Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction

<div><p>Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for <i>in vivo</i> stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs) immortalized by the human telomerase reverse transcriptase (hTERT) gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM) cells were injected into adult (4–6-week-old) Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1–2-week-old) NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL) were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL), they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.</p></div

Comparison of overall median survivals according to strain, age and route.

<p>Comparison of overall median survivals according to strain, age and route.</p

<i>NTRK1</i> Fusion in Glioblastoma Multiforme

<div><p>Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3′ exons of neurotrophic tyrosine kinase receptor type 1 (<i>NTRK1</i>, encoding TrkA) are fused to 5′ exons of the genes that are highly expressed in neuronal tissues, neurofascin (<i>NFASC</i>) and brevican (<i>BCAN</i>). The fusions preserved both the transmembrane and kinase domains of <i>NTRK1</i> in frame. <i>NTRK1</i> is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked <i>NTRK1</i> expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated <i>NTRK1</i>-pathway activity. Lentiviral transduction of the <i>NFASC-NTRK1</i> fusion gene in NIH 3T3 cells increased proliferation <i>in vitro</i>, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.</p></div

<i>BCAN-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage of genomic regions along <i>BCAN</i> and <i>NTRK1</i>. The dotted line marks approximate positions where the fusion has occurred. (<b>B</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p

Tumorigenic activities of <i>NFASC-NTRK1</i> fusion gene.

<p>(<b>A</b>) <i>NFASC-NTRK1</i> and <i>EGFR</i> vIII mRNA expression in NIH 3T3 cells, determined by RT-PCR. (<b>B</b>) Proliferation of NIH 3T3 cells lentivirally infected with the indicated viruses. Error bars are 95% confidence intervals. (<b>C</b>) Number of colonies in a unit microscopic field, formed by NIH 3T3 cells infected with the indicated viruses. Red lines are the average within each group. (<b>D</b>) Morphology of individual colonies in soft agar, formed by NIH 3T3 cells infected with the indicated viruses. (<b>E</b>) Incidences of subcutaneous tumor formation in the mice injected with NIH 3T3 cells infected with the indicated viruses. (<b>F</b>) Inhibition of proliferation by three independent shRNAs targeting the <i>NTRK1</i> fusion transcripts. Error bars are standard deviations of five-replicate experiments.</p

<i>In vivo</i> tumor formation of primarily cultured 578T human GBM cells in various animal models.

<p>578T human GBM cells primary cultured from a GBM surgical sample were transplanted into various immune-deficient mouse strains via the subcutaneous (SC) or intracranial (IC) routes. (A) Tumor volume was calculated after 2 × 10⁶ 578T cells were injected into the SC tissue until a volume of 1,500 mm³. (B) An aliquot of 2 × 10⁵ cells was stereotactically injected into the brains of mice. Total body weight was monitored, and > 20% total body weight reduction was counted as mortality. *P < 0.05.</p

<i>In vivo</i> tumorigenic potential of ahMNCs.

<p><i>In vivo</i> tumorigenic potential of ahMNCs.</p

Histological validation of 578T human GBM cell-derived tumors.

<p>To validate tumor formation after subcutaneous (A) and intracranial (B) injections, tissue sections were stained with hematoxylin and eosin (H&E) or immunostained against Ki-67, a cell proliferation marker.</p

Molecular consequences of <i>NTRK1</i>-fusion.

<p>(<b>A</b>) <i>NTRK1</i> expression in 170 TCGA GBM samples (from 162 patients) with RNA-Seq data. Samples bearing <i>NTRK1</i>-fusion genes are marked and labeled. (<b>B</b>) Relationship between <i>NTRK1</i> expression and NGF/TrkA-downstream pathway activity in 526 TCGA GBM samples (from 526 patients) with microarray gene expression data. Samples with <i>NTRK1</i>-fusion are marked with red circles. Two other samples with outlier <i>NTRK1</i> expression are marked with blue circles (TCGA-32-4209, TCGA-19-5947).</p

<i>In vitro</i> characteristics of ahMNCs and hTERT-ahMNCs.

<p>The hTERT gene was transduced into 682TL and 779TL ahMNCs using a lentiviral vector. (A) Morphology of the ahMNCs was compared with that of hTERT-ahMNCs. (B) ahMNCs and hTERT-ahMNCs were cultured <i>in vitro</i> for a long time (>18 <i>in vitro</i> passages), and the numbers of cells were traced. (C) hTERT expression was confirmed by RT-PCR. (D) Telomere length was analyzed by qPCR and compared. (E) Expression of the neural stem cell marker Nestin and ahMNCs differentiation potential <i>in vitro</i> was compared with those of hTERT-ahMNCs. ahMNCs or hTERT-ahMNCs were cultured under differentiating conditions for 12 days. MAP2 for neurons and GFAP for astrocytes. BD; Before differentiation, AD; After differentiation.</p