Low Initial Cell Density Promotes the Differentiation and Maturation of Human Pluripotent Stem Cells into Erythrocytes.

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. The possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK), and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from hPSCs.

Discriminative analysis of schizophrenia patients using graph convolutional networks: A combined multimodal MRI and connectomics analysis.

Introduction: Recent studies in human brain connectomics with multimodal magnetic resonance imaging (MRI) data have widely reported abnormalities in brain structure, function and connectivity associated with schizophrenia (SZ). However, most previous discriminative studies of SZ patients were based on MRI features of brain regions, ignoring the complex relationships within brain networks. Methods: We applied a graph convolutional network (GCN) to discriminating SZ patients using the features of brain region and connectivity derived from a combined multimodal MRI and connectomics analysis. Structural magnetic resonance imaging (sMRI) and resting-state functional magnetic resonance imaging (rs-fMRI) data were acquired from 140 SZ patients and 205 normal controls. Eighteen types of brain graphs were constructed for each subject using 3 types of node features, 3 types of edge features, and 2 brain atlases. We investigated the performance of 18 brain graphs and used the TopK pooling layers to highlight salient brain regions (nodes in the graph). Results: The GCN model, which used functional connectivity as edge features and multimodal features (sMRI + fMRI) of brain regions as node features, obtained the highest average accuracy of 95.8%, and outperformed other existing classification studies in SZ patients. In the explainability analysis, we reported that the top 10 salient brain regions, predominantly distributed in the prefrontal and occipital cortices, were mainly involved in the systems of emotion and visual processing. Discussion: Our findings demonstrated that GCN with a combined multimodal MRI and connectomics analysis can effectively improve the classification of SZ at an individual level, indicating a promising direction for the diagnosis of SZ patients. The code is available at https://github.com/CXY-scut/GCN-SZ.git.

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

Preferential Hematopoietic Differentiation in Induced Pluripotent Stem Cells Derived From Human Umbilical Cord Arterial Endothelial Cells.

BACKGROUND: The major obstacle for applications of human induced pluripotent stem cells (hiPSCs) is efficient and controlled lineage-specific differentiation. Hence, a deeper understanding of the initial populations of hiPSCs is required to instruct proficient lineage commitment. METHODS: hiPSCs were generated from somatic cells by transduction of 4 human transcription factors (OCT4, SOX2, KLF4, and C-MYC) using Sendai virus vectors. Genome-wide DNA methylation analysis and transcriptional analysis were performed to evaluate the pluripotent capacity and somatic memory state of hiPSCs. Flow cytometric analysis and colony assays were performed to assess the hematopoietic differentiation capacity of hiPSCs. RESULTS: Here, we reveal human umbilical arterial endothelial cell-derived induced pluripotent stem cells (HuA-iPSCs) exhibit indistinguishable pluripotency in comparison with human embryonic stem cells and hiPSCs derived from other tissues of origin (umbilical vein endothelial cells, cord blood, foreskin fibroblasts, and fetal skin fibroblasts). However, HuA-iPSCs retain a transcriptional memory typical of the parental human umbilical cord arterial endothelial cells, together with a strikingly similar DNA methylation signature to umbilical cord blood-derived induced pluripotent stem cells that distinguishes them from other human pluripotent stem cells. Ultimately, HuA-iPSCs are most efficient in targeted differentiation toward hematopoietic lineage among all human pluripotent stem cells based on the functional and quantitative evaluation of both flow cytometric analysis and colony assays. Application of the Rho-kinase activator significantly reduces the effects of preferential hematopoietic differentiation in HuA-iPSCs, reflected in CD34+ cell percentage of day 7, hematopoietic/endothelial-associated gene expression, and even colony-forming unit numbers. CONCLUSIONS: Collectively, our data suggest that somatic cell memory may predispose HuA-iPSCs to differentiate more amenably into hematopoietic fate, bringing us closer to generating hematopoietic cell types in vitro from nonhematopoietic tissue for therapeutic applications.

Protective effects of resveratrol on the ethanol-induced disruption of retinogenesis in pluripotent stem cell-derived organoids.

Prenatal alcohol exposure-induced fetal alcohol syndrome (FAS) can lead to serious maldevelopment in many organ systems, including the eyes. In the present study, the effects of alcohol exposure on early development of the human retina and the therapeutic effects of resveratrol on alcohol-induced neural retinal damage were observed for the first time in an in vitro retinal organoid model. We report that the number of proliferating and apoptotic cells decreased and increased, respectively, following ethanol treatment. In addition, the number of PAX6+ cells and migrating TUJ1+ cells decreased after ethanol exposure. However, pretreatment with resveratrol prevented all of these negative effects. Using RNA sequencing and immunofluorescence, we identified activation of the PI3K-AKT signalling pathway as the possible mechanism through which resveratrol protects the retina from alcohol-induced damage. These results suggest that while ethanol exposure can restrict the growth of the human retina and impede the development of specific retinal cells, pretreatment with resveratrol may be a feasible method for preventing these effects.

The accumulation of miR-125b-5p is indispensable for efficient erythroblast enucleation.

Erythroblast enucleation is a precisely regulated but not clearly understood process. Polycythemia shows pathological erythroblast enucleation, and we discovered a low miR-125b-5p level in terminal erythroblasts of patients with polycythemia vera (PV) compared to those of healthy controls. Exogenous upregulation of miR-125b-5p levels restored the enucleation rate to normal levels. Direct downregulation of miR-125b-5p in mouse erythroblasts simulated the enucleation issue found in patients with PV, and miR-125b-5p accumulation was found in enucleating erythroblasts, collectively suggesting the importance of miR-125b-5p accumulation for erythroblast enucleation. To elucidate the role of miR-125b-5p in enucleation, gain- and loss-of-function studies were performed. Overexpression of miR-125b-5p improved the enucleation of erythroleukemia cells and primary erythroblasts. Infused erythroblasts with higher levels of miR-125b-5p also exhibited accelerated enucleation. In contrast, miR-125b-5p inhibitors significantly suppressed erythrocyte enucleation. Intracellular imaging revealed that in addition to cytoskeletal assembly and nuclear condensation, miR-125b-5p overexpression resulted in mitochondrial reduction and depolarization. Real-time PCR, western blot analysis, luciferase reporter assays, small molecule inhibitor supplementation and gene rescue assays revealed that Bcl-2, as a direct target of miR-125b-5p, was one of the key mediators of miR-125b-5p during enucleation. Following suppression of Bcl-2, the activation of caspase-3 and subsequent activation of ROCK-1 resulted in cytoskeletal rearrangement and enucleation. In conclusion, this study is the first to reveal the pivotal role of miR-125b-5p in erythroblast enucleation.

UHPLC/QTOF-MS-based metabolomics reveal the effect of Melastoma dodecandrum extract in type 2 diabetic rats.

Context: Melastoma dodecandrum Lour. (Melastomataceae) is a traditional Chinese medicine. This is the first study to report a protective effect of the ethanol extract from M. dodecandrum (MDE) in type 2 diabetic (T2DM) rats.Objective: To investigate the therapeutic mechanism of MDE in T2DM rats.Materials and methods: Sprague-Dawley rats were fed a high-fat diet for 6 consecutive weeks, followed by intraperitoneal injection of streptozotocin (STZ) (30 mg/kg) to induce diabetes. T2DM rats were divided into untreated diabetic, metformin-treated and MDE-treated groups. Additionally, normal rats without treatment served as the control group (n = 6). Metformin (250 mg/kg) and MDE (600 mg/kg) were intragastrically administered to T2DM rats for 5 consecutive weeks. Serum samples were evaluated via ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS), followed by principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA).Results: The 17 identified potential biomarkers were mainly involved in lipid, amino acid, arachidonic acid, taurine and nicotinic acid metabolism. MDE also significantly reduced the level of fasting blood glucose (FBG), oral glucose tolerance, insulin, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen (BUN) in T2DM rats. The high-density lipoprotein (HDL), serum creatinine (Scr), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) levels were elevated in MDE-treated group.Discussion and conclusion: MDE possesses substantial antidiabetic activity, especially in lipid disorder regulation. This suggests that the use of MDE can be generalized to broader pharmacological studies, such as obesity and hyperlipidaemia.

[Honokiol inhibits the invasion and angiogenesis of U937 leukemia cells].

OBJECTIVE: To investigate the inhibiting effect of Honokiol (HNK) on the invasion and angiogenesis in U937 leukemia cells and the molecular mechanism. METHODS: After treated with different concentrations of HNK, the growth inhibition rate of U937 cells was determined by MTT assay, and for the adhesion and invasion abilities were assessed using cell matrix adhesion technique and Transwell(TM); assay, respectively. VEGF, VEGFR1 and MMP-9 mRNA expression levels were detected by real-time quantitative RT-PCR (qRT-PCR). VEGF protein levels were determined by ELISA. RESULTS: HNK could significantly inhibit the proliferation of U937 cells in a time- and dose-dependent manner. The adhesion and invasion abilities of U937 cells were suppressed after treated with a low concentration of HNK. The expressions of VEGF, VEGFR1 and MMP-9 were down-regulated by HNK in a dose-dependent manner. CONCLUSION: HNK can inhibit the invasion and angiogenesis of U937 cells via down-regulating VEGF, VEGFR1 and MMP-9 expressions.

[Effect of tyrosine kinase inhibitor imatinib mesylate on K562 cell invasion by PTEN pathway].

AIM: To investigate the effect of tyrosine kinase inhibitor imatinib mesylate on the PTEN signaling pathway and the cell invasion in K562 cells. METHODS: K562 cells were treated with different concentrations of imatinib mesylate. After different time periods, the mRNA levels of BCR/ABL, PTEN and FAK were detected by real-time fluorescent quantitative PCR (FQ-PCR) to analyze their relationships. The protein level of FAK was detected by immunocytochemistry. The cell invasive ability was examined by Transwell (Boyden chamber) assay. RESULTS: In the initial 36 h, the expression level of PTEN mRNA was up-regulated and the FAK mRNA was down-regulated with the reduction of BCR/ABL fusion gene expression and the cell invasive ability of K562 cells was inhibited by 2 µg/mL imatinib mesylate. 48 h later, the PTEN mRNA expression level decreased and the FAK mRNA expression level was elevated with the restore of BCR/ABL fusion gene. BCR/ABL mRNA level presented a positive correlation with PTEN mRNA expression level, and a negative correlation with FAK mRNA. CONCLUSION: Tyrosine kinase inhibitor imatinib mesylate can regulate PTEN/FAK pathway and inhibit the leukemia K562 cell invasive ability via restraining BCR/ABL fusion gene.

[Regulation of wild type PTEN gene on Survivin, Xiap and Smac in chronic leukemia cells].

OBJECTIVE: To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation, apoptosis and the possible regulations of apoptosis-related molecules Survivin, Xiap and Smac gene in human chronic myeloid leukemia (CML) and cell line K562 cells. METHODS: (1) The recombinant adenovirus containing green fluorescent protein (GFP) and PTEN (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 cells. The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry (FCM). PTEN, Survivin, Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) while PTEN protein levels analyzed by Western blot. (2) The expression levels of PTEN, Survivin, Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal control marrow mononuclear cells (MMNC). RESULTS: The growth of K562 cells was suppressed markedly. And the maximal growth inhibition rate was 38.6% after the transfection of PTEN. Survivin, Xiap, Smac mRNA expression levels were down-regulated by around 6.14, 7.44 and 2.95 folds respectively (0.0700 ± 0.0059, 0.0089 ± 0.0006, 0.0600 ± 0.0039 vs 0.4370 ± 0.0790, 0.0661 ± 0.0072, 0.1580 ± 0.0078 vs 0.4530 ± 0.0810, 0.0700 ± 0.0079, 0.1770 ± 0.0085, all P < 0.01). The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control. But Survivin, Xiap, Smac mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control. CONCLUSION: The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin, Xiap and Smac genes.