MicroRNA-18a prevents senescence of mesenchymal stem cells by targeting CTDSPL.

Stem cell therapy requires massive-scale homogeneous stem cells under strict qualification control. However, Prolonged ex vivo expansion impairs the biological functions and results in senescence of mesenchymal stem cells (MSCs). We investigated the function of CTDSPL in the premature senescence process of MSCs and clarified that miR-18a-5p played a prominent role in preventing senescence of long-term cultured MSCs and promoting the self-renewal ability of MSCs. Over-expression of CTDSPL resulted in an enlarged morphology, up-regulation of p16 and accumulation of SA-ß-gal of MSCs. The reduced phosphorylated RB suggested cell cycle arrest of MSCs. All these results implied that CTDSPL induced premature senescence of MSCs. We further demonstrated that miR-18a-5p was a putative regulator of CTDSPL by luciferase reporter assay. Inhibition of miR-18a-5p promoted the expression of CTDSPL and induced premature senescence of MSCs. Continuous overexpression of miR-18a-5p improved self-renewal of MSCs by reducing ROS level, increased expression of Oct4 and Nanog, and promoted growth rate and differentiation capability. We reported for the first time that the dynamic interaction of miR-18a-5p and CTDSPL is crucial for stem cell senescence.

Origin of dendrite-free lithium deposition in concentrated electrolytes.

The electrolyte solvation structure and the solid-electrolyte interphase (SEI) formation are critical to dictate the morphology of lithium deposition in organic electrolytes. However, the link between the electrolyte solvation structure and SEI composition and its implications on lithium morphology evolution are poorly understood. Herein, we use a single-salt and single-solvent model electrolyte system to systematically study the correlation between the electrolyte solvation structure, SEI formation process and lithium deposition morphology. The mechanism of lithium deposition is thoroughly investigated using cryo-electron microscopy characterizations and computational simulations. It is observed that, in the high concentration electrolytes, concentrated Li+ and anion-dominated solvation structure initiate the uniform Li nucleation kinetically and favor the decomposition of anions rather than solvents, resulting in inorganic-rich amorphous SEI with high interface energy, which thermodynamically facilitates the formation of granular Li. On the contrary, solvent-dominated solvation structure in the low concentration electrolytes tends to exacerbate the solvolysis process, forming organic-rich mosaic SEI with low interface energy, which leads to aggregated whisker-like nucleation and growth. These results are helpful to tackle the long-standing question on the origin of lithium dendrite formation and guide the rational design of high-performance electrolytes for advanced lithium metal batteries.

Strongly Solvating Ether Electrolytes for High-Voltage Lithium Metal Batteries.

Ethers are promising electrolytes for lithium (Li) metal batteries (LMBs) because of their unique stability with Li metal. Although intensive research on designing anion-enriched electrolyte solvation structures has greatly improved their electrochemical stabilities, ether electrolytes are approaching an anodic bottleneck. Herein, we reveal the strong correlation between electrolyte solvation structure and oxidation stability. In contrast to previous designs of weakly solvating solvents for enhanced anion reactivities, the triglyme (G3)-based electrolyte with the largest Li+ solvation energy among different linear ethers demonstrates greatly improved stability on Ni-rich cathodes under an ultrahigh voltage of 4.7 V (93% capacity retention after 100 cycles). Ether electrolytes with a stronger Li+ solvating ability could greatly suppress deleterious oxidation side reactions by decreasing the lifetime of free labile ether molecules. This study provides critical insights into the dynamics of the solvation structure and its significant influence on the interfacial stability for future development of high-efficiency electrolytes for high-energy-density LMBs.

Balancing activation and co-stimulation of CAR tunes signaling dynamics and enhances therapeutic potency.

CD19-targeting chimeric antigen receptors (CARs) with CD28 and CD3ζ signaling domains have been approved by the US FDA for treating B cell malignancies. Mutation of immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3ζ generated a single-ITAM containing 1XX CAR, which displayed superior antitumor activity in a leukemia mouse model. Here, we investigated whether the 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their wild-type (WT) counterparts. 1XX CARs showed better antitumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells persisted longer in vivo and had a higher percentage of central memory cells. With fluorescence resonance energy transfer (FRET)-based biosensors, we found that decreased ITAM numbers in 1XX resulted in similar 70-kDa zeta chain-associated protein (ZAP70) activation, while 1XX induced higher Ca2+ elevation and faster extracellular signal-regulated kinase (Erk) activation than WT CAR. Thus, our results confirmed the superiority of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical applications of 1XX CARs against solid tumors.

Structural insights into Ras regulation by SIN1.

Over the years it has been established that SIN1, a key component of mTORC2, could interact with Ras family small GTPases through its Ras-binding domain (RBD). The physical association of Ras and SIN1/mTORC2 could potentially affect both mTORC2 and Ras-ERK pathways. To decipher the precise molecular mechanism of this interaction, we determined the high-resolution structures of HRas/KRas-SIN1 RBD complexes, showing the detailed interaction interface. Mutation of critical interface residues abolished Ras-SIN1 interaction and in SIN1 knockout cells we demonstrated that Ras-SIN1 association promotes SGK1 activity but inhibits insulin-induced ERK activation. With structural comparison and competition fluorescence resonance energy transfer (FRET) assays we showed that HRas-SIN1 RBD association is much weaker than HRas-Raf1 RBD but is slightly stronger than HRas-PI3K RBD interaction, providing a possible explanation for the different outcome of insulin or EGF stimulation. We also found that SIN1 isoform lacking the PH domain binds stronger to Ras than other longer isoforms and the PH domain appears to have an inhibitory effect on Ras-SIN1 binding. In addition, we uncovered a Ras dimerization interface that could be critical for Ras oligomerization. Our results advance our understanding of Ras-SIN1 association and crosstalk between growth factor-stimulated pathways.

Inhibition of RIPK1 by ZJU-37 promotes oligodendrocyte progenitor proliferation and remyelination via NF-κB pathway.

Receptor interacting serine/threonine protein kinase 1 (RIPK1) activation and necroptosis have been genetically and mechanistically linked with human multiple sclerosis and neurodegenerative diseases for which demyelination is a common key pathology. Demyelination can be healed through remyelination which is mediated by new oligodendrocytes derived from the adult oligodendrocyte progenitor cells (OPCs). Unfortunately, the efficiency of remyelination declines with progressive aging partially due to the depletion of OPCs following chronic or repeated demyelination. However, to our knowledge, so far there is no drug which enhances proliferation of OPCs, and it is unknown whether inhibiting RIPK1 activity directly affect OPCs, the central player of remyelination. Using TNFα induced RIPK1-dependent necroptosis in Jurkat FADD-/- cells as a cell death assay, we screened from 2112 FDA-approved drugs and the drug candidates of new RIPK1 inhibitors selected by ourselves, and identified ZJU-37, a small molecule modified by introducing an amide bond to Nec-1s, is a new RIPK1 kinase inhibitor with higher potency than Nec-1s which has the best reported potency. We unveil in addition to protecting myelin from demyelination and axons from degeneration, ZJU-37 exhibits a new role on promoting proliferation of OPCs and enhancing remyelination by inhibiting RIPK1 kinase activity with higher potency than Nec-1s. Mechanistically, ZJU-37 promotes proliferation of OPCs by enhancing the transcription of platelet derived growth factor receptor alpha via NF-κB pathway. This work identifies ZJU-37 as a new drug candidate which enhances remyelination by promoting proliferation of OPCs, paving the way for a potential drug to enhance myelin repair.

Signaling Dynamics of TSHR-Specific CAR-T Cells Revealed by FRET-Based Biosensors.

Although most patients with thyroid cancers have good prognosis and long-term survival, some patients are refractory to traditional therapeutic approaches and face a high risk of mortality. CAR-T therapy provides an attractive strategy to treat these patients. Considering the limited expression in thyroid tissues, thyroid-stimulating hormone receptor (TSHR) has been considered as a promising candidate as CAR-T target. However, it is still a challenge to find the optimal CAR design for the treatment of thyroid cancers. Dynamic signaling cascade is initiated by CAR molecules during CAR-T cell activation. The development of FRET-based biosensors enables us to detect the signaling dynamics of key kinases during CAR-T cell activation with high spatiotemporal resolution. Here using the ZAP70 and ERK biosensors, we visualized the dynamics of ZAP70 and ERK activities in TSHR-specific CAR-T cells upon antigen stimulation. We first constructed several TSHR-targeting CARs for the treatment of advanced thyroid cancers. The TSHR CAR-T cells with CD28 or 4-1BB co-stimulatory signaling domains exhibited potent cytotoxicity in vitro. By FRET imaging, we observed rapid increase of ZAP70 and ERK activities in TSHR CAR-T cells upon target cell binding. Even though CD28-based CAR-T cells had similar ZAP70 activation dynamics as 4-1BB-based CAR-T cells, they displayed slightly enhanced ERK activation, which may contribute to their faster anti-tumor kinetics in vivo. These results demonstrated the efficacy of TSHR CAR-T cells to treat advanced thyroid cancers. Our study indicated the potential of applying FRET biosensors to optimize the design of CAR for effective CAR-T therapy.

Conditioned medium from human cord blood mesenchymal stem cells attenuates age-related immune dysfunctions.

Aging is accompanied with progressive deterioration of immune responses and tissue's function. Using 12-month-old mice as model, we showed that conditioned medium of human cord blood mesenchymal stem cells (CBMSC-CM) significantly reduced the population percentage of CD3-CD335+ NK and CD4+CD25+ regulatory T-cells in peripheral blood. The CBMSC-CM administration also increased naïve T-cells number and restored the ratio of naïve to memory T-cells in CD4+ T-cells population. These results indicated that CBMSC-CM improved the immune response efficiency of aged mice. Moreover, we also found CBMSC-CM treatment significantly reduced the number of senescenT-cells in kidney tissues. Finally, we demonstrated that CBMSC-CM remarkably attenuated hydrogen peroxide triggered T-cell response and ameliorated oxidative stress induced cellular senescence. All of these data suggest a prominent anti-aging effect of secretome of CBMSCs.

Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells.

T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult-to-produce and expensive viral vectors. Here, small double-stranded plasmid DNA modified to mediate high-efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)-T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR-T cells generated using adeno-associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.

Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy.

How single-chain variable fragments (scFvs) affect the functions of chimeric antigen receptors (CARs) has not been well studied. Here, the components of CAR with an emphasis on scFv were described, and then several methods to measure scFv affinity were discussed. Next, scFv optimization studies for CD19, CD38, HER2, GD2 or EGFR were overviewed, showing that tuning the affinity of scFv could alleviate the on-target/off-tumor toxicity. The affinities of scFvs for different antigens were also summarized to designate a relatively optimal working range for CAR design. Last, a synthetic biology approach utilizing a low-affinity synthetic Notch (synNotch) receptor to achieve ultrasensitivity of antigen-density discrimination and murine models to assay the on-target/off-tumor toxicity of CARs were highlighted. Thus, this review provides preliminary guidelines of choosing the right scFvs for CARs.

Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries.

The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.

Exploiting Ca2+ signaling in T cells to advance cancer immunotherapy.

Decades of basic research has established the importance of Ca2+ to various T cell functions, such as cytotoxicity, proliferation, differentiation and cytokine secretion. We now have a good understanding of how proximal TCR signaling initiates Ca2+ influx and how this influx subsequently changes transcriptional activities in T cells. As chimeric antigen receptor (CAR)-T therapy has achieved great clinical success, is it possible to harness Ca2+ signaling to further advance CAR-T research? How is CAR signaling different from TCR signaling? How can functional CARs be identified in a high-throughput way? Quantification of various Ca2+ signals downstream of CAR/TCR activation might help answer these questions. Here we first summarized recent studies that used Ca2+ dye, genetically-encoded Ca2+ indicators (GECI) or transcriptional activity reporters to understand CAR activation in vitro and in vivo. We next reviewed several proof-of-concept reports that manipulate Ca2+ signaling by light or ultrasound to achieve precise spatiotemporal control of T cell functions. These efforts, though preliminary, opened up new avenues to solve the on-target/off-tumor problem of therapeutic T cells. Other modalities to regulate Ca2+ signaling, such as radio wave and electrical pulse, were also discussed. Thus, monitoring or manipulating Ca2+ signaling in T cells provides us many opportunities to advance cancer immunotherapy.

[Detection algorithm of paroxysmal atrial fibrillation with sparse coding based on Riemannian manifold].

In order to solve the problem that the early onset of paroxysmal atrial fibrillation is very short and difficult to detect, a detection algorithm based on sparse coding of Riemannian manifolds is proposed. The proposed method takes into account that the nonlinear manifold geometry is closer to the real feature space structure, and the computational covariance matrix is used to characterize the heart rate variability (RR interval variation), so that the data is in the Riemannian manifold space. Sparse coding is applied to the manifold, and each covariance matrix is represented as a sparse linear combination of Riemann dictionary atoms. The sparse reconstruction loss is defined by the affine invariant Riemannian metric, and the Riemann dictionary is learned by iterative method. Compared with the existing methods, this method used shorter heart rate variability signal, the calculation was simple and had no dependence on the parameters, and the better prediction accuracy was obtained. The final classification on MIT-BIH AF database resulted in a sensitivity of 99.34%, a specificity of 95.41% and an accuracy of 97.45%. At the same time, a specificity of 95.18% was realized in MIT-BIH NSR database. The high precision paroxysmal atrial fibrillation detection algorithm proposed in this paper has a potential application prospect in the long-term monitoring of wearable devices.

Engineering Cytoplasmic Signaling of CD28ζ CARs for Improved Therapeutic Functions.

Chimeric antigen receptor modified T cells (CAR-T) have yielded impressive clinical outcomes in treating hematopoietic malignancies. However, relapses have occurred in a substantial number of patients and limited the development of CAR-T therapy. Most underlying reasons for these relapses can be attributed to poor persistence and rapid exhaustion of CAR-T cells in vivo. Despite multiple strategies having been developed, how to improve CAR-T persistence or resist exhaustion while maintaining sufficient cytotoxic functions is still a great challenge. Here we discuss engineering cytoplasmic signaling as an important strategy for CAR optimization. This review summarizes recent advances showing that the anti-tumor function of CAR-T cells can be improved by optimizing the CD3ζ domain or downstream signaling of CD28ζ CAR.

Comparison in transcriptome and cytokine profiles of mesenchymal stem cells from human umbilical cord and cord blood.

Human umbilical cord (UC) and cord blood (CB) provide attractive sources of mesenchymal stem cells (MSCs) for cell therapy. Both UCMSCs and CBMSCs have been demonstrated to play prominent roles in clinical therapy. However, little is known about their functional differences in clinical application. Our transcriptome analysis uncovered high activity of insulin secretion related signaling pathways for CBMSCs and cell adhesion related signaling pathways for UCMSCs. Expression of a large number of immune related signaling pathways also showed the difference in both cells, implying their distinct immune modulatory functions. As the therapeutic effects of MSCs mainly dependent on the cytokines and growth factors produced by transplanted MSCs, we further compared the cytokine profiles of UCMSCs and CBMSCs using antibody array. By evaluating the expression of 106 cytokines, we found both MSCs abundantly secreted TSP-1, TSG-14, TIMP-1, IL-8, IL-6, CXCL1, GIF and IGFBP3. However, the expression of CCL2 in UCMSCs showed significantly higher than CBMSCs. IGFBP1 and IGFBP2 were secreted by CBMSCs with higher abundance than UCMSCs. Overall, these results suggest that UCMSCs and CBMSCs preserve different functional potentials, which have to be carefully considered before clinical treatment.

MicroRNA profiling analysis revealed different cellular senescence mechanisms in human mesenchymal stem cells derived from different origin.

Mesenchymal stem cells (MSCs) from human umbilical cord (UC) and cord blood (CB) share many common properties and exhibit promising clinical potential. Cellular senescence, which induces the loss of stem cells characters and disrupts their therapeutic functions, has been demonstrated to be under the regulation of microRNAs (miRNAs). In this study, we compared the miRNA profiles in early and late passage UCMSCs and CBMSCs based on deep sequencing. 224 and 170 miRNAs were significantly altered in UCMSCs and CBMSCs respectively. A functional annotation of the predicted miRNA targets revealed a series of common senescence pathways. However, Functional enrichment analysis revealed different bioprocesses involved in cellular senescence of UC- and CB-MSCs. The common miRNAs shared by the two kinds of MSCs also exert different function in terms of GO enrichment analysis. Our results supported MSCs derived from different origin may undergo senescence through different path.

MiR-218 Induces Neuronal Differentiation of ASCs in a Temporally Sequential Manner with Fibroblast Growth Factor by Regulation of the Wnt Signaling Pathway.

Differentiation of neural lineages from mesenchymal stem cells has raised the hope of generating functional cells as seed cells for nerve tissue engineering. As important gene regulators, microRNAs (miRNAs) have been speculated to play a vital role in accelerating stem cell differentiation and repairing neuron damage. However, miRNA roles in directing differentiation of stem cells in current protocols are underexplored and the mechanisms of miRNAs as regulators of neuronal differentiation remain ambiguous. In this study, we have determined that miR-218 serves as crucial constituent regulator in neuronal differentiation of adipose stem cells (ASCs) through Wnt signaling pathway based on comprehensive annotation of miRNA sequencing data. Moreover, we have also discovered that miR-218 and Fibroblast Growth Factor-2 (FGF2) modulate neuronal differentiation in a sequential manner. These findings provide additional understanding of the mechanisms regulating stem cell neuronal differentiation as well as a new method for neural lineage differentiation of ASCs.

Comparative analysis of microRNA expression in human mesenchymal stem cells from umbilical cord and cord blood.

Human mesenchymal stem cells (MSCs) derived from both umbilical cord (UC) and cord blood (CB) share similar characteristics, and their differences are largely unknown. Besides the significant difference in cell morphology, differentiation ability and development processes of the two different origin MSCs, a different expression pattern of microRNAs between the two kinds of MSCs was also obtained. By comprehensively annotating the differently expressed global microRNAs (miRNAs), a series of biological pathways were predicted. We found that miRNAs significantly repressed insulin signaling in UCMSCs, while neural related processes were more repressed in CBMSCs. Particularly, TGF-ß and Notch signaling were differently activated in both MSCs, unveiling their distinct angiogenesis potentials. Taken together, this study illustrates that MSCs from UC and CB display distinct properties, which indicates different potentials for clinical usage.

Mechanism study for hypoxia induced differentiation of insulin-producing cells from umbilical cord blood-derived mesenchymal stem cells.

Recently, we have successfully obtained functional IPCs efficiently from umbilical cord blood-derived mesenchymal stem cells by using hypoxia treatment. In this study, we further elaborated that the improved function and viability of IPCs are the result of the interaction ß cell development pathway and c-Met/HGF axis induced by hypoxia. We found that hypoxia induced c-MET elevation is efficiently initiated the early stage differentiation IPCs from MSCs, and HGF improved the fully differentiation of IPCs by inducing the expression of NGN3. This finding may contribute to understanding ß cell development and the development of stem cell therapy for diabetes.