Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals.

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.

Safety of direct injection of oligodendrocyte progenitor cells into the spinal cord of uninjured Göttingen minipigs.

OBJECTIVE: This study was conducted as a final proof-of-safety direct injection of oligodendrocyte progenitor cells into the uninjured spinal cord prior to translation to the human clinical trials. METHODS: In this study, 107 oligodendrocyte progenitor cells (LCTOPC1, also known as AST-OPC1 and GRNOPC1) in 50-µL suspension were injected directly into the uninjured spinal cords of 8 immunosuppressed Göttingen minipigs using a specially designed stereotactic delivery device. Four additional Göttingen minipigs were given Hanks' Balanced Salt Solution and acted as the control group. RESULTS: Cell survival and no evidence of histological damage, abnormal inflammation, microbiological or immunological abnormalities, tumor formation, or unexpected morbidity or mortality were demonstrated. CONCLUSIONS: These data strongly support the safety of intraparenchymal injection of LCTOPC1 into the spinal cord using a model anatomically similar to that of the human spinal cord. Furthermore, this research provides guidance for future clinical interventions, including mechanisms for precise positioning and anticipated volumes of biological payloads that can be safely delivered directly into uninjured portions of the spinal cord.

Immunological properties of human embryonic stem cell-derived oligodendrocyte progenitor cells.

A major concern in the use of allotransplantation of human embryonic stem cell (hESC)-based therapies is the possibility of allogeneic rejection by the host's immune system. In this report, we determined the immunological properties of hESC-derived oligodendrocyte progenitor cells (OPC) that have the potential for clinical application for the treatment of patients with spinal cord injury. In vitro immunological studies suggest that hESC-derived OPCs are poor targets for both the innate and the adaptive human immune effector cells as well as resistant to lysis by anti-Neu5Gc antibodies. These results indicate that hESC-derived OPCs retain some of the unique immunological properties of the parental cell line from which they were differentiated.

Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury.

Cervical spinal cord injury (SCI) remains an important research focus for regenerative medicine given the potential for severe functional deficits and the current lack of treatment options to augment neurological recovery. We recently reported the preclinical safety data of a human embryonic cell-derived oligodendrocyte progenitor cell (OPC) therapy that supported initiation of a phase I clinical trial for patients with sensorimotor complete thoracic SCI. To support the clinical use of this OPC therapy for cervical injuries, we conducted preclinical efficacy and safety testing of the OPCs in a nude rat model of cervical SCI. Using the automated TreadScan system to track motor behavioral recovery, we found that OPCs significantly improved locomotor performance when administered directly into the cervical spinal cord 1 week after injury, and that this functional improvement was associated with reduced parenchymal cavitation and increased sparing of myelinated axons within the injury site. Based on large scale biodistribution and toxicology studies, we show that OPC migration is limited to the spinal cord and brainstem and did not cause any adverse clinical observations, toxicities, allodynia, or tumors. In combination with previously published efficacy and safety data, the results presented here supported initiation of a phase I/IIa clinical trial in the U.S. for patients with sensorimotor complete cervical SCI. Stem Cells Translational Medicine 2017;6:1917-1929.

Proceedings: Moving Toward Cell-Based Therapies for Heart Disease.

Heart disease due to myocardial infarction and the ensuing heart failure represent a major unmet medical need. Approved treatments do not prevent loss of cardiac muscle or reduce scar formation, both of which weaken heart function. Cell-based therapies currently being investigated both preclinically and clinically have the potential to address these underlying problems either by actually replacing lost tissue or by supplying paracrine growth factors that may have multiple beneficial effects such as reduction of inflammation, increase of blood supply, improvement in cell survival, and reduction of scar size. The best cell types, stage of disease to target, and delivery method to improve heart function are currently unclear. The California Institute for Regenerative Medicine supports multiple different cell-therapy strategies for heart disease, offering hope that improved treatments will be available for patients in the future.

Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury.

AIM: To characterize the preclinical safety profile of a human embryonic stem cell-derived oligodendrocyte progenitor cell therapy product (AST-OPC1) in support of its use as a treatment for spinal cord injury (SCI). MATERIALS & METHODS: The phenotype and functional capacity of AST-OPC1 was characterized in vitro and in vivo. Safety and toxicology of AST-OPC1 administration was assessed in rodent models of thoracic SCI. RESULTS: These results identify AST-OPC1 as an early-stage oligodendrocyte progenitor population capable of promoting neurite outgrowth in vitro and myelination in vivo. AST-OPC1 administration did not cause any adverse clinical observations, toxicities, allodynia or tumors. CONCLUSION: These results supported initiation of a Phase I clinical trial in patients with sensorimotor complete thoracic SCI.

Functional circuitry involved in the regulation of whisker movements.

Neuroanatomical tract-tracing methods were used to identify the oligosynaptic circuitry by which the whisker representation of the motor cortex (wMCx) influences the facial motoneurons that control whisking activity (wFMNs). Injections of the retrograde tracer cholera toxin subunit B into physiologically identified wFMNs in the lateral facial nucleus resulted in dense, bilateral labeling throughout the brainstem reticular formation and in the ambiguus nucleus as well as predominantly ipsilateral labeling in the paralemniscal, pedunculopontine tegmental, Kölliker-Fuse, and parabrachial nuclei. In addition, neurons in the following midbrain regions projected to the wFMNs: superior colliculus, red nucleus, periaqueductal gray, mesencephalon, pons, and several nuclei involved in oculomotor behaviors. Injections of the anterograde tracer biotinylated dextran amine into the wMCx revealed direct projections to the brainstem reticular formation as well as multiple brainstem and midbrain structures shown to project to the wFMNs. Regions in which retrograde labeling and anterograde labeling overlap most extensively include the brainstem parvocellular, gigantocellular, intermediate, and medullary (dorsal and ventral) reticular formations; ambiguus nucleus; and midbrain superior colliculus and deep mesencephalic nucleus. Other regions that contain less dense regions of combined anterograde and retrograde labeling include the following nuclei: the interstitial nucleus of medial longitudinal fasciculus, the pontine reticular formation, and the lateral periaqueductal gray. Premotoneurons that receive dense inputs from the wMCx are likely to be important mediators of cortical regulation of whisker movements and may be a key component in a central pattern generator involved in the generation of rhythmic whisking activity.

Proceedings: cell therapies for Parkinson's disease from discovery to clinic.

In March 2013, the California Institute for Regenerative Medicine, in collaboration with the NIH Center for Regenerative Medicine, held a 2-day workshop on cell therapies for Parkinson's disease (PD), with the goals of reviewing the state of stem cell research for the treatment of PD and discussing and refining the approach and the appropriate patient populations in which to plan and conduct new clinical trials using stem cell-based therapies for PD. Workshop participants identified priorities for research, development, and funding; discussed existing resources and initiatives; and outlined a path to the clinic for a stem cell-based therapy for PD. A consensus emerged among participants that the development of cell replacement therapies for PD using stem cell-derived products could potentially offer substantial benefits to patients. As with all stem cell-based therapeutic approaches, however, there are many issues yet to be resolved regarding the safety, efficacy, and methodology of transplanting cell therapies into patients. Workshop participants agreed that designing an effective stem cell-based therapy for PD will require further research and development in several key areas. This paper summarizes the meeting.

Proceedings: international regulatory considerations on development pathways for cell therapies.

Regenerative medicine is a rapidly evolving field that faces novel scientific and regulatory challenges. In September 2013, the International Workshop on Regulatory Pathways for Cell Therapies was convened to discuss the nature of these challenges and potential solutions and to highlight opportunities for potential convergence between different regulatory bodies that might assist the field's development. The workshop discussions generated potentially actionable steps in five main areas that could mitigate cell therapy development pathway risk and accelerate moving promising therapies to patients. These included the need for convergence of regulatory guidelines on donor eligibility and suitability of lines for use in clinical trials and subsequent commercialization for cell therapies to move forward on a global basis; the need to challenge and encourage investigators in the regenerative medicine field to share information and provide examples of comparability studies related to master cell banks; the need for convergence of guidelines across regulatory jurisdictions on requirements for tumorigenicity studies, based on particular cell types and on biodistribution studies; the need to increase transparency in sharing clinical trial information more broadly and disseminating results more rapidly; and the need to establish a forum for sharing the experiences of various approaches being developed to expedite regulatory approvals and access for patients to innovative cell and regenerative therapies in the different regulatory jurisdictions and to assess their potential strengths and weaknesses.

Efficient generation and cryopreservation of cardiomyocytes derived from human embryonic stem cells.

AIM: Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study, we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS: hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture, and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition, the cell growth and differentiation process was adaptable to large culture formats. Moreover, the cardiomyocytes survived following cryopreservation, and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION: These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium, a step forward towards the application of these cells to human clinical use or drug discovery.

Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

GABAB receptor expression and function in olfactory receptor neuron axon growth.

Neurotransmitters have been implicated in regulating growth cone motility and guidance in the developing nervous system. Anatomical and electrophysiological studies show the presence of functional GABAB receptors on adult olfactory receptor neuron (ORN) nerve terminals. Using antisera against the GABAB R1a/b receptor isoforms we show that developing mouse olfactory receptor neurons express GABAB receptors from embryonic day 14 through to adulthood. GABAB receptors are present on axon growth cones from both dissociated ORNs and olfactory epithelial explants. Neurons in the olfactory bulb begin to express glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, from E16 through to adulthood. When dissociated ORNs were cultured in the presence of the GABAB receptor agonists, baclofen or SKF97541, neurite outgrowth was significantly reduced. Concurrent treatment of the neurons with baclofen and the GABAB receptor antagonist CGP54626 prevented the inhibitory effects of baclofen on ORN neurite outgrowth. These results show that growing ORN axons express GABAB receptors and are sensitive to the effects of GABAB receptor activation. Thus, ORNs in vivo may detect GABA release from juxtaglomerular cells as they enter the glomerular layer and use this as a signal to limit their outgrowth and find synaptic targets in regeneration and development.