Cardiac Bmi1(+) cells contribute to myocardial renewal in the murine adult heart

Introduction: The mammalian adult heart maintains a continuous, low cardiomyocyte turnover rate throughout life. Although many cardiac stem cell populations have been studied, the natural source for homeostatic repair has not yet been defined. The Polycomb protein BMI1 is the most representative marker of mouse adult stem cell systems. We have evaluated the relevance and role of cardiac Bmi1(+) cells in cardiac physiological homeostasis. Methods: Bmi1(CreER/+); Rosa26(YFP/+) (Bmi1-YFP) mice were used for lineage tracing strategy. After tamoxifen (TM) induction, yellow fluorescent protein (YFP) is expressed under the control of Rosa26 regulatory sequences in Bmi1(+) cells. These cells and their progeny were tracked by FACS, immunofluorescence and RT-qPCR techniques from 5 days to 1 year. Results: FACS analysis of non-cardiomyocyte compartment from TM-induced Bmi1-YFP mice showed a Bmi1 (+)-expressing cardiac progenitor cell (Bmi1-CPC: B-CPC) population, SCA-1 antigen-positive (95.9 +/- 0.4 \%) that expresses some stemness-associated genes. B-CPC were also able to differentiate in vitro to the three main cardiac lineages. Pulse-chase analysis showed that B-CPC remained quite stable for extended periods (up to 1 year), which suggests that this Bmi1(+) population contains cardiac progenitors with substantial self-maintenance potential. Specific immunostaining of Bmi1-YFP hearts serial sections 5 days post-TM induction indicated broad distribution of B-CPC, which were detected in variably sized clusters, although no YFP+ cardiomyocytes (CM) were detected at this time. Between 2 to 12 months after TM induction, YFP+ CM were clearly identified (3 +/- 0.6 \% to 6.7 +/- 1.3 \%) by immunohistochemistry of serial sections and by flow cytometry of total freshly isolated CM. B-CPC also contributed to endothelial and smooth muscle (SM) lineages in vivo. Conclusions: High Bmi1 expression identifies a non-cardiomyocyte resident cardiac population (B-CPC) that contributes to the main lineages of the heart in vitro and in vivo.We wish to thank M. Torres, J.M. Perez-Pomares and B.G. Galvez for critical discussions of the manuscript, A. M. Santos for assistance with confocal microscopy and dynamic imaging, R.M. Carmona for help with the animal colony management, F.S. Cabo for bioinformatics and statistical support, J.M Ligos for the sorting strategy, and K. McCreath and C. Mark for editorial support. This study was supported by grants to A.B. from the Ministry of Science and Innovation (SAF2012-34327; PLE2009-0147 and PSE-010000-2009-3), the Research Program of the Comunidad Autonoma de Madrid (S2010/BMD-2420), the Instituto de Salud Carlos III (RETICS-RD12/0019/0018 and RETICS-RD12/0019/0023) and the European Commission (Proposal 242038). The CNB-CSIC and CNIC are supported by the Spanish Ministry of Economy and Competitiveness.S

Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells

Background Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. Aim and scope A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. Methods We compared hADSC cultures at short (P S ) and prolonged (P L ) passages, both in standard and low [O 2 ] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. Results Comparison of hADSCs cultured at P L or P S surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in P L cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O 2 ], was also significantly higher in P L than in P S passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. Conclusion A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Autónoma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH- 2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundación Científica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formación del Profesorado Universitari

Additional file 1: Table S1. of Bmi1 + cardiac progenitor cells contribute to myocardial repair following acute injury

Antibodies used in immunodetection. Antibodies used in this study. (JPG 199 kb

Additional file 4: Figure S2. of Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

Valiente-Alandi.jpg. Flow cytometry analysis in B-CPC throughout mouse lifespan. Flow cytometry characterization of non-CM YFP+ cells derived from Bmi1-YFP hearts at 5d-postTM and 1y-postTM. (JPEG 926 kb

Additional file 6: Figure S4. of Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

Valiente-Alandi.jpg. Cardiac explant model. GFP immunostaining of cardiac explants from 5d-postTM Bmi1-YFP mice. (JPEG 860 kb

Additional file 1: Table S1. of Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

Valiente-Alandi.jpg. Antibodies used in immunodetection. Antibodies used in this study. (JPEG 292 kb

Additional file 8: Figure S6. of Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

Valiente-Alandi.jpg. Calcium transients in freshly isolated YFP+ and YFP- adult CM provide evidence of functional coupling. Contractility and transient Ca2+ efflux of freshly isolated YFP+ and YFP- adult CM. (JPEG 513 kb

Additional file 9: Figure S7. of Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

Valiente-Alandi.jpg. X-Gal-stained heart tissue of Bmi1-lacZ mice 5d-postTM. X-Gal staining showed LacZ+ cells in clusters between sarcomeres and in perivascular locations. (JPEG 546 kb