The Years of the Monkey

The fact that mammals are diploid sets a barrier to rapidly understand the function of non-coding and coding genes in the genome. Recently, Yang et al. reported successful derivation of monkey haploid embryonic stem cells from parthenotes, which provide an effective platform for studying mammalian gene function and enable reverse genetic screening of genes for recessive phenotypes in monkeys.According to the Zodiac in the Chinese Calendar, the next year of the monkey is not slated until February 2016, but a recent paper in this month''s Cell Research suggests that it may have arrived early for the field of stem cell biology. In a stunning technical “Tour de Force”, Jinsong Li and his colleagues report for the first time the generation of several independent haploid monkey embryonic stem (ES) cell lines¹, building on the previous work from their lab and others that described the generation of murine haploid ES cell lines^2,3,4,5 (Figure 1). They first activated metaphase II monkey oocytes with ionomycin followed by cycloheximide treatment. These activated oocytes could develop into blastocysts in vitro and haploid ES cells (haESCs) can be derived by culturing the inner cell mass in a standard monkey ES cell culture system and using Hoechst FACS technique. Remarkably, one of the cell lines remained stable during long term passage, obviating the need for FACS sorting for the haploid cell lines during subsequent propagation. The cell lines can be genetically manipulated by insertional mutagenesis or by PiggyBac transposon technology, suggesting the possibility of genome-wide screening strategies. In this regard, a series of parallel scientific advances suggest that this technology platform may be particularly timely as the field of stem cell biology moves towards regenerative medicine and therapeutics.Open in a separate windowFigure 1The scheme of parthenogenetic (PG) and androgenetic (AG) haploid embryonic stem cells (haESCs) derivation. (A) For the generation of PG-haESCs, metaphase II oocytes were activated with either strontium chloride (SrCl₂) for mice or ionomycin/cycloheximide (CHX) for monkeys and further cultivated to the blastocyst stage. With the help of Hoechst FACS technique, PG-haESCs can be derived. (B) For the generation of AG-haESCs, metaphase II oocytes were enucleated followed by sperm injection. In addition, the reconstructed oocytes were activated with SrCl₂ for mice and further developed to the blastocyst stage in vitro. AG-haESCs can be derived by several rounds of Hoechst FACS based on DNA contents. The derivation of non-human primate AG-haESCs has not been reported yet.For many years, it has proven quite difficult to engineer site-specific mutations, knock-ins, and knock-outs in human ES or induced pluripotent stem (iPS) cells, and only a handful of genetically engineered lines have been created by conventional homologous recombination strategies⁶. However, recent advances in RNA-guided nuclease technology has led to a marked improvement in the efficiency of the knockout of genes in human pluripotent stem cells⁷, suggesting that it may be possible to create knock-out haploid non-human primate (NHP) ES cell lines that harbor specific disease genes and surrogate reporter readouts, and then to look for genetic complementation that could identify critical genes that could be potential drug targets. A library of individual NHP haploid ES cell lines that harbor a loss-of-function mutation across the entire NHP genome could find multiple uses in quickly identifying signaling pathways in differentiated cell types. Given recent advances in screening in human ES and iPS cell lines⁸, direct drug screening on the haploid monkey ES cell lines should also be possible. In addition, it will likely be possible to set up genome-wide screening to systematically identify entire network of genes that drive specific differentiation events, and early steps of primate organogenesis. If androgenetic NHP haploid cell lines can be developed (see Figure 1), a leap in the efficiency of the generation of monkey KO animal models could be envisioned over the long term. In this regard, the recent generation of chimeric monkeys⁹, as well as future technical advances related to this achievement, could become of significant interest.At the same time, the study indirectly raises the query as to the need for monkey model systems when the technology for genetic manipulation in the mouse is without peer, and human ES and iPS cell lines can now be easily generated and genetically manipulated. The recent pronouncement of the termination of NIH support for primate research (http://news.sciencemag.org/people-events/2013/06/nih-will-retire-most-research-chimps-end-many-projects), along with the growing awareness of the need to re-examine the need for NHP models, suggests that there must be very solid scientific grounds for pursuing NHP model systems in the future.In this regard, a growing body of evidence is now pointing to the lack of fidelity of mouse models of human disease to the in vivo human setting, a problem that has plagued cancer therapeutics for decades. Recently, the lack of predictability of human responses from models of murine sepsis has been cogently made¹⁰, and the divergence in the physiology of mice and humans, particularly in terms of metabolism and cardiovascular, are enormous. The complexity and scalability of primate versus murine organogenesis also may be an issue. For example, the human heart is 10 000 times larger than the murine, has a much larger diversity of cell types, and a level of tertiary morphology that is not found in the murine heart (for review see¹¹). Murine cardiogenesis is largely completed with 48 h, while human cardiogenesis occurs over months, and recent studies that suggest a much larger diversity and markedly extended period of proliferation of the family of heart progenitors in the human fetal versus murine heart¹². To date, there are no approved drugs that have come from genetically engineered murine models of cardiovascular (CV) disease, and the biggest CV drugs have actually been discovered based on human genetics (statins, PCSK9, etc.). The increased importance of CV side effects for new drugs in the diabetes space, as well as for other chronic diseases, points to the importance of their study in more sophisticated primate systems, as all these drugs (Avandia, Vioxx, etc.) had cleared conventional screening in rodent model systems. Given the above, we may have to put the Chinese Calendar on auto-repeat mode, as we enter the “Years of the Monkey” in this decade and the next.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

<Emphasis Type="Italic">Sorting nexin 24</Emphasis> genetic variation associates with coronary artery aneurysm severity in Kawasaki disease patients

Background

The sorting nexin (SNX) family is involved in endocytosis and protein trafficking and plays multiple roles in various diseases. The role of SNX proteins in Kawasaki disease (KD) is not known. We attempted to test whether genetic SNX variation associates with the risk of coronary artery aneurysm (CAA) formation in KD.

Methods and results

Chi-square tests were used to identify SNX24 genetic variants associated with KD susceptibility and CAA formation in KD; models were adjusted for fever duration and time of first administration of intravenous immunoglobulin. We obtained clinical characteristics and genotypes from KD patients (76 with CAA and 186 without CAA) in a population-based retrospective KD cohort study (n?=?262). Clinical and genetic factors were associated with CAA formation in KD. In addition, endothelial cell inflammation was evaluated. Significant correlation was observed between KD with CAA complications and the rs28891 single-nucleotide polymorphism in SNX24. Patients with CC?+?CT genotypes had lesser CAA complications. In lipopolysaccharide-treated human umbilical vein endothelial cells, siRNA knockdown of SNX24 significantly decreased gene expression of the proinflammatory cytokines IL-1 beta, IL-6, and IL-8.

Conclusions

Polymorphisms in SNX24 may be used as genetic markers for the diagnosis and prognosis of CAA formation in KD.

     

Self‐Assembled BiFeO3‐ε‐Fe2O3 Vertical Heteroepitaxy for Visible Light Photoelectrochemistry

Self‐assembled vertical heterostructure with a high interface‐to‐volume ratio offers tremendous opportunities to realize intriguing properties and advanced modulation of functionalities. Here, a heterostructure composed of two visible‐light photocatalysts, BiFeO₃ (BFO) and ε‐Fe₂O₃ (ε‐FO), is designed to investigate its photoelectrochemical performance. The structural characterization of the BFO‐FO heterostructures confirms the phase separation with BFO nanopillars embedded in the ε‐FO matrix. The investigation of band structure of the heterojunction suggests the assistance of photoexcited carrier separation, leading to an enhanced photoelectrochemical performance. The insights into the charge separation are further revealed by means of ultrafast dynamics and electrochemical impedance spectroscopies. This work shows a delicate design of the self‐assembled vertical heteroepitaxy by taking advantage of the intimate contact between two phases that can lead to a tunable charge interaction, providing a new configuration for the optimization of photoelectrochemical cell.     

Do the Preferences of Healthcare Provider Selection Vary among Rural and Urban Patients with Different Income and Cause Different Outcome?

BackgroundEqual access to healthcare facilities and high-level quality of care are important strategies to eliminate the disparity in outcome of care. However, the existing literature regarding how urban or rural dwelling patients with different income level select healthcare providers is insufficient. The purposes of this study were to examine whether differences of healthcare provider selection exist among urban and rural coronary artery bypass surgery (CABG) patients with different income level. If so, we further investigated the associated impact on mortality.MethodsA retrospective, multilevel study design was conducted using claims data from 2007–2011 Taiwan’s Universal Health Insurance Scheme. Healthcare providers’ performance and patients’ travelling distance to hospitals were used to define the patterns of healthcare provider selection. Baron and Kenny’s procedures for mediation effect were conducted.ResultsThere were 10,108 CABG surgeries included in this study. The results showed that urban dwelling and higher income patients were prone to receive care from better-performance providers. The travelling distances of urban dwelling patients was 15 KM shorter, especially when they received better-performance provider’s care. The results also showed that the difference of healthcare provider selection and mortality rate existed between rural and urban dwelling patients with different income levels. After the procedure of mediation effect testing, the results showed that the healthcare provider selection partially mediated the relationships between patients’ residential areas with different income levels and 30-day mortality.ConclusionPreferences of healthcare provider selection vary among rural and urban patients with different income, and such differences partially mediated the outcome of care. Health authorities should pay attention to this issue, and propose appropriate solutions to eliminate the disparity in outcome of CABG care.     

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors

Biomechanics and Modeling in Mechanobiology - Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular...     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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Warmest Congratulations to Dr. Yuan-Cheng Fung at His Centennial Celebration

Professor Y.C. Fung has made tremendous impacts on science, engineering and humanity through his research and its applications, by setting the highest standards, through educating many students and their students, and providing his exemplary leadership. He has applied his profound knowledge and elegant analytical methods to the study of biomedical problems with rigor and excellence. He established the foundations of biomechanics in living tissues and organs. Through his vision of the power of “making models” to explain and predict biological phenomena, Dr. Fung opened up new vista for bioengineering, from organs-systems to molecules-genes, and has provided the foundation of research activities in many institutions in the United States and the world. He has made outstanding contributions to education in bioengineering, service to professional organizations, and translation to industry and clinical medicine. He is widely recognized as the Father of Biomechanics and the leading Bioengineer in the world. His extraordinary achievements and commands in science, engineering and the arts make him a Renaissance Man for the world.