Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1

Adult-Derived Stem Cells from the Liver Become Myocytes in the Heart in Vivo

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli β-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the β-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The β-galactosidase-positive myocytes ranged from <20 μm to 110 μm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes

Adult-Derived Liver Stem Cells Acquire a Cardiomyocyte Structural and Functional Phenotype ex Vivo

We examined the differentiation potential of an adult liver stem cell line (WB F344) in a cardiac microenvironment, ex vivo. WB F344 cells were established from a single cloned nonparenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WB F344 cells that express β-galactosidase and green fluorescent protein or only β-galactosidase were co-cultured with dissociated rat or mouse neonatal cardiac cells. After 4 to 14 days, WB F344-derived cardiomyocytes expressed cardiac-specific proteins and exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Further, rhythmically beating WB F344-derived cardiomyocytes displayed calcium transients. Fluorescent recovery after photobleaching demonstrated that WB F344-derived cardiomyocytes were coupled with adjacent neonatal cardiomyocytes and other WB F344-derived cardiomyocytes. Fluorescence in situ hybridization experiments suggested that fusion between WB F344 cells and neonatal mouse cardiomyocytes did not take place. Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function. The identification ex vivo of microenvironmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the molecular basis of adult stem cell differentiation and phenotypic plasticity

10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension.

BACKGROUND Long-term safety and efficacy of osteoporosis treatment are important because of the chronic nature of the disease. We aimed to assess the long-term safety and efficacy of denosumab, which is widely used for the treatment of postmenopausal women with osteoporosis. METHODS In the multicentre, randomised, double-blind, placebo-controlled, phase 3 FREEDOM trial, postmenopausal women aged 60-90 years with osteoporosis were enrolled in 214 centres in North America, Europe, Latin America, and Australasia and were randomly assigned (1:1) to receive 60 mg subcutaneous denosumab or placebo every 6 months for 3 years. All participants who completed the FREEDOM trial without discontinuing treatment or missing more than one dose of investigational product were eligible to enrol in the open-label, 7-year extension, in which all participants received denosumab. The data represent up to 10 years of denosumab exposure for women who received 3 years of denosumab in FREEDOM and continued in the extension (long-term group), and up to 7 years for women who received 3 years of placebo and transitioned to denosumab in the extension (crossover group). The primary outcome was safety monitoring, comprising assessments of adverse event incidence and serious adverse event incidence, changes in safety laboratory analytes (ie, serum chemistry and haematology), and participant incidence of denosumab antibody formation. Secondary outcomes included new vertebral, hip, and non-vertebral fractures as well as bone mineral density (BMD) at the lumbar spine, total hip, femoral neck, and one-third radius. Analyses were done according to the randomised FREEDOM treatment assignments. All participants who received at least one dose of investigational product in FREEDOM or the extension were included in the combined safety analyses. All participants who enrolled in the extension with observed data were included in the efficacy analyses. The FREEDOM trial (NCT00089791) and its extension (NCT00523341) are both registered with ClinicalTrials.gov. FINDINGS Between Aug 3, 2004, and June 1, 2005, 7808 women were enrolled in the FREEDOM study. 5928 (76%) women were eligible for enrolment in the extension, and of these, 4550 (77%) were enrolled (2343 long-term, 2207 crossover) between Aug 7, 2007, and June 20, 2008. 2626 women (1343 long-term; 1283 crossover) completed the extension. The yearly exposure-adjusted participant incidence of adverse events for all individuals receiving denosumab decreased from 165·3 to 95·9 per 100 participant-years over the course of 10 years. Serious adverse event rates were generally stable over time, varying between 11·5 and 14·4 per 100 participant-years. One atypical femoral fracture occurred in each group during the extension. Seven cases of osteonecrosis of the jaw were reported in the long-term group and six cases in the crossover group. The yearly incidence of new vertebral fractures (ranging from 0·90% to 1·86%) and non-vertebral fractures (ranging from 0·84% to 2·55%) remained low during the extension, similar to rates observed in the denosumab group during the first three years of the FREEDOM study, and lower than rates projected for a virtual long-term placebo cohort. In the long-term group, BMD increased from FREEDOM baseline by 21·7% at the lumbar spine, 9·2% at total hip, 9·0% at femoral neck, and 2·7% at the one-third radius. In the crossover group, BMD increased from extension baseline by 16·5% at the lumbar spine, 7·4% at total hip, 7·1% at femoral neck, and 2·3% at one-third radius. INTERPRETATION Denosumab treatment for up to 10 years was associated with low rates of adverse events, low fracture incidence compared with that observed during the original trial, and continued increases in BMD without plateau. FUNDING Amgen

Bone marrow-derived MSCs in co-culture with cardiomyocytes.

<p>(A) A nascent cardiomyocyte derived from mMSCs of a βMHC-YFP genotype mouse at 8 days in co-culture with cardiomyocytes. The βMHC-YFP cell is pseudo colored green for visualization of the striations. Left panel shows novel endogenous expression of βMHC-YFP fluorescence along stress fibers and striations (magnified in insert). Middle panel shows surrounding rat cardiomyocyte. Merged images in right panel. (B) GFP-hMSCs co-cultured with cardiomyocytes for 16 days and immunostained for α-actinin or troponin T (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454-MullerBorer1" target="_blank">[23]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454.s003" target="_blank">Supporting Information S1</a>). Acquisition of a differentiated cardiac phenotype is demonstrated in the inserts where cardiomyocyte striations are visible. Nuclear DAPI in blue. Scale bar = 20 µm.</p

Protein localization by immunocytochemistry in hMSCs co-cultured with cardiomyocytes for 48 hours.

<p>Left panels show dsRed-hMSCs, middle panels show CAMTA1 or NFAT protein localization. Right panels show merged images. (A) Novel expression of CAMTA1 protein is demonstrated in the nucleus of a dsRed hMSCs co-cultured with rat cardiomyocytes (arrow in middle panel). Rat cardiomyocytes (small nuclei in the background) a priori express CAMTA1. Note the larger human nucleus of the hMSCs compared to the size of the rat nuclei in the background. (B) Novel NFAT2c protein expression in an hMSC co-cultured with cardiomyocytes. NFAT2c is localized throughout the hMSC including the nucleus of the hMSC. NFAT2c was not detected in the nucleus in naïve hMSCs.</p

Regulation of cardiac transcription factors in liver stem cells co-cultured with cardiomyocytes after minimizing CAMTA1 expression.

<p>Except for the control conditions (A and B) the stem cells were pre-transfected with specific siRNA 24 hrs before they were added to the co-culture with cardiomyocytes. A: control, naive stem cells in monoculture for 72 hrs. B: naive stem cells cultured for 24 hrs, then co-cultured with cardiomyocytes for 48 hrs. C: Stem cells pre-transfected with a scrambled siRNA 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. D: Stem cells were pre-transfected with a human pool of CAMTA1 siRNAs for 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. The graphs illustrate expression of the transcription factors CAMTA1, CAMTA2, Nk×2.5, Myocardin, cTnT and BetaMHC. A significant decrease in the expression of transcription factors Nkx2.5, Myocardin, cTnT and BetaMHC were measured when CAMTA1 expression was minimized. Note that no significant change in CAMTA2 expression was observed. The bars show mean ± SEM, *p<0.05.</p

Regulation of expression of cardiac transcription factors following the minimization of CAMTA1 expression in hMSCs monocultures.

<p>Human MSCs were pre-transduced with specific lentiviral vectors 24 hrs before the up-regulation of CAMTA1 expression was induced with ionomycin. A: control, naive hMSCs grown in monoculture, B: hMSCs pre-transduced with a GFP-lentiviral vector, then stimulated with ionomycin. C: hMSCs pre-transduced with CAMTA1 shRNA lentiviral vector, then stimulated with ionomycin. The graphs illustrate expression of the transcription factors CAMTA1, Mef2C, and Gata4. Note condition B shows increased expression of Mef2C and Gata 4 with increased CAMTA1 expression. With minimization of CAMTA1 expression (condition C) expression of Mef2C and Gata4 was significantly decreased (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone-0038454-g002" target="_blank">Figure 2D</a> for CAMTA1 control). The bars show mean ± SEM, *p<0.05.</p

Time course of RNA expression levels of cardiac transcription factors and contractile protein genes in hMSCs co-cultured with rat neonatal cardiomyocytes.

<p>Control levels of CAMTA1, RCAN1, GATA4, Nk×2.5, Mef2c, Tb×5, cTnT, and BetaMHC in the stem cells in monoculture at day 0 (0d), or co-cultured with cardiomyocytes for 2 days(2d) and 4 days (4d). The bars show mean ± SEM, *p<0.05. # denotes novel expression in Nkx2.5.</p