A Perfused Microfluidic System to Study the Differentiation of Neural Stem Cells in vitro.

INTRODUCTION: Due to the ability to mimic in vivo cellular microenvironments, the development of multicell culture systems has received increasing interest for use as research models and serving as platforms for drug evaluation. METHODS: In this study, we developed a perfused microfluidic system to resemble the in vivo intercellular environment and applied it to study the differentiation from neural stem cells into neurons. RESULTS: As determined by immunofluorescence chemistry and quantitative real-time PCR, the neural stem cells grown in this microfluidic system showed an elevated differentiation rate toward the formation of neurons as determined by the increased level of ßIII-tubulin production, which is 4 times higher than that of culturing neural stem cells only. CONCLUSION: These results revealed that some factors secreted into the intercellular microenvironment by adult neuron cells can stimulate the differentiation of neural stem cells, pointing to the importance of developing multicellular culture systems such as the perfused microfluidic system we report here to better resemble the in vivo situation.

Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1.

Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on chimeric antigen receptor modified T cell (CAR-T) immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase and programmed cell death-ligand 1 which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial reactive oxygen species release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the stanniocalcin-1 (STC1) gene, which encodes the glycoprotein hormone STC-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo, and STC1 played a critical role. These data revealed a novel function of MSC and STC-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response.

Immunotherapy is a type of cancer treatment that helps the immune system fight cancer. For example, chimeric antigen receptor T cell (CAR-T) therapy is used to target several types of blood cancer. It works by reprogramming patients' immune cells to target specific tumor cells. In blood cancers, CAR-T therapy works very well, but it can cause extreme responses from the patient's immune system, which can be life threatening. In solid tumors, CAR-T therapy is much less successful because the tumors secrete molecules into the space surrounding them, which weaken the immune processes that attack cancerous cells. Stem cells are the master cells of the body. Originating in the bone marrow, they can repair and regenerate the body's cells. Cancer stem cells play a role in resistance to CAR-T therapy, due ­ in part ­ to their ability to renew themselves, but the role of another type of stem cell, called mesenchymal stem cells, was less clear. Mesenchymal stem cells develop into tissues that line organs and blood vessels. Although it is known that mesenchymal stem cells are present in most cancers and play a role in shaping and influencing the space around tumors, their impact on CAR-T therapy has not been studied in depth. To find out more, Zhang et al. looked at the influence of a protein, called staniocalcin-1 (STC1), on CAR-T therapy, by studying cells grown in the laboratory and human tumor cells that had been implanted in mice. Zhang et al. found that mesenchymal stem cells reduce the ability of CAR-T therapy to destroy cancer cells and that they needed STC1 to do this successfully. They also increased the expression of molecules that dampen the immune system, and suppressed molecules called inflammasomes, which are an important part of the way the immune system detects disease. Moreover, reducing the amount of STC1 that mesenchymal stem cells expressed restored the effectivity of CAR-T therapy. This study increases our understanding of the way that mesenchymal stem cells affect CAR-T therapy. It has the potential to open up a new way of improving the efficiency of this treatment and of reducing the harmful side effects that it can cause.

Engineering CAR-NK cells targeting CD33 with concomitant extracellular secretion of anti-CD16 antibody revealed superior antitumor effects toward myeloid leukemia.

Acute myeloid leukemia (AML) is a common form of acute leukemia, and the currently available treatments are unsatisfactory. In the present study, we report an immune cell therapeutic strategy that employed genetically modified bifunctional CAR-NK cells. These cells combined the efficient targeting of AML cells by the CD33 molecule with the concomitant stimulation of NK cell-mediated cytotoxicity via the expression and extracellular secretion of anti-CD16 antibody (B16) that binds back to the FC receptor of NK cells. Compared to CAR-NK cells that target CD33 only, the bifunctional CD33/B16 CAR-NK cells showed superior killing efficiency toward AML cells in vitro. The increase in efficiency was approximately four-fold, as determined based on the number of cells needed to achieve 80% killing activity. An in vivo study using a xenograft model also revealed the effective clearance of leukemic cells and much longer survival, with no relapse or death for at least 60 days. In addition, the safety of CAR-NK cells did not change with additional expression of B16, as determined by the release of cytokines. These data revealed the development of a promising CAR-NK approach for the treatment of patients with AML, which may improve CAR-NK-based treatment strategy in general and may potentially be used to treat other tumors as well.

Knockdown SENP1 Suppressed the Angiogenic Potential of Mesenchymal Stem Cells by Impacting CXCR4-Regulated MRTF-A SUMOylation and CCN1 Expression.

The angiogenic potential of mesenchymal stem cells (MSCs) is critical for adult vascular regeneration and repair, which is regulated by various growth factors and cytokines. In the current study, we report that knockdown SUMO-specific peptidase 1 (SENP1) stimulated the SUMOylation of MRTF-A and prevented its translocation into the nucleus, leading to downregulation of the cytokine and angiogenic factor CCN1, which significantly impacted MSC-mediated angiogenesis and cell migration. Further studies showed that SENP1 knockdown also suppressed the expression of a chemokine receptor CXCR4, and overexpression of CXCR4 could partially abrogate MRTF-A SUMOylation and reestablish the CCN1 level. Mutation analysis confirmed that SUMOylation occurred on three lysine residues (Lys-499, Lys-576, and Lys-624) of MRTF-A. In addition, SENP1 knockdown abolished the synergistic co-activation of CCN1 between MRTF-A and histone acetyltransferase p300 by suppressing acetylation on histone3K9, histone3K14, and histone4. These results revealed an important signaling pathway to regulate MSC differentiation and angiogenesis by MRTF-A SUMOylation involving cytokine/chemokine activities mediated by CCN1 and CXCR4, which may potentially impact a variety of cellular processes such as revascularization, wound healing, and progression of cancer.

Chemotherapeutic drugs induce oxidative stress associated with DNA repair and metabolism modulation.

Bulky DNA damage inducing chemotherapeutic cancer drugs such as cisplatin (CIS) and doxorubicin (DOX) are commonly used in the treatment of a variety of cancers. However, they often cause multi-organ toxicity, and the mechanisms underlying are not clear. Using cellular model, the present study showed that persistent endogenous reactive oxygen species (ROS) were stimulated after a single dose short treatment with CIS and DOX. ROS level correlated with the formation of DNA double-strand breaks (DSBs). Knockdown BRCA1, a key player involved in homologous recombination (HR), enhanced ROS accumulation. Whereas knockdown DNA-PKcs and overexpress BRCA1 to inhibit nonhomologous end-joining (NHEJ) repair pathway and restore HR can partially suppress ROS levels. These data indicated that ROS production is associated with DSB formation and repair which is likely a downstream event of DNA repair. Further studies showed that knockdown DNA repair regulators PP2A but not ATM, could partially reduce ROS too. The induction of ROS affected the level of proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Collectively, the present study reveals that DNA repair associated metabolism change and oxidative stress may be a direct cause of the severe side effects associated with genotoxic chemotherapy cancer drugs.

Myocardin-related transcription factor-A induces cardiomyocyte hypertrophy.

Myocardin is a remarkably potent transcriptional coactivator expressed specifically in cardiac muscle lineages and smooth muscle cells during postnatal development. Myocardin shares homology with myocardin-related transcription factor-A (MRTF-A), which are expressed in a broad range of embryonic and adult tissues. Our previous results show that myocardin induces cardiac hypertrophy. However, the effects of MRTF-A in cardiac hypertrophy remain poorly understood. Our present work further demonstrates that myocardin plays an important role in inducing hypertrophy. At the same time, we find that overexpression of MRTF-A in neonatal rat cardiomyocytes might induce cardiomyocyte hypertrophy. Furthermore, MRTF-A expression is induced in phenylephrine, angiotensin-II, and transforming growth factor-ß-stimulated cardiac hypertrophy, whereas a dominant-negative form of MRTF-A or MRTF-A siRNA strongly inhibited upregulation of hypertrophy genes in response to hypertrophic agonists in neonatal rat cardiomyocytes. Our studies indicate that besides myocardin, MRTF-A might play an important role in cardiac hypertrophy. Our findings provide novel evidence for the future studies to explore the roles of MRTFs in cardiac hypertrophy.

Estrogen enhances the cytotoxicity of PARP inhibitors on breast cancer cells through stimulating nitric oxide production.

Inhibition of Poly(ADP-ribose) polymerase (PARP) is effective for breast cancer susceptibility genes 1 (BRCA1)-deficient breast cancers. Although hormones play critical roles on the occurrence as well as being used in conventional therapies of breast cancer, their impacts on PARP-targeted therapy have been poorly addressed. Here, we showed that addition of estrogen could enhance the cytotoxicity of PARP inhibitors on estrogen receptor (ER)-positive breast cancer cells, causing significant suppression of cell growth. Further analysis revealed that the impact was due to estrogen's stimulating the production of nitric oxide (NO), which could be abrogated when blocking NO formation. Moreover, the effect of estrogen can be resembled by two exogenous nitric oxide donors (SNAP and GSNO). Using ER-negative cell line MDA-MB231, estrogen could not enhance the cell killing of PARP inhibitors any more, but addition of NO donors re-established the enhancing effects. The increased NO level led to accumulation of DNA double strand breaks (DSBs) based on the formation of H2AX foci. Consistent with earlier studies, we demonstrated that NO suppressed the expression of BRCA1, a key player involved in DSB recombination repair. Taken together, these data reveal an important role of estrogen on the treatment of PARP inhibitors, which may affect its clinical treatment and should be considered in precision therapies for ER-positive and negative cancers.

[Research progress of phase separation of intracellular biological macromolecules].

The phenomenon of phase separation of intracellular biological macromolecules is an emerging research field that has received great attention in recent years. As an aggregation and compartment mechanism of cell biochemical reactions, it widely exists in nature and participates in important physiological processes such as gene transcription and regulation, as well as influences organism's response to external stimuli. Disequilibrium of phase separation may lead to the occurrence of some major diseases. Researchers in cross-cutting fields are trying to examine dementia and other related diseases from a new perspective of phase separation, exploring its molecular mechanism and the potential possibility of intervention and treatment. This review intends to introduce the latest research progress in this field, summarize the major research directions, biochemical basis, its relationship with disease occurrence, and giving a future perspective of key problems to focus on.

Slowly Repaired Bulky DNA Damages Modulate Cellular Redox Environment Leading to Premature Senescence.

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a "molecular scar" leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.

[Isolation of cancer stem cells and the establishment of a H 2O 2-resistant cancer stem cell model].

OBJECTIVE: To isolate cancer stem cells (CST) from human breast cancer cell line (MCF-7) and study their sensitivity toward oxidative stress. METHODS: MCF-7 cells were cultured in serum-free suspension culture medium to identify cells forming the sphere phenotype. The morphological changes of MCF-7 cells were observed by inverted phase contrast microscope (compared with MCF-7 cells cultured in serum-free suspension culture medium). The expression of CST marker CD133 was detected by immunocytochemical staining in CST cell spheres (experimental group) with a diameter of 100 µm and MCF-7 cells (control group) with a fusion degree of 70%. The positive rate of CD133 was detected by flow cytometry in the third generation of tumor cells with diameter of 150 µm. The second generation of tumor globular cells (experimental group) with diameter of 150 µm and corresponding MCF-7 cells (control group) were taken to be damaged by 50 mol/L H 2O 2 for 120 minutes. The expression of DNA damage marker histone H2AX phosphorylation (γH2AX) was detected by immunocytochemical staining. RESULTS: Inverted phase contrast microscopy showed that MCF-7 cells grew initially in a single-cell adherent state, then aggregated and grew in serum-free suspension culture medium, and finally formed CST cell spheres, while the control MCF-7 cells cultured in MCF-7 cell culture medium grew extensively and could not grow in suspension. Fluorescence microscopy showed that the expression of CD133 in MCF-7 cells of control group was negative, while that in experimental group was positive. Flow cytometry showed that CD133 was positive in CST cells, and the positive rate was 92%. Inverted fluorescence microscopy showed that the expression of γH2AX in CST tumor spheres of experimental group was significantly lower than that in MCF-7 cells of control group after 120 minutes of H 2O 2 injury. CONCLUSION: Serum-free suspension culture medium can produce globular CST cells from MCF-7 tumor cell line, which have strong antioxidant damage.

[Effect of serum on the differentiation of neural stem cells].

Objective: To investigate the effect of serum on the differentiation of neural stem cells. Methods: The neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (ß-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture. Results: Based on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of ß-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and ß-Ⅲ Tubulin at 4 and 8 days between groups ( P<0.05). Conclusion: Serum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.

Cancer cells growing on perfused 3D collagen model produced higher reactive oxygen species level and were more resistant to cisplatin compared to the 2D model.

INTRODUCTION: Three-dimensional (3D) collagen scaffold models, due to their ability to mimic the tissue and organ structure in vivo, have received increasing interest in drug discovery and toxicity evaluation. METHODS: In this study, we developed a perfused 3D model and studied cellular response to cytotoxic drugs in comparison with traditional 2D cell cultures as evaluated by cancer drug cisplatin. RESULTS: Cancer cells grown in perfused 3D environments showed increased levels of reactive oxygen species (ROS) production compared to the 2D culture. As determined by growth analysis, cells in the 3D culture, after forming a spheroid, were more resistant to the cancer drug cisplatin compared to that of the 2D cell culture. In addition, 3D culturing cells showed elevated level of ROS, indicating a physiological change or the formation of a microenvironment that resembles tumor cells in vivo. CONCLUSIONS: These data revealed that cellular response to drugs for cells growing in 3D environments are dramatically different from that of 2D cultured cells. Thus, the perfused 3D collagen scaffold model we report here might be a potentially very useful tool for drug analysis.

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived-flavonoid monomers using a three-dimensional stem cell-derived neural model.

An in vitro three-dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell-derived neural co-culture model. Rat neural stem cells were differentiated into co-culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two-dimensional (2D) model using a two-step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase-3 activity in a dose-dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co-cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735-744, 2016.

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

[Distribution characteristics of dissolved oxygen and its affecting factors in the Pearl River Estuary during the summer of the extremely drought hydrological year 2011].

More and more attention has focused on assessing impacts of extreme hydrologic events on estuarine ecosystem under the background of climate change. Based on a summer cruise conducted in the Pearl River Estuary in 2011 (extreme drought event), we have investigated the spatial distribution of dissolved oxygen (DO) and its relationships to water column stability, nutrient concentrations, and organic matter; besides, the major reason which caused the oxygen depletion was discussed. Under the influence of the extreme drought event, low bottom water dissolved oxygen was apparent in regions characterized by great depths, with an oxygen minimum value of 1.38 mg x L(-1). Statistical analysis shows significant correlations among deltaDO, deltaT, deltaS and deltaPOC. A comparison was conducted to show the mechanisms of oxygen depletion during the summers of 1999, 2009 and 2011, respectively. The result indicates that prolonged residence time of water due to the extremely low discharge and the subsequently decomposition of organic substance are major factors causing the formation of hypoxia during the summer drought in 2011. Despite the changing nutrient and organic matter regime in the Pearl River Estuary, there was no apparent trend in the minimum values of DO over the past 2 decades.