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Beneficial reciprocal effects of bone marrow stromal cells and Schwann cells from adult rats in a dynamic co‑culture system in vitro without intercellular contact.

Zhou LN, Cui XJ, Su KX, Wang XH, Guo JH - Mol Med Rep (2015)

Bottom Line: In order to examine how implanted bone marrow stromal cells (BMSCs) encourage peripheral nerve regeneration, the present study investigated the interaction of BMSCs and Schwann cells (SCs) using an indirect in vitro co‑culture model.On the 3rd day after co‑culture, only a few co‑cultured BMSCs showed the typical SC‑like morphology, while most BMSCs still kept their native appearance.These results indicated that BMSCs may interact synergistically with SCs with regard to promoting peripheral nerve regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Guangdong Medical University, Zhanjiang, Guangdong 524023, P.R. China.

ABSTRACT
In order to examine how implanted bone marrow stromal cells (BMSCs) encourage peripheral nerve regeneration, the present study investigated the interaction of BMSCs and Schwann cells (SCs) using an indirect in vitro co‑culture model. SCs and BMSCs were obtained from adult Sprague‑Dawley rats. The passaged BMSCs were CD29‑ and CD44‑positive but CD45‑negative and were co‑cultured with the primary SCs using a Millicell system, which allows BMSCs and SCs to grow in the same culture medium but without direct contact. Expression of the typical SC markers S‑100 and glial fibrillary acidic protein (GFAP) of the treated BMSCs as well as the proliferation capacity of the co‑cultured SCs was evaluated by immunocytochemical staining on the 3rd and 5th day of co‑culture. Immunocytochemical staining showed that >75% of the BMSCs in the indirect co‑culture model were GFAP‑ and S‑100‑positive on the 3rd and 5th day after co‑culture, as opposed to <5% of the BMSCs in the control group. On the 3rd day after co‑culture, only a few co‑cultured BMSCs showed the typical SC‑like morphology, while most BMSCs still kept their native appearance. By contrast, on the 5th day after co‑culture, almost all of the co‑cultured BMSCs appeared with the typical SC‑like morphology. Furthermore, 70.71% of the SCs in the indirect co‑culture model were S‑100‑positive on the 5th day of co‑culture, as opposed to >30.43% of the SCs in the control group. These results indicated that BMSCs may interact synergistically with SCs with regard to promoting peripheral nerve regeneration.

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Induction of BMSC differentiation by SC diffusible factors on the 3rd day of co-culture. The glial differentiation of BMSCs was assessed by (A and E) S-100 (red) and (B and F) GFAP (green) immunofluorescence and (C and G) Hoechst nuclear staining (blue). (A–D) Glial differentiation was more evident for BMSCs co-cultured with SCs than for (E–H) BMSCs co-cultured without SCs (scale bar, 50 µm). Quantification of the images provided the percentages of (I) S-100-positive or (J) GFAP-positive cells relative to the number of Hoechst-positive nuclei. Results were averaged for three wells and repeated four times. Each of the four repeats was from a different culture, with each culture in turn being derived from a different animal. Quantitative study showed that the percentage of S-100-positive (76.3±5.47%, n=6) as well as GFAP-positive (88.7±3.35%, n=6) cells in BMSCs co-cultured with SCs was significantly higher than that in BMSCs cultured without SCs (S100, 3.51±2.44%, n=6; GFAP, 4.43±0.99%, n=6) (*P<0.05). BMSC, bone marrow stromal cell; SC, Schwann cell; GFAP, glial fibrillary acidic protein.
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f3-mmr-12-04-4931: Induction of BMSC differentiation by SC diffusible factors on the 3rd day of co-culture. The glial differentiation of BMSCs was assessed by (A and E) S-100 (red) and (B and F) GFAP (green) immunofluorescence and (C and G) Hoechst nuclear staining (blue). (A–D) Glial differentiation was more evident for BMSCs co-cultured with SCs than for (E–H) BMSCs co-cultured without SCs (scale bar, 50 µm). Quantification of the images provided the percentages of (I) S-100-positive or (J) GFAP-positive cells relative to the number of Hoechst-positive nuclei. Results were averaged for three wells and repeated four times. Each of the four repeats was from a different culture, with each culture in turn being derived from a different animal. Quantitative study showed that the percentage of S-100-positive (76.3±5.47%, n=6) as well as GFAP-positive (88.7±3.35%, n=6) cells in BMSCs co-cultured with SCs was significantly higher than that in BMSCs cultured without SCs (S100, 3.51±2.44%, n=6; GFAP, 4.43±0.99%, n=6) (*P<0.05). BMSC, bone marrow stromal cell; SC, Schwann cell; GFAP, glial fibrillary acidic protein.

Mentions: Most of the BMSCs in the co-culture system were GFAP- and S-100-positive on the third and fifth day of co-culture, while the BMSCs in the control culture system hardly showed any GFAP- and S-100-positive populations. On the third day after co-culture, the BMSCs in the co-culture system became dense and grew in a longitudinal, parallel fashion (Fig. 3A and B). Only a few co-cultured BMSCs showed spindle-shaped or triangular soma with two or three slender processes (Fig. 3), the typical morphology of glial differentiation, and most of the co-cultured BMSCs still kept their native appearance. By contrast, on the 5th day after co-culture, almost all of the co-cultured BMSCs appeared with typical SC-like morphology, featuring spindle-shaped or triangular soma with two or three slender processes on the 5th day of co-culture (Fig. 4), the characteristic morphology of glial differentiation. The densities of co-cultured BMSCs were significantly higher in SC co-cultures than in acellular control cultures, whether on the 3rd or 5th day of co-culture, as shown by S-100 (Fig. 3A and E, and Fig. 4A and E), GFAP (Fig. 3B and F, and Fig. 4B and F) and Hoechst 33258 (Fig. 3C and G and Fig. 4C and G) triple labelling. Quantification of the number of S-100-positive cells as a percentage of the number of Hoechst-labeled nuclei on the 3rd day of co-culture showed that 76.3±5.47% of BMSCs in SC co-cultures were S-100-positive (Fig. 3D), which was significantly higher than the 3.51±2.44% of BMSCs in the acellular control cultures (P<0.05) (Fig. 3H and I). Quantification of GFAP-positive cells as a percentage of the number of Hoechst-labeled nuclei showed that 88.7±3.35% of BMSCs in SC co-cultures were GFAP-positive, which was significantly higher than the 4.43±0.99% of BMSCs in the acellular control cultures (P<0.05) (Fig. 3J). Quantitative analysis of the number of S-100-positive cells as a percentage of the number of Hoechst-labeled nuclei on the 5th day after co-culture (Fig. 4) showed that 85.6±1.69% of BMSCs in SC co-cultures were S-100-positive (Fig. 4D), which was significantly higher than the 3.01±4.87% of BMSCs in the acellular control cultures (Fig. 4H). The difference in the percentage of S-100-positive cells was statistically significant at the P<0.05 level (Fig. 4I). Quantification of GFAP-positive cells as a percentage of the number of Hoechst-labeled nuclei showed that 93.1±2.27% of BMSCs in SC co-cultures were GFAP-positive, which was higher than the 4.12±0.23% of BMSCs in the acellular control cultures. This difference in GFAP frequency was statistically significant at the P<0.05 level (Fig. 4J).


Beneficial reciprocal effects of bone marrow stromal cells and Schwann cells from adult rats in a dynamic co‑culture system in vitro without intercellular contact.

Zhou LN, Cui XJ, Su KX, Wang XH, Guo JH - Mol Med Rep (2015)

Induction of BMSC differentiation by SC diffusible factors on the 3rd day of co-culture. The glial differentiation of BMSCs was assessed by (A and E) S-100 (red) and (B and F) GFAP (green) immunofluorescence and (C and G) Hoechst nuclear staining (blue). (A–D) Glial differentiation was more evident for BMSCs co-cultured with SCs than for (E–H) BMSCs co-cultured without SCs (scale bar, 50 µm). Quantification of the images provided the percentages of (I) S-100-positive or (J) GFAP-positive cells relative to the number of Hoechst-positive nuclei. Results were averaged for three wells and repeated four times. Each of the four repeats was from a different culture, with each culture in turn being derived from a different animal. Quantitative study showed that the percentage of S-100-positive (76.3±5.47%, n=6) as well as GFAP-positive (88.7±3.35%, n=6) cells in BMSCs co-cultured with SCs was significantly higher than that in BMSCs cultured without SCs (S100, 3.51±2.44%, n=6; GFAP, 4.43±0.99%, n=6) (*P<0.05). BMSC, bone marrow stromal cell; SC, Schwann cell; GFAP, glial fibrillary acidic protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581791&req=5

f3-mmr-12-04-4931: Induction of BMSC differentiation by SC diffusible factors on the 3rd day of co-culture. The glial differentiation of BMSCs was assessed by (A and E) S-100 (red) and (B and F) GFAP (green) immunofluorescence and (C and G) Hoechst nuclear staining (blue). (A–D) Glial differentiation was more evident for BMSCs co-cultured with SCs than for (E–H) BMSCs co-cultured without SCs (scale bar, 50 µm). Quantification of the images provided the percentages of (I) S-100-positive or (J) GFAP-positive cells relative to the number of Hoechst-positive nuclei. Results were averaged for three wells and repeated four times. Each of the four repeats was from a different culture, with each culture in turn being derived from a different animal. Quantitative study showed that the percentage of S-100-positive (76.3±5.47%, n=6) as well as GFAP-positive (88.7±3.35%, n=6) cells in BMSCs co-cultured with SCs was significantly higher than that in BMSCs cultured without SCs (S100, 3.51±2.44%, n=6; GFAP, 4.43±0.99%, n=6) (*P<0.05). BMSC, bone marrow stromal cell; SC, Schwann cell; GFAP, glial fibrillary acidic protein.
Mentions: Most of the BMSCs in the co-culture system were GFAP- and S-100-positive on the third and fifth day of co-culture, while the BMSCs in the control culture system hardly showed any GFAP- and S-100-positive populations. On the third day after co-culture, the BMSCs in the co-culture system became dense and grew in a longitudinal, parallel fashion (Fig. 3A and B). Only a few co-cultured BMSCs showed spindle-shaped or triangular soma with two or three slender processes (Fig. 3), the typical morphology of glial differentiation, and most of the co-cultured BMSCs still kept their native appearance. By contrast, on the 5th day after co-culture, almost all of the co-cultured BMSCs appeared with typical SC-like morphology, featuring spindle-shaped or triangular soma with two or three slender processes on the 5th day of co-culture (Fig. 4), the characteristic morphology of glial differentiation. The densities of co-cultured BMSCs were significantly higher in SC co-cultures than in acellular control cultures, whether on the 3rd or 5th day of co-culture, as shown by S-100 (Fig. 3A and E, and Fig. 4A and E), GFAP (Fig. 3B and F, and Fig. 4B and F) and Hoechst 33258 (Fig. 3C and G and Fig. 4C and G) triple labelling. Quantification of the number of S-100-positive cells as a percentage of the number of Hoechst-labeled nuclei on the 3rd day of co-culture showed that 76.3±5.47% of BMSCs in SC co-cultures were S-100-positive (Fig. 3D), which was significantly higher than the 3.51±2.44% of BMSCs in the acellular control cultures (P<0.05) (Fig. 3H and I). Quantification of GFAP-positive cells as a percentage of the number of Hoechst-labeled nuclei showed that 88.7±3.35% of BMSCs in SC co-cultures were GFAP-positive, which was significantly higher than the 4.43±0.99% of BMSCs in the acellular control cultures (P<0.05) (Fig. 3J). Quantitative analysis of the number of S-100-positive cells as a percentage of the number of Hoechst-labeled nuclei on the 5th day after co-culture (Fig. 4) showed that 85.6±1.69% of BMSCs in SC co-cultures were S-100-positive (Fig. 4D), which was significantly higher than the 3.01±4.87% of BMSCs in the acellular control cultures (Fig. 4H). The difference in the percentage of S-100-positive cells was statistically significant at the P<0.05 level (Fig. 4I). Quantification of GFAP-positive cells as a percentage of the number of Hoechst-labeled nuclei showed that 93.1±2.27% of BMSCs in SC co-cultures were GFAP-positive, which was higher than the 4.12±0.23% of BMSCs in the acellular control cultures. This difference in GFAP frequency was statistically significant at the P<0.05 level (Fig. 4J).

Bottom Line: In order to examine how implanted bone marrow stromal cells (BMSCs) encourage peripheral nerve regeneration, the present study investigated the interaction of BMSCs and Schwann cells (SCs) using an indirect in vitro co‑culture model.On the 3rd day after co‑culture, only a few co‑cultured BMSCs showed the typical SC‑like morphology, while most BMSCs still kept their native appearance.These results indicated that BMSCs may interact synergistically with SCs with regard to promoting peripheral nerve regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Guangdong Medical University, Zhanjiang, Guangdong 524023, P.R. China.

ABSTRACT
In order to examine how implanted bone marrow stromal cells (BMSCs) encourage peripheral nerve regeneration, the present study investigated the interaction of BMSCs and Schwann cells (SCs) using an indirect in vitro co‑culture model. SCs and BMSCs were obtained from adult Sprague‑Dawley rats. The passaged BMSCs were CD29‑ and CD44‑positive but CD45‑negative and were co‑cultured with the primary SCs using a Millicell system, which allows BMSCs and SCs to grow in the same culture medium but without direct contact. Expression of the typical SC markers S‑100 and glial fibrillary acidic protein (GFAP) of the treated BMSCs as well as the proliferation capacity of the co‑cultured SCs was evaluated by immunocytochemical staining on the 3rd and 5th day of co‑culture. Immunocytochemical staining showed that >75% of the BMSCs in the indirect co‑culture model were GFAP‑ and S‑100‑positive on the 3rd and 5th day after co‑culture, as opposed to <5% of the BMSCs in the control group. On the 3rd day after co‑culture, only a few co‑cultured BMSCs showed the typical SC‑like morphology, while most BMSCs still kept their native appearance. By contrast, on the 5th day after co‑culture, almost all of the co‑cultured BMSCs appeared with the typical SC‑like morphology. Furthermore, 70.71% of the SCs in the indirect co‑culture model were S‑100‑positive on the 5th day of co‑culture, as opposed to >30.43% of the SCs in the control group. These results indicated that BMSCs may interact synergistically with SCs with regard to promoting peripheral nerve regeneration.

Show MeSH
Related in: MedlinePlus