Characterization of human skeletal stem and bone cell populations using dielectrophoresis

Ismail, Abdel Azim A.; Hughes, Michael; Mulhall, Hayley J.; Oreffo, Richard O.C.; Labeed, Fatima H.

doi:10.1002/term.1629

Cited by 38 publications

(31 citation statements)

References 31 publications

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“…3.1 HL-60, MG-63, MSCs and skeletal stem cells MG-63, MSCs and SSCs are large cells with considerable heterogeneity. 47 The channel in the RT-DC device needed to be wide enough to allow the larger cells to flow through, but also not so large that smaller cells would not deform. 36 HL-60 are generally measured using 20 mm Â 20 mm cross-section channels, but for this work 30 mm Â 30 mm channels were used to accommodate the larger MG-63 and SSCs.…”

Section: Resultsmentioning

confidence: 99%

Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry

et al. 2016

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Skeletal stem cells (SSCs) are a sub-population of mesenchymal stromal cells (MSCs) present in bone marrow with multipotent differentiation potential. A current unmet challenge hampering their clinical translation remains the isolation of homogeneous populations of SSCs, in vitro, with consistent regeneration and differentiation capacities. Cell stiffness has been shown to play an important role in cell separation using microfluidic techniques such as inertial focusing or deterministic lateral displacement. Here we report that the mechanical properties of SSCs, and of a surrogate human osteosarcoma cell line (MG-63), differ significantly from other cell populations found in the bone marrow. Using real-time deformability cytometry, a recently introduced method for cell mechanical characterization, we demonstrate that both MG-63 and SSCs are stiffer than the three primary leukocyte lineages (lymphocytes, monocytes and granulocytes) and also stiffer than HL-60, a human leukemic progenitor cell line. In addition, we show that SSCs form a mechanically distinct sub-population of MSCs. These results represent an important step towards finding the bio-physical fingerprint of human SSCs that will allow their label-free separation from bone marrow with significant physiological and therapeutic implications.

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Section: Resultsmentioning

confidence: 99%

Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry

et al. 2016

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“…C mem is an important factor in understanding exocytosis, endocytosis and stimulus secretion mechanism that vary with biochemical cell cycle. Conductivity is a measure of the ability to conduct an electric field while the permittivity is a measure of cytoplasm or cell membrane to transmit the electric field [18]. …”

Section: Resultsmentioning

confidence: 99%

“…In cell biology, DEP can provide a quantitative measure of cellular properties and in our previous studies it was used to investigate cell separation and characterization along with identifying suitable buffers that could be used for cell studies [17]. DEP has been employed in a variety of cancer cell studies and is often used to characterizing cell sub populations [18]. We have also incorporated DEP as a tool to study and characterize dielectric properties of cancer cells at different stages of cell cycle.…”

Section: Introductionmentioning

confidence: 99%

Monitoring drug induced apoptosis and treatment sensitivity in non-small cell lung carcinoma using dielectrophoresis

Kumar

Liu

Minna

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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Non-invasive real time methods for characterizing biomolecular events that contribute towards apoptotic kinetics would be of significant importance in the field of cancer biology. Effective drug-induced apoptosis is an important factor for establishing the relationship between cancer genetics and treatment sensitivity. The objective of this study was to develop a non-invasive technique to characterize cancer cells that are undergoing drug-induced apoptosis. We used dielectrophoresis to determine apoptotic cells as early as 2 h post drug treatment as compared to 24 h with standard flow cytometry method using non-small cell lung cancer (NSCLC) adenocarcinoma cell line (HCC1833) as a study model. Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where treated with a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over 24 h period after exposure to ABT-263 at clinically relevant concentrations. The dielectrophoretic studies were compared to Annexin-V FITC flow assay for the detection of PS in mid-stage apoptosis using flow cytometry. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance. In addition, zeta potential of these fixed isolated cells was measured to obtain direct correlation to biomolecular events.

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“…In addition, it has been shown that CSCs are highly resistant to standard therapy 13 . Stemness-related markers in OS are ABCG2, Nestin, CD44, STRO-1, and Endoglin/CD105 [14][15][16][17] . Previously, we identified a novel protein doublecortin calmodulin-like kinase 1 (DCLK1) that is present in reserve quiescent stem cells in both the normal intestinal epithelium and in colon cancers 18,19 .…”

Section: Introductionmentioning

confidence: 99%

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

et al. 2020

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Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

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Characterization of human skeletal stem and bone cell populations using dielectrophoresis

Cited by 38 publications

References 31 publications

Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry

Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry

Monitoring drug induced apoptosis and treatment sensitivity in non-small cell lung carcinoma using dielectrophoresis

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

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