Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells

In neuro-oncology, the biology of neural stem cells (NSCs) has been pursued in two ways: as tumor-initiating cells (TICs) and as a potential cell-based vehicle for gene therapy. NSCs as well as mesenchymal stem cells (MSCs) have been reported to possess tumor tropism capacities. However, there is little data on the migratory capacity of MSCs toward brain tumor-initiating cells (BTICs). This study focuses on the ability of human adipose tissue derived MSCs (hAT-MSCs) to target BTICs and their crosstalk in the microenvironment. BTICs were isolated from three different types of brain tumors. The migration capacities of hAT-MSCs toward BTICs were examined using an in vitro migration assay and in vivo bioluminescence imaging analysis. To investigate the crosstalk between hAT-MSCs and BTICs, we analyzed the mRNA expression patterns of cyto-chemokine receptors by RT-qPCR and the protein level of their ligands in co-cultured medium. The candidate cyto-chemokine receptors were selectively inhibited using siRNAs. Both in vitro and in vivo experiments showed that hAT-MSCs possess migratory abilities to target BTICs isolated from medulloblastoma, atypical teratoid/rhabdoid tumors (AT/RT) and glioblastoma. Different types of cyto-chemokines are involved in the crosstalk between hAT-MSCs and BTICs (medulloblastoma and AT/RT: CXCR4/SDF-1, CCR5/RANTES, IL6R/IL-6 and IL8R/IL8; glioblastoma: CXCR4/SDF-1, IL6R/IL-6, IL8R/IL-8 and IGF1R/IGF-1). Our findings demonstrated the migratory ability of hAT-MSCs for BTICs, implying the potential use of MSCs as a delivery vehicle for gene therapy. This study also confirmed the expression of hAT-MSCs cytokine receptors and the BTIC ligands that play roles in their crosstalk.     

Antitumor activities of human placenta-derived mesenchymal stem cells expressing endostatin on ovarian cancer

Endostatin is an important endogenous inhibitor of neovascularization that has been widely used in anti-angiogenesis therapy for the treatment of cancer. However, its clinical application is largely hampered by its low efficacy. Human placenta-derived mesenchymal stem cells (hpMSCs) are particularly attractive cells for clinical use in cell-based therapies. In the present study, hpMSCs were isolated and characterized. We then evaluated the tumor targeting properties and antitumor effects of hpMSCs as gene delivery vehicles for ovarian cancer therapy. We efficiently engineered hpMSCs to deliver endostatin via adenoviral transduction mediated by Lipofectamine 2000. The tropism capacity of the engineered hpMSCs toward tumor cells was then confirmed by in vitro migration assays and in vivo by intraperitoneal injection of hpMSCs into nude mice. The hpMSCs expressing the human endostatin gene demonstrated preferential homing to the tumor site and significantly decreased the tumor volume without apparent systemic toxic effects. These observations were associated with significantly decreased blood sprouts and tumor cell proliferation as well as a dramatically increased tumor apoptosis index. These results suggested that hpMSCs are potentially an effective delivery vehicle for therapeutic genes for the treatment of ovarian cancer.     

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.     

Anticancer effects of the engineered stem cells transduced with therapeutic genes via a selective tumor tropism caused by vascular endothelial growth factor toward HeLa cervical cancer cells

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-β) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-β may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.     

Osteogenic and neurogenic differentiation of EGFP gene transfected neural stem cells derived from the brain of porcine fetuses at intermediate and late gestational age

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of porcine fetuses at intermediate and late gestational age and (ii) to determine if these stem cells could be differentiated in vitro into osteogenic and neurogenic lineages following transfection with a reporter gene, EGFP (enhanced green fluorescence protein). The NSCs were isolated from the brains of porcine fetuses at intermediate and late gestational age and transfected with EGFP gene using lipofection. The transfected NSCs cells were induced to differentiate into cells of osteogenic and neurogenic lineages. Markers associated with NSCs and their osteogenic and neurogenic derivatives were tested by PCR. The results demonstrated that NSCs could be isolated from the brain of porcine fetus at intermediate and late gestational age and that transfected NSCs expressed EGFP and could be induced to differentiate in vitro. NSCs expressed CD‐90, Hes1, Oct4, Sox2 and Nestin, while following differentiation cells expressed markers for osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)], oligodendrocyte [GALC (galactosylceramide)] and neuron [NF (neurofilament), ENO2 (enolase 2) and MAP (microtubule‐associated protein)].     

Dynamics of focal adhesions and reorganization of F‐actin in VEGF‐stimulated NSCs under varying differentiation states

Precise migration of neural stem/progenitor cells (NSCs) is crucially important for neurogenesis and repair in the nervous system. However, the detailed mechanisms are not clear. Our previous results showed that NSCs in varying differentiation states possess different migratory ability to vascular endothelial growth factor (VEGF). In this study, we demonstrate the different dynamics of focal adhesions (FAs) and reorganization of F‐actin in NSCs during spreading and migration stimulated by VEGF. We found that the migrating NSCs of 0.5 and 1 day differentiation possess more FAs at leading edge than cells of other states. Moreover, the phosphorylation of focal adhesion kinase (FAK) and paxillin in NSCs correlates closely with their differentiation states. VEGF promotes FA formation with broad lamellipodium generation at the leading edge in chemotaxing cells of 0, 0.5, and 1 day differentiation, but not in cells of 3 days differentiation. Furthermore, cells of 1 day differentiation show a maximal asymmetry of FAs between lamella and cell rear, orchestrating cell polarization and directional migration. Time‐lapse video analysis shows that the disassembly of FAs and the cell tail detachment in NSCs of 1 day differentiation are more rapid, along with the concurrent enlarged size of FAs at the leading edge, leading to the most effective chemotactic response to VEGF. Collectively, these results indicate that the dynamics of FAs and reorganization of F‐actin in NSCs that undergo directional migration correlate closely with their differentiation states, contributing to the different chemotactic responses of these cells to VEGF. J. Cell. Biochem. 114: 1744–1759, 2013. © 2013 Wiley Periodicals, Inc.     

L‐type voltage‐gated calcium channels in stem cells and tissue engineering

L‐type voltage‐gated calcium ion channels (L‐VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L‐VGCCs in non‐excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L‐VGCCs and stem cells, and filling this gap is prescient considering the contribution of L‐VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L‐VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored.     

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.     

Mesenchymal stem cells promote cell invasion and migration and autophagy‐induced epithelial‐mesenchymal transition in A549 lung adenocarcinoma cells

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment‐induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial‐mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E‐cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture‐mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture‐mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell‐containing microenvironments and MSC‐induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion.     

MiR‐485‐3p modulates neural stem cell differentiation and proliferation via regulating TRIP6 expression

Recent references have showed crucial roles of several miRNAs in neural stem cell differentiation and proliferation. However, the expression and role of miR‐485‐3p remains unknown. In our reference, we indicated that miR‐485‐3p expression was down‐regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up‐regulated during NSCs differentiation to neural and astrocytes cell. We carried out the dual‐luciferase reporter and found that overexpression of miR‐485‐3p decreased the luciferase activity of pmirGLO‐TRIP6‐wt but not the pmirGLO‐TRIP6‐mut. Ectopic expression of miR‐485‐3p decreased the expression of TRIP6 in NSC. Ectopic miR‐485‐3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs. Moreover, elevated expression of miR‐485‐3p decreased the ki‐67 and cyclin D1 expression in NSCs. Furthermore, we indicated that miR‐485‐3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR‐485‐3p in self‐proliferation and differentiation of NSCs. Thus, altering miR‐485‐3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.     

Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12

Primary malignant brain tumors have a poor prognosis. This report investigates the potential for gene therapy of experimental brain tumors using neural stem cells (NSCs) expressing IL-12. In this study NSCs were isolated from the hippocampi of 3-5-month human embryos and used for lipofectamine mediated transfer of the IL-12 gene. Positive clones of anti-G418 were obtained and were proliferated in culture and expression of IL-12 was demonstrated by RT-PCR. For the in vivo studies three groups of rats were used and stereotactic injections were made into the striatum. In the first group C6 tumor cells were injected, in the second C6 cells and hNSCs. IL-12, and in the third C6 cells on Day 0 followed by hNSCs.IL-12 on day 5. The growth of the resulting tumors was monitored by magnetic resonance imaging (MRI) and after sacrifice by immunohistochemistry. Rats injected with C6 cells and hNSCs.IL-12 had a significantly prolonged survival. Injections of hNSCs.IL-12 were also made into established gliomas. The survival time was also significantly prolonged compared to controls. MR imaging demonstrated that there was initial growth of tumor followed by shrinkage and then disappearance. After sacrifice, tumor areas were studied by histochemistry. NSCs were often seen intermingled with tumor cells, particularly when they had been injected into established tumors; they were also present at the boundaries of the tumor mass. The immunohistochemical analysis showed that these infiltrates were mostly constituted by CD4(+) and CD8(+) T-lymphocytes, the CD8(+) being more numerous than the CD4(+). These findings indicated that NSCs engineered to release IL-12 could have a strong antitumor effect. Neural stem/precursor cells could be useful vectors in genetic approaches to brain tumors.     

Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood–brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self‐renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase‐like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down‐regulation of ROR1 suppressed the expression of epithelial‐mesenchymal transition‐related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

神经干细胞迁移的研究进展

神经干细胞的定向迁移是胚胎神经系统发育的先决条件,同时在成体组织的许多生理、病理过程中也起着重要作用;研究发现,许多神经退行性疾病都与神经干细胞迁移的缺陷相关。近年来,越来越多的证据表明,无论是内源性的还是移植的神经干细胞都有向大脑损伤部位迁移的特性,显示出神经干细胞用于神经再生及损伤修复治疗的潜能。该文着重在神经干细胞的基本特性以及神经干细胞定向迁移的细胞与分子机制研究等方面进行了综述。     

Neural stem cells: involvement in adult neurogenesis and CNS repair

Recent advances in stem cell research, including the selective expansion of neural stem cells (NSCs) in vitro, the induction of particular neural cells from embryonic stem cells in vitro, the identification of NSCs or NSC-like cells in the adult brain and the detection of neurogenesis in the adult brain (adult neurogenesis), have laid the groundwork for the development of novel therapies aimed at inducing regeneration in the damaged central nervous system (CNS). There are two major strategies for inducing regeneration in the damaged CNS: (i) activation of the endogenous regenerative capacity and (ii) cell transplantation therapy. In this review, we summarize the recent findings from our group and others on NSCs, with respect to their role in insult-induced neurogenesis (activation of adult NSCs, proliferation of transit-amplifying cells, migration of neuroblasts and survival and maturation of the newborn neurons), and implications for therapeutic interventions, together with tactics for using cell transplantation therapy to treat the damaged CNS.     

Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

Nanotechnology‐based molecular photoacoustic and photothermal flow cytometry platform for in‐vivo detection and killing of circulating cancer stem cells

In‐vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem‐like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet‐induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor‐initiating cells in circulation before they form metastasis. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The culture of neural stem cells

A stem cell has three important features. Firstly, the ability of self‐renewal: making identical copies of itself. Secondly, multipotency, generating all the major cell lineages of the host tissue (in the case of embryonic stem cells—pluripotency). Thirdly, the ability to generate/regenerate tissues. Thus, the study of stem cells will help unravel the complexity of tissue development and organisation, and will also have important clinical applications. Neural stem cells (NSCs) are present during embryonic development and in certain regions of the adult central nervous system (CNS). Mobilizing adult NSCs to promote repair of injured or diseased CNS is a promising approach. Since NSCs may give rise to brain tumor, they represent in vitro models for anti‐cancer drug screening. To facilitate the use of NSCs in clinical scenarios, we need to explore the biology of these cells in greater details. One clear goal is to be able to definitively identify and purify NSCs. The neurosphere‐forming assay is robust and reflects the behavior of NSCs. Clonal analysis where single cells give rise to neurospheres need to be used to follow the self‐renewal and multipotency characteristics of NSCs. Neurosphere formation in combination with other markers of NSC behavior such as active Notch signaling represents the state of the art to follow these cells. Many issues connected with NSC biology need to be explored to provide a platform for clinical applications. Important future directions that are highlighted in this review are; identification of markers for NSCs, the use of NSCs in high‐throughput screens and the modelling of the central nervous development. There is no doubt that the study of NSCs is crucial if we are to tackle the diseases of the CNS such as Parkinson's and Alzheimer's. J. Cell. Biochem. 106: 1–6, 2009. © 2008 Wiley‐Liss, Inc.