Identification of the Transgene Integration Site and Host Genome Changes in MRP8-Cre/ires-EGFP Transgenic Mice by Targeted Locus Amplification.

The MRP8-Cre-ires/EGFP transgenic mouse (Mrp8creTg, on C57BL/6J genetic background) is popular in immunological and hematological research for specifically expressing Cre recombinase and an EGFP reporter in neutrophils. It is often crossed with other transgenic lines carrying loxP-flanked genes to achieve restricted gene knockout in neutrophils. However, due to the way in which the line was created, basic knowledge about the MRP8-Cre-ires/EGFP transgene in the host genome, such as its integration site(s) and flanking sequences, remains largely unknown, hampering robust experimental design and data interpretation. Here we used a recently developed technique, targeted locus amplification (TLA) sequencing, to fill these knowledge gaps. We found that the MRP8-Cre-ires/EGFP transgene was integrated into chromosome 5 (5qG2) of the host mouse genome. This integration led to a 44 kb deletion of the host genomic sequence, resulting in complete deletion of Serpine1 and partial deletion of Ap1s1. Having determined the flanking sequences of the transgene, we designed a new genotyping protocol that can distinguish homozygous, heterozygous, and wildtype Mrp8creTg mice. To our surprise, crossing heterozygous mice produced no homozygous Mrp8creTg mice, most likely due to prenatal lethality resulting from disrupted Ap1s1 gene expression.

Dppa2/4 Facilitate Epigenetic Remodeling during Reprogramming to Pluripotency.

As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.

Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration.

AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells (MSCs). METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues (both human and murine) were minced with scalpels into small pieces (< 1 mm(3)) and aliquoted in portions of 200 mm(3). These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for (immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining (hematoxylin-phloxin-saffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect ß-galactosidase-positive cells and myofibers. RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45 (P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, ß-galactosidase-positive myofibers were identified early after grafting at the well-vascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively. CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of species-specific factors in the microenvironment.

Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes.

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.

Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones.

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.

Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease.

BACKGROUND: Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na(+) current (I(Na)) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na(+) channel mutation causing a cardiac Na(+) channel overlap syndrome. METHOD AND RESULTS: Induced PSC (iPSC) lines were generated from mice carrying the Scn5a(1798insD/+) (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in I(Na) density and a larger persistent I(Na) compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A(1795insD/+) mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. CONCLUSION: Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na(+) channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na(+) channel disease.

Cardiomyocyte differentiation of pluripotent stem cells and their use as cardiac disease models.

More than 10 years after their first isolation, human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable, and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever, biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper, we review the cutting edge of this exciting area of translational research.

Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom.

OBJECTIVES: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. METHODS: Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined using microtitre plates and in vitro killing assays. RESULTS: Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from biofilms became susceptible to vancomycin and clindamycin, provided that the medium was refreshed. Daptomycin dose-dependently eliminated the biofilm-derived bacteria immediately. Furthermore, it was significantly more effective against bacteria derived from ES-exposed biofilms than those from control biofilms. ES did not affect the activity of the antibiotics against log-phase S. aureus. CONCLUSIONS: Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived therefrom.

Genome-wide linkage scan in Dutch hereditary non-BRCA1/2 breast cancer families identifies 9q21-22 as a putative breast cancer susceptibility locus.

Breast cancer accounts for over 20% of all female cancers. A positive family history remains one of the most important risk factors for the disease, with first-degree relatives of patients having a twofold elevated risk. Known breast cancer susceptibility genes such as BRCA1 and BRCA2 explain only 20-25% of this risk, suggesting the existence of other breast cancer susceptibility genes. Here, we report the results of a genome-wide linkage scan in 55 high-risk Dutch breast cancer families with no mutations in BRCA1 and BRCA2. Twenty-two of these families were also part of a previous linkage study by the Breast Cancer Linkage Consortium. In addition, we performed CGH analyses in 61 tumors of these families and 31 sporadic tumors. Three regions were identified with parametric HLOD scores >1, and three with nonparametric LOD scores >1.5. Upon further marker genotyping for the candidate loci, and the addition of another 30 families to the analysis, only the locus on chromosome 9 (9q21-22, marker D9S167) remained significant, with a nonparametric multipoint LOD score of 3.96 (parametric HLOD 0.56, alpha = 0.18). With CGH analyses we observed preferential copy number loss at BAC RP11-276H19, containing D9S167 in familial tumors as compared to sporadic tumors (P < 0.001). Five candidate genes were selected from the region around D9S167 and their coding regions subjected to direct sequence analysis in 16 probands. No clear pathogenic mutations were found in any of these genes.