Antje D. Ebert

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

Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model

RATIONALE Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

A Direct Role for Phosphatidylinositol‐4,5‐bisphosphate in Unconventional Secretion of Fibroblast Growth Factor 2

Fibroblast growth factor 2 (FGF‐2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgi‐independent secretory pathway. Recent findings have shown that FGF‐2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF‐2 contains a binding site for phosphatidylinositol‐4,5‐bisphosphate [PI(4,5)P2], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF‐2 and PI(4,5)P2 is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P2 is critically important for FGF‐2 secretion as experimental conditions reducing cellular levels of PI(4,5)P2 resulted in a substantial drop in FGF‐2 export efficiency. Likewise, we have identified FGF‐2 variant forms deficient for binding to PI(4,5)P2 that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P2 associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF‐2.

Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance

The exact nature of the immune response elicited by autologous induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic contexture of intra-graft characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B, and perforin intra-graft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.

Induced pluripotent stem cells as a disease modeling and drug screening platform.

Abstract: Induced pluripotent stem cells (iPSCs) hold great hopes for therapeutic application in various diseases. Although ongoing research is dedicated to achieving clinical translation of iPSCs, further understanding of the mechanisms that underlie complex pathogenic conditions is required. Compared with other classical models for studying diseases, iPSCs provide considerable advantages. A newly emerging application of iPSCs is in vitro disease modeling, which can significantly improve the never-ending search for new pharmacological cures. Here, we will discuss current efforts to create iPSC-dependent patient-specific disease models. Furthermore, we will review the use of iPSCs for development and testing of new therapeutic agents and the implications for high-throughput drug screening.

The Golgi-Associated Protein p115 Mediates the Secretion of Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a leaderless protein that is secreted from cells by a specialized, nonclassical export pathway. The release of MIF nevertheless is regulated and its production in response to different inflammatory, mitogenic, and hormonal stimuli plays an important role in diverse physiologic and pathologic processes. We report herein the identification of the Golgi complex-associated protein p115 as an intracellular binding partner for MIF. MIF interacts with p115 in the cytoplasm and the stimulated secretion of MIF results in the accumulation of both proteins in supernatants, which is consistent with MIF release from cells in conjunction with p115. The depletion of p115 from monocytes/macrophages decreases the release of MIF but not other cytokines following inflammatory stimulation or intracellular bacterial infection. Notably, the small molecule MIF inhibitor 4-iodo-6-phenylpyrimidine inhibits MIF secretion by targeting the interaction between MIF and p115. These data reveal p115 to be a critical intermediary component in the regulated secretion of MIF from monocytes/macrophages.

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes as an In Vitro Model for Coxsackievirus B3–Induced Myocarditis and Antiviral Drug Screening Platform

Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon&bgr;1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferon&bgr;1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system

The decrease of function in the ALDH2*2 genotype disrupts an important cardioprotective oxidative stress regulatory circuit, thus increasing cardiac cell death after ischemic insult. Personalized Heart Healing In poetry, we welcome assaults to the heart that leave one breathless. But depriving actual heart tissue of oxygen—through decreased blood flow—can cause irreparable damage. The human genome houses ALDH2, a gene that encodes the heart-protective metabolic enzyme aldehyde dehydrogenase 2. But ~8% of the human population carries an inactivating gene polymorphism (ALDH2*2) that has been linked to enhanced severity of damage from cardiac ischemia—a shortage in the heart’s oxygen supply—and an increased risk of coronary artery disease (CAD). Now, Ebert et al. investigate the mechanisms underlying these ALDH2*2-associated maladies using a human cellular model of the ALDH2*2 genotype made with induced pluripotent stem cell–derived cardiomyocytes generated from patient fibroblasts. The authors found that ALDH2 regulated cell survival by modulating oxidative stress, a circuit that was dysfunctional in ALDH2*2 cells. This aberration induced cell cycle arrest and enhanced apoptosis in cardiomyocytes after ischemic insult, illuminating a new function for ALDH2 in cell survival decisions. Such mechanistic insights may spur the development of new diagnostic methods for and improved risk management of CAD as well as genotype-specific cardiac therapies. Now, if we can only find a cure for the poetic broken heart…. Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.