Joseph C. Wu

Global Epigenomic Reconfiguration During Mammalian Brain Development

Introduction Several lines of evidence point to a key role for dynamic epigenetic changes during brain development, maturation, and learning. DNA methylation (mC) is a stable covalent modification that persists in post-mitotic cells throughout their lifetime, defining their cellular identity. However, the methylation status at each of the ~1 billion cytosines in the genome is potentially an information-rich and flexible substrate for epigenetic modification that can be altered by cellular activity. Indeed, changes in DNA methylation have been implicated in learning and memory, as well as in age-related cognitive decline. However, little is known about the cell type–specific patterning of DNA methylation and its dynamics during mammalian brain development. The DNA methylation landscape of human and mouse neurons is dynamically reconfigured through development. Base-resolution analysis allowed identification of methylation in the CG and CH context (H = A, C, or T). Unlike other differentiated cell types, neurons accumulate substantial mCH during the early years of life, coinciding with the period of synaptogenesis and brain maturation. Methods We performed genome-wide single-base resolution profiling of the composition, patterning, cell specificity, and dynamics of DNA methylation in the frontal cortex of humans and mice throughout their lifespan (MethylC-Seq). Furthermore, we generated base-resolution maps of 5-hydroxymethylcytosine (hmC) in mammalian brains by TAB-Seq at key developmental stages, accompanied by RNA-Seq transcriptional profiling. Results Extensive methylome reconfiguration occurs during development from fetal to young adult. In this period, coincident with synaptogenesis, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. We uncovered surprisingly complex features of brain cell DNA methylation at multiple scales, first by identifying intragenic methylation patterns in neurons and glia that distinguish genes with cell type–specific activity. Second, we report a novel mCH signature that identifies genes escaping X-chromosome inactivation in neurons. Third, we find >100,000 developmentally dynamic and cell type–specific differentially CG-methylated regions that are enriched at putative regulatory regions of the genome. Finally, whole-genome detection of 5-hydroxymethylcytosine (hmC) at single-base resolution revealed that this mark is present in fetal brain cells at locations that lose CG methylation and become activated during development. CG-demethylation at these hmC-poised loci depends on Tet2 activity. Discussion Whole-genome single-base resolution methylcytosine and hydroxymethylcytosine maps revealed profound changes during frontal cortex development in humans and mice. These results extend our knowledge of the unique role of DNA methylation in brain development and function, and offer a new framework for testing the role of the epigenome in healthy function and in pathological disruptions of neural circuits. Overall, brain cell DNA methylation has unique features that are precisely conserved, yet dynamic and cell-type specific. Epigenetic Brainscape Epigenetic modifications and their potential changes during development are of high interest, but few studies have characterized such differences. Lister et al. (1237905, published online 4 July; see the Perspective by Gabel and Greenberg) report whole-genome base-resolution analysis of DNA cytosine modifications and transcriptome analysis in the frontal cortex of human and mouse brains at multiple developmental stages. The high-resolution mapping of DNA cytosine methylation (5mC) and one of its oxidation derivatives (5hmC) at key developmental stages provides a comprehensive resource covering the temporal dynamics of these epigenetic modifications in neurons compared to glia. The data suggest that methylation marks are dynamic during brain development in both humans and mice. A genome-wide map shows that DNA methylation in neurons and glial cells changes during development in humans and mice. [Also see Perspective by Gabel and Greenberg] DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity.

In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery

Background— Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. Methods and Results— Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1×107 of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7×107±5.8×106 photons · s−1 · cm−2 per steradian (sr) and 0.08±0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg). Conclusion— This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

Chemically defined generation of human cardiomyocytes

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule–based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and ricederived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Addresses for Correspondence: Joseph C. Wu, MD, PhD, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Room G1120B, Stanford, CA 94305-5454. joewu@stanford.edu or Paul W. Burridge, PhD, burridge@stanford.edu. Author Contributions P.W.B. conceived, performed, and interpreted the experiments and wrote the manuscript; E.M. performed cardiomyocyte immunofluorescence, single-cell RT-PCR, and electrophysiology data assessment; P.S., Z.L., and A.J.O. performed electrophysiology experiments and assessed data; S.D. provided CoMiP reprogrammed cells; B.H. performed teratoma assay; J.M.C. A.D.E, F.L., N.M.M., and J.R.P tested differentiation; B.C., J.D.G. provided experimental advice; and J.C.W. provided experimental advice, manuscript writing, and funding support. Competing Financial Interests JCW is a co-founder of Stem Cell Theranostics. Other authors declare no competing financial interests. HHS Public Access Author manuscript Nat Methods. Author manuscript; available in PMC 2015 February 01. Published in final edited form as: Nat Methods. 2014 August ; 11(8): 855–860. doi:10.1038/nmeth.2999. A uhor M anscript

Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.

Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy

Human induced pluripotent stem cells generated from patients with familial dilated cardiomyopathy model cardiovascular disease in these patients. iPSCs Make the Heart Beat Faster Mutations in genes expressed in the heart can cause dilated cardiomyopathy (DCM), a form of heart disease in which a weakened and enlarged heart is unable to pump sufficient blood for the body’s needs. DCM can lead to progressive heart failure that eventually requires heart transplantation. This disease has been challenging to study because cardiomyocytes from the hearts of DCM patients are difficult to obtain and do not survive long. Mouse models of DCM are established and have provided important clues about the disease mechanisms for DCM. However, the mouse heart is very different in physiology compared to the human heart; for example, the mouse heart rate is 10 times faster than that of human. In a new study, Sun et al. generated induced pluripotent stem cells (iPSCs) from skin cells of patients in a family with inherited DCM. This family carries a deleterious mutation in TNNT2, a gene that is expressed specifically in the heart and regulates cardiomyocyte contraction. Using iPSCs, the authors generated a large number of individual-specific cardiomyocytes carrying the specific TNNT2 mutation and analyzed their functional properties. Compared to cardiomyocytes derived from iPSCs of healthy controls in the same family, cardiomyocytes derived from iPSCs of DCM patients exhibited an increased heterogeneous myofilament organization, susceptibility to stress, compromised ability to regulate calcium flux, and decreased contraction force. These results suggest that the mutation in TNNT2 causes abnormalities in the cardiomyocytes and contributes to the development of DCM disease. Using these DCM iPSC–derived cardiomyocytes, the researchers also showed that several current treatments that clinically benefit DCM disease improved DCM cardiomyocyte function in culture. The current study shows that human iPSC-derived cardiomyocytes could provide an important platform to investigate the specific disease mechanisms of DCM as well as other inherited cardiovascular disorders and for screening new drugs for cardiovascular disease. Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca2+), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC–derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

Cerebral Metabolism during Propofol Anesthesia in Humans Studied with Positron Emission Tomography

BACKGROUND Although the effects of propofol on cerebral metabolism have been studied in animals, these effects have yet to be directly examined in humans. Consequently, we used positron emission tomography (PET) to demonstrate in vivo the regional cerebral metabolic changes that occur in humans during propofol anesthesia. METHODS Six volunteers each underwent two PET scans; one scan assessed awake-baseline metabolism, and the other assessed metabolism during anesthesia with a propofol infusion titrated to the point of unresponsiveness (mean rate +/- SD = 7.8 +/- 1.5 mg.kg-1.h-1). Scans were obtained using the 18fluorodeoxyglucose technique. RESULTS Awake whole-brain glucose metabolic rates (GMR) averaged 29 +/- 8 mumoles.100 g-1.min-1 (mean +/- SD). Anesthetized whole-brain GMR averaged 13 +/- 4 mumoles.100 g-1.min-1 (paired t test, P < or = 0.007). GMR decreased in all measured areas during anesthesia. However, the decrease in GMR was not uniform. Cortical metabolism was depressed 58%, whereas subcortical metabolism was depressed 48% (P < or = 0.001). Marked differences within cortical regions also occurred. In the medial and subcortical regions, the largest percent decreases occurred in the left anterior cingulate and the inferior colliculus. CONCLUSION Propofol produced a global metabolic depression on the human central nervous system. The metabolic pattern evident during anesthesia was reproducible and differed from that seen in the awake condition. These findings are consistent with those from previous animal studies and suggest PET may be useful for investigating the mechanisms of anesthesia in humans.

Drug Screening Using a Library of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes Reveals Disease-Specific Patterns of Cardiotoxicity

Background— Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results— Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go–related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations. Conclusions— We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go–related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti–stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs—the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

Molecular Imaging of Cardiac Cell Transplantation in Living Animals Using Optical Bioluminescence and Positron Emission Tomography

Background—The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques. Methods and Results—For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3×106 to 5×105) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3×106 cells: day 1 (627 000±15%), day 2 (346 100±21%), day 4 (112 800±20%), day 8 (78 860±24%), day 12 (67 780±12%), and day 16 (62 200±5% p · s−1 · cm2−1 · sr−1). For micro–positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3×106) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48±0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01±0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays. Conclusions—The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.