Essays on Science and SocietyREGENERATIVE MEDICINE

Hope for the brokenhearted

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Science  17 Jun 2016:
Vol. 352, Issue 6292, pp. 1400-1401
DOI: 10.1126/science.aag1213
Reprogrammed iCMs in a dish.

Green, cardiac reporter aMHC-GFP; red, cardiac TroponinT (cTnT); blue, DAPI-labeled nuclei.

PHOTO: L. WANG

Cellular reprogramming, the conversion of one type of somatic cell into another, has substantially affected the field of stem cells and regenerative medicine in the past decade. Today, it holds great promise as a novel approach for the treatment of various human diseases and as a tool for disease modeling to advance personalized medicine.

Inspired by the paradigm-shifting method, developed by Shinya Yamanaka, of creating induced pluripotent stem cells (iPSCs), and with the full support of my postdoctoral mentor Deepak Srivastava, in the fall of 2009 I undertook a study to determine whether resident cardiac fibroblasts could be converted into functional cardiomyocytes. After overcoming numerous technical and conceptual obstacles, I demonstrated that endogenous cardiac fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs) in their native environment, and that this conversion leads to a functional improvement associated with scar size reduction in an infarcted heart (1). This research was ranked number 2 in the 2012 list of Top 10 Advances in Heart Disease and Stroke Research, compiled by the American Heart Association. From this research, we developed a system in which cardiac reprogramming could be rigorously studied and implemented (13).

Trained as a basic developmental and cell biologist under the mentorship of Rolf Bodmer, I have always believed in the power of basic science to advance translational research. I started my own laboratory at the University of North Carolina (UNC)–Chapel Hill in the fall of 2012. Now as a principal investigator of my own team, I rely on my training in both translational and basic science to tackle reprogramming questions by exploring the fundamental molecular mechanisms of this fascinating process.

Leveraging the knowledge that faithful cell fate conversion requires a precise dosage and temporal expression of transcriptional factors, my laboratory recently identified the optimal ratio of cardiac reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) for more complete and efficient iCM generation (4). In this study, we generated a complete set of polycistronic constructs containing G, M, and T in all possible splicing orders with identical 2A sequences in a single mRNA. We found that each splicing order gave rise to a distinct ratio of G, M, and T protein expression and resulted in significantly different reprogramming efficiencies. The most desirable combination promoted a significant increase in the generation of beating iCMs when compared with G, M, and T delivered via pooled viruses. Importantly, at the molecular level the most optimal G, M, and T stoichiometry, defined by higher protein expression level of M and lower levels of G and T, promoted significantly increased expression of mature cardiomyocyte markers. We went on to demonstrate that when the polycistronic vector that induced the optimal ratio of G, M, and T—which we named MGT—is delivered in vivo to an infarcted heart, it improves iCM reprogramming efficiency and results in a further improvement in ventricular contractile function (5). This work resulted in a series of recent publications and numerous inquiries from around the world to obtain our new tool set for cardiac reprogramming (46).

With the optimal MGT system, we continued our efforts to identify roadblocks of iCM reprogramming. Because cell reprogramming involves substantial chromatin reorganization, it will be of major importance to identify the epigenetic mechanisms that govern this process. Using our polycistronic system, we were able to determine the dynamics of histone marks such as H3K27me3 and H3K4me3 in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that CpG sites were unequally demethylated during early stages of iCM reprogramming. Thus, we propose that there are specific CpGs, whose demethylation states correlate with transcription activation, that play more prominent roles than others. Our study also revealed that the reprogramming fibroblasts rapidly activated the cardiac program while progressively suppressing fibroblast fate at both epigenetic and transcriptional levels. This research provides the first insight into epigenetic repatterning events during cardiac reprogramming (7).

Efficiency of cardiac reprogramming can be further boosted by removing a key epigenetic barrier, which we recently identified through a novel loss-of-function screen. Knockdown of the polycomb ring finger oncogene Bmi1 early in the reprogramming process resulted in the most significant enhancement of iCM induction. This inhibitory role was not associated with its well-known function in regulating cell proliferation through genes such as p16Ink4a, p19Arf, and p53. Instead, we found that Bmi1 directly bound to the regulatory regions of several cardiogenic genes, including Gata4, Nkx2-5, Isl1, Pitx2, and Tbx20. Knockdown of Bmi1 resulted in a marked decrease in repressive histone mark H2AK119ub and an increase in active histone mark H3K4me3 at these loci and derepressed these cardiogenic genes during iCM reprogramming. We further demonstrated that Bmi1 depletion substituted for Gata4 in reprogramming fibroblasts into beating iCMs. Thus, our findings implicate Bmi1 as a critical epigenetic barrier to cardiac reprogramming. Furthermore, our studies reveal a previously uncharacterized function of Bmi1 in repressing the cardiogenic program and demonstrate that removing a specific epigenetic barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes (8).

My team and I will continue to develop and use new reprogramming approaches to advance personalized medicine. I believe that our efforts and the efforts of others will one day lead to tailored therapies designed for individual patients.

Winner

Li Qian

Li Qian received her undergraduate degree from Fudan University and a Ph.D. from the University of Michigan, Ann Arbor. She completed her postdoctoral training at Gladstone Institutes, University of California, San Francisco, in July 2012. As an Assistant Professor at UNC–Chapel Hill, Qian is currently exploring the reprogramming approaches for cardiac regeneration and disease modeling.

PHOTO: C. GOODFELLOW
Finalist

Yosef Buganim

Yosef Buganim received undergraduate degrees from Bar-Ilan University and a Ph.D. from the Weizmann Institute of Science. As a postdoctoral fellow at the Whitehead Institute for Biomedical Research at MIT, he used single-cell technologies and bioinformatic approaches to shed light on the molecular mechanisms that underlie the reprogramming of somatic cells to iPSCs. Currently the leader of his own laboratory at The Hebrew University of Jerusalem, Buganim uses somatic cell conversion models to identify and investigate the elements that facilitate safe and complete nuclear reprogramming.

http://science.sciencemag.org/content/352/6292/1401

PHOTO: SHAY HERMAN

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