Cell & Molecular Biology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30432


Recent Submissions

Now showing 1 - 4 of 4
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    Characterization of adipose tissue and adipose-tissue derived stem cells in Lipedema
    (2021) Al-Ghadban, Sara; Bunnell, Bruce
    Purpose: Lipedema is a painful adipose tissue (AT) disorder that occurs almost exclusively in women, with onset manifesting at puberty or at times of hormonal change. This disorder is characterized by a symmetrical increase of fat deposition in the legs and the arms, sparing the hands and the feet. The goal of this study was to determine the histology of the skin and fat of lipedema tissue and characterize the adipose tissue-derived stem cells (ASCs) obtained from the stromal vascular fraction (SVF) of thigh AT of non-lipedema and lipedema patients. Methods: Histological sections from AT were stained with H&E. Adipocyte area was quantified using ImageJ software. Markers for macrophages (CD68) was investigated by immunohistochemistry. ASCs were characterized by the expression of stemness markers and their multi-differentiation potential. Flow cytometry, RT-PCR and immunofluorescence assays were performed. Results: The data show hypertrophic adipocytes, increased numbers of macrophages and blood vessels in thigh tissue of women with lipedema compared to non-lipedema patients. Additionally, at the cellular level, an increase in the adipogenic differentiation potential with no change in the expression of mesenchymal markers (CD73, CD90 and CD105) or extracellular markers (collagen, fibronectin and laminin) was detected in lipedema ASCs compared to non-lipedema ASCs. Conclusion: Infiltration of immune cells, increase in adipocyte size and adipogenesis stimulates angiogenesis and fibrosis in lipedema AT. Defining the structure and the components of AT will provide insights into the pathophysiology of lipedema and will help researchers develop potential treatment for the disease.
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    Store-Operated Calcium Entry regulated IL6 expression and matrix proteins in glomerular mesangial cells
    (2021) Chaudhari, Sarika; Tao, Yu; Yazdizadeh Shotorbani, Parisa; Ma, Rong
    Background: Glomerular mesangial cells (MCs) produce various cytokines in response to multiple stimuli. MCs also contribute to matrix expansion in kidney diseases. The aim of this study was to determine whether interleukin-6 (IL6) participated in matrix protein turnover and if Orai1-mediated store-operated calcium entry (SOCE) in MCs regulate IL6. Methods: In cultured human MCs, expression of target proteins was examined in culture media and whole cell lysates using ELISA and Western blot analysis, respectively. Overexpression of IL6 was achieved using the expression plasmid for IL6 while knockdown of Orai1 using Orai1 siRNA. Orai1 in MCs was knocked down using the targeted nanoparticle-siRNA delivery system in wild type C57BLKS/J mice at the age of 16 weeks. Glomerular IL6 expression was evaluated using immunohistochemistry of kidney sections. Results: In human MCs, overexpression of IL6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. Thapsigargin (activator of SOCE, 1µM) significantly increased IL6 level in supernatant media and in whole cell lysates of MCs at 15 hrs, and this effect was attenuated by GSK 7975A (selective inhibitor of SOCE). Inhibition of NFкB pathway significantly blunted the thapsigargin-induced increase in IL6 protein abundance. Moreover, thapsigargin stimulated the nuclear translocation of p65 in human MCs. IL6 expression was reduced in the glomeruli of the mice treated with nanoparticle/Orai1 siRNA for 2 weeks compared to control mice. Conclusion: SOCE positively regulates IL6 production by MCs through activation of NFкB pathway and IL6 in turn inhibits matrix proteins.
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    Molecular characterization of adipose-tissue derived stem cells (ASCs) from the breast
    (2021) Artiles, Maria; Bunnell, Bruce; Al-Ghadban, Sara
    Purpose: It has been reported that both the health of the donor, as well as the site of collection can alter the proliferation and differentiation properties of adipose tissue-derived stem cells (ASCs) . Obesity alters ASCs and induces them to enhance the tumorigenic properties of breast cancer cell lines both in vitro and in vivo. Abdominal ASCs (aASCs) from obese donors show increased expression of leptin and estrogen. Leptin signaling cascades and estrogen-mediated pathways can increase breast cancer proliferation, tumorigenesis and metastasis. The aim of this study is to characterize ASCs derived from breast tissue (bASCs) of human donors and compare their proliferation and differentiation properties to those of aASCs, and to understand if bASCs are affected in the same way. Methods: bASCs will be characterized and compared to aASCs. Adipogenic and osteogenic differentiation will be induced and assessed with Oil Red O and Alizarin Red S staining, respectively. Colony forming capabilities will be assessed with crystal violet staining. Surface markers will be evaluated with flow cytometry. Expression of adipogenic genes will be assessed by real-time PCR. Results: bASCs are expected to display CD90, CD105 and CD73 surface markers. They are also expected to successfully undergo adipogenic and osteogenic differentiation and to be capable to form colonies. Finally, it is expected to observe differences in proliferation rates between aASCs and bASCs. Conclusion: Characterizing bASCs, and comparing them to aASCs, will provide us an understanding of the unique properties of this subpopulation and their potential applications.
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    In-vitro Blood-Brain Barrier Models: An Emerging Platform for Central Nervous System Drug Discovery and Delivery
    (2021) Shah, Brijesh; Dong, Xiaowei
    Purpose: Blood-brain barrier (BBB) and its components protects the brain, but drug delivery sufficient to induce clinical effects is hindered causing challenges for therapeutic translation. To study drugs and their delivery systems across the BBB, numerous in-vitro BBB models have been studied. However, the limitations among them are still unanswered causing poor in-vivo imitation. Therefore, we performed an exhaustive literature study on in-vitro BBB models/methods to summarize which model/method could be futuristic. Method: Literature search in PubMed showed that nearly 400 research and review reports were published in last two decades. Three major models including two-dimensional (2D), stem cell and three-dimensional (3D) models were discussed and compared. Each type of model was reviewed in depth to identify an ideal in-vitro model that could closely mimic the in-vivo BBB microvasculature. Results: In 2D models, while cocultures over monocultures showed improved barrier integrity none of them mimicked shear stress. Additionally, the results between double and triple cocultures were controversial. Irrespective of model types, human stem cells revealed an excellent barrier phenotype against other cellular sources. 3D models such as chip-based and microfluidic encompassing multiple compartments and shear stress imitated the BBB microenvironment more realistically than 2D models. However, cost, complex design and poor scalability are few setbacks of 3D models. Conclusion: Considering key limitations of current in-vitro BBB models, the microfluidic technology in combination with stem cells would be the next generation in-vitro models that could best mimic the BBB phenotype.