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Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors.

Mo IF, Yip KH, Chan WK, Law HK, Lau YL, Chan GC - BMC Cell Biol. (2008)

Bottom Line: We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12.But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression.In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China. irene_hku@yahoo.com

ABSTRACT

Background: Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.

Results: We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria.

Conclusion: In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

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Related in: MedlinePlus

Effects of LPS and LTA on the proliferation of human MSCs and PBMCs. Cultured cells were incubated with either LPS (left panel) or LTA (right panel) for 3 days or 7 days. On Day 3 and Day 7, the cell proliferation responses of (A) MSCs and (B) PBMCs were assessed by XTT assay respectively. LPS and LTA did not affect the proliferation of MSCs (pooled data from three experiments, each done in triplicates) but increased proliferation of PBMCs (one representative experiment done in triplicates). The data are expressed as the relative proliferation of untreated controls, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 versus control.
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Figure 1: Effects of LPS and LTA on the proliferation of human MSCs and PBMCs. Cultured cells were incubated with either LPS (left panel) or LTA (right panel) for 3 days or 7 days. On Day 3 and Day 7, the cell proliferation responses of (A) MSCs and (B) PBMCs were assessed by XTT assay respectively. LPS and LTA did not affect the proliferation of MSCs (pooled data from three experiments, each done in triplicates) but increased proliferation of PBMCs (one representative experiment done in triplicates). The data are expressed as the relative proliferation of untreated controls, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 versus control.

Mentions: The multipotency of MSCs used in this investigation was confirmed by standard differentiation assays along three lineages including osteogenic, adipogenic and chondrogenic. All the MSCs from the three donors showed similar differentiating functions. We then tested whether the MSCs responded to bacterial toxins LPS or LTA. As shown in Figure 1A, LPS (left panel) and LTA (right panel) did not affect the proliferation of MSCs during either the 3- or 7-day period, even after incubation with a relatively high dose of toxin (1 μg/mL of LPS and 10 μg/mL of LTA). As expected, these toxins evoked a marked increase in proliferation of peripheral mononuclear cells (PBMCs) in compared to untreated controls (Figure 1B) (n = 3, P < 0.01). For the response of PBMCs to bacterial toxins, 1 ng/mL LPS was sufficient to induce a significant increase in proliferation on Day 3 and Day 7. Since LTA is less potent as compared to LPS, higher dosages of LTA, 10 ng/mL and 100 ng/mL, were needed to generate a significant response on Day 7 and Day 3 respectively.


Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors.

Mo IF, Yip KH, Chan WK, Law HK, Lau YL, Chan GC - BMC Cell Biol. (2008)

Effects of LPS and LTA on the proliferation of human MSCs and PBMCs. Cultured cells were incubated with either LPS (left panel) or LTA (right panel) for 3 days or 7 days. On Day 3 and Day 7, the cell proliferation responses of (A) MSCs and (B) PBMCs were assessed by XTT assay respectively. LPS and LTA did not affect the proliferation of MSCs (pooled data from three experiments, each done in triplicates) but increased proliferation of PBMCs (one representative experiment done in triplicates). The data are expressed as the relative proliferation of untreated controls, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567970&req=5

Figure 1: Effects of LPS and LTA on the proliferation of human MSCs and PBMCs. Cultured cells were incubated with either LPS (left panel) or LTA (right panel) for 3 days or 7 days. On Day 3 and Day 7, the cell proliferation responses of (A) MSCs and (B) PBMCs were assessed by XTT assay respectively. LPS and LTA did not affect the proliferation of MSCs (pooled data from three experiments, each done in triplicates) but increased proliferation of PBMCs (one representative experiment done in triplicates). The data are expressed as the relative proliferation of untreated controls, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 versus control.
Mentions: The multipotency of MSCs used in this investigation was confirmed by standard differentiation assays along three lineages including osteogenic, adipogenic and chondrogenic. All the MSCs from the three donors showed similar differentiating functions. We then tested whether the MSCs responded to bacterial toxins LPS or LTA. As shown in Figure 1A, LPS (left panel) and LTA (right panel) did not affect the proliferation of MSCs during either the 3- or 7-day period, even after incubation with a relatively high dose of toxin (1 μg/mL of LPS and 10 μg/mL of LTA). As expected, these toxins evoked a marked increase in proliferation of peripheral mononuclear cells (PBMCs) in compared to untreated controls (Figure 1B) (n = 3, P < 0.01). For the response of PBMCs to bacterial toxins, 1 ng/mL LPS was sufficient to induce a significant increase in proliferation on Day 3 and Day 7. Since LTA is less potent as compared to LPS, higher dosages of LTA, 10 ng/mL and 100 ng/mL, were needed to generate a significant response on Day 7 and Day 3 respectively.

Bottom Line: We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12.But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression.In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China. irene_hku@yahoo.com

ABSTRACT

Background: Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.

Results: We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria.

Conclusion: In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

Show MeSH
Related in: MedlinePlus