Development of Next-generation Tumor-homing Induced Neural Stem Cells to Enhance Treatment of Metastatic Cancers
Ontology highlight
ABSTRACT: Engineered tumor-homing neural stem cells (NSCs) have shown promise in treating cancer. Recently, we transdifferentiated skin fibroblasts into tumor-homing early-stage human induced NSCs (hiNSC) as personalized NSC drug carriers. Here, using a SOX2 and spheroidal culture-based reprogramming strategy, we generated a new hiNSC variant, hiNeuroS, that were genetically distinct from fibroblasts and first-generation hiNSCs and possessed significantly enhanced tumor-homing and anti-tumor properties. To understand the transcriptomic profile of cells during our sphere-based generation of hiNeuroS, we performed single-cell RNA sequencing (scRNA-seq). The results showed that, while some of the hiNeuroS cells shared a similar trajectory states with the fibroblasts, there was a unique cell group at the far end of hiNeuroS trajectory towards the latest pseudotime, and suggested our new sphere-based culture methods generate hiNeuroS that are a genetically distinct NSC sub-populations that expresses tumor-homing pathways at levels greater than hiNSC.
Project description:Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted.
Project description:Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. Additionally, the single seeded induced NSCs were able to form NSC colonies with efficiency comparable to control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating and attaining neural phenotypes after transplantation into neonatal mouse- and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.
Project description:An organized network of beating cilia transports cerebrospinal fluid (CSF) through the ventricles of the brain. CSF takes along diverse solutes including extracellular vesicles (EV), which arise from the choroid plexus, a secretory epithelium that reaches into the ventricles. Along their path through the ventricles, EVs have the opportunity to contact neural stem cell (NSC) niches. We purified EVZ310 produced by a choroid plexus-cell line, by differential centrifugation and flotation in iodixanol density gradients. We co-cultured suspensions of EVZ310 with NSC from the lateral and third ventricle. Alternatively, EVZ310 were dried-down on a polymer substrate and a suspension of NSCs was added. In both cases, EV Z310 induced, in a dose dependent manner, the NSC to rapidly form cellular networks accompanied by the expression of genes characteristic for neurons (Tuj1) and astrocytes (Gfap). NSCs from either origin responded to EVZ310 qualitatively and quantitatively in a very similar way. By contrast, EVMEF purified from mouse embryonic fibroblasts had little effect on both types of NSC. Mass spectrometry revealed significant differences in composition between EVMEF and EVZ310 proteomes. Notably, EVZ310 were enriched for the membrane or membrane associated proteins known to be involved in neurogenesis, cell differentiation, membrane trafficking and membrane organization.
Project description:Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted. We generated induced neural stem cells (iNS) and compared the gene expressions with control cells, which included primary human dermal fibroblast cultured in DMEM media (FB), primary human dermal fibroblast cultured in neurosphere-forming condition (FB_con), and neural stem cell line (F3). The four cell lines (iNS, FB, FB_con, and F3) showed different gene expression patterns. In detail, RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions, and was labeled and hybridized to an Affimetrix GeneChip human gene 1.0 ST array (Affimetrix, Santa Clara, CA, USA) as described according to the manufacturer’s instructions. Data were analyzed using Expression Console software version 1.1 (Affimetrix), in which gene expression ratios were normalized by Robust Multichip Analysis according to the manufacturer’s protocol.
Project description:Neural stem cells (NSCs) are valuable for both basic research and clinical application. We previously reported a chemical cocktail that could reprogram somatic cells into neural progenitor cells. However, the process of chemical induction is complex and the underlying mechanism remains largely elusive. Here, we identified a new culture condition that greatly promotes the efficiency of NSC generation directly from mouse fibroblasts based on our reported chemical cocktail. Transcriptome and epigenome analyses during the reprogramming process demonstrated that growth factors including IL6, FGF5 and LIF were dynamically activated and contributed to the chemical cocktail induced cell fate changes. Interestingly, fibroblast-to-NSC conversion requires the synergistic action of these growth factors. Moreover, the reprogramming capacity of both chemical cocktail and growth factors require nucleoporin Nup210 to activate endogenous neural transcription factors SoxB1 family to initiate NSC fate. Our study not only provides a novel protocol to generate NSC directly from mouse fibroblasts, but also reveals an important role of growth factors in chemical-induced cellular reprogramming.
Project description:Neural stem cells can migrate towards tumors of both neural and non-neural origins, which is crucial for the success in treating disseminated tumors. Although the understanding of the molecular mechanisms underlying NSC tumor tropism is limited, it has been noted that several cytokines, growth factors and receptors direct the migration in vitro. A proper understanding of the basic molecular mechanisms of NSC migration towards tumors, especially identification of key cellular regulators of the migration, will have important implications in improving the effectiveness of engineering and employing NSCs as tumor therapy agents. We compared gene expression profiles between migratory and non-migratory hiPSC-NSCs towards cancer cells using cDNA microarray profiling. We collected human iPSCs derived NSCs migrating and not migrating towards mouse 4T1 breast cancer cells in an in vitro migration system for total RNA extraction and hybridization to Affymetrix microarrays
Project description:Neural stem cells can migrate towards tumors of both neural and non-neural origins, which is crucial for the success in treating disseminated tumors. Although the understanding of the molecular mechanisms underlying NSC tumor tropism is limited, it has been noted that several cytokines, growth factors and receptors direct the migration in vitro. A proper understanding of the basic molecular mechanisms of NSC migration towards tumors, especially identification of key cellular regulators of the migration, will have important implications in improving the effectiveness of engineering and employing NSCs as tumor therapy agents. We compared gene expression profiles between migratory and non-migratory hiPSC-NSCs towards cancer cells using cDNA microarray profiling. We collected human iPSCs derived NSCs migrating and not migrating towards human U87 glioma cells in an in vitro migration system for total RNA extraction and hybridization to Affymetrix microarrays
Project description:Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to enhance stem cell function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term (ST-NSC) and long-term NSCs (LT-NSCs). ST-NSC divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months, but are pushed out of homeostasis by lengthening quiescence. Single cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC pro-aging factor. Treatment with the Abl-inhibitor Imatinib increased NSC proliferation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable to cellular aging, yet NSC function can be partially restored.
Project description:Neural stem cells (NSCs) constitute the reservoir for new cells and might be harnessed for stem cell-based regenerative therapies. Zebrafish has remarkable ability to regenerate its brain by inducing NSC plasticity upon Alzheimer’s pathology. We recently identified that NSCs enhance their proliferation and neurogenic outcome in an Amyloid-beta42-based (Aβ42) experimental Alzheimer’s disease model in zebrafish brain and Interleukin-4 (IL4) is a critical molecule for inducing NSC proliferation in AD conditions. However, the mechanisms by which Aβ42 and IL4 affect NSCs remained unknown. Using single cell transcriptomics, we determined distinct subtypes of NSCs and neurons in adult zebrafish brain, identified differentially expressed genes after Aβ42 and IL4 treatments, analyzed the gene ontology and pathways that are affected by Aβ42 and IL4, and investigated how cell-cell communication is altered through secreted molecules and their receptors. Our results constitute the most extensive resource in the Alzheimer’s disease model of adult zebrafish brain, are likely to provide unique insights into how Aβ42/IL4 affects NSC plasticity and yield in novel drug targets for mobilizing neural stem cells for endogenous neuro-regeneration.
Project description:Transplantation of neural stem cells (NSCs) has been proved to promote functional rehabilitation of brain lesions including ischemic stroke. However, the therapeutic effects of NSC transplantation is limited by the low survival and differentiation rates of NSCs due to the harsh environment in the brain after ischemic stroke. Here, we employed NSCs derived from human induced pluripotent stem cells (iPSCs) together with exosomes extracted from NSCs to treat cerebral ischemia induced by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice. The results showed that NSC-derived exosomes significantly reduced the inflammatory response, alleviated oxidative stress after NSC transplantation, and facilitated NSCs differentiation in vivo. The combination of NSCs with exosomes ameliorated the injury of brain tissue including cerebral infarct, neuronal death and glial scarring, and promoted the motor function recovery. To explore the underlying mechanisms, we analyzed the miRNA profiles of NSC-derived exosomes and the potential downstream genes. Our study provided the rationale for the clinical application of NSC-derived exosomes as a supportive adjuvant for NSC transplantation after stroke.