Development and Validation of a Standardized Adipogenesis Assay Using Human Bone Marrow Mesenchymal Stem Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE283759 | GEO | 2025/12/31
REPOSITORIES: GEO
Dataset's files
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| GSE283759_Count_Matrix_genes_expected_count.tsv.gz | Tabular |
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Project description:Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing and potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products (ATMPs) for cellular therapies. Since the healing of bone defects in larger bone grafts depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. We tested this by assessing the lineage (i.e. adipogenic {A}, osteogenic {O} and/or chondrogenic {C}) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic {O} potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation.
2018-12-26 | GSE117844 | GEO
Project description:The Recombinant human bone morphogenetic protein 2 (rhBMP-2) and the human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied with research and translational bone regenerative purposes. rhBMP-2 induces in vivo the endochondral ossification and bone formation processes, while hBM-MSCs are considered its target, bone forming cells. The in vitro ability of rhBMP-2 to induce hBM-MSC osteogenic differentiation has been previously tested. However, bone formation implies mesenchymal cell differentiation to multiple phenotypes, including chondrogenesis, adipogenesis and also osteogenesis. In this article, we wondered whether rhBMP-2 may drive not only osteogenic, but also multilineage differentiation of hBM-MSCs, both in vivo and in vitro. aiming to model all the differentiation processes observed in the rhBMP-2 induced in vivo bone formation. First, the rhBMP-2 and hBM-MSCs were tested in an in vivo implantation model to assess their ability to induce mature bone formation and mutilineage differentiation of implanted cells. Then, hBM-MSCs were in vitro treated with rhBMP-2 at short- and long-term cell culture periods, alone or in combination with osteogenic, adipogenic and chondrogenic media, aiming to define the role of rhBMP-2 in these differentiation processes. Data indicate that hBM-MSCs respond to rhBMP-2 at short term, but fail to differentiate in long term cultures, which was linked to the induction of rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, when rhBMP-2 is used in combination with other differentiation signals in in vitro long term cell cultures, the mentioned negative feedback loop mechanism disappears and rhBMP-2 acts as potentiator of multilineage differentiation, not only osteogenesis, but also adipogenesis and chondrogenesis. Altogether, data indicate rhBMP-2 alone is unable to induce hBM-MSC terminal differentiation in in vitro settings, but synergizes with other signals to potentiate multiple differentiation phenotypes. When using rhBMP-2 and hBM-MSCs for translational bone formation applications, they should be considered that these regenerative agents are acting in a system together with other signals, triggering multiple phenotypes depending on the signaling environment, to yield the formation of mature bone and bone marrow tissues. In vivo implantation assay and sample testing: All animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes) and internal guidelines. Experimental procedures were approved by the ethical committee and local competent authorities (Project number PRO-AE-SS-171). All animals were housed in ventilated cages and fed with standard diet ad libitum. E. coli produced recombinant BMP-2 (rhBMP-2) was obtained from commercial providers (Noricum SL, Cat.# BMP2). RNA Sequencing and data processing : The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855) and Agilent RNA 6000 Nano Chips (Agilent Technologies, Cat.# 5067-1511), respectively. Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E)” using the “TruSeq Stranded mRNA Library Prep” kit (Illumina Inc. Cat. # 20020594) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (Illumina Inc. Cat. # 20019792). Starting from 700 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat. # 18064-014) for 10 min at 25°C, 15 min at 42°C, 15 min at 70°C and pause at 4°C. cDNA second strand was synthesized with Illumina reagents at 16°C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98°C, 15 cycles of 10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C, 5 min at 72°C and pause at 4°C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626 ) and quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854).
2024-05-24 | GSE221289 | GEO
Project description:Mesenchymal stem cells (MSC) maintain the musculoskeletal system by differentiating into multiple cell types including osteocytes and adipocytes. Mechanical signals, including strain and low intensity vibration (LIV), are important regulators of MSC differentiation. Lamin A/C is a vital nucleoskeleton protein that provides mechanical properties to the nucleus and mechanical competency to MSCs. Loss of Lamin A/C has been associated with reduced structural integrity and genomic regulation of the nucleus, but direct involvement of Lamin A/C in mechanical regulation of MSC differentiation is unknown. We investigated the effects of Lamin A/C depletion on the nucleus and regulation of adipogenesis during mechanical stimulation. Upon Lamin A/C depletion, the nucleus experienced decreased area, height, volume, and stiffness while the focal adhesion phosphorylation in response to LIV and dynamic substrate strain remained intact. Cells experiencing Lamin A/C depletion undergo slower adipogenesis than control cells indicating a loss of genomic regulation due to the depletion of Lamin A/C. Mechanical stimulation via daily LIV application induced a reduction of adiponectin protein levels in both control and Lamin A/C depleted cells. RNA-seq data indicated a large adipogenic mRNA shift in control cells while Lamin A/C depleted cells showed a blunted adipogenic mRNA profile. Treatment with LIV did not induce large trascriptome changes in ether control or Lamin A/C depleted MSCs except for lowered intereferon response, suggested that LIV effects on adipogenesis may not occur at the transcriptional level. In summary, focal adhesion activation by dynamic mechanical signals and LINC complex elements largely remain unaltered under Lamin A/C depletion. While the adipogenic commitment was dependent upon Lamin A/C, the reduction of adiponectin protein in response to LIV was independent of Lamin A/C indicating that the Lamin A/C depletion and mechanical regulation of adipogenesis may not utilize similar pathways to elicit a response in MSCs.
2020-11-15 | GSE157056 | GEO
Project description:Bone marrow mesenchymal stromal cells (MSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by MSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of MSCs and thus indirectly maintaining HSCs. Bmi1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how Bmi1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of MSCs. Bmi1 is highly expressed in MSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from MSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that Bmi1 repressed multiple developmental programs in MSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which Bmi1 repressed in MSCs. Our results establish Bmi1 as a critical regulator of MSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs.
2019-04-12 | GSE121289 | GEO
Project description:Bone marrow mesenchymal stromal cells (MSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by MSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of MSCs and thus indirectly maintaining HSCs. Bmi1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how Bmi1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of MSCs. Bmi1 is highly expressed in MSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from MSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that Bmi1 repressed multiple developmental programs in MSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which Bmi1 repressed in MSCs. Our results establish Bmi1 as a critical regulator of MSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs.
2019-04-12 | GSE121288 | GEO
Project description:Background: Obesity is characterized as a disease that directly affects the whole-body metabolism and is associated with excess fat mass and several related comorbidities. Dynamics of the adipocyte hypertrophy and hyperplasia play an important role in health and disease, especially in obesity. Human adipose-derived stem cells (hASC) represent an important source for understanding the entire adipogenic differentiation process. However, little is known about the triggering step of adipogenesis in hASC. Here, we performed a proteogenomic approach for understanding the protein abundance alterations expression changes during the initiation of the adipogenic differentiation process. Methods: hASC were isolated from adipose tissue from three donors, characterized and expanded. Cells were cultured for 24 hours in adipogenic differentiation medium followed to protein extraction. We used shotgun proteomics to compare the proteomic profile of 24h-adipogenic differentiated and undifferentiated hASC. Besides, we used our previously next-generation sequencing data (RNA-seq) from the total and polysomal mRNA fractions of hASC to study post-transcriptional regulation during the initial steps of adipogenesis. Results: We identified a total of 3,.420 proteins out of and 48,.336 peptides, being 92 exclusively identified proteins in the undifferentiated hASC and 53 exclusive proteins in 24h-differentiated cells. Using a stringent criterion, we identified 33 differentially abundant proteins when compare 24h-differentiated versus undifferentiated hASC (14 upregulated and 19 downregulated, respectively). Among the upregulated proteins, we shortlist identified several adipogenic-related proteins. Combined analysis of the proteome and the transcriptome allowed the identification of positive correlation coefficients between proteins and mRNAs. Conclusions: These results demonstrate a specific proteome profile related to adipogenesis at the very beginning (24h) of the differentiation process in hASC, which represents an important piece for a better understanding of human adipogenesis and obesity. In addition, the adipogenic differentiation is fine-tuning regulated at transcriptional, post-transcriptional and post-translational levels.
2022-02-17 | PXD026299 | Pride
Project description:Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers, that direct differentiation toward soft or stiff tissue lineages. Even under controlled conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of naïve, matrix-conditioned, and early differentiation state cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We showed that soft matrices support adipogenesis of multipotent cells and endochondral ossification of non-adipogenic cells, whereas intramembranous ossification and pre-osteoblast proliferation are enhanced by stiff matrices. Using diffusion pseudotime mapping, we delineated hierarchical matrix-directed differentiation and identified mechanoresponsive genes. We found that tropomyosin-1 (TPM1) is highly sensitive to stiffness cues both at RNA and protein levels and that changes in expression of TPM1 determine adipogenic or osteogenic fates. Thus, cell-to-cell variation in tropomyosin-mediated matrix-sensing contributes to impaired differentiation with implications to the biomedical potential of MSCs.
2021-05-01 | GSE166824 | GEO
Project description:Mechanisms involved in adipose tissue expansion is of great interest in development of obesity and associated metabolic complications. Mesenchymal stem cell (MSC) differentiation in to adipocytes provides a model system to understand various gene expression pattern involved at different stages of adipogenesis. In this project we used bulk RNASeq to identify the genes that are selectively expressed at 3 different time points of adipocyte differentaition from MSCs.
2019-11-04 | GSE135775 | GEO
Project description:Here we show through genome-wide binding studies that transcription factor 7-like 1 (TCF7L1) represses structure-related genes during adipogenesis. Intriguingly, TCF7L1 is induced in a cell contact-dependent manner by confluency in preadipocytes and is required for adipocyte differentiation by repressing transcription of cell structure genes. TCF7L1 is also sufficient to bestow adipogenic potential upon non-adipogenic cells. These results implicate TCF7L1 as a novel adipogenic competency factor that uniquely determines adipogenic fate through cell structure organization required for adipocyte gene activation.
2011-09-02 | GSE31867 | GEO
Project description:Human bone marrow mesenchymal stem cells (hBM-MSCs) hold promise for treating diseases for which currently no therapeutic options exist. High quantities of MSCs are needed, requiring extensive cell expansion in long-term culture. However, the fundamental biological properties of MSCs can be altered by culture conditions. In this study, hBM-MSCs were isolated from residual human bone marrow (hBM) material and expanded to clinically relevant numbers at passage 3-4. The cells had normal morphology, proliferation rate, immunophenotype and karyotype. Despite the chromosomal stability, after additional three passages (P6-P7) hBM-MSCs entered senescence state, confirmed by SA-β-galactosidase staining and paralleling the slower proliferation, altered morphology and imunophenotype. qRT-PCR array profiling revealed 3 out of 86 genes significantly (p <0.05) differentially (â¥2 fold) expressed in the late passage cells compared to the early passage cells. qPCR gene expression profiling. hBM-MSCs from 3 (1 male and 2 females) adult donors (age 30±7.21 years) were used. Each sample was tested in technical duplicate. 6 samples of early passage (P3-P4) hBM-MSCs were grouped to CONTROL group and 6 samples of late passage (P6-P7) hBM-MSCs were grouped to TEST group. The data were analyzed using web-based RT2 Profiler PCR Array Data Analysis v3.5 software (Qiagen)
2016-02-29 | E-GEOD-68926 | biostudies-arrayexpress