Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.

Isolation and characterization of equine endometrial mesenchymal stromal cells.

BACKGROUND: Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. METHODS: The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. RESULTS: Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. CONCLUSIONS: We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

Expression of the Helicobacter pylori virulence factor vacuolating cytotoxin A (vacA) is influenced by a potential stem-loop structure in the 5' untranslated region of the transcript.

The vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogen Helicobacter pylori. Certain vacA genotypes are strongly associated with disease risk, but the association is not absolute. The factors determining vacA gene expression are not fully understood, and the mechanisms of its regulation are elusive. We have identified a potential mRNA stem-loop forming structure in the 5' untranslated region (UTR) of the vacA transcript. Using site-directed mutagenesis, we found that disruption of the stem-loop structure reduced steady-state mRNA levels between two- and sixfold (P = 0.0005) and decreased mRNA half-life compared with wild type (P = 0.03). This led to a marked reduction in VacA protein levels and overall toxin activity. Additionally, during stressful environmental conditions of acid pH or high environmental salt concentrations, when general transcription of vacA was decreased or increased respectively, the stabilising effects of the stem-loop were even more pronounced. Our results suggest that the stem-loop structure in the vacA 5' UTR is an important determinant of vacA expression through stabilisation of the vacA mRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress.

A role for the vacuolating cytotoxin, VacA, in colonization and Helicobacter pylori-induced metaplasia in the stomach.

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA(+) background.