Formation Of Stem Cells Research Articles

Carbon Quantum Dots for Stem Cell Imaging and Deciding the Fate of Stem Cell Differentiation

Nanotechnology advancements and applications have paved the way for new possibilities in regenerative medicine and tissue engineering. It is a relatively new field that has the potential to improve stem cell differentiation and therapy greatly. Numerous studies have demonstrated that nanomaterials can function as a physiological niche for the formation and differentiation of stem cells. However, quantum dots (QDs), such as carbon quantum dots (CQDs) and graphene quantum dots (GQDs), have shown considerable promise in the field of regenerative medicine. To date, most research has focused on stem cell tracking and imaging using CQDs. However, their interaction with stem cells and the associated possibility for differentiation by selectively focusing chemical signals to a particular lineage has received scant attention. In this mini-review, we attempt to categorize a few pathways linked with the role of CQDs in stem cell differentiation.

New insight on the role of Helicobacter pylori cagA in the expression of cell surface antigens with important biological functions in gastric carcinogenesis.

Expression of cluster of differentiation (CD) antigens changes according to disease status and inflammation. Profiles of CD antigens expression in gastric cancer patients are different based on the status of H.pylori infection. We conducted this study to profile CD antigen markers in gastric adenocarcinoma cells (AGS cell line) infected with distinct cytotoxin-associated gene A (cagA) genotypes of H.pylori clinical isolates. The AGS cells were infected with H.pylori isolates with different cagA genotypes, and CD antigens expression was determined using DotScan™ antibody microarray. Formation of "hummingbird" phenotype was determined, and the percentage was calculated. H.pylori strains harboring cagA upregulated the expression of CD antigen involved in cancer stem cell formation (CD55), but downregulated CD antigens involved in immune regulation (CD40 and CD186) and cell adhesion (CD44). CD54 (neutrophil adhesion) and CD71 (iron transfer) were highly downregulated in the gastric cells infected with Western cagA isolates compared with East Asian isolates. CD antigen expression was different in the cells infected with H.pylori harboring different CagA EPIYA (Glu-Pro-Ile-Tyr-Ala) numbers, in which higher repression of CD54 and CD15 (Lewis x antigen) were observed in the isolate with the highest number of EPIYA motif. Furthermore, higher downregulation of CD15 was observed in the infected gastric cells with high percentage of "hummingbird" phenotype than that of low percentage of "hummingbird" phenotype. Our study demonstrated the critical roles of CD antigens in the CagA pathogenesis and should be investigated further.

Prediction of Human Induced Pluripotent Stem Cell Formation Based on Deep Learning Analyses Using Time-lapse Brightfield Microscopy Images.

We use deep learning methods to predict human induced pluripotent stem cell (hiPSC) formation using time-lapse brightfield microscopy images taken from a cell identified as the beginning of entered into the reprogramming process. A U-net is used to segment cells and a CNN is used to classify the segmented cells into eight types of cells during the reprogramming and hiPSC formation based on cellular morphology on the microscopy images. The numbers of respective types of cells in cell clusters before the hiPSC formation stage are used to predict if hiPSC regions can be well formed lately. Experimental results show good prediction by the criteria using the numbers of different cells in the clusters. Time-series images with respective types of classified cells can be used to visualize and quantitatively analyze the growth and transition among dispersed cells not in cell clusters, various types of cells in the clusters before the hiPSC formation stage and hiPSC cells.

High AUF1 level in stromal fibroblasts promotes carcinogenesis and chemoresistance and predicts unfavorable prognosis among locally advanced breast cancer patients

BackgroundLocally advanced breast cancer (LABC), the most aggressive form of the disease, is a serious threat for women's health worldwide. The AU-rich RNA-binding factor 1 (AUF1) promotes the formation of chemo-resistant breast cancer stem cells. Thereby, we investigated the power of AUF1 expression, in both cancer cells and their stromal fibroblasts, as predictive biomarker for LABC patients’ clinical outcome following neoadjuvant treatment.MethodsWe have used immunohistochemistry to assess the level of AUF1 on formalin-fixed paraffin-embedded tissues. Immunoblotting was utilized to show the effect of AUF1 ectopic expression in breast stromal fibroblasts on the expression of various genes both in vitro and in orthotopic tumor xenografts. Cytotoxicity was evaluated using the WST1 assay, while a label-free real-time setting using the xCELLigence RTCA technology was utilized to assess the proliferative, migratory and invasive abilities of cells.ResultsWe have shown that high AUF1 immunostaining (≥ 10%) in both cancer cells and their adjacent cancer-associated fibroblasts (CAFs) was significantly associated with higher tumor grade. Kaplan–Meier univariate analysis revealed a strong correlation between high AUF1 level in CAFs and poor patient’s survival. This correlation was highly significant in patients with triple negative breast cancer, who showed poor disease-free survival (DFS) and overall survival (OS). High expression of AUF1 in CAFs was also associated with poor OS of ER+/Her2− patients. Similarly, AUF1-positive malignant cells tended to be associated with shorter DFS and OS of ER+/Her2+ patients. Interestingly, neoadjuvant therapy downregulated AUF1 to a level lower than 10% in malignant cells in a significant number of patients, which improved both DFS and OS. In addition, ectopic expression of AUF1 in breast fibroblasts activated these cells and enhanced their capacity to promote, in an IL-6-dependent manner, the epithelial-to-mesenchymal transition and stemness processes. Furthermore, these AUF1-expressing cells enhanced the chemoresistance of breast cancer cells and their growth in orthotopic tumor xenografts.ConclusionsThe present findings show that the CAF-activating factor AUF1 has prognostic/predictive value for breast cancer patients and could represent a great therapeutic target in order to improve the precision of cancer treatment.

Promising Stem Cell therapy in the Management of HIV and AIDS: A Narrative Review.

Stem cell therapies are becoming a major topic in biomedical research all over the planet. It may be a viable treatment choice for people suffering from a wide range of illnesses and injuries. It has recently emerged as an extremely intriguing and well-established science and research topic. Expectations have risen due to advancements in therapeutic approaches. Multiple laboratory testing of regulated stem cell culture and derivation is carried out before the formation of stem cells for the use of therapeutic process. Whereas HIV infection is contagious and can last a lifetime. Researchers are still working to develop a comprehensive and effective treatment for HIV and its associated condition, as well as AIDS. HIV propagation is primarily restricted to the immune system, notably T lymphocytes, as well as macrophages. Large numbers of research studies have contributed to a plethora of data about the enigmatic AIDS life cycle. This vast amount of data provides potential targets for AIDS therapies. Currently, stem cell transplantation, along with other procedures, provided novel insights into HIV pathogenesis and offered a glimpse of hope for the development of a viable HIV cure technique. One of its existing focus areas in HIV and AIDS research is to develop a novel therapeutic strategic plan capable of providing life-long complete recovery of HIV and AIDS without regular drug treatment and, inevitably, curative therapy for HIV and AIDS. The current paper tries to address the possibilities for improved stem cell treatments with “bone marrow, Hematopoietic, human umbilical cord mesenchymal, Genetical modifications with CRISPR/Cas9 in combination of stem cells, induced pluripotent stem cells applications” are discussed which are specifically applied in the HIV and AIDS therapeutic management advancement procedures.

Abstract 1029: The anti-androgen seviteronel sensitizes triple-negative breast cancer to chemotherapy

Abstract Therapeutic strategies that improve survival outcomes for advanced-stage breast cancers have proven a major clinical challenge. Here, we define an androgen receptor (AR) signalling network that governs the maintenance and de novo formation of cancer stem cells in triple-negative breast cancer. In response to chemotherapy, AR activation switches cells into a cancer stem cell state, while AR antagonism suppresses cancer stem cell formation and function. In vivo, we validate that the AR antagonist, seviteronel, significantly improves chemotherapy-mediated inhibition of primary and metastatic tumour growth. Analysing three independent triple-negative breast cancer patient cohorts we identify that cytoplasmic AR expression prognosticates poor survival in treatment-naïve patients and predicts poor response to chemotherapy. Additionally, Phase II clinical trial data demonstrates that seviteronel treatment followed by chemotherapy improved survival outcomes in triple-negative breast cancer patients expressing high cytoplasmic AR levels. Seviteronel combined with chemotherapy represents a promising therapeutic strategy for triple-negative breast cancer patients. Citation Format: Beatriz P. San Juan, Soroor Hediyah-Zadeh, Laura Rangel, Vanina Rodriguez, Heloisa H. Milioli, Felix Kohane, Carley A. Purcell, Therese E. Hickey, Leonard D. Goldstein, John G. Lock, Melissa J. Davis, Christine L. Chaffer. The anti-androgen seviteronel sensitizes triple-negative breast cancer to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1029.

Abstract 1039: TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most commonly diagnosed cancer in American women and accounts for ~15% of cancer-related deaths. Despite the current standard of care, metastatic HER2-enriched breast cancer and triple-negative breast cancer remain difficult to treat due to poor response to treatment, lack of actionable targets, or eventual acquired resistance. The high mortality rate of metastatic HER2-enriched breast cancers and triple-negative breast cancers highlights the need for novel actionable targets for improved response to therapeutic intervention. Through datamining of publicly available breast cancer patient datasets, we recently found that Tropomyosin receptor kinase A (TrkA) and Janus kinase 2 (JAK2)-STAT3 pathways are co-activated and co-enriched in HER2-enriched breast cancers and triple-negative breast cancers (Cancers 10:2340-2360, 2021). We also found that TrkA, a receptor tyrosine kinase, directly interacts with and phosphorylates STAT3 on Y705 residue, resulting in STAT3 activation, nuclear translocation, and increased transcription of STAT3 target genes SOX2 and c-MYC, which are associated with breast cancer stem cell formation as well as tumor recurrence and metastasis. In this study, we aimed to further characterize the effects of the TrkA-JAK2/STAT3 pathway crosstalk on breast cancer stem cells. Our data showed that overexpression of TrkA enhances mammosphere formation and ALDH activity of breast cancer cells, which are further enhanced upon co-overexpression of STAT3. Our study further revealed that TrkA and JAK2 inhibitors synergize to reduce breast cancer stemness since co-inhibition of TrkA and JAK2 significantly reduces the CD44high/CD24low cell subpopulation, mammosphere formation, and ALDH activity to a greater extent compared to vehicle or monotherapies. Western blot analysis of breast cancer cells treated with TrkA and/or JAK2 inhibitors showed that co-inhibition of these two kinases significantly reduces levels of p-STAT3 (Y705), as well as stemness markers SOX2, MYC, and CD44. Taken together, these findings suggest that TrkA cooperates with the JAK2-STAT3 signaling pathway to promote breast cancer stem cells through increasing expression of SOX2, c-MYC, and CD44, and that co-inhibition of TrkA and JAK2 significantly suppressed breast cancer stemness, suggesting its future utility as a promising new treatment modality for patients with HER2-enriched breast cancers and triple-negative breast cancers. Citation Format: Angelina T. Regua, Dongqin Zhu, Daniel L. Doheny, Grace L. Wong, Sara G. Manore, Calvin J. Wagner, Austin Arrigo, Mariana Najjar, Hui-Wen Lo. TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1039.

The role of selected molecular factors in ovarian cancer metastasis.

The main reason for treatment failure in ovarian cancer is chemoresistance and the presence of metastasis. Ascites, whichallows the physical movement of cancer cells, the lymphovascular pathway, and several molecular factors and signalling axes, are involved in metastasis. Ascites, with the involvement of cytokines and chemokines, MAPK/STAT1 and NOTCH as well as CXCL12/CXCR4 signaling pathways and circulating anoikis induces cancer dissemination, in particular to the peritoneum and omentum. The spread of lymphatic and bloodstream cancer cells is a multi-stage process. Tumour infiltration of the stroma and lymphovascular space (LVSI) produces biologically active cancer-associated fibroblasts and macrophages (CAFs, TAMs) that secrete numerous cytokines, chemokines and growth factors, inhibit NK function, induce epithelial-mesenchymal transition (EMT), resulting in an increase of the metastatic potential of cancer cells and the formation of cancer stem cells (CSCs). Overexpression of some genes, and microRNAs, in LVSI-(LMGS) associated with metastasis has been identified. The role of extracellular vesicles (EVs) transporting metastasis-associated factors has been described as has the role of cancer stem cells (CSCs) in chemotherapy resistance and metastasis. Sirtuins, enzymes involved in metastasis formation, have also been detected. Certain types of microRNAs (miR-509-3p, microRNA-506-3p) and melatonin have been shown to inhibit metastasis.

The Effect of Matrix for Regeneration of Enamel, Dentin and Pulp: A Systematic Review

The tooth structure does not have ability to regenerate on it’s own until there is introduction of some inducing factors. Several inducing factors present in the form of stem cells, scaffolds and growth factors which thereby, plays role in the formation of enamel, dentin and pulp. In order to stimulate these regenerative factors, certain amount of temperature, pressure and atmospheric humidity should be in control. Specific knowledge of proteins which is present in each of the part of tooth structure (enamel, dentin) is important in order to achieve the final outcome. The present review elaborates about the achievement of tooth structure matrix and organs by usage of specific amount of proteins, growth factors, stem cells

M6A regulator expression patterns predict the immune microenvironment and prognosis of non-small cell lung cancer.

The m6A methylation modification is one of the most common mRNA modifications, and involved in a variety of biological processes, such as cell death, cancer stem cell formation and tumorigenesis. Increasing evidences have demonstrated that the expression patterns of m6A regulators are significantly correlated with PD-L1 level some solid tumors, but few study has explored the function of m6A regulators in the immune microenvironment and prognosis in non-small cell lung cancer (NSCLC). Survival analysis was independently conducted for 20 m6A regulators to explore their prognostic value in NSCLC, and then the prognostic risk model based on m6A regulator expression profiles is built to stratify NSCLC patients. Also, the correlation analysis between immune infiltrating cells and m6A regulators is used to reveal the impact of m6A on immune microenvironment of NSCLC. Furthermore, to explore the function of m6A as biomarker of anit-PD-L1 therapeutic effect, we explored the associations of tumor mutation burden (TMB) and PD-L1 levels to 20 m6A regulator expression patterns in NSCLC. First, the expressions of 20 m6A regulators in NSCLC tissues were significantly increased compared to normal tissues. Survival analysis revealed that three genes, METTL3, HNRNPC and VIRMA, were markedly correlated to the prognosis of NSCLC patients. In particular, cox regression analysis verified that METTL3 could be used as an independent prognostic factor to predict the survival rate of NSCLC patients. Second, the risk prognostic model built on seven m6A regulators can effectively stratify NSCLC patients, and the low-risk subgroup had better prognosis compared to high-risk group. Finally, a few m6A regulators showed significant associations with immune microenvironment, as well as TMB and PD-L1 level, suggesting that the m6A RNA methylation is indicative of therapeutic effect of anti-PD-L1 treatment. Our study identified some m6A regulatory factors as independent risk factors for the prognosis of NSCLC, and the expression patterns of m6A regulators are also correlated to the immune infiltration, as well as TMB and PD-L1 level in NSCLC. The m6A regulators could be used as biomarkers indicative of immunotherapy to NSCLC patients.

Role of the Ubiquitin Ligase RNF149 in the Development of Rat Neonatal Gonocytes.

Male reproductive function depends on the formation of spermatogonial stem cells from their neonatal precursors, the gonocytes. Previously, we identified several UPS enzymes dynamically altered during gonocyte differentiation. The present work focuses on understanding the role of the RING finger protein 149 (RNF149), an E3 ligase that we found to be strongly expressed in gonocytes and downregulated in spermatogonia. The quantification of RNF149 mRNA from postnatal day (PND) 2 to 35 (puberty) in rat testis, brain, liver, kidney, and heart indicated that its highest levels are found in the testis. RNF149 knock-down in PND3 rat gonocytes was performed to better understand its role in gonocyte development. While a proliferative cocktail of PDGF-BB and 17β-estradiol (P+E) increased both the expression levels of the cell proliferation marker PCNA and RNF149 in mock cells, the effects of P+E on both genes were reduced in cells treated with RNF149 siRNA, suggesting that RNF149 expression is regulated during gonocyte proliferation and that there might be a functional link between RNF149 and PCNA. To examine RNF149 subcellular localization, EGFP-tagged RNF149 vectors were constructed, after determining the rat testis RNF149 mRNA sequence. Surprisingly, two variant transcripts were expressed in rat tissues, predicting truncated proteins, one containing the PA and the other the RING functional domains. Transfection in mouse F9 embryonal carcinoma cells and C18-4 spermatogonial cell lines showed differential subcellular profiles of the two truncated proteins. Overall, the results of this study support a role for RNF149 in gonocyte proliferation and suggest its transcription to variant mRNAs resulting in two proteins with different functional domains. Future studies will examine the respective roles of these variant proteins in the cell lines and isolated gonocytes.

MiR-2392 functions as tumour suppressor and inhibits malignant progression of hepatocellular carcinoma via directly targeting JAG2.

Dysregulation of microRNA (miRNA) expression in various cancers and their vital roles in malignant progression of cancers are well investigated. Our previous studies have analysed miRNAs that promote malignant progression in hepatocellular carcinoma (HCC); this study aims to systematically elucidate the mechanism of metastasis suppressor miRNAs in HCC. High-throughput RNA sequencing was used to identify anti-metastatic miRNAs. The relative expression levels of miRNAs were confirmed by qRT-PCR. The biological functions of miRNAs were detected in vitro and in vivo. Circulating tumour cells (CTCs) were enriched from blood samples of HCC patients and cultured by three-dimensional (3D) system. Kaplan-Meier and Cox regression were used to analyse the value of potential target mRNAs on overall survival. miR-2392 was significantly down-regulated in HCC. Overexpression of miR-2392 suppressed proliferation, clonogenicity, mobility, spheroid formation and maintenance of cancer stem cells (CSC)-like characteristics in HCC cells. CTCs from HCC patients with lower serum miR-2392 level had stronger cell spheroid formation ability. A negative correlation between the content of miR-2392 in serum and the number of CTC spheroids had been found. We identified Jagged2 (JAG2) as a direct target of miR-2392. miR-2392 inhibited the expression of JAG2 by targeting 3'-UTR of JAG2. Down-regulation of JAG2 inhibited the overexpression effects of miR-2392 in vitro and in vivo. JAG2 is highly expressed in HCC and is closely related to poor prognosis and survival of patients. miR-2392 may play a role as a tumour suppressor to guide the individualized precise treatment of HCC.

Salvia miltiorrhiza in Breast Cancer Treatment: A Review of Its Phytochemistry, Derivatives, Nanoparticles, and Potential Mechanisms.

Breast cancer is one of the most deadly malignancies in women worldwide. Salvia miltiorrhiza, a perennial plant that belongs to the genus Salvia, has long been used in the management of cardiovascular and cerebrovascular diseases. The main anti-breast cancer constituents in S. miltiorrhiza are liposoluble tanshinones including dihydrotanshinone I, tanshinone I, tanshinone IIA, and cryptotanshinone, and water-soluble phenolic acids represented by salvianolic acid A, salvianolic acid B, salvianolic acid C, and rosmarinic acid. These active components have potent efficacy on breast cancer in vitro and in vivo. The mechanisms mainly include induction of apoptosis, autophagy and cell cycle arrest, anti-metastasis, formation of cancer stem cells, and potentiation of antitumor immunity. This review summarized the main bioactive constituents of S. miltiorrhiza and their derivatives or nanoparticles that possess anti-breast cancer activity. Besides, the synergistic combination with other drugs and the underlying molecular mechanisms were also summarized to provide a reference for future research on S. miltiorrhiza for breast cancer treatment.

Functional Importance of Mini-Puberty in Spermatogenic Stem Cell Formation

Primordial germ cells nesting in the fetal testis give rise to gonocytes. The gonocytes then transform into spermatogenic stem cells (SSCs) during the neonatal period and thereafter serve as a lifetime source of spermatogenesis. Therefore, gonocyte to SSC transformation is quite an important process that supports fertility in males. During the gonocyte to SSC transformation, morphological and transcriptomic changes sequentially occur and gonocytes migrate from the center to the peripheral region of the seminiferous tubules. However, extrinsic signals which trigger the transcriptomic changes as well as the migration are not yet fully clarified. Recent studies have drawn attention to the temporal activation of the hypothalamic-pituitary-gonadal axis during the neonatal stage which occurs concurrently with SSC formation. This phenomenon is called mini-puberty, and recent studies on human cryptorchid patients as well as animal models partially support the hypothesis that mini-puberty plays pivotal roles in gonocyte-to-SSC transformation. Focusing on this point, here, we aimed to discuss the latest knowledge on the importance of mini-puberty in spermatogenesis in this review.

Meristem Initiation and de novo Stem Cell Formation.

Plant aerial development relies on meristem activity which ensures main body plant axis development during plant life. While the shoot apical meristem (SAM) formed in the embryo only contributes to the main stem, the branched structure observed in many plants relies on axillary meristems (AMs) formed post-embryonically. These AMs initiate from a few cells of the leaf axil that retain meristematic characteristics, increase in number, and finally organize into a structure similar to the SAM. In this review, we will discuss recent findings on de novo establishment of a stem cell population and its regulatory niche, a key step essential for the indeterminate fate of AMs. We stress that de novo stem cell formation is a progressive process, which starts with a transient regulatory network promoting stem cell formation and that is different from the one acting in functional meristems. This transient stage can be called premeristems and we discuss whether this concept can be extended to the formation of meristems other than AMs.

Characterization of transcriptome diversity and in vitro behavior of primary human high-risk breast cells

Biology and transcriptomes of non-cancerous human mammary epithelial cells at risk for breast cancer development were explored following primary isolation utilizing conditional reprogramming cell technology from mastectomy tissue ipsilateral to invasive breast cancer. Cultures demonstrated consistent categorizable behaviors. Relative viability and mammosphere formation differed between samples but were stable across three different mammary-specific media. E2F cell cycle target genes expression levels were positively correlated with viability and advancing age was inversely associated. Estrogen growth response was associated with Tissue necrosis factor signaling and Interferon alpha response gene enrichment. Neoadjuvant chemotherapy exposure significantly altered transcriptomes, shifting them towards expression of genes linked to mammary stem cell formation. Breast cancer prognostic signature sets include genes that in normal development are limited to specific stages of pregnancy or the menstrual cycle. Sample transcriptomes were queried for stage specific gene expression patterns. All cancer samples and a portion of high-risk samples showed overlapping stages reflective of abnormal gene expression patterns, while other high-risk samples exhibited more stage specific patterns. In conclusion, at-risk cells preserve behavioral and transcriptome diversity that could reflect different risk profiles. It is possible that prognostic platforms analogous to those used for breast cancer could be developed for high-risk mammary cells.

ING4 Promotes Stemness Enrichment of Human Renal Cell Carcinoma Cells Through Inhibiting DUSP4 Expression to Activate the p38 MAPK/type I IFN-Stimulated Gene Signaling Pathway

Renal cell carcinoma (RCC) recurs frequently due to high metastatic spread, resulting in a high mortality. Cancer stem cells play a critical role in initiating the tumor metastasis. Inhibitor of growth 4 (ING4) is a member of the ING family, but its impact on cancer stem cells in RCC is still unknown. In this study, we found that ING4 significantly promoted the sphere-forming size and number of RCC cells under an ultralow-attachment culture condition in vitro, tumor growth and metastasis in vivo, and the expression of some stem-like or pluripotent biomarkers CD44, MYC, OCT4, and NANOG, indicating that ING4 increased the stemness enrichment of RCC cells. Mechanistically, the ING4-activated p38 MAPK pathway possibly upregulated the expression of type I IFN-stimulated genes to promote the formation of RCC stem cells. ING4 could inhibit the expression of DUSP4 to activate p38 MAPK. In addition, selective pharmacological p38 MAPK inhibitors could significantly inhibit stemness enrichment only in ING4-overexpressed RCC cells, suggesting that the p38 MAPK inhibitors might be effective in patients with high ING4 expression in RCC tissue. Taken together, our findings proposed that ING4 might serve as a potential therapeutic target for metastatic RCC, particularly RCC stem cells.

Angiogenin and plexin-B2 axis promotes glioblastoma progression by enhancing invasion, vascular association, proliferation and survival.

Angiogenin is a multifunctional secreted ribonuclease that is upregulated in human cancers and downregulated or mutationally inactivated in neurodegenerative diseases. A role for angiogenin in glioblastoma was inferred from the inverse correlation of angiogenin expression with patient survival but had not been experimentally investigated. Angiogenin knockout mice were generated and the effect of angiogenin deficiency on glioblastoma progression was examined. Angiogenin and plexin-B2 genes were knocked down in glioblastoma cells and the changes in cell proliferation, invasion and vascular association were examined. Monoclonal antibodies of angiogenin and small molecules were used to assess the therapeutic activity of the angiogenin-plexin-B2 pathway in both genetic and xenograft animal models. Deletion of Ang1 gene prolonged survival of PDGF-induced glioblastoma in mice in the Ink4a/Arf-/-:Pten-/- background, accompanied by decreased invasion, vascular association and proliferation. Angiogenin upregulated MMP9 and CD24 leading to enhanced invasion and vascular association. Inhibition of angiogenin or plexin-B2, either by shRNA, monoclonal antibody or small molecule inhibitor, decreases sphere formation of patient-derived glioma stem cells, reduces glioblastoma proliferation and invasion and inhibits glioblastoma growth in both genetic and xenograft animal models. Angiogenin and its receptor, plexin-B2, are a pair of novel regulators that mediate invasion, vascular association and proliferation of glioblastoma cells. Inhibitors of the angiogenin-plexin-B2 axis have therapeutic potential against glioblastoma.

Retinoic acid promotes formation of chicken (Gallus gallus) spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation

Human induced pluripotent stem cell (iPSC)-derived neuronal and glial cell models are suitable to assess the effects of environmental chemicals on the developing brain. Such test systems can recapitulate several key neurodevelopmental features, such as neural stem cell formation and differentiation towards different neuronal subtypes and astrocytes, neurite outgrowth, synapse formation and neuronal network formation and function, which are crucial for brain development. While monolayer, two-dimensional (2D) cultures of human iPSC-neuronal or glial derivatives are generally suited for high-throughput testing, they also show some limitations. In particular, differentiation towards myelinating oligodendrocytes can only be achieved after extended periods in differentiation. In recent years, the implementation of three-dimensional (3D) neuronal and glial models obtained from human iPSCs has been shown to compensate for such limitations, enabling robust differentiation towards both neuronal and glial cell populations, myelination and formation of more mature neuronal network activity. Here we compared the differentiation capacity of human iPSC-derived neural stem cells cultured either as 2D monolayer or as 3D neurospheres, and assessed chlorpyrifos (CPF) effects. Data indicate that 3D neurospheres differentiate towards neurons and oligodendroglia more rapidly than 2D cultures; however, the 2D model is more suitable to assess neuronal functionality by analysis of spontaneous electrical activity using multielectrode array. Moreover, 2D and 3D test systems are diversely susceptible to CPF treatment. In conclusion, the selection of the most suitable in vitro test system (either 2D or 3D) should take into account the context of use and intended research goals (‘fit for purpose’ principle).