NK92 cells and peripheral blood NK cells respond oppositely upon dasatinib treatment.

Natural killer (NK) cell is a valuable tool for immunotherapy in cancer treatment, both the cultured cell line NK92 and primary NK cells are widely studied and used in research and clinical trials. Clinical observations witnessed the improvement of patients' NK cells in terms of cell counts and cytotoxic activity upon dasatinib treatment, an approved drug for chronic myeloid leukaemia and Ph+ acute lymphocytic leukaemia. Several studies supported the clinical observations, yet others argued a detrimental effect of dasatinib on NK cells. Due to the complex conditions in different studies, the definite influence of dasatinib on NK92 and primary NK cells remains to be settled. Here, we used a well-defined in vitro system to evaluate the effects of dasatinib on NK92 cells and peripheral blood (PB)-NK cells. By co-culturing NK cells with dasatinib to test the cell counts and target cell-killing activities, we surprisingly found that the chemical influenced oppositely on these two types of NK cells. While dasatinib suppressed NK92 cell proliferation and cytotoxic activity, it improved PB-NK-killing tumour cells. RNA sequencing analysis further supported this finding, uncovering several proliferating and cytotoxic pathways responding invertedly between them. Our results highlighted an intrinsic difference between NK92 and PB-NK cells and may build clues to understand how dasatinib interacts with NK cells in vivo.

1-Naphthaleneacetic Acid Improved the In Vitro Cell Culturing by Inhibiting Apoptosis.

In vitro cell culturing witnessed its applications in scientific research and industrial activities. Attempts to shorten the doubling time of cultured cells have never ceased. In plants, auxin is applied to promote plant growth, the synthetic derivative 1-Naphthaleneacetic acid (NAA) is a good example. Despite the auxin's naturally occurring receptors are not present in mammalian cells, studies suggested they may affect cell culturing. Yet the effects and mechanisms are still unclear. Here, an up to 2-fold increase in the yield of in vitro cultured human cells is observed. Different types of human cell lines and primary cells are tested and found that NAA is effective in all the cells tested. The PI staining followed by FACS suggested that NAA do not affect the cell cycling. Apoptosis-specific dye staining analysis implicated that NAA rescued cell death. Further bulk RNA sequencing is done and it is identified that the lipid metabolism-engaging and anti-apoptosis gene, ANGPTL4, is enhanced in expression upon NAA treatment. Studies on ANGPTL4 knockout cells indicated that ANGPTL4 is required for NAA-mediated response. Thus, the data identified a beneficial role of NAA in human cell culturing and highlighted its potency in in vitro cell culturing.

Epigenetics alternation in lung fibrosis and lung cancer.

Respiratory disease including interstitial lung diseases (ILDs) and lung cancer is a group of devastating diseases that linked with increased morbidity and healthcare burden. However, respiratory diseases cannot be fully explained by the alternation of genetic information. Genetic studies described that epigenetic mechanisms also participate to transmit genetic information. Recently, many studies demonstrated the role of altered epigenetic modification in the pathogenesis of lung cancer and pulmonary fibrosis. Due to lacking effective medication, the underlying pathophysiological processes and causal relationships of lung diseases with epigenetic mechanisms still need to be better understood. Our present review provided a systematic revision of current knowledge concerning diverse epigenetic aberrations in major lung diseases, with special emphasis on DNA methylation, histone modifications, lncRNAs profiles, telomere patterns, as well as chromatin-remodelling complexes. We believed that a new target therapy for lung disease based on findings of the involved epigenetic pathway is a promising future direction.

Current Progress of CAR-NK Therapy in Cancer Treatment.

CD8+ T cells and natural killer (NK) cells eliminate target cells through the release of lytic granules and Fas ligand (FasL)-induced target cell apoptosis. The introduction of chimeric antigen receptor (CAR) makes these two types of cells selective and effective in killing cancer cells. The success of CAR-T therapy in the treatment of acute lymphoblastic leukemia (ALL) and other types of blood cancers proved that the immunotherapy is an effective approach in fighting against cancers, yet adverse effects, such as graft versus host disease (GvHD) and cytokine release syndrome (CRS), cannot be ignored for the CAR-T therapy. CAR-NK therapy, then, has its advantage in lacking these adverse effects and works as effective as CAR-T in terms of killing. Despite these, NK cells are known to be hard to transduce, expand in vitro, and sustain shorter in vivo comparing to infiltrated T cells. Moreover, CAR-NK therapy faces challenges as CAR-T therapy does, e.g., the time, the cost, and the potential biohazard due to the use of animal-derived products. Thus, enormous efforts are needed to develop safe, effective, and large-scalable protocols for obtaining CAR-NK cells. Here, we reviewed current progress of CAR-NK therapy, including its biological properties, CAR compositions, preparation of CAR-NK cells, and clinical progresses. We also discussed safety issues raised from genetic engineering. We hope this review is instructive to the research community and a broad range of readers.

Multifaceted modifications for a cell size-based circulating tumor cell scope technique hold the prospect for large-scale application in general populations.

Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear-cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.

Immunity-and-matrix-regulatory cells derived from human embryonic stem cells safely and effectively treat mouse lung injury and fibrosis.

Lung injury and fibrosis represent the most significant outcomes of severe and acute lung disorders, including COVID-19. However, there are still no effective drugs to treat lung injury and fibrosis. In this study, we report the generation of clinical-grade human embryonic stem cells (hESCs)-derived immunity- and matrix-regulatory cells (IMRCs) produced under good manufacturing practice requirements, that can treat lung injury and fibrosis in vivo. We generate IMRCs by sequentially differentiating hESCs with serum-free reagents. IMRCs possess a unique gene expression profile distinct from that of umbilical cord mesenchymal stem cells (UCMSCs), such as higher expression levels of proliferative, immunomodulatory and anti-fibrotic genes. Moreover, intravenous delivery of IMRCs inhibits both pulmonary inflammation and fibrosis in mouse models of lung injury, and significantly improves the survival rate of the recipient mice in a dose-dependent manner, likely through paracrine regulatory mechanisms. IMRCs are superior to both primary UCMSCs and the FDA-approved drug pirfenidone, with an excellent efficacy and safety profile in mice and monkeys. In light of public health crises involving pneumonia, acute lung injury and acute respiratory distress syndrome, our findings suggest that IMRCs are ready for clinical trials on lung disorders.

Therapeutic Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells on Canine Radiation-Induced Lung Injury.

PURPOSE: To investigate the effect of human umbilical cord-derived mesenchymal stem cell (MSC) transplantation on canine radiation-induced lung injury. METHODS AND MATERIALS: Beagle dogs received localized 15-Gy x-ray radiation to the right lower lung to establish the model of radiation-induced lung injury. After 180 days, dogs were divided into 2 groups (4 per group). The MSC group received intratracheal MSC transplantation, and the saline group received the same volume of normal saline by lavage. The effect of MSC transplantation on lung injury was then evaluated 180 days after transplantation. RESULTS: At 180 days after 15-Gy radiation, canine arterial blood oxygen partial pressure was significantly decreased, and the levels of hydroxyproline and transforming growth factor (TGF)-ß in peripheral blood were significantly increased, whereas that of TGF-α was significantly decreased. Computed tomography evaluation revealed visible honeycomb shadows in the right middle and lower pulmonary pleurae. Blood oxygen partial pressure of the MSC group gradually increased over time, whereas the levels of hydroxyproline and TGF-ß in the peripheral blood showed a decreasing trend; TGF-α levels gradually increased, which differed significantly from the results observed in the saline group. In addition, computed tomography and pathologic examination showed that the degree of lung injury in the MSC group was milder. The MSC group also showed significantly increased pulmonary superoxide dismutase levels and significantly decreased tumor necrosis factor-α, Interleukein-1, and hyaluronic acid levels. Further study confirmed that MSC transplantation inhibited the activation of TGF-ß-Smad2/3 in lung tissues, and in vitro experiments showed that medium conditioned with MSCs effectively inhibited the increase in Smad2 and 3 levels induced by TGF-ß1. CONCLUSION: Canine radiation-induced lung injury could be observed at 180 days after radiation at 15 Gy. MSC transplantation can reduce oxidative stress, inflammatory reactions, and TGF-ß-Smad2/3 pathway activation, thereby reducing lung injury.

Generation of clinical-grade functional cardiomyocytes from human embryonic stem cells in chemically defined conditions.

A highly efficient cardiac differentiation from human pluripotent stem cells (hPSCs) is achievable using existing methods, especially with the standard B27 induction system. However, bovine serum albumin (BSA), one of the essential ingredients in B27, may pose significant complications for clinical studies owing to its animal origin and potential risks of virus contamination. Furthermore, the high cost of the B27 induction system also limits the applications of hPSCs-derived cardiomyocytes. Here, a BSA-free and chemically defined medium has been developed for differentiating hPSCs to clinical-grade cardiomyocytes, which generated over 80% cardiac troponin T (cTNT)-positive cardiomyocytes with high yield. When engrafting the cardiomyocytes into the hearts of myocardial infarction model rats, the rats survived with significantly improved heart functions in Δ ejection fraction and Δ fractional shortening. Importantly, the human embryonic stem cell (hESC) line (Q-CTS-hESC-2) chosen for differentiation was of a clinical-grade maintained in defined xeno-free conditions. Compliant with the biological safety requirements, the Q-CTS-hESC-2-derived cardiomyocytes have passed the sterility and pathogen criteria tests for clinical applications. This study reports, for the first time, the generation of clinical-grade and functional cardiomyocytes from hPSCs where BSA-free and chemically defined conditions were maintained throughout the whole process. This provides the possibility of future therapeutic use of clinical-grade hPSCs-derived cardiomyocytes in treating heart diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Accreditation of Biosafe Clinical-Grade Human Embryonic Stem Cells According to Chinese Regulations.

Human embryonic stem cells (hESCs) are promising in regenerative medicine. Although several hESC-based clinical trials are under way, a widely accepted standard of clinical-grade cells remains obscure. To attain a completely xeno-free clinical-grade cell line, the system must be free of xenogenic components, the cells must have a comprehensive set of functions, and good manufacturing practice conditions must be used. In this study, following these criteria, we successfully derived two hESC lines, which were thereby considered "clinical-grade embryonic stem cells". In addition to the primary capacity for pluripotency, these two cell lines were efficiently differentiated into various types of clinical-grade progeny. Importantly, the cells were recognized by the National Institutes for Food and Drug Control of China for further eligible accreditation. These data indicate that we have established completely xeno-free clinical-grade hESC lines and their derivatives, which will be valuable for the foundation of an international standard for clinical-grade cells for therapy.

Binding induced colocalization activated hybridization chain reaction on the surface of magnetic nanobead for sensitive detection of adenosine.

Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10(-7)mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.

Durable pluripotency and haploidy in epiblast stem cells derived from haploid embryonic stem cells in vitro.

Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

Hairpin assembly circuit-based fluorescence cooperative amplification strategy for enzyme-free and label-free detection of small molecule.

Here, we developed an enzyme-free, label-free, and sensitive fluorescence cooperative amplification strategy based on a hairpin assembly circuit which coupled catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR) for small molecule adenosine. A double-strand DNA probe with aptamer-catalysis strand (Apt-C) and inhibit strand (Inh) was designed for adenosine recognition and signal transduction which was named as Apt-C/Inh. Hairpins H1 and H2 were employed for constructing the CHA, and hairpins H3 and H4 for the HCR. Through the binding of adenosine and the Apt-C, the Inh was released from the Apt-C/Inh. Then the free Apt-C initiated the CHA through successively opening H1 and H2, generating H1/H2 complex and recyclable Apt-C. Next, the released Apt-C entered another CHA cycle, and the H1/H2 complex further initiated the HCR of H3 and H4 which induced the formation of the concatemers of H3/H4 complex. Such a process brought the two ends of hairpins H3 into close proximity, yielding numerous integrated G-quadruplexes which were initially sequestered in the stem and two terminals of H3. Finally, N-methyl mesoporphyrin IX (NMM) was added to generate an enhanced fluorescence signal. In the proposed strategy, driven only by the energy from hybridization, one target could trigger multiple HCR events via CHA-based target-cycle, leading to a remarkable enzyme-free amplification for adenosine. The detection limit could achieve as low as 9.7 × 10(-7) mol L(-1). Furthermore, G-quadruplexes were applied to construct label-free hairpin assembly circuit, which made it more simple and cost-effective. The satisfactory recoveries were obtained when detecting adenosine in spiked human serum and urine samples, demonstrating the feasibility of this detection strategy in biological samples.

Generation of fertile offspring from Kit(w)/Kit(wv) mice through differentiation of gene corrected nuclear transfer embryonic stem cells.

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit(w)/Kit(wv) mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit(w)/Kit(wv) somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit(w)/Kit(wv) infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.

Quantitative detection of tumor necrosis factor-α by single molecule counting based on a hybridization chain reaction.

This work reports a novel and sensitive quantitative method for detection of tumor necrosis factor-α (TNF-α) based on single molecule counting and hybridization chain reaction (HCR). In the presence of TNF-α, sandwich-type immunocomplex was formed on the surface of glass substrate. The streptavidin acted as a bridge bounded to the biotinylated immunocomplex, which provided three sites to fixate the biotinylated initiator strands. The initiator strands triggered the chain reaction of hybridization to form a long double-helix polymer and SYBR Green I, acted as the fluorescence label, intercalated into the grooves of the long dsDNA polymer. Then, the quantitative detection of TNF-α was realized by single molecule counting. Under the optimal conditions, HCR-based single molecule counting quantitative method could successfully detect TNF-α in the range of 50 fM to 1 pM, and it revealed a reliable result for TNF-α detection in real serum. Moreover, the proposed immunosensor exhibited excellent specificity. These results greatly demonstrated that the proposed method possessed the potentiality in clinical application and it was suitable for quantification of biomarker under low concentration.

Optical aptasensors for quantitative detection of small biomolecules: a review.

Aptasensors are aptamer-based biosensors with excellent recognition capability towards a wide range of targets. Specially, there have been ever-growing interests in the development of aptasensors for the detection of small molecules. This phenomenon is contributed to two reasons. On one hand, small biomolecules play an important role in living organisms with many kinds of biological function, such as antiarrhythmic effect and vasodilator activity of adenosine. On the other hand, the concentration of small molecules can be an indicator for disease diagnosis, for example, the concentration of ATP is closely associated with cell injury and cell viability. As a potential analysis tool in the construction of aptasensors, optical analysis has attracted much more interest of researchers due to its high sensitivity, quick response and simple operation. Besides, it promises the promotion of aptasensors in performance toward a new level. Review the development of optical aptasensors for small biomolecules will give readers an overall understanding of its progress and provide some theoretical guidelines for its future development. Hence, we give a mini-review on the advance of optical aptasensors for small biomolecules. This review focuses on recent achievements in the design of various optical aptasensors for small biomolecules, containing fluorescence aptasensors, colorimetric aptasensors, chemiluminescence aptasensors and other optical aptasensors.

Genetic modification and screening in rat using haploid embryonic stem cells.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.

Pluripotency of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated by forced expression of four pluripotency factors in somatic cells. This has received much attention in recent years since it may offer us a promising donor cell source for cell transplantation therapy. There has been great progress in iPS cell research in the past few years. However, several issues need to be further addressed in the near future before the clinical application of iPS cells, like the immunogenicity of iPS cells, the variability of differentiation potential and most importantly tumor formation of the iPS derivative cells. Here, we review recent progress in research into the pluripotency of iPS cells.