Edward C. Hsiao

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1s effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Canonical Wnt signaling is a positive regulator of mammalian cardiac progenitors

Guiding multipotent cells into distinct lineages and controlling their expansion remain fundamental challenges in developmental and stem cell biology. Members of the Wnt pathway control many pivotal embryonic events, often promoting self-renewal or expansion of progenitor cells. In contrast, canonical Wnt ligands are thought to negatively regulate cardiomyogenesis in several species. However, the cell-autonomous role of canonical Wnt signaling within precardiac mesoderm, through its obligatory transcriptional mediator, β-catenin, is unknown. Using tissue-specific in vivo genetic manipulation, we found that β-catenin is required for development of cardiac progenitors and is a positive regulator of proliferative expansion of such progenitor cells. At discrete windows of development in embryonic stem cells, activation of canonical Wnt signaling promoted expansion of cardiac progenitors after initial commitment and was required for cardiac differentiation. Together, these data provide in vivo and in vitro evidence that canonical Wnt signaling promotes the expansion of cardiac progenitors and differentiation of cardiomyocytes.

Characterization of Growth-Differentiation Factor 15, a Transforming Growth Factor β Superfamily Member Induced following Liver Injury

ABSTRACT We have identified a new murine transforming growth factor β superfamily member, growth-differentiation factor 15 (Gdf15), that is expressed at highest levels in adult liver. As determined by Northern analysis, the expression ofGdf15 in liver was rapidly and dramatically up-regulated following various surgical and chemical treatments that cause acute liver injury and regeneration. In situ hybridization analysis revealed distinct patterns of Gdf15 mRNA localization that appeared to reflect the known patterns of hepatocyte injury in each experimental treatment. In addition, treatment of two hepatocyte-like cell lines with either carbon tetrachloride or heat shock inducedGdf15 mRNA expression, indicating that direct cellular injury can induce Gdf15 expression in the absence of other cell types, such as inflammatory cells. In order to investigate the potential functions of Gdf15, we created Gdf15 null mice by gene targeting. Homozygous null mice were viable and fertile. Despite the dramatic regulation of Gdf15 expression observed in the partial-hepatectomy and carbon tetrachloride injury models, we found no differences in the injury responses between homozygous null mutants and wild-type mice. Our findings suggest either that Gdf15 does not have a regulatory role in liver injury and regeneration or that Gdf15 function within the liver is redundant with that of other signaling molecules.

Engineering GPCR signaling pathways with RASSLs

We are creating families of designer G protein–coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi and Gq). We review these advances here to facilitate the use of these powerful and diverse tools.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury.

The immunoregulatory cytokine macrophage inhibitory cytokine-1 (MIC-1), a divergent TGF-β family member, and its murine ortholog, growth/differentiation factor-15 (GDF-15) are induced in hepatocytes by surgical and chemical injury and heat shock. To better understand the in vivo role this factor plays in organ injury, we examined the regulation of GDF-15 in murine models of kidney and lung injury. We demonstrate herein induction of GDF-15/MIC-1 after surgical, toxic/genotoxic, ischemic, and hyperoxic kidney or lung injury. Gdf15 induction was independent of protein synthesis, a hallmark of immediate-early gene regulation. Although TNF induced GDF-15 expression, injury-elicited Gdf15 expression was not reduced in mice deficient for both TNF receptor subtype. Furthermore, although the stress sensor p53 is known to induce GDF-15/MIC-1 expression, injury-elicited Gdf15 expression was unchanged in p53-null mice. Our results demonstrate that GDF-15 induction after organ injury is a hallmark of many tissues. These data demonstrate that GDF-15/MIC-1 is an early mediator of the injury response in kidney and lung that might regulate inflammation, cell survival, proliferation, and apoptosis in a variety of injured tissues and disease processes.

Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

Astrocytes express a variety of G protein–coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimers disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.

Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass

Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use “Rs1,” an engineered GPCR with constitutive Gs signaling, to evaluate the temporal and skeletal effects of Gs signaling in murine osteoblasts. In vivo, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.

Cytokine-Responsive Gene-2/IFN-Inducible Protein-10 Expression in Multiple Models of Liver and Bile Duct Injury Suggests a Role in Tissue Regeneration

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl4 treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl4, d-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl4 injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.

Reporter-Based Isolation of Induced Pluripotent Stem Cell– and Embryonic Stem Cell–Derived Cardiac Progenitors Reveals Limited Gene Expression Variance

Rationale: Induced pluripotent stem (iPS) cells can differentiate into multiple cell types, including cardiomyocytes and have tremendous potential for drug discovery and regenerative therapies. However, it is unknown how much variability exists between differentiated lineages from independent iPS cell lines and, specifically, how similar iPS cell–derived cardiomyocytes (iPS-CMs) are to embryonic stem (ES) cell–derived cardiomyocytes (ES-CMs). Objective: We investigated how much variability exists between differentiated lineages from independent iPS cell lines and how similar iPS-CMs are to ES-CMs. Methods and Results: We generated mouse iPS cells in which expression of NKX2-5, an early cardiac transcription factor, was marked by transgenic green fluorescent protein (GFP). Isolation of iPS- and ES-derived NKX2-5–GFP+ cardiac progenitor pools, marked by identical reporters, revealed unexpectedly high similarity in genome-wide mRNA expression levels. Furthermore, the variability between cardiac progenitors derived from independent iPS lines was minimal. The NKX2-5–GFP+ iPS cells formed cardiomyocytes by numerous induction protocols and could survive upon transplantation into the infarcted mouse heart without formation of teratomas. Conclusions: Despite the line-to-line variability of gene expression in the undifferentiated state of ES and iPS cells, the variance narrows significantly in lineage-specific iPS-derived cardiac progenitors, and these progenitor cells can be isolated and used for transplantation without generation of unwanted cell types.

Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation

BackgroundAbnormal activation of endochondral bone formation in soft tissues causes significant medical diseases associated with disability and pain. Hyperactive mutations in the bone morphogenetic protein (BMP) type 1 receptor ACVR1 lead to fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterized by progressive ossification in soft tissues. However, the specific cellular mechanisms are unclear. In addition, the difficulty obtaining tissue samples from FOP patients and the limitations in mouse models of FOP hamper our ability to dissect the pathogenesis of FOP.MethodsTo address these challenges and develop a “disease model in a dish”, we created human induced pluripotent stem cells (iPS cells) derived from normal and FOP dermal fibroblasts by two separate methods, retroviral integration or integration-free episomal vectors. We tested if the ability to contribute to different steps of endochondral bone formation was different in FOP vs. control iPS cells.ResultsRemarkably, FOP iPS cells showed increased mineralization and enhanced chondrogenesis in vitro. The mineralization phenotypes could be suppressed with a small-molecule inhibitor of BMP signaling, DMH1. Our results indicate that the FOP ACVR1 R206H mutation favors chondrogenesis and increases mineral deposition in vitro.ConclusionsOur findings establish a FOP disease cell model for in vitro experimentation and provide a proof-of-concept for using human iPS cell models to understand human skeletal disorders.