Project description:A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin ?4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.
Project description:BACKGROUND:Murine kobuviruses (MuKV) are newly recognized picornaviruses first detected in murine rodents in the USA in 2011. Little information on MuKV epidemiology in murine rodents is available. Therefore, we conducted a survey of the prevalence and genomic characteristics of rat kobuvirus in Guangdong, China. RESULTS:Fecal samples from 223 rats (Rattus norvegicus) were collected from Guangdong and kobuviruses were detected in 12.6% (28) of samples. Phylogenetic analysis based on partial 3D and complete VP1 sequence regions showed that rat kobuvirus obtained in this study were genetically closely related to those of rat/mouse kobuvirus reported in other geographical areas. Two near full-length rat kobuvirus genomes (MM33, GZ85) were acquired and phylogenetic analysis of these revealed that they shared very high nucleotide/amino acids identity with one another (95.4%/99.4%) and a sewage-derived sequence (86.9%/93.5% and 87.5%/93.7%, respectively). Comparison with original Aichivirus A strains, such human kobuvirus, revealed amino acid identity values of approximately 80%. CONCLUSION:Our findings indicate that rat kobuvirus have distinctive genetic characteristics from other Aichivirus A viruses. Additionally, rat kobuvirus may spread via sewage.
Project description:TLDA miRNA profiling on purified rat cardiomyocytes (Myo) (Ctl) and myocyte-derived progenitor cells (MDCs) demonstrated significant dedifferentiation of myocytes and identity of stemness, cell cycle progression and proliferation in MDCs after continuous culture in mitogen-rich medium for about 2 weeks. Total RNA was extracted from 3 batches of myocytes or MDCs; 100ug each was subjected to TLDA microRNA profiling after preamplification using ABI's kit. Cardiomyocyte (Myo, Ctl) was used as calibrator sample.
Project description:TLDA miRNA profiling on purified rat cardiomyocytes (Myo) (Ctl) and myocyte-derived progenitor cells (MDCs) demonstrated significant dedifferentiation of myocytes and identity of stemness, cell cycle progression and proliferation in MDCs after continuous culture in mitogen-rich medium for about 2 weeks. Overall design: Total RNA was extracted from 3 batches of myocytes or MDCs; 100ug each was subjected to TLDA microRNA profiling after preamplification using ABI's kit. Cardiomyocyte (Myo, Ctl) was used as calibrator sample.
Project description:Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals. Here we describe a detailed protocol that describes how to initiate the in vitro differentiation of mouse and human PSCs into cardiac progenitor cells, followed by intramyocardial delivery of the progenitor cells into neonatal rat hearts, in vivo incubation and analysis. The entire process takes ?6 weeks, and the resulting cardiomyocytes can be analyzed for morphology, function and gene expression. The neonatal system provides a valuable tool for understanding the maturation and pathogenesis of adult human heart muscle cells, and this concept may be expanded to maturing other PSC-derived cell types, including those containing mutations that lead to the development of diseases in the adult.
Project description:The therapeutic potential of mesenchymal stem cells (MSCs) for restoring cardiac function after cardiomyocyte loss remains controversial. Engineered cardiac tissues (ECTs) offer a simplified three-dimensional in vitro model system to evaluate stem cell therapies. We hypothesized that contractile properties of dysfunctional ECTs would be enhanced by MSC treatment. ECTs were created from neonatal rat cardiomyocytes with and without bone marrow-derived adult rat MSCs in a type-I collagen and Matrigel scaffold using custom elastomer molds with integrated cantilever force sensors. Three experimental groups included the following: (1) baseline condition ECT consisting only of myocytes, (2) 50% myocyte-depleted ECT, modeling a dysfunctional state, and (3) 50% myocyte-depleted ECT plus 10% MSC, modeling dysfunctional myocardium with intervention. Developed stress (DS) and pacing threshold voltage (VT) were measured using 2-Hz field stimulation at 37°C on culture days 5, 10, 15, and 20. By day 5, DS of myocyte-depleted ECTs was significantly lower than baseline, and VT was elevated. In MSC-supplemented ECTs, DS and VT were significantly better than myocyte-depleted values, approaching baseline ECTs. Findings were similar through culture day 15, but lost significance at day 20. Trends in DS were partly explained by changes in the cell number and alignment with time. Thus, supplementing myocyte-depleted ECTs with MSCs transiently improved contractile function and compensated for a 50% loss of cardiomyocytes, mimicking recent animal studies and clinical trials and supporting the potential of MSCs for myocardial therapy.
Project description:PURPOSE:Group B Streptococcus (S. agalactiae, GBS) is a Gram-positive opportunistic pathogen that inhabits the respiratory, urogenital and gastrointestinal tracts of humans and animals. Maternal colonization of GBS is a risk factor for a spectrum of clinical diseases in humans and a principle cause of neonatal meningitis and septicaemia. METHODOLOGY:We describe polymicrobial sepsis including GBS in two gravid adult female Long-Evans rats experiencing acute mortality from a colony of long-term breeding pairs. Fluorescent in situ hybridization confirmed GBS association with pathological changes in affected tissues, including the heart and uterus. RESULTS:Characterization of seven GBS strains obtained from clinically affected and non-affected animals indicated similar antibiotic resistance and susceptibility patterns to that of human strains of GBS. The rat strains have virulence factors known to contribute to pathogenicity, and shared serotypes with human invasive isolates. Phylogenetic analyses revealed that one rat-derived GBS strain was more closely related to human-derived strains than other rat-derived strains, strengthening the notion that interspecies transmission is possible. CONCLUSIONS:To our knowledge, this is the first investigation of genotypic and phenotypic features of rat-derived GBS strains and their comparison to human- and other animal-derived GBS strains. Since GBS commonly colonizes commercially available rats, its exclusion as a potential pathogen for immunocompromised or stressed animals is recommended.
Project description:Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.