Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs).

Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility.

3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs.

Compartmentation of cGMP Signaling in Induced Pluripotent Stem Cell Derived Cardiomyocytes during Prolonged Culture.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs).

Rostrocaudal patterning and neural crest differentiation of human pre-neural spinal cord progenitors in vitro.

The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by WNT/fibroblast growth factor (FGF) signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which compromises production of the full range of rostrocaudal spinal cord identities in vitro.

Modelling Metabolic Shifts during Cardiomyocyte Differentiation, Iron Deficiency and Transferrin Rescue Using Human Pluripotent Stem Cells.

Cardiomyocytes rely on specialised metabolism to meet the high energy demand of the heart. During heart development, metabolism matures and shifts from the predominant utilisation of glycolysis and glutamine oxidation towards lactate and fatty acid oxidation. Iron deficiency (ID) leads to cellular metabolism perturbations.

Vulnerability of progeroid smooth muscle cells to biomechanical forces is mediated by MMP13.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease in children that leads to early death. Smooth muscle cells (SMCs) are the most affected cells in HGPS individuals, although the reason for such vulnerability remains poorly understood. In this work, we develop a microfluidic chip formed by HGPS-SMCs generated from induced pluripotent stem cells (iPSCs), to study their vulnerability to flow shear stress.

Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state.

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling.

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors.

Stable methylation at promoters distinguishes epiblast stem cells from embryonic stem cells and the in vivo epiblasts.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq.

Directed differentiation of embryonic origin-specific vascular smooth muscle subtypes from human pluripotent stem cells.

Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may, in part, be attributed to this inherent heterogeneity in SMC lineage. Therefore, systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease.

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis.

Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility.

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin-specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm.

BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells.

Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell.

Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic beta cell.

In recent years major progress has been made in understanding the role of transcription factors in the development of the endocrine pancreas in the mouse. Here we describe how a number of these transcription factors play a role in maintaining the differentiated phenotype of the beta cell, and in the mechanisms that allow the beta cell to adapt to changing metabolic demands that occur throughout life. Amongst these factors, Pdx1 plays a critical role in defining the region of the primitive gut that will form the pancreas, Ngn3 expression drives cells towards an endocrine lineage, and a number of additional proteins including Pdx1, in a second wave of expression, Pax4, NeuroD1/beta2, and MafA act as beta cell differentiation factors.

Stem cells and metabolic diseases.

Obesity is a metabolic disorder, which has been recognized as a global epidemic. It contributes to insulin resistance, the major cause of Type 2 diabetes, as well as to the development of other related diseases. Our basic premise is that a better understanding of how adult stem cells of the pancreas contribute to the maintenance of the pancreatic beta-cell pool against the increased metabolic demands associated with obesity may provide new therapeutic targets for treating diabetes.

Embryonic stem cell therapy for diabetes mellitus.

There is a compelling need to develop novel therapies for diabetes mellitus. Recent successes in the transplantation of islets of Langerhans are seen as a major breakthrough. However, there is huge disparity between potential recipients and the availability of donor tissue.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.