Primary Choroid Plexus Tissue for Use in Cellular Therapy.

The choroid plexus (CP) has been explored as a cellular therapeutic due to its broad-ranging secretome and demonstrated longevity in a variety of encapsulation modalities. While the CP organ is normally involved in disease repair processes in the brain, the range of indications that could potentially be ameliorated with exogenous CP therapy is widespread, including diseases of the central nervous system, hearing loss, chronic wounds, and others. The CP can be isolated from animal sources and digested into a highly purified epithelial culture that can withstand encapsulation and transplantation. Its epithelium can adapt to different microenvironments, and depending on culture conditions, can be manipulated into various three-dimensional configurations with distinct gene expression profiles. The cocktail of proteins secreted by the CP can be harvested in culture, and purified forms of these extracts have been evaluated in topical applications to treat poorly healing wounds. When encapsulated, the epithelial clusters can be maintained for extended durations in vitro with minimal impact on potency. A treatment for Parkinson's disease utilizing encapsulated porcine CP has been developed and is currently being evaluated in a Phase I clinical trial. The current chapter serves to summarize recent experience with CP factor delivery, and provides a description of the relevant materials and methods employed in these studies.

The development of encapsulated cell technologies as therapies for neurological and sensory diseases.

Cell encapsulation therapies involve the implantation of cells that secrete a therapeutic factor to provide clinical benefits. The transplanted cells are protected from immunorejection via encapsulation in a semipermeable membrane. This treatment strategy was originally investigated as a method for protecting pancreatic islets from immunorejection, thus allowing them to secrete insulin as a chronic treatment for diabetes. Since then a significant body of work has been conducted in developing cell encapsulation therapies to treat a variety of different diseases. Many of these conditions involve neurodegeneration, such as Alzheimer's and Parkinson's disease, as cell encapsulation therapies have proven to be particularly suitable for delivering therapeutics to the central nervous system. This is mainly because they offer chronic delivery of a therapeutic and can be implanted proximal to the affected tissue, bypassing the blood brain barrier, which is impermeable to many agents. Whilst these therapies are not yet widely available in the clinic, promising results have been obtained in several advanced clinical trials and further developmental work is currently underway. This review specifically examines the development of encapsulated cell therapies as treatments for neurological and sensory diseases and evaluates the challenges that are yet to be overcome before they can be made available for clinical use.

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer.

Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer's disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modified human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to significantly higher NGF output, which in both cases correlate with highly improved potency.

Functional modulation of choroid plexus epithelial clusters in vitro for tissue repair applications.

One of the primary obstacles in the restoration or repair of damaged tissues is the temporospatial orchestration of biological and physiological events. Cellular transplantation is an important component of tissue repair as grafted cells can serve as replacement cells or as a source of secreted factors. But few, if any, primary cells can perform more than a single tissue repair function. Epithelial cells, derived from the choroid plexus (CP), are an exception to this rule, as transplanted CP is protective and regenerative in animal models as diverse as CNS degeneration and dermal wound repair. They secrete a myriad of proteins with therapeutic potential as well as matrix and adhesion factors, and contain responsive cytoskeletal components potentially capable of precise manipulation of cellular and extracellular niches. Here we isolated CP from neonatal porcine lateral ventricles and cultured the cells under a variety of conditions to specifically modulate tissue morphology (2D vs. 3D) and protein expression. Using qRT-PCR analysis, transmission electron microscopy, and gene microarray studies we demonstrate a fine level of control over CP epithelial cell clusters opening further opportunities for exploration of the therapeutic potential of this unique tissue source.

Biomaterial-based technologies for brain anti-cancer therapeutics and imaging.

Treating malignant brain tumors represents one of the most formidable challenges in oncology. Contemporary treatment of brain tumors has been hampered by limited drug delivery across the blood-brain barrier (BBB) to the tumor bed. Biomaterials are playing an increasingly important role in developing more effective brain tumor treatments. In particular, polymer (nano)particles can provide prolonged drug delivery directly to the tumor following direct intracerebral injection, by making them physiochemically able to cross the BBB to the tumor, or by functionalizing the material surface with peptides and ligands allowing the drug-loaded material to be systemically administered but still specifically target the tumor endothelium or tumor cells themselves. Biomaterials can also serve as targeted delivery devices for novel therapies including gene therapy, photodynamic therapy, anti-angiogenic and thermotherapy. Nanoparticles also have the potential to play key roles in the diagnosis and imaging of brain tumors by revolutionizing both preoperative and intraoperative brain tumor detection, allowing early detection of pre-cancerous cells, and providing real-time, longitudinal, non-invasive monitoring/imaging of the effects of treatment. Additional efforts are focused on developing biomaterial systems that are uniquely capable of delivering tumor-associated antigens, immunotherapeutic agents or programming immune cells in situ to identify and facilitate immune-mediated tumor cell killing. The continued translation of current research into clinical practice will rely on solving challenges relating to the pharmacology of nanoparticles but it is envisioned that novel biomaterials will ultimately allow clinicians to target tumors and introduce multiple, pharmaceutically relevant entities for simultaneous targeting, imaging, and therapy in a unique and unprecedented manner.

Secreted products from the porcine choroid plexus accelerate the healing of cutaneous wounds.

The choroid plexus (CP), located at the blood-brain interface, is partially responsible for maintaining the composition of cerebrospinal fluid. Epithelial cell clusters isolated from the CP secrete numerous biologically active molecules, and are neuroprotective when transplanted in animal models of Huntington's disease and stroke. The transcriptomic and proteomic profiles of CP may extend beyond CNS applications due to an abundance of trophic and regenerative factors, including vascular endothelial growth factor, transforming growth factor-beta, and others. We used microarray to investigate the transcriptome of porcine CP epithelium, and then assessed the in vitro and in vivo regenerative capability of secreted CP products in cell monolayers and full-thickness cutaneous wounds. In vitro, CP reduced the void area of fibroblast and keratinocyte scratch cultures by 70% and 33%, respectively, compared to empty capsule controls, which reduced the area by only 35% and 6%, respectively. In vivo, after 10 days of topical application, CP conditioned medium lyophilate dispersed in antibiotic ointment produced a twofold improvement in incision tensile strength compared to ointment containing lyophilized control medium, and an increase in the regeneration of epidermal appendages from roughly 50-150 features per wound. Together, these data identify the CP as a source of secreted regenerative molecules to accelerate and improve the healing of superficial wounds and potentially other similar indications.

Formulating the alginate-polyornithine biocapsule for prolonged stability: evaluation of composition and manufacturing technique.

Alginate encapsulation is one of the most widely used techniques for introducing cell-based therapeutics into the body. Numerous encapsulation methodologies exist, utilizing a variety of alginates, purification technologies, and unique polycationic membrane components. The stability of a conventional alginate formulation encapsulated using a commercially available technique and apparatus has been characterized extensively. The current study employs an encapsulation protocol and ultra-pure alginate pioneered at the University of Perugia. The enhanced microcapsules were produced, characterized, and implanted into the brain, peritoneal cavity, and subcutaneous space of Long-Evans rats. After 14, 28, 60, 90, 120, and 180 or 215 days, capsules were explanted and the surface was analyzed using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Image analysis was carried out to measure changes in diameter and wall thickness. FTIR peak analysis and surface morphology from SEM indicated that the enhanced encapsulation technique and formulation produced a stable biocapsule capable of survival in all sites, including the harsh peritoneal environment, for at least 215 days. Preimplant analysis showed a marked increase in the structural integrity of the enhanced formulation with improved elasticity and burst strength compared with the baseline formulation, which remained stable for less than 60 days. The enhanced microcapsule composition showed advantages in physical strength and longevity, indicating that small changes in encapsulation methodologies and materials selection can dramatically impact the stability and longevity of alginate microcapsules and their contents.

Stability of alginate-polyornithine microcapsules is profoundly dependent on the site of transplantation.

Alginate encapsulation is a form of cell-based therapy with numerous preclinical successes but recalcitrant complications related to stability and reproducibility. Understanding how alginate stability varies across different transplant sites will help identify indications that might benefit most from this approach. Alginate stability has been quantified in the peritoneum, but there are no systematic studies comparing its relative stability across transplant sites. This study compares the stability of alginate-polycation microcapsules implanted in the peritoneum to those implanted in the brain and subcutaneous space at 14, 28, 60, 90, 120, and 180 days in-life. Using Fourier-Transform Infrared Spectroscopy (FTIR), the surface of explanted capsules was analyzed for the relative proportion of alginate (outer coat) and the polycationic polyornithine (middle coat). Using a mathematic relationship between FTIR peaks related to these two material components, an index was generated to compare the stability of four different alginates. A notable difference was observed with rapid breakdown in the peritoneum. Conversely, identical alginate capsules transplanted into the brain or subcutaneous space were stable for the 6 month study. These data suggest that (1) successful intraperitoneal transplantation requires modifications of the capsule configuration, the host environment, or both and (2) that sites such as the brain and subcutaneous space are inherently less hostile to conventional alginate capsule configurations.

Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates.

BACKGROUND: A nonhuman primate model of diabetes is valuable for assessing porcine pancreatic islet transplants that might have clinical benefits in humans. METHODS: Neonatal porcine islets, microencapsulated in alginate-polyornithine-alginate, were injected intraperitoneally (10,000 IEQs/kg islets) into eight adult male cynomolgus monkeys rendered diabetic with streptozotocin. Eight diabetic controls were given an equivalent dose of empty placebo capsules. All subjects received a repeat transplant 3 months after the first. RESULTS: The transplant was well tolerated and no adverse or hypoglycemic events occurred. There were two deaths from nontransplant treatment or diabetic complications unrelated to the transplants. After transplantation, the average insulin dose was reduced in the islet-treated group and increased in the control group. At 12 weeks after the first transplant there was a mean 36% (95% CI: 6% to 65%, P = .02) drop in daily insulin dose compared with the control group. After 24 weeks the difference increased to a mean of 43% (95% CI: 12% to 75%, P = .01) without significant differences in blood glucose values between the two groups. Individual responses after islet transplant varied and one monkey was weaned off insulin by 36 weeks. At terminal autopsy, organs appeared normal and there was no visible peritoneal reaction. No animal had polymerase chain reaction (PCR)-amplified signals of porcine endogenous retrovirus or exogenous virus infections in blood or tissues. CONCLUSION: Repeated intraperitoneal transplantation of microencapsulated neonatal porcine islets is a safe procedure in diabetic primates. It was shown to result in a significant reduction in insulin dose requirement in the majority of animals studied, whereas insulin requirement increased in controls.

Xenotransplantation of neonatal porcine liver cells.

Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

CNS grafts of rat choroid plexus protect against cerebral ischemia in adult rats.

The present study examined the neuroprotective effects of choroid plexus isolated from adult rats and encapsulated within alginate microcapsules. In vitro, conditioned media from cultured choroid plexus produced a marked, dose-dependent protection of embryonic cortical neurons against serum deprivation-induced cell death. In vivo studies demonstrated that a one-hour middle cerebral artery occlusion in adult Wistar rats produced profound motor and neurological impairments 1-3 days after stroke. In contrast, stroke animals transplanted with encapsulated choroid plexus cells displayed a significant reduction in both motor and neurological abnormalities. Histological analysis 3 days post-transplantation revealed that choroid plexus transplants significantly decreased the volume of striatal infarction. This is the first report demonstrating the therapeutic potential of transplanted choroid plexus for stroke.

Genetically engineered Sertoli cells are able to survive allogeneic transplantation.

The immunoprotective nature of the testis has led to numerous investigations for its ability to protect cellular grafts. Sertoli cells (SCs) are at least partially responsible for this immunoprotective environment and survive allogeneic and xenogeneic transplantation. The ability of SCs to survive transplantation leads to the possibility that they could be engineered to deliver therapeutic proteins. As a model to test this hypothesis, we examined the ability of SCs that produce green fluorescent protein (GFP) to survive transplantation and continue expressing GFP. SCs were isolated from transgenic mice engineered to express GFP and transplanted as aggregates under the kidney capsule of severe combined immunodeficient (SCID) and Balb/c mice. Using this paradigm, it was possible to compare the survival of transgenic SCs directly in both immunodeficient and immunocompetent recipients. Fluorescence microscopy of the kidney capsule and immunohistochemistry of the grafts for GFP and GATA-4 revealed the presence of GFP-expressing SCs under the kidney capsule of SCID and Balb/c mice at both 30 and 60 days post-transplantation. In contrast, islets transplanted to Balb/c mice were rejected. Thus, SCs survive transplantation and continue to express GFP raising the possibility that SCs can be engineered using transgenic technology to produce proteins, such as insulin, factor VIII, or dopamine for the treatment of diabetes, hemophilia or Parkinson's disease, respectively.

Facilitation of drug entry into the CNS via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, Cereport.

One novel approach of transporting drugs into the central nervous system (CNS) involves the activation of receptors on the endothelial cells comprising the blood brain barrier (BBB). Recently the selective B(2) bradykinin receptor agonist, Cereport (also called RMP-7), has been shown to transiently increase permeability of the BBB. Although initially developed to increase the permeability of the vasculature feeding glioma, recent studies have demonstrated that Cereport also increases the delivery of pharmacological agents across the normal (i.e. nontumor) BBB. In this review paper, we discuss evidence of enhanced CNS delivery of carboplatin, loperamide, and cyclosporin-A, which are accompanied by enhanced chemotherapeutic, analgesic and neuroprotective effects, respectively. These observations suggest feasibility of Cereport as an adjunct therapy to pharmacological treatments that require drug availability in the CNS to exert therapeutic efficacy. Because many potential drugs for CNS disorders normally do not cross the BBB, Cereport-induced transient permeation of BBB stands as an efficacious strategy for enhancing pharmacotherapy.

Article Commentary: Cell Transplantation for Parkinson's Disease.

Hype slang. n. 1. Excessive publicity and the ensuing commotion. 2. Exaggerated or extravagant claims made especially in advertising or promotional material. (Source: The American Heritage Dictionary of the English Language, third edition).

Neuroprotective possibilities for Huntington's disease.

Huntington's disease (HD) is a devastating genetic disorder. Despite the absence of effective therapy, there has been an explosion in interest for developing treatment strategies aimed at lessening or preventing the neuronal death that occurs in this disease. In large part, the renewed interest in neuroprotective strategies has been spurred by our increasing understanding of the genetic and molecular events that drive the underlying neuropathology of HD. This escalating understanding of the biological underpinnings of HD is derived from several convergent sources represented by investigators with clinical, genetic, molecular, physiological and neurobehavioural backgrounds. The diversity of data being generated has, in turn, produced a unique time in HD research where an impressive number of potential therapeutics are coming to the forefront. This review outlines several of these possibilities including the use of intracerebrally delivered neurotrophic factors, pharmacologically altering cellular energy production, the use of antiglutamatergic drugs, the use of caspase inhibitors and inhibitors of protein aggregation. This review also touches on the interesting possibility of whether or not the neurodegeneration in HD is at least partially reversible in nature. All of these possibilities are highlighted in the context that HD is a neurodegenerative disorder in which genetic detection provides a clear and unequivocal opportunity for neuroprotection.

Immunoisolation cell therapy for CNS diseases.

Delivery of potentially therapeutic drugs to the brain is hindered by the blood-brain barrier (BBB), which restricts the diffusion of drugs from the vasculature to the brain parenchyma. One means of overcoming the BBB is with cellular implants that produce and deliver therapeutic molecules directly into the CNS region of interest. In this paper we describe the current status of one iteration of cell-based therapy that uses xenogeneic cells encased within a selectively permeable polymeric membrane; this is known as immunoisolation. For the purposes of this review, cell immunoisolation for treating CNS diseases is presented in terms of device configurations, membrane manufacturing, characterization in relevant preclinical model systems, and the current status of clinical trials.

Update on immunoisolation cell therapy for CNS diseases.

Delivery of potentially therapeutic drugs to the brain is hindered by the blood-brain barrier (BBB), which restricts the diffusion of drugs from the vasculature to the brain parenchyma. One means of overcoming the BBB is with cellular implants that produce and deliver therapeutic molecules. Polymer encapsulation, or immunoisolation, provides a means of overcoming the BBB to deliver therapeutic molecules directly into the CNS region of interest. Immunoisolation is based on the observation that xenogeneic cells can be protected from host rejection by encapsulating, or surrounding, them within an immunoisolatory, semipermeable membrane. Cells can be enclosed within a selective, semipermeable membrane barrier that admits oxygen and required nutrients and releases bioactive cell secretions, but restricts passage of larger cytotoxic agents from the host immune defense system. The selective membrane eliminates the need for chronic immunosuppression of the host and allows the implanted cells to be obtained from nonhuman sources. In this review, cell immunoisolation for treating CNS diseases is updated from considerations of device configurations, membrane manufacturing and characterization in preclinical models of Alzheimer's and Huntington's disease.

The development of the bradykinin agonist labradimil as a means to increase the permeability of the blood-brain barrier: from concept to clinical evaluation.

Labradimil (Cereport; also formerly referred to as RMP-7) is a 9-amino-acid peptide designed for selectivity for the bradykinin B2 receptor and a longer plasma half-life than bradykinin. It has been developed to increase the permeability of the blood-brain barrier (BBB) and is the first compound with selective bradykinin B2 receptor agonist properties to progress from concept design through to tests of efficacy in patients. In vitro studies demonstrate that labradimil has a longer half-life than bradykinin and selectively binds to bradykinin B2 receptors, initiating typical bradykinin-like second messenger systems, including increases in intracellular calcium and phosphatidylinositol turnover. Initial proof of principle studies using electron microscopy demonstrated that intravenous labradimil increases the permeability of the BBB by disengaging the tight junctions of the endothelial cells that comprise the BBB. Autoradiographic studies in rat models further demonstrated that labradimil increases the permeability of the BBB in gliomas. Intravenous or intra-arterial labradimil increases the uptake of many different radiolabelled tracers and chemotherapeutic agents into the tumour in a dose-related fashion. These effects are selective for the tumour and for the brain surrounding the tumour, and are particularly robust in tumour areas that are normally relatively impermeable. The increased chemotherapeutic concentrations are maintained for at least 90 minutes, well beyond the transient effects on the BBB. The increase in permeability with labradimil occurs rapidly but is transient, in that restoration of the BBB occurs very rapidly (2 to 5 minutes) following cessation of infusion. Even with continuous infusion of labradimil, spontaneous restoration of the barrier begins to occur within 10 to 20 minutes. Collectively, these data demonstrate that the B2 receptor system that modulates permeability of the BBB is highly sensitive and autoregulated and that careful attention to the timing of labradimil and the chemotherapeutic agent is important to achieve maximal effects. Survival studies in rodent models of both gliomas and metastatic tumours in the brain demonstrate that the enhanced uptake observed with the combination of labradimil and water-soluble chemotherapeutics enhances survival to a greater extent than achieved with chemotherapy alone. Finally, preliminary clinical trials in patients with gliomas provide confirmatory evidence that labradimil permeabilises the blood-brain tumour barrier and might, therefore, be used to increase delivery of agents such as carboplatin to tumours without the toxicity typically associated with dose escalation.

Bradykinin modulation of tumor vasculature: I. Activation of B2 receptors increases delivery of chemotherapeutic agents into solid peripheral tumors, enhancing their efficacy.

Delivery of chemotherapeutic agents to solid peripheral tumors is compromised because the impaired microvasculature within and surrounding tumors limits diffusion and convection of agents from the vasculature to the tumor. Using a variety of rat tumor models, we show that intravenous administration of a vasoactive bradykinin B2 receptor agonist (Cereport, or labradimil; formerly RMP-7) enhances by nearly 3 times the delivery of the chemotherapeutic agent carboplatin, as well as the larger 70-kDa marker dextran, into ectopic and orthotopic solid tumors. This effect was selective for tumor tissue, with little or no increase seen in nontumor tissues and organs. Additionally, the increased carboplatin levels observed in tumors persisted for at least 90 min (the longest time point measured). In contrast to the consistent effects with hydrophilic compounds, delivery of the lipophilic, high protein-binding chemotherapeutics paclitaxel and 1,3-bis[2-chloroethyl]-1-nitrourea (BNCU) was not enhanced. Administration of Cereport with either carboplatin or another hydrophilic chemotherapeutic agent, doxorubicin, significantly increased efficacy of both agents, manifested by suppression of tumor growth and prolonged survival in tumor-bearing rats. These data demonstrate that delivery of chemotherapeutics to tumors can be pharmacologically increased (by stimulating bradykinin B2 receptors) without increasing the systemic exposure, or therefore, the toxic liability associated with higher chemotherapeutic doses.