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Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells.

ABSTRACT: Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated osteoblasts), and the average membrane tether length measured with laser optical tweezers (10.6 +/- 1.1 microm for stem cells, and 4.0 +/- 1.1 microm for osteoblasts). These differences in cell elasticity and membrane mechanics result primarily from differential actin cytoskeleton organization in these two cell types, whereas microtubules did not appear to affect the cellular mechanics. The membrane-cytoskeleton linker proteins may contribute to a stronger interaction of the plasma membrane with F-actins and shorter membrane tether length in osteoblasts than in stem cells. Actin depolymerization or ATP depletion caused a two- to threefold increase in the membrane tether length in osteoblasts, but had essentially no effect on the stem-cell membrane tethers. Actin remodeling in the course of a 10-day osteogenic differentiation of hMSC mediates the temporally correlated dynamical changes in cell elasticity and membrane mechanics. For example, after a 10-day culture in osteogenic medium, hMSC mechanical characteristics were comparable to those of mature bone cells. Based on quantitative characterization of the actin cytoskeleton remodeling during osteodifferentiation, we postulate that the actin cytoskeleton plays a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics at the early stage of stem-cell osteodifferentiation.

SUBMITTER: Titushkin I

PROVIDER: S-EPMC2072058 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells.

Titushkin Igor I Cho Michael M

Biophysical journal 20070803 10

Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated os ...[more]

PMID: 17675345

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Project description:Bone marrow mesenchymal stem cells (BMSCs) nowadays are regarded as promising candidates in cell-based therapy for the regeneration of damaged bone tissues that are either incurable or intractable due to the insufficiency of current therapies. Recent studies suggest that BMSCs differentiate into osteoblasts, and that this differentiation is regulated by some specific patterns of epigenetic modifications, such as DNA methylation. However, the potential role of DNA methylation modification in BMSC osteogenic differentiation is unclear. In this study, we performed a genome-wide study of DNA methylation between the noninduced and induced osteogenic differentiation of BMSCs at day 7. We found that the majority of cytosines in a CpG context were methylated in induced BMSCs. Our results also revealed that, along with the induced osteogenic differentiation in BMSCs, the average genomic methylation levels and CpG methylation in transcriptional factor regions (TFs) were increased, the CpG methylation level of various genomic elements was mainly in the medium-high methylation section, and CpG methylation levels in the repeat element had highly methylated levels. The GO analysis of differentially methylated region- (DMR-) associated genes (DMGs) showed that GO terms, including cytoskeletal protein binding (included in Molecular Function GO terms), skeletal development (included in Biological Process GO terms), mesenchymal cell differentiation (included in Biological Process GO terms), and stem cell differentiation (included in Biological Process), were enriched in the hypermethylated DMGs. Then, the KEGG analysis results showed that the WNT pathway, inositol phosphate metabolism pathway, and cocaine addiction pathway were more correlative with the DMRs during the induced osteogenic differentiation in BMSCs. In conclusion, this study revealed the difference of methylated levels during the noninduced and induced osteogenic differentiation of BMSCs and provided useful information for future works to characterize the important function of epigenetic mechanisms on BMSCs' differentiation.

Project description:Human bone marrow mesenchymal stem cells (hBMSCs) are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP) activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions). The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient's own cell images to predict a new patient's cellular potential. The prediction accuracy was found to be greatly enhanced by incorporation of patients' own cell features in the modeling, indicating the practical strategy for clinical usage. Consequently, our results provide strong evidence for the feasibility of using a quantitative time series of phase-contrast cellular morphology for non-invasive cell quality prediction in regenerative medicine.

Dataset Information

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells.

Publications

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells.

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