Inhibitory Nerve Cell Precursors: Dosing, Safety and Efficacy

Advances in stem cell research and regenerative medicine have led to the potential use of stem cell therapies for neurodegenerative, developmental and acquired brain disease. The Alvarez-Buylla lab at UCSF is part of a collaboration that is pioneering the investigation of therapeutic interneuron replacement for the correction of neurological disorders arising from defects in neural excitation/inhibition. Our preliminary data suggests that grafting interneuron precursors into the postnatal rodent brain allows for up to a 35% increase in the number of cortical interneurons. Interneuron replacement has been used in animal models to modify plasticity, prevent spontaneous epileptic seizures, ameliorate hemiparkinsonian motor symptoms, and prevent PCP-induced cognitive deficits. Transplantation of interneuron precursors therefore holds therapeutic potential for treatment of human neurological diseases involving an imbalance in circuit inhibition/excitation. The goal of the research in progress here is to ultimately prepare human interneuron precursors for clinical trials. Towards the therapeutic development of inhibitory neuron precursor transplantation for human neurological disorders, we have made significant progress in the differentiation of these cells from human ESCs and will complete optimization of this protocol. We will continue our investigation of rodent-derived interneuron transplantation to obtain relevant preclinical data for dose response, safety and efficacy in animal models. These dosing and safety data will then serve as the baseline for comparison with human interneuron precursors and inform design of preclinical studies of these cells in immunosuppressed mice. Together, these data will provide essential information for developing a plan for clinical trials using human interneuron precursors. During this first year, we have made considerable headway in the optimization of the human interneuron precursor differentiation protocol, verified functional engraftment of these cells in mice, and begun to collect dose, safety and efficacy data for rodent-derived interneuron transplantation. Importantly, we have achieved the development of a protocol that robustly generates interneuron-like progenitor cells from human ES cells and demonstrated that these progenitors mature in vitro and in vivo into GABAergic inhibitory interneurons with functional potential. We have also compared the behavior of primary fetal cells to these human interneuron precursor-like cells both in vivo and in vitro. As we continue to optimize our ES cell differentiation protocol, these primary interneuron precursors will enable initial human cell dose response and behavior experiments and, along with rodent-derived cells, will provide important baseline measures. In sum, this work will provide essential knowledge for the therapeutic development of inhibitory neuron transplantation. The experiments underway will yield insights that will be critical to the development of a clinical trial using human interneuron precursors.

During the reporting period, we have developed methods to enable the optimization of inhibitory nerve precursor cell, or MGE cell, derivation from human pluripotent stem cells (hPSCs). Optimization encompassed increasing MGE cell motility, enhancing MGE cell maturation into inhibitory nerve subtypes, and elimination of tumors post-transplantation into the rodent brain. Furthermore, we demonstrated that the injected hPSC-MGE cells functionally matured into inhibitory nerves with advanced physiological properties that integrated into the rodent brain. In addition, we determined an optimum dose of injected mouse MGE cells in rodent. Moreover, following injection of either the optimum dose or a 10-fold higher dose of mouse MGE cells, we found no detectable behavioral side effects from MGE cell transplantation.

In this reporting period, we continued to improve the acquisition of migratory medial ganglionic eminence (MGE)-type interneurons from human embryonic stem cells (hESC). We compared alternative procedures by testing MGE marker expression, and we developed additional tests to measure cell division and migration of MGE cell made from hESCs. We also extended our methods to clinical-grade hESC lines. With these optimized procedures, both research and clinical grade lines were transplanted into the rodent brain. In addition, we completed an evaluation of human fetal MGE transplants after-injection into the rodent brain. Finally, we report safety, survival, and neuronal differentiation of both hESC-MGE and human Fetal-MGE grafts at three months post-injection into the brain of the non-human primate rhesus macaque. A manuscript is in preparation concerning this work. Our work has continued to show the viability of using a cell therapy technique in the potential treatment of brain-related disease.