Dataset Information

Evaluation of metabolomic changes as a biomarker of chondrogenic differentiation in 3D-cultured human mesenchymal stem cells using proton (1H) nuclear magnetic resonance spectroscopy.

ABSTRACT:

Purpose

The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using (1)H nuclear magnetic resonance (NMR) spectroscopy.

Materials and methods

hMSCs (2×10(6) cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (600-MHz) NMR with the water suppression sequence, zgpr. Real-Time polymerase chain reaction (PCR) was performed to confirm that that the hMSCs differentiated into chondrocytes and to analyze the metabolomic changes indicated by the NMR spectra.

Results

During chondrogenesis, changes were detected in several metabolomes as hMSC chondrogenesis biomarkers, e.g., fatty acids, alanine, glutamate, and phosphocholine. The metabolomic changes were compared with the Real-Time PCR results, and significant differences were determined using statistical analysis. We found that changes in metabolomes were closely related to biological reactions that occurred during the chondrogenesis of hMSCs.

Conclusions

In this study, we confirm that metabolomic changes detected by (1)H-NMR spectroscopy during chondrogenic differentiation of 3D-cultured hMSCs in alginate beads can be considered as biomarkers of stem cell differentiation.

SUBMITTER: Jang MY

PROVIDER: S-EPMC3804484 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Evaluation of metabolomic changes as a biomarker of chondrogenic differentiation in 3D-cultured human mesenchymal stem cells using proton (1H) nuclear magnetic resonance spectroscopy.

Jang Moo-Young MY Chun Song-I SI Mun Chi-Woong CW Hong Kwan Soo KS Shin Jung-Woog JW

PloS one 20131021 10

<h4>Purpose</h4>The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using (1)H nuclear magnetic resonance (NMR) spectroscopy.<h4>Materials and methods</h4>hMSCs (2×10(6) cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (60 ...[more]

PMID: 24205199

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Project description:Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa?Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface.Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm.Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm.DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87.Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm.1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility.This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Dataset Information

Evaluation of metabolomic changes as a biomarker of chondrogenic differentiation in 3D-cultured human mesenchymal stem cells using proton (1H) nuclear magnetic resonance spectroscopy.

Purpose

Materials and methods

Results

Conclusions

Publications

Evaluation of metabolomic changes as a biomarker of chondrogenic differentiation in 3D-cultured human mesenchymal stem cells using proton (1H) nuclear magnetic resonance spectroscopy.

Similar Datasets

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