Preclinical Study on Biodistribution of Mesenchymal Stem Cells after Local Transplantation into the Brain.

Therapeutic efficacy of mesenchymal stem cells (MSCs) is determined by biodistribution and engraftment in vivo. Compared to intravenous infusion, biodistribution of locally transplanted MSCs are partially understood. Here, we performed a pharmacokinetics (PK) study of MSCs after local transplantation. We grafted human MSCs into the brains of immune-compromised nude mice. Then we extracted genomic DNA from brains, lungs, and livers after transplantation over a month. Using quantitative polymerase chain reaction with human Alu-specific primers, we analyzed biodistribution of the transplanted cells. To evaluate the role of residual immune response in the brain, MSCs expressing a cytosine deaminase (MSCs/CD) were used to ablate resident immune cells at the injection site. The majority of the Alu signals mostly remained at the injection site and decreased over a week, finally becoming undetectable after one month. Negligible signals were transiently detected in the lung and liver during the first week. Suppression of Iba1-positive microglia in the vicinity of the injection site using MSCs/CD prolonged the presence of the Alu signals. After local transplantation in xenograft animal models, human MSCs remain predominantly near the injection site for limited time without disseminating to other organs. Transplantation of human MSCs can locally elicit an immune response in immune compromised animals, and suppressing resident immune cells can prolong the presence of transplanted cells. Our study provides valuable insights into the in vivo fate of locally transplanted stem cells and a local delivery is effective to achieve desired dosages for neurological diseases.

THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor prognosis and limited treatment options. While 5-fluorouracil (5-FU) has not been widely employed in GBM therapy, emerging research indicates its potential for effectiveness when combined with advanced drug delivery systems to improve its transport to brain tumors. This study aims to investigate the role of THOC2 expression in 5-FU resistance in GBM cell lines. We evaluated diverse GBM cell lines and primary glioma cells for 5-FU sensitivity, cell doubling times, and gene expression. We observed a significant correlation between THOC2 expression and 5-FU resistance. To further investigate this correlation, we selected five GBM cell lines and developed 5-FU resistant GBM cells, including T98FR cells, through long-term 5-FU treatment. In 5-FU challenged cells, THOC2 expression was upregulated, with the highest increase in T98FR cells. THOC2 knockdown in T98FR cells reduced 5-FU IC50 values, confirming its role in 5-FU resistance. In a mouse xenograft model, THOC2 knockdown attenuated tumor growth and extended survival duration after 5-FU treatment. RNA sequencing identified differentially expressed genes and alternative splicing variants in T98FR/shTHOC2 cells. THOC2 knockdown altered Bcl-x splicing, increasing pro-apoptotic Bcl-xS expression, and impaired cell adhesion and migration by reducing L1CAM expression. These results suggest that THOC2 plays a crucial role in 5-FU resistance in GBM and that targeting THOC2 expression could be a potential therapeutic strategy for improving the efficacy of 5-FU-based combination therapies in GBM patients.

Enhanced antitumor efficacy of mesenchymal stem cells expressing cytosine deaminase and 5-fluorocytosine combined with α-galactosylceramide in a colon cancer model.

Cancer immunotherapy has emerged as a promising approach for treating various malignancies. In this study, we investigated the combined therapeutic effects of mesenchymal stem cells expressing cytosine deaminase (MSC/CD) and 5-fluorocytosine (5-FC) with α-galactosylceramide (α-GalCer) in a colon cancer model. Our findings demonstrated that the combination of MSC/CD, 5-FC, and α-GalCer resulted in enhanced antitumor activity compared to the individual treatments. This was evidenced by increased infiltration of immune cells, such as natural killer T (NKT) cells, antigen-presenting cells (APCs), T cells, and natural killer (NK) cells, in the tumor microenvironment, as well as elevated expression of proinflammatory cytokines and chemokines. Furthermore, we observed no significant hepatotoxicity following the combined treatment. Our study highlights the potential therapeutic benefits of combining MSC/CD, 5-FC, and α-GalCer for colon cancer treatment and contributes valuable insights to the field of cancer immunotherapy. Future research should focus on elucidating the underlying mechanisms and exploring the applicability of these findings to other cancer types and immunotherapy strategies.

Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-Vivo Therapy.

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

Molecular characteristics of incidental lower-grade glioma for treatment decision-making.

OBJECTIVE: Several limitations are associated with the early diagnosis and treatment of incidental lower-grade glioma (iLGG), and due to its unknown molecular features, its management is categorized as either the "wait-and-see" strategy or immediate treatment. Therefore, in this study the authors explored iLGG's clinical and molecular landscape to improve its management. METHODS: The authors retrospectively assessed the differences between the molecular and clinical characteristics of iLGG and symptomatic lower-grade glioma (sLGG) samples filtered based on symptom data corresponding to The Cancer Genome Atlas cohort with mutations. Thereafter, genomic and transcriptomic analysis was performed. RESULTS: There was no significant difference between iLGG and sLGG with respect to mutation status; however, there was an increase in the interaction between major mutations in sLGG, depending on the histological subtype and the IDH1 mutation status. Furthermore, the IDH1 mutation characteristics corresponding to wild-type glioma were much more obvious in sLGG than in iLGG. Additionally, in sLGG, genes associated with malignancy, including cell proliferation-related, cell migration-related, epithelial-to-mesenchymal transition-related, and negative regulation of cell death-related genes, were significantly upregulated, and groups showing higher expression levels of these genes were associated with worse prognosis. Also, 8 of the 75 identified upregulated genes showed positive correlation with resistance to the drugs that are normally used for glioma treatment, including procarbazine, carmustine, vincristine, and temozolomide. CONCLUSIONS: The new insights regarding the different molecular features of iLGG and sLGG indicated that the immediate management of iLGG could result in better prognosis than the wait-and-see strategy.

Improving the Safety of Mesenchymal Stem Cell-Based Ex Vivo Therapy Using Herpes Simplex Virus Thymidine Kinase.

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC50) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.

Combined effects of mesenchymal stem cells carrying cytosine deaminase gene with 5-fluorocytosine and temozolomide in orthotopic glioma model.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, and current standard therapy provides modest improvements in progression-free and overall survival of patients. Innate tumor resistance and presence of the blood-brain barrier (BBB) require the development of multi-modal therapeutic regimens. Previously, cytosine deaminase (CD)-expressing mesenchymal stem cells (MSC/CD) were found to exhibit anticancer activity with a wide therapeutic index by converting 5-fluorocytosine (5-FC), a nontoxic prodrug into 5-fluorouracil (5-FU), a potent anticancer drug. In this study, we evaluated the efficacy of MSC/CD in a multi-modal combination regimen with temozolomide (TMZ). Cell viability test, cell cycle, and normalized isobologram analyses were performed. In vivo anticancer effects were tested in a mouse orthotopic glioma model. TMZ and MSC/CD with 5-FC synergistically interacted and suppressed U87 glioma cell line growth in vitro. Combined treatment with TMZ and 5-FU increased cell cycle arrest and DNA breakage. In an orthotopic xenograft mouse model, treatment with TMZ alone suppressed tumor growth; however, this effect was more intense with MSC/CD transplantation followed by the sequential treatment with 5-FC and TMZ. Therefore, we propose that sequential treatment with 5-FC and MSC/CD can be used in patients with GBM during the immediate postoperative period to sensitize tumors to subsequent adjuvant chemo- and radiotherapy.

CXCR4-STAT3 Axis Plays a Role in Tumor Cell Infiltration in an Orthotopic Mouse Glioblastoma Model.

Glioblastoma multiforme (GBM) is a fatal malignant tumor that is characterized by diffusive growth of tumor cells into the surrounding brain parenchyma. However, the diffusive nature of GBM and its relationship with the tumor microenvironment (TME) is still unknown. Here, we investigated the interactions of GBM with the surrounding microenvironment in orthotopic xenograft animal models using two human glioma cell lines, U87 and LN229. The GBM cells in our model showed different features on the aspects of cell growth rate during their development, dispersive nature of glioma tumor cells along blood vessels, and invasion into the brain parenchyma. Our results indicated that these differences in the two models are in part due to differences in the expression of CXCR4 and STAT3, both of which play an important role in tumor progression. In addition, the GBM shows considerable accumulation of resident microglia and peripheral macrophages, but polarizes differently into tumor-supporting cells. These results suggest that the intrinsic factors of GBM and their interaction with the TME determine the diffusive nature and probably the responsiveness to non-cancer cells in the TME.

Effects of Adenoviral Gene Transduction on the Stemness of Human Bone Marrow Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are currently being evaluated as a cell-based therapy for tissue injury and degenerative diseases. Recently, several methods have been suggested to further enhance the therapeutic functions of MSCs, including genetic modifications with tissue- and/or disease-specific genes. The objective of this study was to examine the efficiency and stability of transduction using an adenoviral vector in human MSCs. Additionally, we aimed to assess the effects of transduction on the proliferation and multipotency of MSCs. The results indicate that MSCs can be transduced by adenoviruses in vitro, but high viral titers are necessary to achieve high efficiency. In addition, transduction at a higher multiplicity of infection (MOI) was associated with attenuated proliferation and senescence-like morphology. Furthermore, transduced MSCs showed a diminished capacity for adipogenic differentiation while retaining their potential to differentiate into osteocytes and chondrocytes. This work could contribute significantly to clinical trials of MSCs modified with therapeutic genes.

Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy.

Three-dimensional assessment of bystander effects of mesenchymal stem cells carrying a cytosine deaminase gene on glioma cells.

Stem cells carrying a suicide gene have emerged as therapeutic candidates for their cytotoxic bystander effects on neighboring cancers, while being non-toxic to other parts of the body. However, traditional cytotoxicity assays are unable to adequately assess the therapeutic effects of bystander cells. Here, we report a method to assess bystander effects of therapeutic stem cells against 3-dimensionally grown glioma cells in real time. U87 glioma cells were stably transduced to express a green fluorescence protein and co-cultivated with mesenchymal stem cells engineered to carry a bacterial cytosine deaminase gene (MSC/CD). Following addition of a 5-fluorocytine (5-FC) prodrug to the co-culture, fluorescence from U87 cells was obtained and analyzed in real time. Notably, the IC50 of 5-FC was higher when U87 cells were grown 3-dimensionally in soft agar medium for 3 weeks, as compared to those grown for one week in two-dimensional monolayer cultures. Additionally, more MSC/CD cells were required to maintain a similar level of efficacy. Since three-dimensional growth of glioma cells under our co-culture condition mimics the long-term expansion of cancer cells in vivo, our method can extend to an in vitro assay system to assess stem cell-mediated anti-cancer effects before advancing into preclinical animal studies.

Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis.

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-ß1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.

Immune following suppression mesenchymal stem cell transplantation in the ischemic brain is mediated by TGF-ß.

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the recovery of brain functions following ischemic injury. Although immune modulation has been suggested to be one of the mechanisms, the molecular mechanisms underlying improved recovery has not been clearly identified. Here, we report that MSCs secrete transforming growth factor-beta (TGF-ß) to suppress immune propagation in the ischemic rat brain. Ischemic stroke caused global death of resident cells in the infarcted area, elevated the monocyte chemoattractant protein-1 (MCP-1) level, and evoked massive infiltration of circulating CD68+ immune cells through the impaired blood-brain barrier. Transplantation of MSCs at day 3 post-ischemia blocked the subsequent upregulation of MCP-1 in the ischemic area and the infiltration of additional CD68+ immune cells. MSC-conditioned media decreased the migration and MCP-1 production of freshly isolated immune cells in vitro, and this effect was blocked by an inhibitor of TGF-ß signaling or an anti-TGF-ß neutralizing antibody. Finally, transplantation of TGF-ß1-silenced MSCs failed to attenuate the infiltration of CD68+ cells into the ischemic brain, and was associated with only minor improvements in motor function. These results indicate that TGF-ß is key to the ability of MSCs to beneficially attenuate immune reactions in the ischemic brain. Our findings offer insight into the interactions between allogeneic MSCs and the host immune system, reinforcing the prospective clinical value of using MSCs in the treatment of neurological disorders involving inflammation-mediated secondary damage.

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.

The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase.

Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments.

Neural induction with neurogenin1 increases the therapeutic effects of mesenchymal stem cells in the ischemic brain.

Mesenchymal stem cells (MSCs) have been shown to ameliorate a variety of neurological dysfunctions. This effect is believed to be mediated by their paracrine functions, since these cells rarely differentiate into neuronal cells. It is of clinical interest whether neural induction of MSCs is beneficial for the replacement therapy of neurological diseases. Here we report that expression of Neurogenin1 (Ngn1), a proneural gene that directs neuronal differentiation of progenitor cells during development, is sufficient to convert the mesodermal cell fate of MSCs into a neuronal one. Ngn1-expressing MSCs expressed neuron-specific proteins, including NeuroD and voltage-gated Ca2+ and Na+ channels that were absent in parental MSCs. Most importantly, transplantation of Ngn1-expressing MSCs in the animal stroke model dramatically improved motor functions compared with the parental MSCs. MSCs with Ngn1 populated the ischemic brain, where they expressed mature neuronal markers, including microtubule associated protein 2, neurofilament 200, and vesicular glutamate transporter 2, and functionally connected to host neurons. MSCs with and without Ngn1 were indistinguishable in reducing the numbers of Iba1+, ED1+ inflammatory cells, and terminal deoxynucleotidyl transferase dUTP nick-end labeling(+) apoptotic cells and in increasing the numbers of proliferating Ki67+ cells. The data indicate that in addition to the intrinsic paracrine functions of MSCs, motor dysfunctions were remarkably improved by MSCs able to transdifferentiate into neuronal cells. Thus, neural induction of MSCs is advantageous for the treatment of neurological dysfunctions.