optimization-wound-scratch-assay-to-detect-changes-murine-mesenchymal

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

College of The Holy Cross

The use of an evidence-based approach allows veterinary clinicians to assess questions that are clinically relevant to the diagnosis and treatment of equine gastrointestinal tract disease. This approach involves formulating a clinical question, searching the literature, and answering the question with the best available evidence, with the results summarized as a clinical "bottom line." This article is organized to reinforce the principle that the cornerstone of evidence-based medicine is the clinical question. Specific questions are further categorized as to topic, with epidemiologic risk factors, diagnostic process, clinical examination, differential diagnosis, diagnostic tests, treatment, harm, prognosis, and prevention as general themes. The topics covered in this article are by no means exhaustive but give an example of how the veterinary literature can be used to answer clinically important questions in an evidence-based manner.

Evidence-based gastrointestinal medicine in horses: it's not about your gut instincts.

Adult mice have a remarkable capacity to regenerate functional alveoli following either lung resection or injury that exceeds the regenerative capacity observed in larger adult mammals. The molecular basis for this unique capability in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling mechanisms used in this species during lung regeneration.

Global gene expression patterns in the post-pneumonectomy lung of adult mice.

While multipotent mesenchymal stromal cells have been recently isolated from adult lung (L-MSCs), there is very limited data on their biological properties and therapeutic potential in vivo. How L-MSCs compare with bone marrow-derived MSCs (BM-MSCs) is also unclear. In this study, we characterized L-MSC phenotype, clonogenicity, and differentiation potential, and compared L-MSCs to BM-MSCs in vivo survival, retention, paracrine gene expression, and repair or elastase injury after transplantation. L-MSCs were highly clonogenic, frequently expressed aldehyde dehydrogenase activity, and differentiated into osteocytes, chondrocytes, adipocytes, myofibroblasts, and smooth muscle cells. After intravenous injection (2 h), L-MSCs showed greater survival than BM-MSCs; similarly, L-MSCs were significantly more resistant than BM-MSCs to anchorage independent culture (4 h) in vitro. Long after transplantation (4 or 32 days), a significantly higher number of CD45(neg) L-MSCs were retained than BM-MSCs. By flow cytometry, L-MSCs expressed more intercellular adhesion molecule-1 (ICAM-1), platelet derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs; these proteins were found to modulate endothelial adherence, directional migration, and migration across Matrigel in L-MSCs. Further, L-MSCs with low ICAM-1 showed poorer lung retention and higher phagocytosis in vivo. Compared with BM-MSCs, L-MSCs expressed higher levels of several transcripts (e.g., Ccl2, Cxcl2, Cxcl10, IL-6, IL-11, Hgf, and Igf2) in vitro, although gene expression in vivo was increased by L-MSCs and BM-MSCs equivalently. Accordingly, both L-MSCs and BM-MSCs reduced elastase injury to the same extent. This study demonstrates that tissue-specific L-MSCs possess mechanisms that enhance their lung retention after intravenous transplantation, and produce substantial healing of elastase injury comparable to BM-MSCs.

Lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of elastase-injured lung.

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.

Age-dependent decline in mouse lung regeneration with loss of lung fibroblast clonogenicity and increased myofibroblastic differentiation.

Aging is a critical determinant of regenerative capacity in many organ systems, but it remains unresolved in the lung. This study examines murine lung cell dynamics during age-dependent lung regeneration. Proliferation of lung progenitor cells (EpCAM(neg)/Sca-1(high) lung mesenchymal stromal cells - LMSCs, EpCAM(pos)/Sca-1(low) epithelial progenitor cells, proSP-C(pos) alveolar type II epithelial cells - AECII, and CD31(pos) - endothelial cells) was tracked to day 3 or 7 after pneumonectomy (PNX) or SHAM surgery in 3, 9, and 17 month mice. In 3 month mice, post-PNX LMSC proliferation peaked early (3 days), with 50%-80% more BrdU-positive cells than the other cell types, which peaked later (4-7 days). In older mice (9 and 17 month), abundance and post-PNX proliferation of LMSCs at day 3 were reduced (40%-80%). In both young and old mice, LMSCs were isolated and compared phenotypically with whole lung non-LMSCs. Donor age had no qualitative effect on the phenotype (LMSC vs. non-LMSC), with increased expression of CD90/Thy1, CD105/Eng, CD106/Vcam, CD146/Mcam, and Pdgfrα, and up-regulation of mRNA encoding Fap, Eln, Col1a1, Col3a1, Aldh1a3, Arhgef25, Dner, Fgfr1, and Midkine. However, compared with LMSCs isolated from young mice, LMSCs from older mice exhibited reduced mRNA expression of retinoic acid (Aldh1a3, Rbp4), Fgf/Wnt (Fgfr1, Sfrp1, Wnt2, and Ctnnb1), and elastogenesis (Col1a1, Eln, Fbn1, and Sdc2) pathway genes. Isolated LMSCs from older mice also demonstrated lower colony-forming units (-67%), growth potential (-60% by day 7), ALDH activity (-49%), and telomerase activity (-47%). Therefore, age is associated with declining proliferative potential and regenerative functions of LMSCs in the lung.

Age dependence of lung mesenchymal stromal cell dynamics following pneumonectomy.

Inflammatory airway disease (IAD) in horses, similar to asthma in humans, is a common cause of chronic poor respiratory health and exercise intolerance due to airway inflammation and exaggerated airway constrictive responses. Human rhinovirus is an important trigger for the development of asthma; a similar role for viral respiratory disease in equine IAD has not been established yet.

Julia Paxson

Department of Biology

College of The Holy Cross

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Julia Paxson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

College of The Holy Cross

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Department of Biology