Induced pluripotent stem cell-derived exosome attenuates the inflammatory response in microglia / 中华急诊医学杂志

Objective:To investigate the effects of induced pluripotent stem cell-derived exosome (iPSC-Exo) on releasing inflammatory factors from microglia induced by lipopolysaccharide (LPS).Methods:iPSC derived from the tubular epithelial cells of sepsis encephalopathy patients were resuscitated and cultured. The iPSC-Exo was isolated by low-temperature ultracentrifugation and analyzed by transmission electron microscopy, Western blot and high sensitivity flow cytometry (HSFCM). Based on the concentration of iPSC-Exo, human microglia line HMO6 cells activated by LPS (100 ng/mL) were divided into four groups randomly: LPS+ phosphate buffer solution (PBS) group, LPS+iPSC-Exo 10 5 group, LPS+iPSC-Exo 10 6 group and LPS+iPSC-Exo 10 7 group. The control group was added equal PBS but not LPS. After culture for 24 h, the concentrations of malondialdehyde in cells were detected. Quantitative RT-PCR was used to measure the mRNA expression levels of macrophage inflammatory protein 2 (MIP2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in the cells and enzyme-linked immunosorbent assay (ELISA) was used to assess the concentration of these cytokines in the supernatant. Under the same concentration of iPSC-Exo, one-way ANOVA and SNK- q test were used for comparison between groups. Results:The extracts showed spherical membrane structure by transmission electron microscopy. HSFCM showed the mean diameter of the extracts was (74.66±15.60) nm and the concentration around 2.98×10 10/mL. Western blot analysis showed high expression of exosome markers CD63, Alix and TSG101, but not GM130. Intracellular MDA concentration and mRNA expression levels and protein concentration of MIP2, TNF-α, IL-1β and IL-6 in the LPS+PBS group were significantly higher than those in the control group (all P<0.01). With the increase of iPSC-Exo concentration, the intracellular MDA concentration decreased gradually ( P<0.01), the mRNA expression levels of inflammatory factors showed a gradual downward trend (all P<0.05). Each inflammatory cytokine in the supernatant declined in a manner that was almost consistent with mRNA. Concentrations of MDA remained constant in the control group. Conclusions:iPSC-Exo derived from the tubular epithelial cells of sepsis encephalopathy patients alleviate oxidative stress and inflammation effect of microglia induced by LPS, and the modulatory effect is dose-dependent.

Developmental comparison between cerebral organoids in vitro and body's cortices in vivo / 解剖学报

Objective To understand the characteristics and developmental differences between cerebral organoids in vitro and normal cerebral cortices in vivo. Methods 1. Grouping: cerebral cortices in vivo group and cultured cerebral organoids in vitro group. 2. Sample collection: cortical tissues were collected from Kunming mouse embryos at embryonic day 7.5(E7.5), E9.5, E11.5, E14.5, and postnatal day 3 (P3) or P7. Three specimens were taken from each group. Meanwhile, cerebral organoids were cultured with mouse induced pluripotent stem cells (iPSCs), and samples at different culture time point were collected, and more than 3 samples were collected at each time point. 3. Detection method: the distribution of different types of cells in each group of specimens was analyzed by immunofluorescent staining. Results While relative similarities between in vivo cerebral cortical development and the cerebral organoids in vitro were observed, including the histogenesis, and the morphological differentiation of nerve cells and glial cells, the lamellar architecture of cerebral cortex in mouse brain was not observed in cerebral organoids. Conclusion The development of cerebral organoids in vitro has some similarity with body's cortical development. Therefore, cerebral organoids can be used to a substitution of cortex and diseases' models, but improvement of the existing technologies is necessary.

Advances and Applications of Brain Organoids / 神经科学通报·英文版

Understanding the fundamental processes of human brain development and diseases is of great importance for our health. However, existing research models such as non-human primate and mouse models remain limited due to their developmental discrepancies compared with humans. Over the past years, an emerging model, the "brain organoid" integrated from human pluripotent stem cells, has been developed to mimic developmental processes of the human brain and disease-associated phenotypes to some extent, making it possible to better understand the complex structures and functions of the human brain. In this review, we summarize recent advances in brain organoid technologies and their applications in brain development and diseases, including neurodevelopmental, neurodegenerative, psychiatric diseases, and brain tumors. Finally, we also discuss current limitations and the potential of brain organoids.

Research progress of methodological techniques of generation of platelets from induced pluripotent stem cells in vitro / 中国输血杂志

Platelets play a role in hemostasis in vivo, and platelet transfusion is the main means to treat bleeding diseases caused by thrombocytopenia or platelet dysfunction. However, platelets are in short supply due to the increasing demand for platelet products in clinical, the limited number of blood donors and the disadvantages of platelet products such as short shelf life and bacteria contamination. Currently, induced pluripotent stem cells are considered an ideal source for producing platelets in vitro. They have the potential for self-renewal and differentiation into any cell type, and can be obtained and manipulated easily. Given the recent advances in megakaryocytic series, bioreactors, feeder-free cell production and large-scale propagation research, platelet preparations derived from induced pluripotent stem cells have gradually shown great potential for clinical applications. Considering the minimal risk of alloimmunization and tumorigenesis with these blood products, they are promising to become the standard source of future blood transfusions. This paper reviews the research progress of the methodological techniques of in vitro generation of platelets from induced pluripotent stem cells.

Current status and reflections on stem cell transplantation in the treatment of testicular aging / 器官移植

Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.

Advances in research of heart organoid and its application in drug discovery / 药学学报

Cardiovascular diseases are fatal threats to human health and also important fields in drug discovery. Organoid is a miniature with the structure and function similar to the organ, which is formed by the self-updating and specific differentiation of stem cells during the in vitro culture. Considering its characteristics of human origin, physical features, self-assembling and genetic stability, heart organoid has attracted much attention in the study of cardiogenesis, cardiovascular diseases modeling and related drug research. Hence, this article will review the development of heart organoids and its construction strategies, highlighting its application and prospects in drug discovery.

Role of PI3K in systolic dysfunction of hiPSC-CMs induced by sunitinib / 中国药房

OBJECTIVE To study the role of phosphatidylinositol-3-kinase （PI3K） on sunitinib-induced myocardial systolic dysfunction. METHODS Using human-induced pluripotent stem cell-derived cardiomyocytes （hiPSC-CMS） as objects， the contractile force of cardiomyocytes was measured by CardioExcyte 96 system， and IC50 of sunitinib was calculated after hiPSC- CMS were treated with sunitinib at different concentrations [0 （control）， 0.5， 1， 3， 5， 10 μmol/L] for 24 hours. The effects of sunitinib （3.14 μmol/L） on the contractile frequency of cardiomyocytes， calcium transient amplitude and calcium transient recovery time course， mRNA expression of myocardial injury markers atrial natriuretic peptide （ANP）， brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected. PI3K activator 3，4，5-triphosphate phos-phatidylinositol （PIP3， 1 μmol/L） and sunitinib were used to intervene in hiPSC-CMs jointly， so as to investigate the role of PI3K in the myocardial systolic dysfunction induced by sunitinib. RESULTS Sunitinib inhibited the contractile force of hiPSC-CMs in a concentration-dependent manner. IC50 of sunitinib was 3.14 μmol/L. After intervention with 3.14 μmol/L sunitinib， the contractile frequency of hiPSC-CMs and calcium transient amplitude were decreased significantly （P＜0.05 or P＜0.01）； the duration of calcium transient recovery was prolonged significantly （P＜0.05）， and mRNA expressions of ANP， BNP and β-MHC were significantly increased （P＜0.01）. After PI3K was activated with PIP3， the contractile force of hiPSC-CMs was increased significantly （P＜0.01）. CONCLUSIONS Activating PI3K activity is a potential molecular mechanism to improve myocardial toxicity induced by sunitinib.

Multiplexed single-cell transcriptome analysis reveals molecular characteristics of monkey pluripotent stem cell lines / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Efforts have been made to establish various human pluripotent stem cell lines. However, such methods have not yet been duplicated in non-human primate cells. Here, we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions. The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines. Overall, this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.

Bone regeneration of induced pluripotent stem cells derived from peripheral blood cells in collagen sponge scaffolds

Abstract Stem cell-based regeneration therapy offers new therapeutic options for patients with bone defects because of significant advances in stem cell research. Although bone marrow mesenchymal stem cells are the ideal material for bone regeneration therapy using stem cell, they are difficult to obtain. Induced pluripotent stem cells (iPSCs) are now considered an attractive tool in bone tissue engineering. Recently, the efficiency of establishing iPSCs has been improved by the use of the Sendai virus vector, and it has become easier to establish iPSCs from several type of somatic cells. In our previous study, we reported a method to purify osteogenic cells from iPSCs. Objective: This study aimed to evaluate the osteogenic ability of iPSCs derived from peripheral blood cells. Methodology: Mononuclear cells (MNCs) were obtained from human peripheral blood. Subsequently, T cells were selectively obtained from these MNCs and iPSCs were established using Sendai virus vectors. Established iPSCs were evaluated by the expression of undifferentiated markers and teratoma formation assays. Osteoblasts were induced from these iPSCs and evaluated by the expression of osteoblast markers. Additionally, the induced osteoblasts were transplanted into rat critical size calvaria bone defect models with collagen sponge scaffolds. Samples were evaluated by radiographical and histological assessments. Results: Induced osteoblasts expressed several osteoblast-specific markers. The results of radiographical and histological assessments revealed that the cell transplant group had bone formations superior to those of the control group. Conclusions: This study suggests that peripheral blood MNCs have the potential to differentiate into osteoblasts. Although there are some hurdles in iPSC transplantation, osteoblasts obtained from MNC-iPSCs could be applied to bone regeneration therapy in the future.

Generation of Kidney Directed Differentiation Platform from Human Pluripotent Stem Cells / 中国生物化学与分子生物学报

Kidney is an essential organ in human body with multiple physiological functions. However, there is 10 % population worldwide with renal disease. It is urgent to generate a model which is more similar with kidney at structural and functional level to study renal disease. The rise of in vitro differentiation technology from pluripotent stem cells gives regeneration medicine and precise medicine new energy. This study mimics kidney development in vitro by inducing human pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into kidney progenitor cells, and further forming nephrons, which is the structure and function unit in kidney. Human pluripotent stem cells were differentiated into primitive streak through activating WNT pathway while inhibiting TGF-(B signaling. Afterward, the primitive streak spontaneously differentiated into intermediate mesoderm. Then, we induced intermediate mesoderm cells into kidney progenitor cells through FGF pathway. The FACS analysis data indicated kidney progenitor cells were up to 51. 5%-61. 9% in total cell population. Immuno-stai-ning results showed these structures contained podocytes of glomerulus, proximal tubule, and distal tubule. This kidney differentiation protocol is stable, high-efficient, and well repeatable. This research provides a novel platform for early human kidney development study, kidney disease modeling, and drug screening.

Effects of insulin on the QT interval and induced arrhythmias of human cardiomycytes / 西安交通大学学报(医学版)

【Objective】 To explore the effects of insulin on the QT interval and induced arrhythmias of cardiomyocytes derived from human induced pluripotent stem cells （hiPSC-CMs）. 【Methods】 Immunofluorescence staining and flow cytometry were used to analyze the purity of hiPSC-CMs. Microelectrode array （MEA） was utilized to detect the electrophysiological changes including heart rate （HR）， field potential duration （FPD， which is similar to QT interval in ECG）， FPDc （FPD corrected by HR）， conduction velocity （CV）， and spike amplitude before and after insulin treatment. The effects of E4031 on QT interval prolongation and induced arrhythmias of hiPSC-CMs were evaluated before and after treatment with insulin. 【Results】 hiPSC-CMs highly expressed myocardial specific marker cTnT. The purity of hiPSC-CMs was 97.1%. After 5-day insulin treatment of hiPSC-CMs， HR increased by （11.9±3.3）%， FPD shortened by （22.7±2.8）%， FPDc shortened by （15.6±1.6）%， and spike amplitude increased by （39.1±7.9）% when compared with untreated group， but CV remained unchanged. 10 nmol/L of E4031 could prolong the FPDc of hiPSC-CMs by （37.8±9.0）%， and 30 nmol/L of E4031 could induce arrhythmias. After insulin treatment， 10 nmol/L of E4031 prolonged the FPDc of hiPSC-CMs by （21.8±3.1）% （compared with the untreated group， insulin decreased FPDc prolongation by E4031， 37.8%±9.0% vs. 21.8%±3.1%， P<0.05）， while 30 nmol/L of E4031 did not induce arrhythmias. 【Conclusion】 Insulin can shorten the QT interval of hiPSC-CMs and significantly reduce the QT interval prolongation and the risk of arrhythmias induced by drugs.

Research progress on optimization strategies for microencapsulated islet transplantation / 器官移植

Islet transplantation is one of the effective therapies for diabetes mellitus. Nevertheless, multiple issues still exist, such as shortage of donors and adverse reactions caused by long-term use of immunosuppressants, which limit the islet survival post-transplantation. Microencapsulated islet transplantation may overcome these difficulties to certain extent, whereas many factors, such as the destruction of immune isolation microenvironment within the microcapsules and insufficient supply of oxygen and nutrients, constrain the application of microencapsulated islet transplantation in clinical practice. In recent years, how to enhance the effect of microencapsulated islet transplantation has been gradually studied. The application of stem cells in microencapsulated islet transplantation has steadily become a research hot spot. Therefore, the optimizing strategies for microencapsulated islet transplantation and the application of stem cells in microencapsulated islet transplantation were reviewed, and the potential improvement techniques of microencapsulated islet transplantation were investigated in this article, aiming to provide reference for further clinical application of microencapsulated islet transplantation.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system

ABSTRACT Introduction Sickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD. Objective Generation of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium. Method This study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed. Results The patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium. Conclusion In conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Progress of urine-derived stem cell / 国际儿科学杂志

Urine-derived stem cell(USC) refers to a type of mesenchymal stem cell obtained from urine.Due to its simple and quick extraction, non-invasive access, and no ethical issues, it has many advantages over other stem cells in clinical research, and has attracted the attention of the academic community.This article summarizes recent research progress in the aspects of urine-derived stem cell isolation and culture, cell characterizations, source, application, and exosomes.

Construction of a drug screening model for miscarriage from induced pluripotent stem cells in vitro / 药学学报

Drug use during pregnancy is unavoidable. Therefore, it is vitally important for medical workers to help pregnant women take drugs correctly to reduce the incidence of spontaneous abortion, premature birth, and low birth weight. In our study, drug screening model with induced pluripotent stem cells (iPSCs) was used to find some improper drugs which will result in woman's abortion. With 3D culture in vitro, iPSCs can form embryoid bodies (EBs) and cerebral organoids, which simulated in vitro development of early embryos, from inner cell mass to germ-layer differentiation. In the experiment, EBs were exposed to mifepristone (RU486), and three experimental groups were divided randomly. They were control group (without RU486), low-dose group (L-RU486, 10 μg·mL-1), and high-dose group (H-RU486, 20 μg·mL-1). After mifepristone exposure, EBs were observed at days 5, 8, and 11, including size of EB, cell apoptosis, and differentiation of germ layers, by using inverted optical microscope, TUNEL assay, and immunofluorescent staining. The results showed that through 3D culture, iPSCs could develop into embryoid bodies, neural rosettes, and finally cerebral organoids. After mifepristone exposure, EBs' sizes were decreased (P < 0.01); the levels of cell apoptosis in EBs were increased after mifepristone exposure (P < 0.01); the development of EBs' germ layer was affected. Mifepristone exposure could inhibit the proliferation of embryonic stem cells, reduce the differentiation of ectoderm (P < 0.01) and promote the development of mesoderm (P < 0.05). In conclusion, iPSCs can be used as a screening model for abortion drug, and EBs’ diameter, cell apoptosis, and differentiation changes of the germ layers can serve as criteria of abortion drug screening.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells / Journal of Rural Medicine

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Research status and prospect of basic application of islet organoid / 器官移植

Organoids are tissue structures, generated from pluripotent stem cells and cultured in vitro, which form self-organize and recapitulate tissues with similar structure and function to the original organs. Organoids have similar appearance and function to the original tissues, and have been widely applied in basic research and clinical trial. At present, the organoids of liver, kidney, islet, brain, intestine and other organs have been successfully cultivated. The use of islet organoid is a hotspot in the field of organoid research. However, islet organoid is currently applied in basic research because rejection after organ transplantation and other issues remain unresolved. In this article, the origin, development and basic application of islet organoid were reviewed, aiming to provide reference for the transformation from basic research of islet organoid into clinical application as well as the treatment of diabetes mellitus.

Graded and pan-neural disease phenotypes of Rett Syndrome linked with dosage of functional MeCP2

Rett syndrome (RTT) is a progressive neurodevelopmental disorder, mainly caused by mutations in MeCP2 and currently with no cure. We report here that neurons from R106W MeCP2 RTT human iPSCs as well as human embryonic stem cells after MeCP2 knockdown exhibit consistent and long-lasting impairment in maturation as indicated by impaired action potentials and passive membrane properties as well as reduced soma size and spine density. Moreover, RTT-inherent defects in neuronal maturation could be pan-neuronal and occurred in neurons with both dorsal and ventral forebrain features. Knockdown of MeCP2 led to more severe neuronal deficits as compared to RTT iPSC-derived neurons, which appeared to retain partial function. Strikingly, consistent deficits in nuclear size, dendritic complexity and circuitry-dependent spontaneous postsynaptic currents could only be observed in MeCP2 knockdown neurons but not RTT iPSC-derived neurons. Both neuron-intrinsic and circuitry-dependent deficits of MeCP2-deficient neurons could be fully or partially rescued by re-expression of wild type or T158M MeCP2, strengthening the dosage dependency of MeCP2 on disease phenotypes and also the partial function of the mutant. Our findings thus reveal stable neuronal maturation deficits and unexpectedly, graded sensitivities of neuron-inherent and neural transmission phenotypes towards the extent of MeCP2 deficiency, which is informative for future therapeutic development.

Chemistry, manufacturing, and controls regulatory considerations for human pluripotent stem cell-derived cellular products / 药学学报

In recent years, the number of clinical trials of stem cell products has increased, and the research and development technology and evaluation system have developed rapidly. Human pluripotent stem cell (hPSC)-derived cellular products are in the phase I/II stage of clinical trials. Related products include hPSC-derived neurons, retinal pigment epithelial cells, pancreatic beta cells, etc. They are generally used for the repair and replacement of functional cells related to degenerative diseases and genetic diseases via local transplantation. So far, no similar products have been officially approved on market. As hPSC possesses multi-directional differentiation potential and the ability to form teratoma in vivo, compared with other stem cell products, hPSC-derived cellular products have relatively higher risk of tumorigenicity, longer differentiation induction cycle, more complex production process, together with the rapidly updating quality characterization methods, which pose challenges to the scientific evaluation of their human applications. Based on the problems in the recent review and communication of clinical trial applications of stem cell products, and with reference to the relevant technical guidelines, this paper proposes the chemistry, manufacturing, and controls review considerations on the manufacturing process and quality study of hPSC-derived cellular products. We hope to improve the communications between developers and regulators.

Cellular metabolism and homeostasis in pluripotency regulation

Pluripotent stem cells (PSCs) can immortally self-renew in culture with a high proliferation rate, and they possess unique metabolic characteristics that facilitate pluripotency regulation. Here, we review recent progress in understanding the mechanisms that link cellular metabolism and homeostasis to pluripotency regulation, with particular emphasis on pathways involving amino acid metabolism, lipid metabolism, the ubiquitin-proteasome system and autophagy. Metabolism of amino acids and lipids is tightly coupled to epigenetic modification, organelle remodeling and cell signaling pathways for pluripotency regulation. PSCs harness enhanced proteasome and autophagy activity to meet the material and energy requirements for cellular homeostasis. These regulatory events reflect a fine balance between the intrinsic cellular requirements and the extrinsic environment. A more complete understanding of this balance will pave new ways to manipulate PSC fate.