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Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen YT, Lehenkari P, Hukkanen M, Korhonen M - Stem Cell Res Ther (2015)

Bottom Line: Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks.In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment.As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. eeva.castren@helsinki.fi.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.

Methods: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.

Results: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.

Conclusions: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

No MeSH data available.


Related in: MedlinePlus

Quantitative RT-PCR in two-dimensional (2D) cell culture. The cells were cultured in 2D osteogenic medium up to 21 days. Total RNA was isolated and the expression of Runx2 (a), alkaline phosphatase (ALP) (b), collagen I α1 (Col1α1) (c), and osteocalcin (OCN) (d) was analyzed using quantitative RT-PCR. DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
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Fig2: Quantitative RT-PCR in two-dimensional (2D) cell culture. The cells were cultured in 2D osteogenic medium up to 21 days. Total RNA was isolated and the expression of Runx2 (a), alkaline phosphatase (ALP) (b), collagen I α1 (Col1α1) (c), and osteocalcin (OCN) (d) was analyzed using quantitative RT-PCR. DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma

Mentions: Osteogenic differentiation in 2D cultures was also examined by analyzing the expression of the Runx2, ALP, Col1α1 and OCN genes using quantitative RT-PCR. Increased Runx2 levels were found on days 9 and 14 in FCS but remained low in PLP cultures. In both conditions, Runx2 levels rose until day 21 (Fig. 2a). ALP mRNA levels increased steadily in both FCS and PLP cultures (Fig. 2b). Col1α1 levels increased steeply until day 9 after which the levels decreased in both FCS and PLP cultures (Fig. 2c). OCN mRNA rose until day 21 (Fig. 2d). In FCS cultures, elevated levels of OCN were seen from day 7 onwards, whereas in PLP cultures elevated OCN expression was seen only in late cultures.Fig. 2


Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen YT, Lehenkari P, Hukkanen M, Korhonen M - Stem Cell Res Ther (2015)

Quantitative RT-PCR in two-dimensional (2D) cell culture. The cells were cultured in 2D osteogenic medium up to 21 days. Total RNA was isolated and the expression of Runx2 (a), alkaline phosphatase (ALP) (b), collagen I α1 (Col1α1) (c), and osteocalcin (OCN) (d) was analyzed using quantitative RT-PCR. DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562352&req=5

Fig2: Quantitative RT-PCR in two-dimensional (2D) cell culture. The cells were cultured in 2D osteogenic medium up to 21 days. Total RNA was isolated and the expression of Runx2 (a), alkaline phosphatase (ALP) (b), collagen I α1 (Col1α1) (c), and osteocalcin (OCN) (d) was analyzed using quantitative RT-PCR. DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
Mentions: Osteogenic differentiation in 2D cultures was also examined by analyzing the expression of the Runx2, ALP, Col1α1 and OCN genes using quantitative RT-PCR. Increased Runx2 levels were found on days 9 and 14 in FCS but remained low in PLP cultures. In both conditions, Runx2 levels rose until day 21 (Fig. 2a). ALP mRNA levels increased steadily in both FCS and PLP cultures (Fig. 2b). Col1α1 levels increased steeply until day 9 after which the levels decreased in both FCS and PLP cultures (Fig. 2c). OCN mRNA rose until day 21 (Fig. 2d). In FCS cultures, elevated levels of OCN were seen from day 7 onwards, whereas in PLP cultures elevated OCN expression was seen only in late cultures.Fig. 2

Bottom Line: Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks.In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment.As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. eeva.castren@helsinki.fi.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.

Methods: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.

Results: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.

Conclusions: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

No MeSH data available.


Related in: MedlinePlus