The advancement of human pluripotent stem cell-derived therapies into the clinic.

There was an error published in Development 142, 3077-3084.On p. 3081, it was incorrectly stated that Dr Lorenz Studer's group is supported by the New York Stem Cell Foundation. The correct funding credit is the New York State Stem Cell Science program.The authors apologise to readers for this mistake.

The advancement of human pluripotent stem cell-derived therapies into the clinic.

Human pluripotent stem cells (hPSCs) offer many potential applications for drug screening and 'disease in a dish' assay capabilities. However, a more ambitious goal is to develop cell therapeutics using hPSCs to generate and replace somatic cells that are lost as a result of disease or injury. This Spotlight article will describe the state of progress of some of the hPSC-derived therapeutics that offer the most promise for clinical use. Lessons from developmental biology have been instrumental in identifying signaling molecules that can guide these differentiation processes in vitro, and will be described in the context of these cell therapy programs.

Axin pathway activity regulates in vivo pY654-ß-catenin accumulation and pulmonary fibrosis.

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3ß1-mediated cross-talk between TGFß1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-ß-catenin accumulation, but whether pY654-ß-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of ß-catenin in fibrosis, we explored pY654-ß-catenin generation and function. α3ß1 was required for TGFß1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFß1 stimulated ß-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT ß-catenin as well as Y654E or Y654F ß-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-ß-catenin was sensitive to the axin ß-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-ß-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-ß-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total ß-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFß1-induced pY654-ß-catenin, provide evidence that pY654-ß-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.

Study of the 14Be continuum: identification and structure of its second 2+ state.

The coupling between bound quantum states and those in the continuum is of high theoretical interest. Experimental studies of bound drip-line nuclei provide ideal testing grounds for such investigations since they, due to the feeble binding energy of their valence particles, are easy to excite into the continuum. In this Letter, continuum states in the heaviest particle-stable Be isotope, 14Be, are studied by employing the method of inelastic proton scattering in inverse kinematics. New continuum states are found at excitation energies E*=3.54(16) MeV and E*=5.25(19) MeV. The structure of the earlier known 2(1)+ state at 1.54(13) MeV was confirmed with a predominantly (0d5/2)2 configuration while there is very clear evidence that the 2(2)+ state has a predominant (1s1/2, 0d5/2) structure with a preferential three-body decay mechanism. The region at about 7 MeV excitation shows distinct features of sequential neutron decay via intermediate states in 13Be. This demonstrates that the increasing availability of energetic beams of exotic nuclei opens up new vistas for experiments leading towards a new understanding of the interplay between bound and continuum states.

Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

Novel oxysterols activate the Hedgehog pathway and induce osteogenesis.

Localized induction of bone formation is essential during orthopedic procedures that involve skeletal repair, such as surgical treatment of non-union bone fractures and degenerative disk disease. Herein we disclose the synthesis and biological evaluation of novel oxysterol derivatives designed as anabolic bone growth agents. Structure-activity relationship studies of oxysterol 4 have identified analogues such as 18, 21 and 30. These new analogues are characterized by higher potency in an osteoblast differentiation assay and/or by increased metabolic stability in human liver microsomes. Oxysterols 4, 18 and 21 were evaluated in vivo in a rat spinal fusion model.

Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes.

Heart failure remains a significant cause of morbidity and mortality following myocardial infarction. Cardiac remuscularization with transplantation of human pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). Although transient, the risk posed by EA presents a barrier to clinical translation. We hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. This study uses a porcine model to provide proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively suppress EA. None of the nine treated subjects experienced the primary endpoint of cardiac death, unstable EA, or heart failure compared with five out of eight (62.5%) in the control cohort (hazard ratio = 0.00; 95% confidence interval: 0-0.297; p = 0.002). Pharmacologic treatment of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.

Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9.

Bone morphogenetic proteins (BMPs) have multiple functions in the developing nervous system. A member of this family, BMP-9, was found to be highly expressed in the embryonic mouse septum and spinal cord, indicating a possible role in regulating the cholinergic phenotype. In cultured neurons, BMP-9 directly induced the expression of the cholinergic gene locus encoding choline acetyltransferase and the vesicular acetylcholine transporter and up-regulated acetylcholine synthesis. The effect was reversed upon withdrawal of BMP-9. Intracerebroventricular injection of BMP-9 increased acetylcholine levels in vivo. Although certain other BMPs also up-regulated the cholinergic phenotype in vitro, they were less effective than BMP-9. These data indicate that BMP-9 is a differentiating factor for cholinergic central nervous system neurons.

Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates.

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

The BMP proteins in bone formation and repair.

From recent advances in the fields of bone biology and pattern formation, the first clues to our understanding of embryonic skeletal development are beginning to emerge. This complex process involves an integration of spatial patterning and the differentiation of specialized cells that make up bone and cartilage. The result is a scale model of the mature skeleton which is able to grow in size to fit the adult body plan. In the mature animal, bone repair after injury appears to be similar to bone formation in the embryo, suggesting that analogous mechanisms for the control of bone formation may exist in the adult and embryonic skeletons.

Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors.

Oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem (hES) cells have been reported to remyelinate axons and improve locomotor function in a rodent model of spinal cord injury. Although remyelination would be expected to have a beneficial effect in spinal cord injury, neurotrophic factor expression may also contribute to functional recovery. Neurotrophic factors could impact the survival of axotomized neurons, as well as promote axonal regeneration in interrupted conduction pathways. This study demonstrates that hES cell-derived OPCs express functional levels of midkine, hepatocyte growth factor (HGF), activin A, transforming growth factor-beta2 (TGF-beta2), and brain-derived neurotrophic factor (BDNF), proteins with reported trophic effects on neurons. The neurotrophic activity of hES cell-derived OPCs is further demonstrated by stimulatory effects on neurite outgrowth of adult rat sensory neurons in vitro.

Insulin-receptor autophosphorylation and endogenous substrate phosphorylation in human adipocytes from control, obese, and NIDDM subjects.

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37 degrees C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12 degrees C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of "spare" autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of beta-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was approximately 50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a focused microarray to assess human embryonic stem cell differentiation.

Human embryonic stem cells (hESC) must be differentiated before clinical use. In addition, the extent of contamination of undifferentiated cells and the efficiency of differentiation must also be assessed prior to clinical application. In this manuscript, we describe the development of a focused microarray that may be used to discriminate between hESC and their differentiated progeny. This array contains 755 genes including embryonic stem cell markers as well as markers of differentiation into neural, mesodermal, and endodermal phenotypes. In addition, we have included candidate genes belonging to families of cytokines, chemokines, receptors, signaling pathways, and homeodomain proteins that are likely to be important in the process of differentiation. Testing and validation of the focused array was performed using RNA from hESC, human embryoid body (hEB) outgrowths, and a human embryonal carcinoma (hEC) cell line. We have compared gene expression with negative background, GAPDH, beta-actin positive controls, and human universal RNA (hURNA), showing that such an array can rapidly distinguish between undifferentiated and differentiated hESC-derived cell populations. We expect that the described array will be extremely useful in evaluating the extent of differentiation and the state of the hESC-derived population utilized for therapeutic purposes.

Heart specific expression of mouse BMP-10 a novel member of the TGF-beta superfamily.

Here we report the cloning and expression of murine BMP-10, a novel member of the TGF-beta superfamily. In the mouse embryo, BMP-10 expression begins at 9.0 d.p.c. and is restricted to the developing heart. Initially, BMP-10 expression localizes to the trabeculated part of the common ventricular chamber and to the bulbus cordis region. After 12.5 d.p.c., additional BMP-10 expression is seen in the atrial wall. The data presented here suggest that BMP-10 plays an important role in trabeculation of the embryonic heart.

Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes.

There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast-like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation.

MPSS profiling of human embryonic stem cells.

BACKGROUND: Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. RESULTS: Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. CONCLUSIONS: Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions

Glucose and insulin regulate insulin sensitivity in primary cultured adipocytes without affecting insulin receptor kinase activity.

We have previously shown in primary cultured adipocytes that chronic insulin exposure decreases insulin's subsequent ability to maximally restimulate the glucose transport system, and that extracellular glucose potentiates this ligand-induced defect in maximal insulin responsiveness. To examine whether glucose could also modulate insulin sensitivity (i.e. acute insulin effects at submaximal concentrations), adipocytes were cultured for 5 and 24 h in the absence and presence of various glucose and insulin concentrations. Then, after washing cells to remove any insulin and allow for full deactivation of transport, we assessed the dose response of insulin's acute ability to stimulate 2-deoxyglucose transport, bind to cell surface receptors, and activate insulin receptor tyrosine kinase activity. After 5 h, glucose and insulin alone had no chronic regulatory effects; however, in combination, these agents were able to decrease insulin sensitivity. In cells preincubated with 50 ng/ml insulin, the insulin ED50 for acute stimulation of glucose transport was increased by 65% and 116% as medium glucose was raised to 5 and 20 mM, respectively, relative to that at 0 mM glucose. After 24 h, chronic exposure to either glucose (20 mM) or insulin (50 ng/ml) alone increased the ED50 value by 52%, and, together they acted synergistically to increase the ED50 by 183%. While glucose and insulin independently and synergistically impaired insulin sensitivity, both agents were necessary for coregulation of maximal insulin responsiveness (confirming our previous observation). Insulin receptor down-regulation (18%) was observed after 24 h (but not 5 h) in insulin-treated cells; however, the major portion of the decrease in insulin sensitivity was due to uncoupling of occupied insulin receptors from stimulation of the glucose transport system. To further determine the mechanism for postbinding desensitization, we tested for concordant regulation of insulin receptor kinase activity. Insulin's ability to stimulate the receptor tyrosine kinase was assessed by multiple methods, including 1) receptor autophosphorylation and phosphorylation of Glu4-Tyr1 by solubilized insulin receptors activated in vitro, 2) histone-2B phosphorylation by receptors that were stimulated in intact cells and then solubilized under conditions that preserve the in cellulo phosphorylation state, and 3) receptor autophosphorylation and phosphorylation of pp180 in intact cells. Long term treatment (24 h) with glucose (10 mM) and insulin (50 ng/ml) markedly decreased insulin sensitivity (and receptor coupling), but did not affect insulin receptor kinase activity in any of these studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells.

To better understand the in vivo bone-inductive properties of recombinant human (rh) BMP-2, we examined the ability of the protein to alter the phenotype of a bone marrow stromal cell line. W-20-17. rhBMP-2 increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner in the absence of an effect on proliferation. The induction of alkaline phosphatase activity was not apparent until 12 h after rhBMP-2 treatment had begun and was effectively eliminated by cotreatment with cycloheximide, suggesting a requirement for protein synthesis. Continued treatment of W-20-17 cells with rhBMP-2 for 8 days resulted in a significant increase, compared to control cultures, in the production of cellular cAMP in response to a PTH challenge. In addition, 4-day treatment with rhBMP-2 induced osteocalcin levels in W-20-17 cells. These results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that BMP-2 may be involved in the differentiation of osteoblasts from progenitor cells resident in bone marrow.

Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation.

A new member of the transforming growth factor (TGF)-beta superfamily, BMP-9, has recently been identified and shown to be expressed in the developing mouse liver. This report demonstrates that human HepG2 liver tumor cells bind recombinant human BMP-9 (rhBMP-9) with high affinity. Cross-linking analysis indicates that HepG2 cells express two BMP-9 receptors of approximately 54 and 80 kilodaltons, similar in size to the Type I and Type II receptors reported by others for TGF-beta and BMP-4. However, cross-competition experiments demonstrate that the BMP-9 receptors on HepG2 cells do not bind other BMPs or TGF-beta s, indicating that these are novel receptors with binding specificity for BMP-9. In functional studies, rhBMP-9 stimulates HepG2 cell proliferation as indicated by [3H]thymidine incorporation and cell counting assays. A proliferative effect of rh-BMP-9 was also observed on primary rat hepatocytes. In contrast, TGF-beta had no effect on HepG2 cell proliferation and inhibited proliferation in primary hepatocytes. These results suggest that BMP-9, acting through a novel set of receptors, may play a regulatory role in hepatic growth and function.