Grant Award Details
Optimizing the differentiation and expansion of microglial progenitors from human pluripotent stem cells for the study and treatment of neurological disease.
To generate and use pluripotent fluorescent reporter lines to streamline this process and develop fully-defined microglial differentiation protocols. It is also critical to determine whether human induced pluripotent stem cell-derived microglia can be used to study or potentially treat human neurological disease. As a proof-of-principle, the team will therefore examine the role of the Alzheimer’s disease-associated gene CD33 in human microglial function and use novel xenotransplantation-compatible AD mice to examine the functional effects of human microglia transplantation in vivo.
Microglia are specialized cells that play critical roles in brain development, immune defense, and neuronal plasticity. Importantly, microglia also profoundly influence the development and progression of many of the most common neurological disorders including Alzheimer’s disease, stroke, Parkinson’s disease, traumatic brain injury, and autism. As the resident immune cell of the brain, Microglia inherently migrate toward areas of injury and pathology and may therefore also represent an excellent candidate for cell transplantation based therapies. Despite these important functions, methods to generate microglia from pluripotent human stem cells have yet to be reported. Our goal is to therefore develop a new and highly robust approach to generate microglia from renewable human pluripotent stem cell sources. By developing such an approach, researchers can use these cells to understand the biology of microglia and their role in these various diseases. During the first year of funding our collaborative team has made excellent progress towards our goals. For example, we have developed new ‘reporter’ cell lines to help inform and guide our differentiation approach. We have also successfully developed a ‘fully-defined’ protocol for generating human microglia that uses standard medias and growth factors to generate large numbers of microglia. Importantly, unbiased genetic analysis of these cells demonstrates that they are extremely similar to brain-derived human microglia. These stem cell-derived microglia also respond to stimuli and exhibit characteristic features equivalent to human microglia in several functional assays. As our studies continue, we aim to further refine our differentiation approach and to then test the utility of stem cell-derived microglia to study important questions about the genetics and causes of Alzheimer’s disease (AD). In particular we will focus on a gene called CD33 that has been implicated in AD. This gene is thought to influence the clearance of beta-amyloid, a key protein involved in AD pathogenesis, and yet one cannot study CD33 effectively using existing mouse models of AD as the human version of this gene is unique.
<p>The ultimate goal of this Tools and Technology Grant was to develop methods to differentiate human induced pluripotent stem cells (iPSCs) into microglia, the principle immune cell of the brain. Microglia have implicated in virtually every neurological disorder including Alzheimer's Disease, traumatic brain injury, and stroke and yet studies of human microglia have been greatly limited because of the considerable difficulty of obtaining and growing these cells. By developing methods to coax human iPSCs into becoming microglia, we aimed to provide a renewable and expendable source of these important cells that researchers worldwide could use to study an array of human neurological diseases and injuries. We are delighted to report that our team was successful in this endeavor and established a fully-defined and highly reproducible method to differentiate iPSCs into microglia (Abud et. al., Neuron, 2017). From 1 million starting iPSCs we are able to generate well over 50 million microglia. By closely examining micoglial gene expression and function, we have also confirmed that the resulting microglia are extremely similar to human brain-derived microglia. Importantly, many independent labs have now replicated our approach and are using these methods to perform cutting edge research on a variety of human diseases ranging from Alzheimer's and Parkinson's Disease to Autism, Schizophrenia, and traumatic brain injury. To test the utility of this new model to study Alzheimer's Disease we have used siRNA and CRISPR gene editing technology to manipulate the expression of two genes that are implicated in the development of Alzheimer's Disease and highly expressed in human microglia. Our findings have revealed important new functions for these genes and are helping to clarify how these mutations effect disease risk. </p>
Grant Application Details
- Optimizing the differentiation and expansion of microglial progenitors from human pluripotent stem cells for the study and treatment of neurological disease.
Microglia are a type of immune cell within the brain that profoundly influence the development and progression of many neurological disorders. Microglia also inherently migrate toward areas of brain injury, making them excellent candidates for use in cell transplantation therapies. Despite the widely accepted importance of microglia in neurological disease, methods to produce microglia from stem cells have yet to be reported. Our team has recently developed one of the first protocols to generate microglia from human pluripotent stem cells. We have used several approaches to confirm that the resulting cells are microglia including examination of gene expression and testing of key microglial functions. However, our current protocol uses cell culture supplements that preclude the use of these cells for any future clinical applications in people. The major goal of this proposal is to resolve this problem. We will generate pluripotent human stem cells that have special "reporter" genes that make the cells glow as they become microglia, allowing us to readily monitor and quantify the generation of these important cells. Using these reporter lines we can then streamline the differentiation process and develop improved protocols that could be translated toward eventual clinical use. As a proof-of-principle experiment we will then use the resulting human microglia to study some important questions about the genetic causes and potential treatment of Alzheimer’s disease.
Statement of Benefit to California:
Recent estimates suggest that nearly 2 million Californian adults are currently living with a neurological disorder. While the causes of neurological disease vary widely from Alzheimer’s disease to Stroke to Traumatic Brain Injury, a type of brain cell called microglia has been strongly implicated in all of these disorders. Microglia are often considered the immune cell of the brain, but they play many additional roles in the development and function of the nervous system. In neurological disease, Microglia appear to be involved in a response to injury but they can also secrete factors that exacerbate neurological impairment. Unfortunately, it has been difficult to study human microglia and their role in these diseases because of challenges in producing these cells. Our group recently developed an approach to ‘differentiate’ microglia from human pluripotent stem cells. This enables researchers to now study the role of different genes in human microglial function and disease. Yet our current approach dose not allow these cells to be used for potential clinical testing in patients. Our proposal therefore aims to develop new tools and technology that will allow us to produce clinically-relevant human microglia. These cells will then be used to study the role of a specific microglial gene in Alzheimer’s disease, and may ultimately be useful for developing treatments for the many Californians suffering from neurological disease.
Source URL: https://www.cirm.ca.gov/our-progress/awards/optimizing-differentiation-and-expansion-microglial-progenitors-human