Requirements for human embryonic stem cells

‘Requirements for Human Embryonic Stem Cells’ is the first set of guidelines on human embryonic stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for human embryonic stem cells, which is applicable to the quality control for human embryonic stem cells. It was originally released by the China Society for Cell Biology on 26 February 2019 and was further revised on 30 April 2020. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human embryonic stem cells for applications.     

Neural induction: Historical views and application to pluripotent stem cells

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state‐of‐the‐art stem cell research.     

The secretome from embryonic stem cell cardiomyogenesis: Same signals,different cellular feedbacks

Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC‐derived cardiomyocytes.     

Spawning habits and embryonic development of the banded lampeye killifish Aplocheilichthys spilauchen (Duméril 1861) in ex situ fresh and brackish water environments

Aside from ornamental uses, there is growing interest in using killifishes for a multiplicity of purposes including baitfish and mosquito biocontrol. This experiment explored the spawning habits and embryonic development of the banded lampeye, Aplocheilichthys spilauchen in ex situ freshwater (0.04‰) and brackish water (5.01‰) to ascertain the captive breeding prospects for mosquito control in areas where they occur. Significantly higher number of eggs were laid in the brackish water than the freshwater (X² = 1613.0, P < 0.05), and black mop was the most preferred spawning substrate, followed by green, blue and white mops. Microscopic monitoring of embryos revealed that cleavage occurred within the first 30 min after fertilization, organogenesis commenced on average in the 25th hour and hatching in approximately 230 h. Although freshwater eggs were relatively bigger than brackish water eggs and certain embryonic developmental stages occurred faster in the freshwater than brackish water, these differences were overall not significant and had no effects on the development and hatching. The observed outcome that A. spilauchen can be optimally propagated with black mops in brackish water offers a significant step in its use for the mosquito biocontrol programme, as well as other potential uses not yet explored.     

A new unique form of microRNA from human heart,microRNA-499c,promotes myofibril formation and rescues cardiac development in mutant axolotl embryos

Background

A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.

Results

We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.

Conclusions

This new form, microRNA-499c, plays an important role in cardiac development.     

Resistance to freezing temperatures in Aedes (Ochlerotatus) albifasciatus (Macquart) eggs (Diptera: Culicidae) from two different climatic regions of Argentina

Aedes (Ochlerotatus) albifasciatus (Macquart) has the capacity to proliferate in different kinds of climates within its distribution range in South America. With the aim of studying local thermal adaptations of eggs, we exposed egg stocks from two climatically different localities: temperate humid pampa (Buenos Aires) and cold arid Patagonian (Sarmiento), to freezing conditions and then evaluated the effect on some features at this level. First, we thermally described the substrate where this species lays its eggs in the arid region. A typical thermal condition during winter was 10 h at ?12° C. Second, we evaluated the effect of freezing on primary hatching (vs total hatching) and embryo survival. We also compared the proportion of embryonated eggs from both populations. The proportions of embryonated eggs were not different between localities, with averages of 78% and 83% in Sarmiento and Buenos Aires, respectively. Survival was equally successful after freezing in the two localities with an average range between 94–99%. Whether or not the eggs from Buenos Aires and Sarmiento were under freezing conditions, hatching was more than 98% after the first flooding. The results suggest that eggs of Ae. albifasciatus from Sarmiento and Buenos Aires have the same ability to survive at extreme temperatures (<0° C), showing a regional thermal adaptation rather than a local one.     

Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding

ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC₅₀ of 7.6, 13.6, and 7.0 μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC₅₀ of 18.8 μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [³H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC₅₀ of 1.9 μM, and efficiently competed with [³H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL.