J Vet Sci.  2016 Sep;17(3):289-297. 10.4142/jvs.2016.17.3.289.

Immunologic properties of differentiated and undifferentiated mesenchymal stem cells derived from umbilical cord blood

• Lee HJ
• Kang KS
• Kang SY
• Kim HS
• Park SJ
• Lee SY
• Kim KD
• Lee HC
• Park JK
• Paik WY
• Lee L
• Yeon SC

• KMID: 2413127
• DOI: http://doi.org/10.4142/jvs.2016.17.3.289

Abstract

The expression of immunogenic markers after differentiation of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) has been poorly investigated and requires extensive in vitro and in vivo testing for clinical application. The expression of human leukocyte antigen (HLA) classes on UCB-derived MSC was tested by Fluorescence-activated cell sorting analysis and immunocytochemical staining. The undifferentiated MSC were moderately positive for HLA-ABC, but almost completely negative for HLA-DR. The MSC differentiated to chondrocytes expressed neither HLA-ABC nor HLA-DR. The proliferation of MSC was not significantly affected by the allogeneic lymphocytes stimulated with concanavalin A. The responder lymphocytes showed no significant decrease in proliferation in the presence of the MSC, but the apoptosis rate of the lymphocytes was increased in the presence of MSC. Taken together, these findings indicate that UCB-derived MSC differentiated to chondrocytes expressed less HLA class I and no class II antigens. The MSC showed an immunomodulatory effect on the proliferation and apoptosis of allogeneic lymphocytes. These data suggest that the differentiated and undifferentiated allogeneic MSC derived from umbilical cord blood can be a useful candidate for allogeneic cell therapy and transplantation without a major risk of rejection.

Keyword

apoptosis; human leukocyte antigen; human umbilical cord blood; immunophenotypes; mesenchymal stem cells

MeSH Terms

Animals
*Cell Differentiation
*Cell Proliferation
Cells, Cultured
Fetal Blood/*cytology
Flow Cytometry
HLA Antigens/genetics/metabolism
Humans
Mesenchymal Stem Cell Transplantation/*veterinary
Mesenchymal Stromal Cells/cytology/*immunology
Mice

Figure

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  Fig. 1 FACS analysis of immunophenotypes of human leukocyte antigen (HLA)-ABC and HLA-DR on the undifferentiated mesenchymal stem cells (MSC) (A and D), chondrocytes differentiated from the MSC (B and E) and allogeneic peripheral lymphocytes (C and F).

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  Fig. 2 Expression of human HLA-ABC on the surface of human undifferentiated (A–C) and differentiated (D–F) MSC by immunocytochemical stain (green color). The nuclei of the MSC were counterstained with DAPI (blue color). (A and D) DAPI stain. (B and E) FITC stain. (C and F) Merged. 200× (A–C), 40× (D–F). Sale bars = 20 µm (A–C), 100 µm (D–F).

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  Fig. 3 Expression of human HLA-DR on the surface of human undifferentiated (A–C) and differentiated (D–F) MSC by immunocytochemical stain (green color). The nuclei of the MSC were counterstained with DAPI (blue color). The HLA-DR antigen was negatively expressed on the differentiated and undifferentiated MSC. (A and D) DAPI stain. (B and E) FITC stain. (C and F) Merged. 200× (A–F). Scale bars = 20 µm.

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  Fig. 4 Proliferation of UCB-derived MSC when co-cultured with or without allogeneic lymphocytes at a ratio of 1 : 0 and 1 : 5 in vitro (n = 5). The MSC and Con A-activated lymphocytes were co-cultured in RPMI-1640 medium with 10% FBS, L-glutamine and antibiotics in a CO2 incubator at 37℃ for 3 days. There was no significant (p = 0.05) difference between groups. Allogeneic lymphocytes were pre-activated with 5 µg/mL of Con A for 3 days.

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  Fig. 5 Effect of MSC on apoptosis of allogeneic lymphocytes following co-culture at ratios of 1 : 1 and 1 : 5 for 3 days. The test was conducted by Annexin-V and PI double staining and analyzed by flow cytometry. Apoptosis of MSC gated on size (A–C) and apoptosis of lymphocytes gated on size (D–F) were analyzed following MSC and activated lymphocytes were cultured alone (A and D) or cocultured at a ratio of 1 : 1 (B and E) and 1 : 5 (C and F).

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  Fig. 6 Proliferation of allogeneic lymphocytes when co-cultured with UCB-derived MSC at ratios of 0 : 1, 1 : 1 and 1 : 5 in vitro (n = 5). The Con A-activated allogeneic lymphocytes were cultured with or without MSC at ratios of 0 : 1, 1 : 1 and 1 : 5 in a RPMI-1640 medium with 10% FBS, L-glutamine and antibiotics in a CO2 incubator at 37℃ for 3 days. There was no significant (p > 0.05) inhibition of the proliferation of lymphocytes when they were co-cultured with MSC.

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