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Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering

View Article: PubMed Central - PubMed

ABSTRACT

Umbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-β1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte-specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering.

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Effect of culture conditions on the mRNA steady-state levels of specific markers in mature and hypertrophic chondrocytes and osteoblasts.Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were cultured in collagen sponges at 21% O2 for 14 days, in incomplete chondrogenic medium in the absence (control) or presence of 50 ng/ml of BMP-2 and 10 ng/ml of TGF-β1. Undifferentiated hUCB-MSCs were also cultured as monolayers and used as a control before differentiation (day 0, D0). mRNA extracts obtained from human articular chondrocytes (HACs) released from cartilage after overnight enzymatic digestion were used as controls. (A) Relative mRNA expression of chondrogenic markers. (B) Relative mRNA expression of hypertrophic chondrocyte and osteoblast markers. Bone: mRNA extracted from osteoblast cultures at passage 3 and obtained from human femoral head of osteoarthritis patients. (C) COL2A1:COL1A1 mRNA ratio. All results were normalized to RPL13a mRNA expression, compared with undifferentiated hUCB-MSCs cultured in monolayer, and presented as the relative expression of each gene. Box plots represent six independent experiments performed in triplicate. Statistically significant differences among hUCB-MSCs in monolayer and untreated or treated cells in sponges were determined using the Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001).
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f5: Effect of culture conditions on the mRNA steady-state levels of specific markers in mature and hypertrophic chondrocytes and osteoblasts.Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were cultured in collagen sponges at 21% O2 for 14 days, in incomplete chondrogenic medium in the absence (control) or presence of 50 ng/ml of BMP-2 and 10 ng/ml of TGF-β1. Undifferentiated hUCB-MSCs were also cultured as monolayers and used as a control before differentiation (day 0, D0). mRNA extracts obtained from human articular chondrocytes (HACs) released from cartilage after overnight enzymatic digestion were used as controls. (A) Relative mRNA expression of chondrogenic markers. (B) Relative mRNA expression of hypertrophic chondrocyte and osteoblast markers. Bone: mRNA extracted from osteoblast cultures at passage 3 and obtained from human femoral head of osteoarthritis patients. (C) COL2A1:COL1A1 mRNA ratio. All results were normalized to RPL13a mRNA expression, compared with undifferentiated hUCB-MSCs cultured in monolayer, and presented as the relative expression of each gene. Box plots represent six independent experiments performed in triplicate. Statistically significant differences among hUCB-MSCs in monolayer and untreated or treated cells in sponges were determined using the Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: In the absence of growth factors, hUCB-MSCs seeded in collagen sponge scaffolds exhibited only a slight increase in SOX9, COL2A1, COL2A and ACAN mRNA levels and no statistically significant differences were observed compared with undifferentiated cells (Fig. 5A). In contrast, treatment with growth factors upregulated expression of SOX9 (9-fold), COL2A1 (34,875-fold) and its isoforms COL2A (9695-fold) and COL2B (709-fold), and ACAN (24-fold) compared with the cells seeded in the collagen sponge scaffolds without growth factors. Interestingly, COL2B, a known marker of functionally mature chondrocytes was not expressed without growth factors (Fig. 5A). When, and only when, hUCB-MSCs were incubated with growth factors, the mRNA expression levels were very close to those observed in HACs. The committed hUCB-MSCs incubated in the presence of BMP-2 and TGF-β1 showed very strong potential for stage-specific chondrogenesis.


Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering
Effect of culture conditions on the mRNA steady-state levels of specific markers in mature and hypertrophic chondrocytes and osteoblasts.Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were cultured in collagen sponges at 21% O2 for 14 days, in incomplete chondrogenic medium in the absence (control) or presence of 50 ng/ml of BMP-2 and 10 ng/ml of TGF-β1. Undifferentiated hUCB-MSCs were also cultured as monolayers and used as a control before differentiation (day 0, D0). mRNA extracts obtained from human articular chondrocytes (HACs) released from cartilage after overnight enzymatic digestion were used as controls. (A) Relative mRNA expression of chondrogenic markers. (B) Relative mRNA expression of hypertrophic chondrocyte and osteoblast markers. Bone: mRNA extracted from osteoblast cultures at passage 3 and obtained from human femoral head of osteoarthritis patients. (C) COL2A1:COL1A1 mRNA ratio. All results were normalized to RPL13a mRNA expression, compared with undifferentiated hUCB-MSCs cultured in monolayer, and presented as the relative expression of each gene. Box plots represent six independent experiments performed in triplicate. Statistically significant differences among hUCB-MSCs in monolayer and untreated or treated cells in sponges were determined using the Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001).
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f5: Effect of culture conditions on the mRNA steady-state levels of specific markers in mature and hypertrophic chondrocytes and osteoblasts.Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were cultured in collagen sponges at 21% O2 for 14 days, in incomplete chondrogenic medium in the absence (control) or presence of 50 ng/ml of BMP-2 and 10 ng/ml of TGF-β1. Undifferentiated hUCB-MSCs were also cultured as monolayers and used as a control before differentiation (day 0, D0). mRNA extracts obtained from human articular chondrocytes (HACs) released from cartilage after overnight enzymatic digestion were used as controls. (A) Relative mRNA expression of chondrogenic markers. (B) Relative mRNA expression of hypertrophic chondrocyte and osteoblast markers. Bone: mRNA extracted from osteoblast cultures at passage 3 and obtained from human femoral head of osteoarthritis patients. (C) COL2A1:COL1A1 mRNA ratio. All results were normalized to RPL13a mRNA expression, compared with undifferentiated hUCB-MSCs cultured in monolayer, and presented as the relative expression of each gene. Box plots represent six independent experiments performed in triplicate. Statistically significant differences among hUCB-MSCs in monolayer and untreated or treated cells in sponges were determined using the Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: In the absence of growth factors, hUCB-MSCs seeded in collagen sponge scaffolds exhibited only a slight increase in SOX9, COL2A1, COL2A and ACAN mRNA levels and no statistically significant differences were observed compared with undifferentiated cells (Fig. 5A). In contrast, treatment with growth factors upregulated expression of SOX9 (9-fold), COL2A1 (34,875-fold) and its isoforms COL2A (9695-fold) and COL2B (709-fold), and ACAN (24-fold) compared with the cells seeded in the collagen sponge scaffolds without growth factors. Interestingly, COL2B, a known marker of functionally mature chondrocytes was not expressed without growth factors (Fig. 5A). When, and only when, hUCB-MSCs were incubated with growth factors, the mRNA expression levels were very close to those observed in HACs. The committed hUCB-MSCs incubated in the presence of BMP-2 and TGF-β1 showed very strong potential for stage-specific chondrogenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Umbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-&beta;1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte-specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering.

No MeSH data available.


Related in: MedlinePlus