A biological study establishing the endotoxin limit for osteoblast and adipocyte differentiation of human mesenchymal stem cells

Abstract

IntroductionMultipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions, such as hypoxia or inflammation after transplantation in vivo. Although endotoxin testing is required for evaluating the quality and safety of transplanted MSCs, no reports on their dose response to endotoxins are available to establish the limits for in vitro MSC culture systems. In the present study, we aimed to accurately quantify the risk of endotoxin contamination in cell culture systems to establish an acceptable endotoxin limit for the differentiation of hMSC osteoblasts and adipocytes.MethodsThree types of bone marrow-derived hMSCs (hMSC-1: 21-year-old, M/B; hMSC-2: 36-year-old, M/B; hMSC-3: 43-year-old, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in osteogenic or adipogenic differentiation media, respectively, from commercial kits, containing various concentrations of endotoxin (0.01–100 ng/ml). The degree of adipocyte and osteoblast differentiation was estimated by fluorescent staining of lipid droplets and hydroxyapatite, respectively. To clarify the molecular mechanism underlying the effect of endotoxin on hMSC differentiation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis.ResultsAlthough endotoxin did not effect the adipocyte differentiation of hMSCs, osteoblast differentiation was enhanced by various endotoxin concentrations: over 1 ng/ml, for hMSC-1; 10 ng/ml, for hMSC-2; and 100 ng/ml, for hMSC-3. Proteomic analysis of hMSC-1 cells revealed up-regulation of many proteins related to bone formation. These results suggested that endotoxin enhances the osteoblast differentiation of MSCs depending on the cell type.ConclusionsSince endotoxins can affect various cellular functions, an endotoxin limit should be established for in vitro MSC cultures. Its no-observed-adverse-effect level was 0.1 ng/ml based on the effect on the hMSC osteoblast differentiation, but it may not necessarily be the limit for ADSCs.

Performance evaluation of bactericidal effect and endotoxin inactivation by low‐temperature ozone/hydrogen peroxide mixed gas exposure

“…It has been reported that the successful pregnancy rate in assisted reproduction decreased from 73 to 33% when the culture medium was contaminated with more than 2 pg/ml of endotoxin 26 . In addition, endotoxin levels greater than 0.1 ng/ml enhanced the proliferative and osteogenic potential of human mesenchymal stem cells 27,28 . These findings indicate that there is a need to minimize endotoxin levels to improve the quality of utensils used in the manufacturing of products for regenerative medicine.…”

Dlx5 Represses the Transcriptional Activity of PPARγ

“…5) MSCs are converted into committed whole adipocytes and then differentiated into mature adipocytes. 6,7) Therefore, inhibiting adipocyte differentiation is an effective strategy for the prevention and treatment of obesity.…”