Silver Nanoparticles Selectively Treat Neurofibromatosis Type 1-Associated Plexiform Neurofibroma Cells at Doses That Do Not Affect Patient-Matched Schwann Cells.

Neurofibromatosis Type 1 (NF1) is a common neurogenic condition characterized by heterozygous loss of function mutations in the neurofibromin gene. NF1 patients are susceptible to the development of neurofibromas, including plexiform neurofibromas (pNFs), which occurs in about half of all cases. Plexiform neurofibroma are benign peripheral nerve sheath tumors originating from Schwann cells after complete loss of neurofibromin; they can be debilitating and also transform into deadly malignant peripheral nerve sheath tumors (MPNSTs). Here, our data indicates that silver nanoparticles (AgNPs) may be useful in the treatment of pNFs. We assessed the cytotoxicity of AgNPs using pNF cells and Schwann cells derived from the same NF1 patient. We found that AgNPs are selectively cytotoxic to pNF cells relative to isogenic Schwann cells. We then examined the role of neurofibromin expression on AgNP-mediated cytotoxicity; restoration of neurofibromin expression in pNF cells decreased sensitivity to AgNP, and knockdown of neurofibromin in isogenic Schwann cells increased sensitivity to AgNP, outlining a correlation between neurofibromin expression and AgNP-mediated cytotoxicity. AgNP was able to selectively remove pNF cells from a co-culture with patient-matched Schwann cells. Therefore, AgNPs represent a new approach for clinical management of NF1-associated pNF to address significant clinical need.

Silver Nanoparticles Selectively Treat Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors in a Neurofibromin-Dependent Manner.

Neurofibromatosis type 1 (NF1) is among the most common neurogenic disorders, characterized by loss of function mutations in the neurofibromin gene (NF1). NF1 patients are extremely susceptible to developing neurofibromas, which can transform into deadly malignant peripheral nerve sheath tumors (MPNSTs). At the center of these tumors are NF1-null Schwann cells. Here, we found that nanomedicine shows promise in the treatment of NF1-associated MPNSTs. We assessed the cytotoxicity of silver nanoparticles (AgNPs) in NF1-null NF1-associated MPNSTs, NF1-wildtype sporadic MPNST, and normal Schwann cells. Our data show that AgNP are selectivity cytotoxic to NF1-associated MPNSTs relative to sporadic MPNST and Schwann cells. Furthermore, we found that sensitivity to AgNPs is correlated with the expression levels of functional neurofibromin. The restoration of functional neurofibromin in NF1-associated MPNSTs reduces AgNP sensitivity, and the knockdown of neurofibromin in Schwann cells increases AgNP sensitivity. This finding is unique to AgNPs, as NF1 restoration does not alter sensitivity to standard of care chemotherapy doxorubicin in NF1-associated MPNSTs. Using an in vitro model system, we then found that AgNP can selectively eradicate NF1-associated MPNSTs in co-culture with Schwann cells at doses tolerable to normal cells. AgNP represents a novel therapy for the treatment of NF1-associated MPNSTs and addresses significant unmet clinical need.

Low Doses of Silver Nanoparticles Selectively Induce Lipid Peroxidation and Proteotoxic Stress in Mesenchymal Subtypes of Triple-Negative Breast Cancer.

Molecular profiling of tumors shows that triple-negative breast cancer (TNBC) can be stratified into mesenchymal (claudin-low breast cancer; CLBC) and epithelial subtypes (basal-like breast cancer; BLBC). Subtypes differ in underlying genetics and in response to therapeutics. Several reports indicate that therapeutic strategies that induce lipid peroxidation or proteotoxicity may be particularly effective for various cancers with a mesenchymal phenotype such as CLBC, for which no specific treatment regimens exist and outcomes are poor. We hypothesized that silver nanoparticles (AgNPs) can induce proteotoxic stress and cause lipid peroxidation to a greater extent in CLBC than in BLBC. We found that AgNPs were lethal to CLBCs at doses that had little effect on BLBCs and were non-toxic to normal breast epithelial cells. Analysis of mRNA profiles indicated that sensitivity to AgNPs correlated with expression of multiple CLBC-associated genes. There was no correlation between sensitivity to AgNPs and sensitivity to silver cations, uptake of AgNPs, or proliferation rate, indicating that there are other molecular factors driving sensitivity to AgNPs. Mechanistically, we found that the differences in sensitivity of CLBC and BLBC cells to AgNPs were driven by peroxidation of lipids, protein oxidation and aggregation, and subsequent proteotoxic stress and apoptotic signaling, which were induced in AgNP-treated CLBC cells, but not in BLBC cells. This study shows AgNPs are a specific treatment for CLBC and indicates that stratification of TNBC subtypes may lead to improved outcomes for other therapeutics with similar mechanisms of action.

Combined Photothermal and Ionizing Radiation Sensitization of Triple-Negative Breast Cancer Using Triangular Silver Nanoparticles.

BACKGROUND: Ionizing radiation (IR) is commonly used in triple-negative breast cancer (TNBC) treatment regimens. However, off-target toxicity affecting normal tissue and grueling treatment regimens remain major limitations. Hyperthermia is one of the greatest IR sensitizers, but only if heat is administered simultaneously or immediately prior to ionizing radiation. Difficulty in co-localizing ionizing radiation (IR) in rapid succession with hyperthermia, and confining treatment to the tumor have hindered widespread clinical adoption of combined thermoradiation treatment. Metal nanoparticle-based approaches to IR sensitization and photothermal heat generation may aid in overcoming these issues and improve treatment specificity. METHODS: We assessed the potential to selectively treat MDA-MB-231 TNBC cells without affecting non-malignant MCF-10A breast cells using a multimodal approach based upon combined photothermal therapy, IR sensitization, and specific cytotoxicity using triangular silver nanoparticles (TAgNPs) with peak absorbance in the near-infrared light (NIR) spectrum. RESULTS: We found that TAgNP-mediated photothermal therapy and radiosensitization offer a high degree of specificity for treatment of TNBC without affecting non-malignant mammary epithelial cells. DISCUSSION: If given at a high enough dose, IR, heat, or TAgNPs alone could be sufficient for tumor treatment. However, when the dose of one or all of these modalities increases, off-target effects also increase. The challenge lies in identifying the minimal doses of each individual treatment such that when combined they provide maximum selectivity for treatment of TNBC cells with minimum off-target effects on non-malignant breast cells. Our results provide proof of concept that this combination is highly selective for TNBC cells while sparing non-malignant mammary epithelial cells. This treatment would be particularly important for patients undergoing breast conservation therapy and for treatment of invasive tumor margins near the periphery where each individual treatment might be at a sub-therapeutic level.

Arginine vasopressin receptor 1a is a therapeutic target for castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) recurs after androgen deprivation therapy (ADT) and is incurable. Reactivation of androgen receptor (AR) signaling in the low androgen environment of ADT drives CRPC. This AR activity occurs through a variety of mechanisms, including up-regulation of AR coactivators such as VAV3 and expression of constitutively active AR variants such as the clinically relevant AR-V7. AR-V7 lacks a ligand-binding domain and is linked to poor prognosis. We previously showed that VAV3 enhances AR-V7 activity to drive CRPC progression. Gene expression profiling after depletion of either VAV3 or AR-V7 in CRPC cells revealed arginine vasopressin receptor 1a (AVPR1A) as the most commonly down-regulated gene, indicating that this G protein-coupled receptor may be critical for CRPC. Analysis of publicly available human PC datasets showed that AVPR1A has a higher copy number and increased amounts of mRNA in advanced PC. Depletion of AVPR1A in CRPC cells resulted in decreased cell proliferation and reduced cyclin A. In contrast, androgen-dependent PC, AR-negative PC, or nontumorigenic prostate epithelial cells, which have undetectable AVPR1A mRNA, were minimally affected by AVPR1A depletion. Ectopic expression of AVPR1A in androgen-dependent PC cells conferred castration resistance in vitro and in vivo. Furthermore, treatment of CRPC cells with the AVPR1A ligand, arginine vasopressin (AVP), activated ERK and CREB, known promoters of PC progression. A clinically safe and selective AVPR1A antagonist, relcovaptan, prevented CRPC emergence and decreased CRPC orthotopic and bone metastatic growth in mouse models. Based on these preclinical findings, repurposing AVPR1A antagonists is a promising therapeutic approach for CRPC.

Silver nanoparticles selectively treat triple-negative breast cancer cells without affecting non-malignant breast epithelial cells in vitro and in vivo.

Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple-negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP-based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non-malignant breast epithelial cells. In contrast, TNBC cells and non-malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC-specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non-malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non-malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non-toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.

Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane.

There is a growing interest in the use of multiwalled carbon nanotubes (MWCNTs) to treat diseases of the brain. Little is known about the effects of MWCNTs on human brain microvascular endothelial cells (HBMECs), which make up the blood vessels in the brain. In our studies, we evaluate the cytotoxicity of MWCNTs and acid oxidized MWNCTs, with or without a phospholipid-polyethylene glycol coating. We determined the cytotoxic effects of MWCNTs on both tissue-mimicking cultures of HBMECs grown on basement membrane and on monolayer cultures of HBMECs grown on plastic. We also evaluated the effects of MWCNT exposure on the capacity of HBMECs to form rings after plating on basement membrane, a commonly used assay to evaluate angiogenesis. We show that tissue-mimicking cultures of HBMECs are less sensitive to all types of MWCNTs than monolayer cultures of HBMECs. Furthermore, we found that MWCNTs have little impact on the capacity of HBMECs to form rings. Our results indicate that relative cytotoxicity of MWCNTs is significantly affected by the type of cell culture model used for testing, and supports further research into the use of tissue-mimicking endothelial cell culture models to help bridge the gap between in vitro and in vivo toxicology.

Large-Pore Functionalized Mesoporous Silica Nanoparticles as Drug Delivery Vector for a Highly Cytotoxic Hybrid Platinum-Acridine Anticancer Agent.

Large-pore mesoporous silica nanoparticles (MSN) were prepared and functionalized to serve as a highly robust and biocompatible delivery platform for platinum-acridine (PA) anticancer agents. The material showed a high loading capacity for the dicationic, hydrophilic hybrid agent [PtCl(en)(N-[acridin-9-ylaminoethyl]-N-methylpropionamidine)] dinitrate salt (P1A1) and virtually complete retention of payload at neutral pH in a high-chloride buffer. In acidic media mimicking the pH inside the cell lysosomes, rapid, burst-like release of P1A1 from the nanoparticles is observed. Coating of the materials in phospholipid bilayers resulted in nanoparticles with greatly improved colloidal stability. The lipid and carboxylate-modified nanoparticles containing 40 wt % drug caused S-phase arrest and inhibited cell proliferation in pancreatic cancer cells at submicromolar concentrations similar to carrier-free P1A1. The most striking feature of nanoparticle-delivered P1A1 was that the payload did not escape from the acidified lysosomal vesicles into the cytoplasm, but was shuttled to the nuclear membrane and released into the nucleus.

Heterogeneous Responses of Ovarian Cancer Cells to Silver Nanoparticles as a Single Agent and in Combination with Cisplatin.

We investigated the effects of silver nanoparticle (AgNP) exposure in three ovarian cancer cell lines (A2780, SKOV3, and OVCAR3). We found that AgNPs were highly cytotoxic toward A2780 and SKOV3 cells but OVCAR3 cells were less sensitive to AgNPs. In agreement with the cytotoxicity data, AgNPs caused DNA damage in A2780 and SKOV3 cells, but not in OVCAR3 cells. A2780 and SKOV3 showed higher levels of basal reactive oxygen species (ROS) relative to OVCAR3 cells. AgNP exposure increased ROS levels in both A2780 and SKOV3 cells, but not in OVCAR3 cells. We found that the heterogeneous cytotoxicity was specific to the uptake of intact particles and was not due to differences in sensitivity to silver ions. Furthermore, the combination of AgNPs and standard-of-care platinum therapy, cisplatin (cis-diamminedichloroplatinum(II), CDDP), was synergistic for treatment of A2780 andOVCAR3 cells and the combination of AgNPs and CDDP showed a favorable dose reduction in all cell lines tested. These results provide insight into potential applications of AgNPs for treatment of ovarian cancer.

Photothermal therapy of glioblastoma multiforme using multiwalled carbon nanotubes optimized for diffusion in extracellular space.

Glioblastoma multiforme (GBM) is the most common and most lethal primary brain tumor with a 5 year overall survival rate of approximately 5%. Currently, no therapy is curative and all have significant side effects. Focal thermal ablative therapies are being investigated as a new therapeutic approach. Such therapies can be enhanced using nanotechnology. Carbon nanotube mediated thermal therapy (CNMTT) uses lasers that emit near infrared radiation to excite carbon nanotubes (CNTs) localized to the tumor to generate heat needed for thermal ablation. Clinical translation of CNMTT for GBM will require development of effective strategies to deliver CNTs to tumors, clear structure-activity and structure-toxicity evaluation, and an understanding of the effects of inherent and acquired thermotolerance on the efficacy of treatment. In our studies, we show that a dense coating of phospholipid-poly(ethylene glycol) on multiwalled CNTs (MWCNTS) allows for better diffusion through brain phantoms, while maintaining the ability to achieve ablative temperatures after laser exposure. Phospholipid-poly(ethylene glycol) coated MWCNTs do not induce a heat shock response (HSR) in GBM cell lines. Activation of the HSR in GBM cells via exposure to sub-ablative temperatures or short term treatment with an inhibitor of heat shock protein 90 (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)), induces a protective heat shock response that results in thermotolerance and protects against CNMTT. Finally, we evaluate the potential for CNMTT to treat GBM multicellular spheroids. These data provide pre-clinical insight into key parameters needed for translation of CNMTT including nanoparticle delivery, cytotoxicity, and efficacy for treatment of thermotolerant GBM.

Design and cellular studies of a carbon nanotube-based delivery system for a hybrid platinum-acridine anticancer agent.

A three-component drug-delivery system has been developed consisting of multi-walled carbon nanotubes (MWCNTs) coated with a non-classical platinum chemotherapeutic agent ([PtCl(NH3)2(L)]Cl (P3A1; L=N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG). The optimized P3A1-MWCNTs are colloidally stable in physiological solution and deliver more P3A1 into breast cancer cells than treatment with the free drug. Furthermore, P3A1-MWCNTs are cytotoxic to several cell models of breast cancer and induce S-phase cell cycle arrest and non-apoptotic cell death in breast cancer cells. By contrast, free P3A1 induces apoptosis and allows progression to G2/M phase. Photothermal activation of P3A1-MWCNTs to generate mild hyperthermia potentiates their cytotoxicity. These findings suggest that delivery of P3A1 to cancer cells using MWCNTs as a drug carrier may be beneficial for combination cancer chemotherapy and photothermal therapy.

Targeting breast cancer with sugar-coated carbon nanotubes.

AIMS: To evaluate the use of glucosamine functionalized multiwalled carbon nanotubes (glyco-MWCNTs) for breast cancer targeting. MATERIALS & METHODS: Two types of glucosamine functionalized MWCNTs were developed (covalently linked glucosamine and non-covalently phospholipid-glucosamine coated) and evaluated for their potential to bind and target breast cancer cells in vitro and in vivo. RESULTS & CONCLUSION: Binding of glyco-MWCNTs in breast cancer cells is mediated by specific interaction with glucose transporters. Glyco-MWCNTs prepared by non-covalent coating with phospholipid-glucosamine displayed an extended blood circulation time, delayed urinary clearance, low tissue retention and increased breast cancer tumor accumulation in vivo. These studies lay the foundation for development of a cancer diagnostic agent based upon glyco-MWCNTs with the potential for superior accuracy over current radiopharmaceuticals.

A novel calcium-dependent mechanism of acquired resistance to IGF-1 receptor inhibition in prostate cancer cells.

Inhibition of the mitogenic insulin-like growth factor receptor 1 (IGF-1R) signaling axis is a compelling treatment strategy for prostate cancer. Combining the IGF-1R inhibitor ganitumab (formerly AMG 479) with standard of care androgen-deprivation therapy greatly delays prostate cancer recurrence in xenograft models; however, a significant proportion of these tumors ultimately acquire resistance to ganitumab. Here we describe the development of a stable and reproducible ganitumab-resistant VCaP human prostate cancer cell derivative termed VCaP/GanR to investigate the mechanism of acquired resistance to IGF-1R inhibition. Unlike parental VCaP, VCaP/GanR did not undergo apoptosis following ganitumab treatment. VCaP/GanR did not express increased levels of IGF-1R, insulin receptor, or phospho-AKT compared to parental VCaP. VCaP/GanR exhibited increased levels of phospho-S6 indicative of increased mTOR activity. However, acquired resistance to ganitumab was not dependent on increased mTOR activity in VCaP/GanR. Phospho-proteomic arrays revealed alterations in several calcium-regulated signaling components in VCaP/GanR compared to VCaP. Reduction of intracellular calcium using cell-permeable calcium-specific chelators restored ganitumab sensitivity to VCaP/GanR through inhibition of cell-cycle progression. These data suggest a new mechanism of resistance to IGF-1R inhibition involving calcium-mediated proliferation effects. Such pathways should be considered in future clinical studies of IGF-1R inhibitors in prostate cancer.

Preclinical efficacy of growth hormone-releasing hormone antagonists for androgen-dependent and castration-resistant human prostate cancer.

Advanced hormone-sensitive prostate cancer responds to androgen-deprivation therapy (ADT); however, therapeutic options for recurrent castration-resistant disease are limited. Because growth hormone-releasing hormone (GHRH) and GHRH receptor (GHRH-R) are regulated in an autocrine fashion in prostate cancer, inhibition of GHRH-R represents a compelling approach to treatment. We investigated the effects of the latest series of improved, highly potent GHRH antagonists--MIA-602, MIA-606, and MIA-690--on the growth of androgen-dependent as well as castration-resistant prostate cancer (CRPC) cells in vitro and in vivo. GHRH-R and its splice variant, SV1, were present in 22Rv1, LNCaP, and VCaP human prostate cancer cell lines. Androgen-dependent LNCaP and VCaP cells expressed higher levels of GHRH-R protein compared with castration-resistant 22Rv1 cells; however, 22Rv1 expressed higher levels of SV1. In vitro, MIA-602 decreased cell proliferation of 22Rv1, LNCaP, and VCaP prostate cancer cell lines by 70%, 61%, and 20%, respectively (all P < 0.05), indicating direct effects of MIA-602. In vivo, MIA-602 was more effective than MIA-606 and MIA-690 and decreased 22Rv1 xenograft tumor volumes in mice by 63% after 3 wk (P < 0.05). No noticeable untoward effects or changes in body weight occurred. In vitro, the VCaP cell line was minimally inhibited by MIA-602, but in vivo, this line showed a substantial reduction in growth of xenografts in response to MIA-602, indicating both direct and systemic inhibitory effects. MIA-602 also further inhibited VCaP xenografts when combined with ADT. This study demonstrates the preclinical efficacy of the GHRH antagonist MIA-602 for treatment of both androgen-dependent and CRPC.

Targeting IGF-IR with ganitumab inhibits tumorigenesis and increases durability of response to androgen-deprivation therapy in VCaP prostate cancer xenografts.

Prostate cancer is the most commonly diagnosed malignancy in men. While tumors initially respond to androgen-deprivation therapy, the standard care for advanced or metastatic disease, tumors eventually recur as castration-resistant prostate cancer (CRPC). Upregulation of the insulin-like growth factor receptor type I (IGF-IR) signaling axis drives growth and progression of prostate cancer by promoting proliferation, survival, and angiogenesis. Ganitumab (formerly AMG 479) is a fully human antibody that inhibits binding of IGF-I and IGF-II to IGF-IR. We evaluated the therapeutic value of ganitumab in several preclinical settings including androgen-dependent prostate cancer, CRPC, and in combination with androgen-deprivation therapy. Ganitumab inhibited IGF-I-induced phosphorylation of the downstream effector AKT and reduced proliferation of multiple androgen-dependent and castration-resistant human prostate cancer cell lines in vitro. Ganitumab inhibited androgen-dependent VCaP xenograft growth and increased tumor-doubling time from 2.3 ± 0.4 weeks to 6.4 ± 0.4 weeks. Ganitumab blocked growth of castration-resistant VCaP xenografts for over 11.5 weeks of treatment. In contrast, ganitumab did not have appreciable effects on the castration-resistant CWR-22Rv1 xenograft model. Ganitumab was most potent against VCaP xenografts when combined with complete androgen-deprivation therapy (castration). Tumor volume was reduced by 72% after 4 weeks of treatment and growth suppression was maintained over 16 weeks of treatment. These data suggest that judicious use of ganitumab particularly in conjunction with androgen-deprivation therapy may be beneficial in the treatment of prostate cancer.

Vav3 enhances androgen receptor splice variant activity and is critical for castration-resistant prostate cancer growth and survival.

Advanced or metastatic prostate cancer is treated by androgen deprivation; however, patients inevitably relapse with castration-resistant prostate cancer (CRPC). CRPC remains dependent on androgen receptor (AR) signaling, which may include constitutive, ligand-independent action of naturally occurring AR splice variants. For example, the AR splice variant AR3 (also termed AR-V7) is expressed in CRPC and is linked to poor prognosis. Vav3, a Rho GTPase guanine nucleotide exchange factor, is an AR coactivator that is up-regulated in human prostate cancer compared with benign tissue and in preclinical models of CRPC. Vav3 confers castration-resistant growth to androgen-dependent human prostate cancer cells. Despite the importance of AR coactivators in promoting CRPC, the potential role of these regulatory proteins in modulating AR splice variant activity is unknown. We examined the contributions of Vav3 to AR activity in two CRPC cell lines that naturally express relatively high levels of Vav3 and AR3. Vav3 or AR3 knockdown greatly attenuated cell proliferation, soft agar growth, and ligand-independent AR activity. Vav3 potently enhanced the transcriptional activity of AR3 and another clinically relevant AR splice variant, ARv567es. Vav3 knockdown resulted in lowered nuclear AR3 levels, whereas total AR3 levels remained similar. Conversely, overexpression of Vav3 resulted in increased nuclear AR3. Coimmunoprecipitation revealed that AR3 and Vav3 interact. These novel data demonstrating physical and functional interactions between Vav3, a unique AR coactivator, and an AR splice variant provide insights into the mechanisms by which Vav3 exploits and enhances AR signaling in the progression to CRPC.

Generation of cultured oligodendrocyte progenitor cells from rat neonatal brains.

The oligodendrocyte progenitor cell (OPC) is one of the most studied progenitor cells of the body. It has been extensively researched in tissue culture and more recently in vivo using a wide range of markers that recognize transcription factors and cell surface markers and identify its earliest development from neural stem cells onward. Isolation of OPCs in large numbers and in purified preparations has been sought after as a source of cells for the repair of human myelin disorders. It has been proposed that such cells could be used as an exogenous source of cells for the treatment of lesions in multiple sclerosis and the less common genetic myelin disorders such as Pelizaeus-Merzbacher disease. Prior to clinical trials, such approaches can be tested in animal models. Here, we describe the isolation of rat OPCs in culture conditions that provide large numbers of purified populations of cells.