The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiate them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled comprehensive analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids and proteins in cell culture is challenging due to the compound’s chemical difference so that most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two–phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C-isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS) and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over 3-4 orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0) and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications.

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The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

Molecules ◽

10.3390/molecules24193615 ◽

2019 ◽

Vol 24 (19) ◽

pp. 3615 ◽

Cited By ~ 6

Author(s):

Evelyn Rampler ◽

Dominik Egger ◽

Harald Schoeny ◽

Mate Rusz ◽

Maria Pires Pacheco ◽

...

Keyword(s):

Stromal Cells ◽

Adipogenic Differentiation ◽

Data Interpretation ◽

Molecular Study ◽

Separate Analysis ◽

Human Mscs ◽

Fat Cell ◽

Two Phase ◽

Chemical Difference ◽

Biological Validation

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiated them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids, and proteins in cell culture is challenging due to the compound’s chemical difference, so most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two–phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS), and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over three to four orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0), and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides, and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications with a high potential to understand the complex pathophysiology of diseases.

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Experimental Type 2 Diabetes Differently Impacts on the Select Functions of Bone Marrow-Derived Multipotent Stromal Cells

Cells ◽

10.3390/cells10020268 ◽

2021 ◽

Vol 10 (2) ◽

pp. 268

Author(s):

Jonathan Ribot ◽

Cyprien Denoeud ◽

Guilhem Frescaline ◽

Rebecca Landon ◽

Hervé Petite ◽

...

Keyword(s):

Type 2 Diabetes ◽

Bone Marrow ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Therapeutic Modality ◽

Multipotent Stromal Cells ◽

Cell Therapies

Bone marrow-derived multipotent stromal cells (BMMSCs) represent an attractive therapeutic modality for cell therapy in type 2 diabetes mellitus (T2DM)-associated complications. T2DM changes the bone marrow environment; however, its effects on BMMSC properties remain unclear. The present study aimed at investigating select functions and differentiation of BMMSCs harvested from the T2DM microenvironment as potential candidates for regenerative medicine. BMMSCs were obtained from Zucker diabetic fatty (ZDF; an obese-T2DM model) rats and their lean littermates (ZL; controls), and cultured under normoglycemic conditions. The BMMSCs derived from ZDF animals were fewer in number, with limited clonogenicity (by 2-fold), adhesion (by 2.9-fold), proliferation (by 50%), migration capability (by 25%), and increased apoptosis rate (by 2.5-fold) compared to their ZL counterparts. Compared to the cultured ZL-BMMSCs, the ZDF-BMMSCs exhibited (i) enhanced adipogenic differentiation (increased number of lipid droplets by 2-fold; upregulation of the Pparg, AdipoQ, and Fabp genes), possibly due to having been primed to undergo such differentiation in vivo prior to cell isolation, and (ii) different angiogenesis-related gene expression in vitro and decreased proangiogenic potential after transplantation in nude mice. These results provided evidence that the T2DM environment impairs BMMSC expansion and select functions pertinent to their efficacy when used in autologous cell therapies.

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Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Scientific Reports ◽

10.1038/s41598-021-85122-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Theresa Weickert ◽

Judith S. Hecker ◽

Michèle C. Buck ◽

Christina Schreck ◽

Jennifer Rivière ◽

...

Keyword(s):

Bone Marrow ◽

Cell Fate ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Cell Types ◽

Bone Marrow Niche ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

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Evaluation of Potential Application of Wharton’s Jelly-Derived Human Mesenchymal Stromal Cells and its Conditioned Media for Dermal Regeneration using Rat Wound Healing Model

Cells Tissues Organs ◽

10.1159/000513895 ◽

2021 ◽

pp. 1-14

Author(s):

Caroline Mathen ◽

Mrunal Ghag Sawant ◽

Raghubansh Gupta ◽

Wilfrid Dsouza ◽

Shilpa G. Krishna

Keyword(s):

Wound Healing ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Wound Care ◽

Free Cell ◽

Conditioned Media ◽

Human Umbilical Cord ◽

Human Mscs ◽

Wound Model

Mesenchymal stromal cells and the derived conditioned media represent an area of tremendous medical interest and, among other clinical applications, are currently being extensively explored for wound healing. The aim of this study was to comparatively evaluate the wound healing potential of xeno-free human umbilical cord-derived mesenchymal stromal cells (MSCs) and the conditioned media (CM) in a full-thickness excision wound model in rats. The evaluation parameters included rate of wound healing, serum cytokine analyses, collagen content, histopathology, and hyperspectral imaging as an independent qualitative and quantitative tool. Both the cell-based and cell-free approaches scored better in lower inflammation, as evidenced in lower IL-10 and stable IL-6 levels, and improved rate of wound healing (<i>p</i> < 0.0001). More importantly, no adverse reaction or rejection was observed although human MSCs and CM were used in a xenogeneic model. The presence of hFGF, hHGF, hGCSF, hIL-1Ra, hVEGF, and hIL-6 in the secretome may elucidate the regenerative potential of the xeno-free cell-based and cell-free approaches which have translational value for advanced wound care. The results revealed the therapeutic potential of both the cell-based and cell-free approaches for wound healing.

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The oncometabolite R-2-hydroxyglutarate dysregulates the differentiation of human mesenchymal stromal cells via inducing DNA hypermethylation

BMC Cancer ◽

10.1186/s12885-020-07744-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lizhen Liu ◽

Kaimin Hu ◽

Jingjing Feng ◽

Huafang Wang ◽

Shan Fu ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Chondrogenic Differentiation ◽

Cell Counting ◽

Human Mscs ◽

Mrna Levels ◽

Dependent Manner ◽

Dna Hypermethylation ◽

Cartilaginous Tumors

Abstract Background Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs. Methods Human bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in. Results R-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition. Conclusions The oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.

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Interferon-γ-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell

Blood ◽

10.1182/blood-2005-07-2793 ◽

2006 ◽

Vol 107 (6) ◽

pp. 2570-2577 ◽

Cited By ~ 221

Author(s):

John Stagg ◽

Sandra Pommey ◽

Nicoletta Eliopoulos ◽

Jacques Galipeau

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Stromal Cells ◽

Matrix Protein ◽

Marrow Stromal Cells ◽

Human Mscs ◽

Dependent Manner ◽

Antigen Presenting ◽

Significant Production

AbstractSeveral studies have demonstrated that marrow stromal cells (MSCs) can suppress allogeneic T-cell responses. However, the effect of MSCs on syngeneic immune responses has been largely overlooked. We describe here that primary MSCs derived from C57BL/6 mice behave as conditional antigen-presenting cells (APCs) and can induce antigen-specific protective immunity. Interferon gamma (IFNγ)-treated C57BL/6 MSCs, but not unstimulated MSCs, cocultured with ovalbumin-specific major histocompatibility (MHC) class II-restricted hybridomas in the presence of soluble ovalbumin-induced significant production of interleukin-2 (IL-2) in an antigen dose-dependent manner (P < .005). IFNγ-treated MSCs could further activate in vitro ovalbumin-specific primary transgenic CD4+ T cells. C57BL/6 MSCs, however, were unable to induce antigen cross-presentation via the MHC class I pathway. When syngeneic mice were immunized intraperitoneally with ovalbumin-pulsed IFNγ-treated MSCs, they developed antigen-specific cytotoxic CD8+ T cells and became fully protected (10 of 10 mice) against ovalbumin-expressing E.G7 tumors. Human MSCs were also studied for antigen-presenting functions. IFNγ-treated DR1-positive human MSCs, but not unstimulated human MSCs, induced significant production of IL-2 when cocultured with DR1-restricted influenza-specific humanized T-cell hybridomas in the presence of purified influenza matrix protein 1. Taken together, our data strongly suggest that MSCs behave as conditional APCs in syngeneic immune responses. (Blood. 2006;107:2570-2577)

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Neonatal IL-4 exposure decreases adipogenesis of male rats into adulthood

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00600.2020 ◽

2021 ◽

Author(s):

Tammy Ying ◽

Thea N. Golden ◽

Lan Cheng ◽

Jeff Ishibashi ◽

Patrick Seale ◽

...

Keyword(s):

Adipose Tissue ◽

Neonatal Period ◽

Uncoupling Protein ◽

Adipogenic Differentiation ◽

Male Rats ◽

Interleukin 4 ◽

Sprague Dawley ◽

Fat Cell ◽

Subcutaneous White Adipose Tissue

The cytokine interleukin 4 (IL-4) can increase beige adipogenesis in adult rodents. However, neonatal animals use a distinct adipocyte precursor compartment for adipogenesis compared to adults. In this study, we address whether IL-4 can induce persistent effects on adipose tissue when administered subcutaneously in the interscapular region during the neonatal period in Sprague Dawley rats. We injected IL-4 into neonatal male rats during postnatal days 1-6, followed by analysis of adipose tissue and adipocyte precursors at 2 weeks and 10 weeks of age. Adipocyte precursors were cultured and subjected to differentiation in vitro. We found that a short and transient IL-4 exposure in neonates upregulated uncoupling protein 1 (Ucp1) mRNA expression and decreased fat cell size in subcutaneous white adipose tissue (WAT). Adipocyte precursors from mature rats that had been treated with IL-4 as neonates displayed a decrease in adiponectin (Adipoq) but no change in Ucp1 expression, as compared to controls. Thus, neonatal IL-4 induces acute beige adipogenesis and decreases adipogenic differentiation capacity long term. Overall, these findings indicate that the neonatal period is critical for adipocyte development and may influence the later onset of obesity.

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