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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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Morphology of adult SCs and BMSCs in vitro. (A–C) Immunofluorescence staining of adult primary SCs: (A) S-100 (red); (B) Hoechst 33258 (blue); (C) merged (scale bar, 50 µm). (D) Phase-contrast images of adult primary BMSCs (scale bar, 100 µm). (E) Flow-cytometric analysis showed CD29- and CD44-positive but CD45-negative signals in P13 BMSCs of adult rats (blue, assay group; red, control group). BMSC, bone marrow stromal cell; SC, Schwann cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FSC, forward scatter; SSC, side scatter.
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f1-mmr-12-04-4931: Morphology of adult SCs and BMSCs in vitro. (A–C) Immunofluorescence staining of adult primary SCs: (A) S-100 (red); (B) Hoechst 33258 (blue); (C) merged (scale bar, 50 µm). (D) Phase-contrast images of adult primary BMSCs (scale bar, 100 µm). (E) Flow-cytometric analysis showed CD29- and CD44-positive but CD45-negative signals in P13 BMSCs of adult rats (blue, assay group; red, control group). BMSC, bone marrow stromal cell; SC, Schwann cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FSC, forward scatter; SSC, side scatter.

Mentions: In the present study, the primary adult SCs were confluent at 4–5 days of culture. Most of the S-100-positive SCs had typical spindle-shaped bodies with two slender processes. Only a few cells showed triangular soma with three projections (Fig. 1A). The proportion of S-100-positive SCs was 69±3.8% at the time of confluence (Fig. 1B and C). At 6–7 days, the adult primary BMSCs were confluent with cells being heterogeneous in morphology (Fig. 1D). They were easily expanded to up to 15 passages, maintaining the undifferentiated state as fibroblastic morphology. Flow-cytometric analysis showed that the P13 BMSCs were positive for the well-defined BMSC markers CD29 and CD44, but negative for the haematopoietic surface antigen CD45 (Fig. 1E).


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)

Morphology of adult SCs and BMSCs in vitro. (A–C) Immunofluorescence staining of adult primary SCs: (A) S-100 (red); (B) Hoechst 33258 (blue); (C) merged (scale bar, 50 µm). (D) Phase-contrast images of adult primary BMSCs (scale bar, 100 µm). (E) Flow-cytometric analysis showed CD29- and CD44-positive but CD45-negative signals in P13 BMSCs of adult rats (blue, assay group; red, control group). BMSC, bone marrow stromal cell; SC, Schwann cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FSC, forward scatter; SSC, side scatter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-mmr-12-04-4931: Morphology of adult SCs and BMSCs in vitro. (A–C) Immunofluorescence staining of adult primary SCs: (A) S-100 (red); (B) Hoechst 33258 (blue); (C) merged (scale bar, 50 µm). (D) Phase-contrast images of adult primary BMSCs (scale bar, 100 µm). (E) Flow-cytometric analysis showed CD29- and CD44-positive but CD45-negative signals in P13 BMSCs of adult rats (blue, assay group; red, control group). BMSC, bone marrow stromal cell; SC, Schwann cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FSC, forward scatter; SSC, side scatter.
Mentions: In the present study, the primary adult SCs were confluent at 4–5 days of culture. Most of the S-100-positive SCs had typical spindle-shaped bodies with two slender processes. Only a few cells showed triangular soma with three projections (Fig. 1A). The proportion of S-100-positive SCs was 69±3.8% at the time of confluence (Fig. 1B and C). At 6–7 days, the adult primary BMSCs were confluent with cells being heterogeneous in morphology (Fig. 1D). They were easily expanded to up to 15 passages, maintaining the undifferentiated state as fibroblastic morphology. Flow-cytometric analysis showed that the P13 BMSCs were positive for the well-defined BMSC markers CD29 and CD44, but negative for the haematopoietic surface antigen CD45 (Fig. 1E).

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