of time (Fig 2). After 10 days in culture, the slices maintained a normal morphology with a thickened epithelium surrounding the blastema mesenchyme (Fig 2A). The cells within the slice appeared healthy and regions of high cell density corresponding to pre-chondrogenic condensations were evident in some sections (Fig 2A, arrows). Although the appearance of an OSC blastema was similar to a blastema that was integrated into the amputated limb (e.g. there was an apical epithelium that covered the blastema mesenchyme), there also were differences. At the proximal boundary of the slice, the epidermis began to migrate over the free surface that was created when the blastema was surgically removed from the animal. This phenomenon referred to as “epiboly”, has been described previously in whole-mount explants of amphibian blastemas and limb buds [42,43]. Many of the cells of the blastema mesenchyme incorporated EdU after 10 days of culture, although cells in the regions of high cell density (visualized by DAPI staining of the nuclei) did not (Fig 2B and 2D). The keratinocytes of the apical epithelium (Apical Epithelial Cap, AEC) in this experiment (cultured with 5% FBS) did not incorporate EdU (Fig 2B), which was consistent with the observation that AEC cells withdraw from the cell cycle in vivo [44,45]. Better understanding how the proliferative response of AEC keratinocytes, particularly the basal keratinocytes [2] is regulated in organotypic slice cultures is a goal of future experiments.


Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

Lehrberg J, Gardiner DM - PLoS ONE (2015)

Organotypic blastema slices survive and proliferate in vitro.(A) Hematoxylin and eosin stained section of a blastema slice that had been cultured for 10 days in the presence of 5% FBS. Regions of pre-cartilage condensations are evident (arrows). The blastema epithelium has begun to migrate over the proximal cut end. (B) EdU labeling of a blastema slice after 10 days in vitro. EdU positive nuclei (green) are present in the mesenchyme but absent from the epithelium. (C) A bright field image of a blastema slice (upper right) and DRG (lower left) after 10 days of co-culture. (D) Immunofluorescence of the blastema slice/DRG co-culture illustrated in (C). Proliferating cells in the slices (green) are restricted to the mesenchyme, nuclei are stained with DAPI (blue), and neurofilaments are labeled with RT97 (red). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414535&req=5

pone.0123186.g002: Organotypic blastema slices survive and proliferate in vitro.(A) Hematoxylin and eosin stained section of a blastema slice that had been cultured for 10 days in the presence of 5% FBS. Regions of pre-cartilage condensations are evident (arrows). The blastema epithelium has begun to migrate over the proximal cut end. (B) EdU labeling of a blastema slice after 10 days in vitro. EdU positive nuclei (green) are present in the mesenchyme but absent from the epithelium. (C) A bright field image of a blastema slice (upper right) and DRG (lower left) after 10 days of co-culture. (D) Immunofluorescence of the blastema slice/DRG co-culture illustrated in (C). Proliferating cells in the slices (green) are restricted to the mesenchyme, nuclei are stained with DAPI (blue), and neurofilaments are labeled with RT97 (red). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.
Mentions: Blastema slices appeared healthy and the cells continued to proliferate in vitro over an extended period of time (Fig 2). After 10 days in culture, the slices maintained a normal morphology with a thickened epithelium surrounding the blastema mesenchyme (Fig 2A). The cells within the slice appeared healthy and regions of high cell density corresponding to pre-chondrogenic condensations were evident in some sections (Fig 2A, arrows). Although the appearance of an OSC blastema was similar to a blastema that was integrated into the amputated limb (e.g. there was an apical epithelium that covered the blastema mesenchyme), there also were differences. At the proximal boundary of the slice, the epidermis began to migrate over the free surface that was created when the blastema was surgically removed from the animal. This phenomenon referred to as “epiboly”, has been described previously in whole-mount explants of amphibian blastemas and limb buds [42,43]. Many of the cells of the blastema mesenchyme incorporated EdU after 10 days of culture, although cells in the regions of high cell density (visualized by DAPI staining of the nuclei) did not (Fig 2B and 2D). The keratinocytes of the apical epithelium (Apical Epithelial Cap, AEC) in this experiment (cultured with 5% FBS) did not incorporate EdU (Fig 2B), which was consistent with the observation that AEC cells withdraw from the cell cycle in vivo [44,45]. Better understanding how the proliferative response of AEC keratinocytes, particularly the basal keratinocytes [2] is regulated in organotypic slice cultures is a goal of future experiments.

Bottom Line: Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo.Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves.Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

No MeSH data available.


Related in: MedlinePlus