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.

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.

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.

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.

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.

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

Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.

To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.

Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line.

Previous studies showed that bone morphogenetic protein 9 (BMP-9) induces the expression of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter, and upregulates ACh synthesis in cultured primary neurons from embryonic mouse septum [I. López-Coviella, B. Berse, R. Krauss, R.S. Thies, J.K. Blusztajn, Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289 (2000) 313-316]. In the present studies we investigated the effects of BMP-9 on ACh synthesis in the cholinergic mouse SN56T17 septal cell line. BMP-9 increased ACh synthesis in these cells up to 2.5-fold in a time- and dose-dependent, saturable manner. The maximal effect of BMP-9 was observed after a 3-day treatment and the median effective concentration of BMP-9 was 0.5 ng/ml. These data show that SN56T17 cells are a useful model for studies of the effects of BMPs on the cholinergic phenotype.

Bone morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCN) participate in processes of learning, memory, and attention. Little is known about the genes expressed by BFCN and the extracellular signals that control their expression. Previous studies showed that bone morphogenetic protein (BMP) 9 induces and maintains the cholinergic phenotype of embryonic BFCN. We measured gene expression patterns in septal cultures of embryonic day 14 mice and rats grown in the presence or absence of BMP9 by using species-specific microarrays and validated the RNA expression data of selected genes by immunoblot and immunocytochemistry analysis of their protein products. BMP9 enhanced the expression of multiple genes in a time-dependent and, in most cases, reversible manner. The set of BMP9-responsive genes was concordant between mouse and rat and included genes encoding cell-cycle/growth control proteins, transcription factors, signal transduction molecules, extracellular matrix, and adhesion molecules, enzymes, transporters, and chaperonins. BMP9 induced the p75 neurotrophin receptor (NGFR), a marker of BFCN, and Cntf and Serpinf1, two trophic factors for cholinergic neurons, suggesting that BMP9 creates a trophic environment for BFCN. To determine whether the genes induced by BMP9 in culture were constituents of the BFCN transcriptome, we purified BFCN from embryonic day 18 mouse septum by using fluorescence-activated cell sorting of NGFR(+) cells and profiled mRNA expression of these and NGFR(-) cells. Approximately 30% of genes induced by BMP9 in vitro were overexpressed in purified BFCN, indicating that they belong to the BFCN transcriptome in situ and suggesting that BMP signaling contributes to maturation of BFCN in vivo.

Gene expression in human embryonic stem cell lines: unique molecular signature.

Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing 16 659 seventy-base pair oligonucleotides to examine gene expression in 6 of the 11 available huES cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient > 0.85).A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4, and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases, reverse transcriptase-polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. Comparison with published "stemness" genes revealed a limited overlap, suggesting little similarity with other stem cell populations. Several novel ES cell-specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells.