Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation

Ayanoğlu, Fatma Betül; Elçin, Ayşe Eser; Elçin, Yaşar Murat

doi:10.1007/s11033-020-05311-y

Cited by 12 publications

(9 citation statements)

References 49 publications

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“…Therefore, its use as a reference would not be optimal for use when comparing changes in gene expression associated with induction of differentiation over time [35]. This finding has been reported from studies of differentiation of human adipose-derived mesenchymal stem cells [36], from human liver samples [37], in non-alcoholic fatty liver disease (NAFLD) animal models [38], different pig tissues [39], and plant samples [40]. Those reports show that when GAPDH was used as an internal control, the investigated GOI had no significant change or had fluctuating patterns of expression, indicating that GAPDH is unreliable for RT-qPCR analysis.…”

Section: Discussionmentioning

confidence: 97%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde-hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

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Section: Discussionmentioning

confidence: 97%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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“…We actually found ACTB to be acceptably stable for hBM-MSC but less consistent for hAT-MSC. We did not agree that GUSB was a poor reference gene for hAT-MSC [70], perhaps reflecting our use of FBS-free culture media. The MIQE guidelines discouraged large discrepancies in transcript abundance between reference and target genes and recommend more than one reference gene [71].…”

Section: Discussionmentioning

confidence: 87%

Qualifying Osteogenic Potency Assay Metrics for Human Multipotent Stromal Cells: TGF-β2 a Telling Eligible Biomarker

Ofiteru

Becheru

Gharbia

et al. 2020

Cells

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Potency assays are critical for regenerative medicine, addressing the known challenge of functional heterogeneity among human multipotent stromal cells (hMSC). Necessary laboratory cell expansion allows analysis before implantation in the patient. Levels of induction of five signature gene biomarkers, ALPL, COL1A2, DCN, ELN and RUNX2, constituted a previously reported proof-of-principle osteogenic potency assay. We tested assay modification to enhance reproducibility using six consistent bone marrow derived hBM-MSC and explored applicability to three adipose tissue derived hAT-MSC. Using a potent proprietary osteogenic induction factor, the GUSB/YWAHZ reference gene pair provided real time PCR consistency. The novel assay conditions supported the concept that genes encoding extracellular matrix proteins one week after osteogenic induction were informative. Nonetheless, relatively low induction of COL1A2 and ELN encouraged search for additional biomarkers. TGFB2 mRNA induction, important for osteogenic commitment, was readily quantifiable in both hBM-MSC and hAT-MSC. Combined with DCN, TGFB2 mRNA induction data provided discriminatory power for resolving donor-specific heterogeneity. Histomorphometric decorin and TGF-β2 protein expression patterns in eight-week heterotopic bone implants also discriminated the two non-bone-forming hMSC. We highlight progress towards prompt osteogenic potency assays, needed by current clinical trials to accelerate improved intervention with enhanced stem cell therapy for serious bone fractures.

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“…For instance, PCS‐500‐011 primary human ADMSCs served as a model for studying mechanisms related to human MSC differentiation (Choi et al, 2015; Gonzalez Suarez et al, 2021; Li et al, 2013, 2019; Yang et al, 2018). They were also employed for evaluating the stability of standard reference genes during the proliferation and differentiation of human ADMSCs (Ayanoglu et al, 2020) and contributing to cell validation in karyotypic analyses of MSCs (Hwang et al, 2013). Beyond MSC differentiation, PCS‐500‐011 has found application in exometabolome research (Bispo et al, 2022) and investigations concerning paracrine secretory factors involved in angiogenesis in humans (Sun et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

The optimized priming effect of FGF‐1 and FGF‐2 enhances preadipocyte lineage commitment in human adipose‐derived mesenchymal stem cells

Tarapongpun,

Onlamoon,

Tabu

et al. 2024

Genes to Cells

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The cell‐assisted lipotransfer technique, integrating adipose‐derived mesenchymal stem cells (ADMSCs), has transformed lipofilling, enhancing fat graft viability. However, the multipotent nature of ADMSCs poses challenges. To improve safety and graft vitality and to reduce unwanted lineage differentiation, this study refines the methodology by priming ADMSCs into preadipocytes—unipotent, self‐renewing cells. We explored the impact of fibroblast growth factor‐1 (FGF‐1), fibroblast growth factor‐2 (FGF‐2), and epidermal growth factor (EGF), either alone or in combination, on primary human ADMSCs during the proliferative phase. FGF‐2 emerged as a robust stimulator of cell proliferation, preserving stemness markers, especially when combined with EGF. Conversely, FGF‐1, while not significantly affecting cell growth, influenced cell morphology, transitioning cells to a rounded shape with reduced CD34 expression. Furthermore, co‐priming with FGF‐1 and FGF‐2 enhanced adipogenic potential, limiting osteogenic and chondrogenic tendencies, and possibly promoting preadipocyte commitment. These preadipocytes exhibited unique features: rounded morphology, reduced CD34, decreased preadipocyte factor 1 (Pref‐1), and elevated C/EBPα and PPARγ, alongside sustained stemness markers (CD73, CD90, CD105). Mechanistically, FGF‐1 and FGF‐2 activated key adipogenic transcription factors—C/EBPα and PPARγ—while inhibiting GATA3 and Notch3, which are adipogenesis inhibitors. These findings hold the potential to advance innovative strategies for ADMSC‐mediated lipofilling procedures.

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Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation

Cited by 12 publications

References 49 publications

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Qualifying Osteogenic Potency Assay Metrics for Human Multipotent Stromal Cells: TGF-β2 a Telling Eligible Biomarker

The optimized priming effect of FGF‐1 and FGF‐2 enhances preadipocyte lineage commitment in human adipose‐derived mesenchymal stem cells

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