Immunohistochemical Analysis of Human Mesenchymal Stem Cells Differentiating into Chondrogenic, Osteogenic, and Adipogenic Lineages

Yang, Zheng; Schmitt, Jacqueline F.; Lee, Eng Hin

doi:10.1007/978-1-60761-999-4_26

Cited by 36 publications

(37 citation statements)

References 18 publications

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“…We found that after treatment with either IL6, IL8 or both differentiation induction into mesenchymal lineages occurred remarkably fast (10 days compared to the 2–3 weeks needed for the differentiation of bone-marrow mesenchymal stem cells [61, 62]), and very efficiently (as judged by the intense and more homogenous staining in the culture dish) (Fig. 6a).…”

Section: Resultsmentioning

confidence: 99%

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

2017

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BackgroundThere is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines.MethodsThe MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231.ResultsOur results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features.ConclusionsThis study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-017-0172-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

2017

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“…Cells were seeded onto modified and unmodified hydrogels as described above. After incubation for 24 h in growth medium, cells were cultured for 21 days in osteogenic (DMEM with 10% FBS, 1 mM sodium pyruvate (Life Technologies), 100 U/100 μg penicillin–streptomycin, 10 –7 M dexamethasone (Sigma-Aldrich), 50 μg/mL ascorbic acid (Sigma-Aldrich), and 10 mM β-glycerophosphate), chondrogenic (high glucose DMEM supplemented with ITS (BD, Franklin Lakes, NJ), 4 mM l -proline (Sigma-Aldrich), 50 μg/mL ascorbic acid, 1% sodium pyruvate, 10 –7 M dexamethasone, and 100 U/100 μg penicillin–streptomycin, and 10 ng/μL TGF-β3 (R&D Systems, Minneapolis, MN)), or adipogenic (DMEM with 10% FBS, 1 mM sodium pyruvate, 100 U/100 μg penicillin–streptomycin, 10 –6 M dexamethasone, 10 μg/mL insulin (Sigma-Aldrich), 0.5 mM IBMX (Life Technologies), and 200 μM indomethacin (Life Technologies)) media as described by Yang et al 26 Growth media as well as groups cultured on TCPS were used as controls. All media were changed every 3 days.…”

Section: Materials and Methodsmentioning

confidence: 99%

Development of a Dynamic Stem Cell Culture Platform for Mesenchymal Stem Cell Adhesion and Evaluation

et al. 2014

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The importance of providing a physiologically relevant environment for cell culture is well recognized. The combination of proper environmental cues which are provided in vivo by the bloodstream and extracellular matrix must be reproduced to properly examine cell response in vitro, and cannot be recapitulated using traditional culture on polystyrene. Here, we have developed a device, the dynamic stem cell culture platform (DSCCP), consisting of a biomimetic scaffold cultured within the dynamic environment of a perfusion bioreactor. By varying scaffold parameters including stiffness and protein inclusion at the material surface, we found that human mesenchymal stem cells (hMSCs) were able to adhere to modified substrates, while still maintaining multipotency. Culture in a perfusion bioreactor showed cell survival and proliferation, particularly on modified substrates. The DSCCP represents a complete platform for cell adhesion and subsequent evaluation, including the response of a cell population to drug treatment.

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“…Optimizing these microenvironmental cues is of particular interest in regenerative medicine where biomaterial substrates may serve as a vehicle for directed cell delivery or as a system for tissue formation in-vivo . Commonly used methods for assessing microenironment modulated osteogenic and adipogenic differentiation include reverse transcriptase PCR (RT-PCR) for detection of osteogenic or adipogenic transcripts, immunohistochemical analysis, enzyme linked immunosorbent assay (ELISA) or western blotting for secreted protein markers, colorimetric assays, and direct staining of matrix components 8, 9 . To aid in identifying optimal microenvironments that efficiently elicit strategic stem cell phenotypes, high-throughput screening (HTS) approaches that allow for the assessment of up to several thousand conditions within one experiment have also been developed 10–12 .…”

Section: Introductionmentioning

confidence: 99%

Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling

et al. 2015

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Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to > 90% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, β-catenin, and FAK could be classified with > 90% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a “fingerprint” of the intracellular mechanotransductive signaling that governs growth factor- and topography-responsive stem cell states.

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Immunohistochemical Analysis of Human Mesenchymal Stem Cells Differentiating into Chondrogenic, Osteogenic, and Adipogenic Lineages

Cited by 36 publications

References 18 publications

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

Development of a Dynamic Stem Cell Culture Platform for Mesenchymal Stem Cell Adhesion and Evaluation

Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling

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