Synthetic probes for in vitro purification and in vivo tracking of hepatocytes derived from human pluripotent stem cells.

Hepatocytes derived from human pluripotent stem cells (hPSCs) are promising candidates for cell therapy and drug discovery. However, it remains challenging to efficiently purify hepatocytes from undesired cell types after differentiation and to accurately monitor grafted cells after transplantation. Indocyanine Green (ICG), an FDA-approved, near-infrared (NIR) dye, has been used for various clinical purposes and is exclusively taken up by hepatocytes. However, ICG has a long emission wavelength (λem > 800 nm) that is beyond the detection range of fluorescence-activated cell sorting (FACS) systems. Moreover, it is easily eliminated from hepatocytes, hindering its application for NIR imaging. Here, we designed and synthesized two different probes based on the properties of ICG; 1) hepatocyte purifying agent (HPA, λem = 562 nm) for in vitro sorting and 2) hepatocyte imaging agent (HIA, λem = 817 nm) for efficient in vivo NIR imaging. We obtained highly enriched populations of hPSC-derived hepatocytes (hPSC-Heps) from various hPSC lines using HPA probe-based FACS purification. In addition, HIA labelling and NIR imaging allowed the direct visualization and tracking of grafted hPSC-Heps in animals with liver injuries. These results demonstrated that these two probes could be used as powerful tools with hPSC-Heps in both cell replacement therapy and drug screening.

Neurons Derived from Human Induced Pluripotent Stem Cells Integrate into Rat Brain Circuits and Maintain Both Excitatory and Inhibitory Synaptic Activities.

The human cerebral cortex is a complex structure with tightly interconnected excitatory and inhibitory neuronal networks. In order to study human cortical function, we recently developed a method to generate cortical neurons from human induced pluripotent stem cells (hiPSCs) that form both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. These cultures and organoids recapitulate neuronal populations representative of the six cortical layers and a balanced excitatory and inhibitory network that is functional and homeostatically stable. To determine whether hiPSC-derived neurons can integrate and retain physiologic activities in vivo, we labeled hiPSCs with red fluorescent protein (RFP) and introduced hiPSC-derived neural progenitors to rat brains. Efficient neural induction, followed by differentiation resulted in a RFP+ neural population with traits of forebrain identity and a balanced synaptic activity composed of both excitatory neurons and inhibitory interneurons. Ten weeks after transplantation, grafted cells structurally integrated into the rat forebrain. Remarkably, these hiPSC-derived neurons were able to fire, exhibiting both excitatory and inhibitory postsynaptic currents, which culminates in the establishment of neuronal connectivity with the host circuitry. This study demonstrates that neural progenitors derived from hiPSCs can differentiate into functional cortical neurons and can participate in neural network activity through functional synaptic integration in vivo, thereby contributing to information processing.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1.

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development.

Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells.

Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals. Here we describe a detailed protocol that describes how to initiate the in vitro differentiation of mouse and human PSCs into cardiac progenitor cells, followed by intramyocardial delivery of the progenitor cells into neonatal rat hearts, in vivo incubation and analysis. The entire process takes ∼6 weeks, and the resulting cardiomyocytes can be analyzed for morphology, function and gene expression. The neonatal system provides a valuable tool for understanding the maturation and pathogenesis of adult human heart muscle cells, and this concept may be expanded to maturing other PSC-derived cell types, including those containing mutations that lead to the development of diseases in the adult.

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy.

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases.

Current Understanding of Stem Cell and Secretome Therapies in Liver Diseases.

Liver failure is one of the main risks of death worldwide, and it originates from repetitive injuries and inflammations of liver tissues, which finally leads to the liver cirrhosis or cancer. Currently, liver transplantation is the only effective treatment for the liver diseases although it has a limitation due to donor scarcity. Alternatively, cell therapy to regenerate and reconstruct the damaged liver has been suggested to overcome the current limitation of liver disease cures. Several transplantable cell types could be utilized for recovering liver functions in injured liver, including bone marrow cells, mesenchymal stem cells, hematopoietic stem cells, macrophages, and stem cell-derived hepatocytes. Furthermore, paracrine effects of transplanted cells have been suggested as a new paradigm for liver disease cures, and this application would be a new strategy to cure liver failures. Therefore, here we reviewed the current status and challenges of therapy using stem cells for liver disease treatments.

Tbx2 regulates anterior neural specification by repressing FGF signaling pathway.

During early embryogenesis, FGF signals regulate the antero-posterior (AP) patterning of the neural plate by promoting posterior cell fates. In particular, BMP signal-mediated attenuation of FGF pathway plays a critical role in the determination of the anterior neural region. Here we show that Tbx2, a T-box transcriptional repressor regulates anterior neural specification by suppressing FGF8 signaling pathway in Xenopus embryo. Tbx2 is expressed in the anterior edge of the neural plate in early neurulae. Overexpression and knockdown of Tbx2 induce expansion and reduction in the expression of anterior neural markers, respectively. It also suppresses FGF8-induced ERK phosphorylation and neural caudalization. Tbx2, which is a target gene of BMP signal, down-regulates FGF8 signaling by inhibiting the expression of Flrt3, a positive regulator of this pathway. We found that Tbx2 binds directly to the T-box element located in the promoter region of Flrt3 gene, thereby interfering with the activity of the promoter. Consistently, Tbx2 augmentation of anterior neural formation is inhibited by co-expression of Flrt3. Furthermore, disruption of the anterior-most structures such as eyes in Tbx2-depleted embryos can be rescued by inhibition of Flrt3 function or FGF signaling. Taken together, our results suggest that Tbx2 mediates BMP signal to down-regulate FGF signaling pathway by repressing Flrt3 expression for anterior tissue formation.

Functional Coupling with Cardiac Muscle Promotes Maturation of hPSC-Derived Sympathetic Neurons.

Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Regenerative medicine for the heart: perspectives on stem-cell therapy.

SIGNIFICANCE: Despite decades of progress in cardiovascular biology and medicine, heart disease remains the leading cause of death, and there is no cure for the failing heart. Since heart failure is mostly caused by loss or dysfunction of cardiomyocytes (CMs), replacing dead or damaged CMs with new CMs might be an ideal way to reverse the disease. However, the adult heart is composed mainly of terminally differentiated CMs that have no significant self-regeneration capacity. RECENT ADVANCES: Stem cells have tremendous regenerative potential and, thus, current cardiac regenerative research has focused on developing stem cell sources to repair damaged myocardium. CRITICAL ISSUES: In this review, we examine the potential sources of cells that could be used for heart therapies, including embryonic stem cells and induced pluripotent stem cells, as well as alternative methods for activating the endogenous regenerative mechanisms of the heart via transdifferentiation and cell reprogramming. We also discuss the current state of knowledge of cell purification, delivery, and retention. FUTURE DIRECTIONS: Efforts are underway to improve the current stem cell strategies and methodologies, which will accelerate the development of innovative stem-cell therapies for heart regeneration.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion.DOI: http://dx.doi.org/10.7554/eLife.02164.001.

BMP signal attenuates FGF pathway in anteroposterior neural patterning.

In vertebrate early development, the neural tissue is specified along the antero-posterior (A-P) axis by the activity of graded patterning signals such as Wnt, Nodal and FGF. Attenuation of these signals has been shown to play critical roles in the determination of anterior neural region, but it remains poorly understood how FGF action is counteracted in the neural plate. Here, we show that BMP signal acts as an antagonist of FGF signaling for AP neural patterning in Xenopus embryo. During the neurula stages, BMP signal was up-regulated in the anterior neural plate, displaying a graded pattern along the AP axis. Inhibition of the late BMP signaling after mid-gastrulation abrogated the expression of anterior neural markers. We found that BMP signaling interfered with FGFs-induced ERK phosphorylation and neural caudalization. This inhibitory action of BMP signal involved repression of the expression of Flrt3, a positive regulator of FGF signaling. Furthermore, the gain- and loss-of-function of Flrt3 inhibited and expanded the expression of forebrain marker genes, respectively. Together, these results demonstrate that BMP signal can down-regulate FGF pathway via inhibition of Flrt3 expression for anterior neural formation, revealing stage-specific roles of BMP signaling and its novel crosstalk with FGF pathway in neural development.

Selective fluorescent detection of RNA in living cells by using imidazolium-based cyclophane.

A water-soluble imidazolium-based fluorescent chemosensor senses RNA selectively through fluorescence enhancement over other biologically relevant biomolecules in aqueous solution at physiological pH 7.4. Fluorescence image detection of RNA in living cells such as onion cells, HeLa cells, and animal model cells was successfully demonstrated which displays a chelation-enhanced fluorescence effect. These affinities can be attributed to the strong electrostatic (C-H)(+)···A(-) ionic H-bonding and the aromatic moiety driven π-stacking of imidazolium-based cyclophane with the size-complementary major groove of RNA.

Role of Tbx2 in defining the territory of the pronephric nephron.

Despite extensive study of the development of the nephron, which is the functional unit of the kidney, the molecular mechanisms underlying the determination of nephron size remain largely unknown. Using the Xenopus pronephros, we demonstrate here that Tbx2, a T-box transcriptional repressor, functions to demarcate the territory of the pronephric nephron. Tbx2 is specifically expressed around three distinct components of the pronephric nephron: the tubule, duct and glomus. Gain of function of Tbx2 inhibits nephric mesoderm formation. Conversely, Tbx2 loss of function expands the boundary of each component of the pronephric nephron, resulting in an enlarged pronephros. BMP signals induce Tbx2 in the non-nephric mesoderm, which inhibits the expression of the nephric markers Hey1 and Gremlin. Importantly, these pronephric molecules repress Tbx2 expression by antagonizing BMP signals in the nephric mesoderm. These results suggest that the negative regulatory loops between BMP/Tbx2 and Gremlin or Hey1 are responsible for defining the territory of the pronephric nephron.

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.