6 research outputs found
Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils
No full textReal-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils
Citation:
Pulikkot, S., Zhao, M., Fan, Z. Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils. J. Vis. Exp. (196), e64971, doi:10.3791/64971 (2023)
ESTABLISHMENT OF A 3D CULTURE MODEL OF GASTRIC STEM CELLS SUPPORTING THEIR DIFFERENTIATION INTO MUCOUS CELLS USING MICROFIBROUS POLYCAPROLACTONE SCAFFOLD
No full textIn the stomach, epithelial stem cells are responsible for glandular homeostasis and continuous production of four main cell lineages secreting mucus, acid, pepsinogen and hormones. While alteration in the proliferation and differentiation program of these stem cells is linked to the origin of gastric cancer, they represent an effective target for chemotherapy and a source for cell therapy or tissue engineering in cases of gastric mucosal damage or loss. The aims of this study were 1) to manufacture various forms of scaffolds using a biodegradable polymer (polycaprolactone), 2) to test the suitability of these scaffolds for growth of mouse gastric stem (mGS) cells, and 3) to evaluate whether this culture system could sustain exposure to acidic environment for possible future applications. Three forms of polycaprolactone scaffold were fabricated: nonporous, microporous and microfibrous. Scanning electron microscopy (SEM) and mechanical testing revealed some similarities between the microfibrous scaffold and extracellular matrix of mouse stomach wall. Examination of mGS cells seeded on different forms of scaffold for 3 days using SEM and calcein viability assay revealed their preferential growth on microfibrous scaffolds fabricated by electrospinning technique. Analysis of the growth pattern of mGS cells on microfibrous scaffolds following 3, 6, 9 and 12 days of culture using SEM and DNA PicoGreen assay demonstrated an initial increase in cell number, followed by reduction by days 9 and 12. To test whether this reduction was associated with cell differentiation, cryosections of cultured mGS cells on scaffolds were probed with gastric epithelial cell differentiation markers. On day 3, none of the markers bound to the cells. However by day 9, approximately, 50% of the cells bound to N-acetyl-Dvii glucosamine-specific lectin (Griffonia simplicifolia II) suggesting differentiation into gland mucous cells. This finding was confirmed by the expression of trefoil factor 2 using immunocytochemisty. In addition, gene expression analysis using quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the expression of transcription factor SPDEF, required for differentiation of mucous cells, was gradually up-regulated with culture of mGS cells from 3 to 12 days. To test whether this 3D culture system could tolerate the acidic environment of the stomach, the mechanical/chemical integrity of microfibrous scaffolds and cultured mGS cells were studied at acidic pH (3.0 to 7.4) using tensile strength measurements, fourier transform infrared spectroscopy, calcein assay, and mRNA/protein expression analysis. The in vitro wound-healing assay was also used to examine effects of acidic pH on cell migration. RPMI culture media at pH 3.0 and 4.5 reduced the mechanical integrity of scaffolds and significantly inhibited cell viability by \u3e70%. However, at pH 5.5 and 6.0, no significant change in cell viability and scaffold integrity was observed, but cell migration was inhibited by more than 50%. Interestingly, only after 3-day culture at pH 5.5, N-acetyl-Dglucosamine- specific lectin binding combined with significant up-regulation in the expression of SPDEF gene confirmed mucous cell differentiation. In conclusion, a 3D culture model of mGS cells using microfibrous PCL scaffold supporting their differentiation into gland mucous cells has been established. Reducing the pH value of culture media to 5.5 modulates proliferation/migration programs of mGS cells and speeds up their differentiation into mucous cells. This study provides important basic information for the possible use of mGS cells and microfibrous PCL scaffolds for future gastric tissue engineering studies and viii regenerative therapy of some stomach diseases involving gastric mucosal damage or loss
Integrin Regulators in Neutrophils
No full textNeutrophils are the most abundant leukocytes in humans and are critical for innate immunity and inflammation. Integrins are critical for neutrophil functions, especially for their recruitment to sites of inflammation or infections. Integrin conformational changes during activation have been heavily investigated but are still not fully understood. Many regulators, such as talin, Rap1-interacting adaptor molecule (RIAM), Rap1, and kindlin, are critical for integrin activation and might be potential targets for integrin-regulating drugs in treating inflammatory diseases. In this review, we outline integrin activation regulators in neutrophils with a focus on the above critical regulators, as well as newly discovered modulators that are involved in integrin activation
Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells
No full textMonitoring Circulating Myeloid Cells in Peritonitis with an In Vivo Imaging Flow Cytometer
No full textPeritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases
