Embryos can achieve immune tolerance, yet the underlying mechanisms remain incompletely understood. Here, we demonstrate that pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), secrete extracellular vesicles (EVs) that markedly outperform mesenchymal stem cell (MSC)-derived EVs in suppressing pro-inflammatory cytokine secretion, inhibiting activated T-cell proliferation, and inducing regulatory T-cell (Treg) formation through CDK8 downregulation. Nuclear magnetic resonance (NMR) analysis reveals distinct molecular fingerprints of PSC EVs compared to those of MSC EVs. Moreover, comparative analyses show that PSC EVs contain unique proteins and microRNAs, such as the pluripotency-associated proteins ROR1 and CD133 and members of the miR-302 family, which are not found in MSC EVs, as determined by proteomic profiling and microRNA sequencing. Notably, the dynamic suspension culture of PSC aggregates significantly increases EV yield, offering a scalable and reproducible source superior to other cell sources. To evaluate their therapeutic potential, we employed an antigen-specific type 1 diabetes model and found that two local injections of iPSC EVs, particularly when delivered via a biomaterial scaffold, significantly enhanced diabetes-free survival. These treatments increased Treg populations in draining lymph nodes, induced systemic immunomodulation, and preserved β-cell mass from immune-mediated destruction. The immunomodulatory capability of PSC EVs suggests broad applications in treating autoimmune diseases and supporting stem cell-derived cell therapies by promoting immune tolerance. Their scalability, consistency, and superior therapeutic properties position PSC EVs as a compelling platform for next-generation immunotherapies and cell-based treatment strategies.

Biomaterials mimicking natural extracellular matrix are necessary to create an optimal microenvironment for cell adhesion, migration, proliferation, and differentiation. These scaffolds must possess bicontinuous interconnected porosity to ensure the effective exchange of oxygen, nutrients, and metabolic waste, which are crucial for developing functional tissues. Here, a novel bicontinuous interfacially jammed emulsion (BIJEL)-Integrated PORous Engineered System (BIPORES) is developed to confer bioinert synthetic polyethylene glycol diacrylate (PEGDA) with unique bicontinuous interconnected porosity and surface topography. This platform is fabricated through controlled phase separation and interfacial stabilization of two continuous phases by nanoparticles. Functional validation using human mesenchymal stem cells, and human induced pluripotent stem cells-derived cardiomyocytes and cardiac fibroblasts, reveals outstanding cell attachment, growth, proliferation, and/or differentiation within tissue-scale BIPORES scaffolds. These findings indicate that bicontinuous interconnected porosity with negative Gaussian curvature in the BIPORES scaffolds plays a key role in organ-scale tissue engineering and regeneration.

We report a large field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13mm 2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction stress into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave.One-Sentence Summary:An optical platform for fast, concurrent measurements of cell mechanics at 83 frames per second, over a large area of 13mm 2 .

Mammalian hearts undergo major changes after birth during the perinatal period. While several extrinsic factors such as mechanical load, electrical stimulation, hormones, and nutrients have been implicated in this process, the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. Importantly, stem-cell derived cardiomyocytes commonly exhibit immaturity which is a major limiting factor for their application as disease models or cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanism of mammalian cardiomyocyte maturation has significant implications in both basic cardiac biology and cell-based therapy and disease modeling. In our recent analysis of the global transcriptome transition from neonatal to adult rat hearts, we found RNA splicing among the top changed pathways. Additionally, we found that expression of RBFox1, a cardiac enriched RNA splicing regulator, was significantly increased during the postnatal transition to adolescence. We further demonstrate that RBFox1 mediated post-transcriptional regulation has a potent effect to promote neonatal and stem-cell derived cardiomyocyte maturation. RNA-seq followed by Gene Ontology enrichment analysis showed that Rbfox1 led to differentially expressed genes enriched in cardiac contraction and conduction, sarcomere organization, K + ion import and muscle filament sliding, as well as differential isoform switches enriched in cardiac contraction and sarcomere structure. An evolutionarily conserved, temporal specific super enhancer exists upstream of cardiac Rbfox1 , which is occupied by active epigenetic markers in the postnatal period. We have identified the tissue and temporal transcriptional activity of this super enhancer in vitro and in vivo . Several binding motifs of transcription factors are predicted within it and a key DNA fragment was identified to be essential for enhancer activity. A transgenic mouse strain has been established to facilitate the tracing of myocyte maturation in vivo by constructing a reporter system driven by a 4xSuperEnhancer-Rbfox1Promoter-tdTomato construct. In conclusion, we propose a mechanism that RBFox1 is an intracellular regulator with tissue- and temporal-specific expression patterns that regulates cardiac maturation by promoting alternative splicing that fine tunes the genetic code.

Colchicine, a tricyclic alkaloid derived from Colchicum autumnale, is well known for its anti-inflammatory properties, and has been used to treat conditions such as gout, familial Mediterranean fever, and pericarditis. Colchicine's inhibition of the NLRP-3 inflammasome and reduction of key pro-inflammatory cytokines has been considered potentially beneficial in managing COVID-19. While early anecdotal reports and small-scale studies suggest potential benefits, including reduced hospital stay and oxygen requirements, larger randomized controlled trials (RCT) have largely failed to demonstrate significant improvement in mortality, the need for mechanical ventilation, or ICU admissions. Meta-analyses of RCT data corroborate these findings, showing no substantial benefit of colchicine in treating COVID-19. In non-hospitalized patients, the data also suggests limited efficacy, with some studies indicating potential benefit in specific subgroups, though these findings have not been consistently replicated. Colchicine for the treatment of cardiac injury in individuals infected with COVID-19 has also been an area of interest; despite early work suggesting benefit, subsequent RCTs have not shown clear benefit in this subgroup of patients. Overall, despite its promising mechanism of action, the evidence does not support the use of colchicine as standard treatment for COVID-19, either in hospitalized or community-based settings, or with evidence of cardiac injury. This review highlights the need for further research to better understand the potential role of colchicine – looking back as well as a look ahead – in the management of COVID-19.

The increased demand for personalized wearable and implantable medical devices has created the need for the generation of electronics that interface with living systems. Current bioelectronics has not fully resolved mismatches between biological systems and engineered circuits, resulting in tissue injury and pain. Thus, there is an unmet need to develop materials for the fabrication of wearable electronics that are biocompatible at the tissue interface. Here, we developed a tailorable gelatin-based bio-ink functionalized with a choline bio-ionic liquid (BIL) for in situ 3D bioprinting of bioelectronics at the tissue interface. The resultant photocrosslinked polymer is programmable, transparent, ion conductive, and flexible. BILs are stably conjugated with a gelatin methacryloyl (GelMA) hydrogel using photocrosslinking to make BioGel, which routes ionic current with high resolution and enables localized electrical stimulation delivery. Controllable crosslinking, achieved by varying reactants composition, allows the BioGel bio-ink platform for easy and rapid in-situ 3D bioprinting of complex designs directly on skin tissue. Bio-ionic modified polymers thus represent a versatile and wide-applicable bio-ink solution for personalized bioelectronics fabrication that minimizes tissue damage.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4–expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy ( r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Despite improvements in the standard of care for patients with heart diseases, including innovation in pharmacotherapy and surgical interventions, none have yet been proven effective to prevent the progression to heart failure. Cardiac transplantation is the last resort for patients with severe heart failure, but donor shortages remain a roadblock. Cardiac regenerative strategies include cell-based therapeutics, gene therapy, direct reprogramming of noncardiac cells, acellular biologics, and tissue engineering methods to restore damaged hearts. Significant advancements have been made over the past several decades within each of these fields. This review focuses on the advancements of: 1) cell-based cardiac regenerative therapies, 2) the use of noncoding RNA to induce endogenous cell proliferation, and 3) application of bioengineering methods to promote retention and integration of engrafted cells. Different cell sources have been investigated, including adult stem cells derived from bone marrow and adipose cells, cardiosphere-derived cells, skeletal myoblasts, and pluripotent stem cells. In addition to cell-based transplantation approaches, there have been accumulating interest over the past decade in inducing endogenous CM proliferation for heart regeneration, particularly with the use of noncoding RNAs such as miRNAs and lncRNAs. Bioengineering applications have focused on combining cell-transplantation approaches with fabrication of a porous, vascularized scaffold using biomaterials and advanced bio-fabrication techniques that may offer enhanced retention of transplanted cells, with the hope that these cells would better engraft with host tissue to improve cardiac function. This review summarizes the present status and future challenges of cardiac regenerative therapies.

After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

HomeCirculationVol. 148, No. 16Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1 No AccessResearch ArticleRequest AccessFull TextAboutView Full TextView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toNo AccessResearch ArticleRequest AccessFull TextRegulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1 Jijun Huang, Josh Z. Lee, Christoph D. Rau, Arash Pezhouman, Tomohiro Yokota, Hiromi Miwa, Matthew Feldman, Tsz Kin Kong, Ziyue Yang, Woan Ting Tay, Ivan Pushkarsky, Kyungsoo Kim, Shan S. Parikh, Shreya Udani, Boon Seng Soh, Chen Gao, Linsey Stiles, Orian S. Shirihai, Bjorn C. Knollmann, Reza Ardehali, Dino Di Carlo and Yibin Wang Jijun HuangJijun Huang Correspondence to: Yibin Wang, PhD, Duke-NUS Medical School, 8 College Rd, Level 8, Singapore 169857, Singapore. Email E-mail Address: [email protected] https://orcid.org/0000-0002-5520-193X Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Division of Endocrinology (J.H.), University of California, Los Angeles. , Josh Z. LeeJosh Z. Lee Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. , Christoph D. RauChristoph D. Rau Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Department of Genetics and Computational Medicine, University of North Carolina, Chapel Hill (C.D.R.). , Arash PezhoumanArash Pezhouman https://orcid.org/0000-0001-9106-7136 Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.). , Tomohiro YokotaTomohiro Yokota Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Department of Medicine, Greater Los Angeles VA Healthcare System, CA (T.Y.). , Hiromi MiwaHiromi Miwa Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles. , Matthew FeldmanMatthew Feldman School of Medicine, Meharry Medical College, Nashville, TN (M.F.). , Tsz Kin KongTsz Kin Kong Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. , Ziyue YangZiyue Yang Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX (Z.Y.). , Woan Ting TayWoan Ting Tay https://orcid.org/0000-0002-4845-287X Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). , Ivan PushkarskyIvan Pushkarsky Forcyte Biotechnologies, Inc, Los Angeles, CA (I.P.). , Kyungsoo KimKyungsoo Kim https://orcid.org/0000-0003-2869-0659 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Shan S. ParikhShan S. Parikh https://orcid.org/0000-0003-1806-9199 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Shreya UdaniShreya Udani Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). , Boon Seng SohBoon Seng Soh https://orcid.org/0000-0001-9134-3081 Institute of Molecular and Cell Biology, The Agency for Science, Technology and Research (A*STAR), Singapore (B.S.S.). , Chen GaoChen Gao Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Department of Pharmacology and System Physiology, University of Cincinnati, OH (C.G.). , Linsey StilesLinsey Stiles Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles. , Orian S. ShirihaiOrian S. Shirihai Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles. , Bjorn C. KnollmannBjorn C. Knollmann https://orcid.org/0000-0003-4956-9735 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Reza ArdehaliReza Ardehali https://orcid.org/0000-0003-1318-4016 Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.). , Dino Di CarloDino Di Carlo Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles. and Yibin WangYibin Wang https://orcid.org/0000-0003-0852-0767 Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). Originally published16 Oct 2023https://doi.org/10.1161/CIRCULATIONAHA.122.061602Circulation. 2023;148:1263–1266Footnotes*J. Huang and J.Z. Lee contributed equally.For Sources of Funding and Disclosures, see page 1266.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Yibin Wang, PhD, Duke-NUS Medical School, 8 College Rd, Level 8, Singapore 169857, Singapore. Email yibinwang@duke-nus.edu.sgREFERENCES1. Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X, Murry CE. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.Nat Rev Cardiol. 2020; 17:341–359. doi: 10.1038/s41569-019-0331-xCrossrefMedlineGoogle Scholar2. Guo Y, Pu WT. Cardiomyocyte maturation: new phase in development.Circ Res. 2020; 126:1086–1106. doi: 10.1161/CIRCRESAHA.119.315862LinkGoogle Scholar3. Gao C, Ren S, Lee JH, Qiu J, Chapski DJ, Rau CD, Zhou Y, Abdellatif M, Nakano A, Vondriska TM, et al. RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.J Clin Invest. 2016; 126:195–206. doi: 10.1172/JCI84015CrossrefMedlineGoogle Scholar4. Wang Y, Yao F, Wang L, Li Z, Ren Z, Li D, Zhang M, Han L, Wang SQ, Zhou B, et al. Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation.Nat Commun. 2020; 11:2585. doi: 10.1038/s41467-020-16204-wCrossrefMedlineGoogle Scholar5. Pushkarsky I. FLECS technology for high-throughput single-cell force biology and screening.Assay Drug Dev Technol. 2018; 16:7–11. doi: 10.1089/adt.2017.825CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails October 17, 2023Vol 148, Issue 16 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.122.061602PMID: 37844148 Originally publishedOctober 16, 2023 KeywordsRNA, messengersequence analysis, RNAmyocytes, cardiacPDF download Advertisement SubjectsMechanismsMyocardial BiologyMyocardial RegenerationStem Cells

Cardiovascular disease remains a leading cause of death worldwide despite important advances in modern medical and surgical therapies. As human adult cardiomyocytes have limited regenerative ability, cardiomyocytes lost after myocardial infarction are replaced by fibrotic scar tissue, leading to cardiac dysfunction and heart failure. To replace lost cardiomyocytes, a promising approach is direct cardiac reprogramming, in which cardiac fibroblasts are transdifferentiated into induced cardiomyocyte-like cells (iCMs). Here we review cardiac reprogramming cocktails (including transcription factors, microRNAs and small molecules) that mediate iCM generation. We also highlight mechanistic studies exploring the barriers to and facilitators of this process. We then review recent progress in iCM reprogramming, with a focus on single-cell '-omics' research. Finally, we discuss obstacles to clinical application.

Immune cells have emerged as powerful regulators of regenerative as well as pathological processes. The vast majority of regenerative immunoengineering efforts have focused on macrophages; however, growing evidence suggests that other cells of both the innate and adaptive immune system are as important for successful revascularization and tissue repair. Moreover, spatiotemporal regulation of immune cells and their signaling have a significant impact on the regeneration speed and the extent of functional recovery. In this review, we summarize the contribution of different types of immune cells to the healing process and discuss ways to manipulate and control immune cells in favor of vascularization and tissue regeneration. In addition to cell delivery and cell-free therapies using extracellular vesicles, we discuss in situ strategies and engineering approaches to attract specific types of immune cells and modulate their phenotypes. This field is making advances to uncover the extraordinary potential of immune cells and their secretome in the regulation of vascularization and tissue remodeling. Understanding the principles of immunoregulation will help us design advanced immunoengineering platforms to harness their power for tissue regeneration.

Cardiomyocytes in the adult mammalian heart have a low turnover during homeostasis. After myocardial injury, there is irreversible loss of cardiomyocytes, which results in subsequent scar formation and cardiac remodeling. In order to better understand and characterize the proliferative capacity of cardiomyocytes, in vivo methods have been developed to track their fate during normal development and after injury. Lineage tracing models are of particular interest due to their ability to record cell proliferation events over a long period of time, either during development or in response to a pathological event. This paper reviews two well-studied lineage-tracing, transgenic mouse models-mosaic analysis with double markers and rainbow reporter system.