Molecular Characterization of hESC and hIPSC-Derived Spinal Motor Neurons

Molecular Characterization of hESC and hIPSC-Derived Spinal Motor Neurons

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01367
Approved funds: 
$1,277,986
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Muscular Atrophy
Spinal Cord Injury
Genetic Disorder
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
One of the main objectives of stem cell biology is to create physiologically relevant cell types that can be used to either facilitate the study of or directly treat human disease. Tremendous progress towards these goals has been made in the area of motor neuron disease and spinal cord injury through the findings that motor neurons can be generated from human embryonic stem cells and induced pluripotent stem cells. These advances have made possible the creation of motor neurons from patients afflicted with neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy that can be studied in the laboratory to determine the root causes of these diseases. In addition, stem cell-derived motor neurons could potentially serve as replacement cells that could be introduced into the spinal cord to recover motor functions in these patients, as well as those suffering from spinal cord injuries. A major assumption, however, is that human embryonic and induced pluripotent cell-derived motor neurons are identical to their normal counterparts. Despite its relevance, few studies of human motor neuron development have been carried out, and little information on the genetic and functional similarities between stem cell- and embryo-derived motor neurons has been obtained. The proposed research will provide important new insights into the profile of human motor neurons that must be recapitulated by stem cell studies. This approach is critical given that most of our knowledge on human motor neuron development is based on animal models. In addition, work with mouse embryonic stem cell-derived motor neurons has revealed limitations in the motor neuron subtypes that can be generated in culture, something others and we have also observed in human embryonic and induced pluripotent stem cell-derived motor neurons. The differences between embryo and stem cell-derived motor neurons are currently unknown, though our preliminary studies suggest that this deficiency may result from the inability of stem cell-derived motor neurons to express key regulators of motor neuron development. We will directly test this hypothesis by examining whether artificially expressing some of these important motor neuron fate determinants can alter the classes of motor neurons formed in culture and thereby broaden their innervation potential. Since most motor neuron diseases tend to affect certain motor neuron populations more than others, and that the pattern of motor innervation is highly specific to the type of cells formed, these studies will significantly advance our understanding of how the full repertoire of motor neuron subtypes may be created from stem cells to build disease models and generate therapeutically beneficial cells.
Statement of Benefit to California: 
Neurological diseases are among the most debilitating medical conditions that affect millions of Californians each year, and many more worldwide. Few effective treatments for these diseases currently exist, in part because we know very little about the mechanisms underlying these conditions. Through the use of human embryonic stem cell and induced pluripotent stem cell technologies, it is now possible to create neurons from patients suffering from a variety of neurological disorders that can serve as the basis for cell culture-based models to study disease pathologies in an experimentally accessible setting. Our proposed research seeks to develop the means to form different classes of neurons, confirm their physiological identities, and establish a system for studying their neurological activity in a cell culture setting. The generation of these models will constitute an important step towards understanding the basis of neurological illnesses and developing a platform for the discovery of drugs that can alter disease progression and improve the productivity and quality of life for many Californians. Moreover, progress in this field will help solidify the leadership role of California in bringing stem cell research to the clinic, and stimulate the future growth of the biotechnology and pharmaceutical industries within the state.
Progress Report: 

Year 1

The main goals of this project are to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the first year of this project, we have confirmed that motor neuron generated from stem cells exhibit many molecular and physiological changes over time that closely mirror the formation of motor neurons during normal human development. There are some subtle differences, however, and our ongoing work will explore whether these discrepancies have any functional relevance. In carrying out these experiments, we also discovered new techniques by which we can create more diverse populations of motor neurons that better match the complexity seen in the spinal cord. Lastly, we have made significant progress in developing experimental assays to study the connections formed between stem cell-derived motor neurons and their muscle targets. We anticipate that these assays will serve as a valuable platform for modeling the pathology of human motor neuron diseases.

Year 2

The main goals of this project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the second year of this project, we have documented that the initial stages of motor neuron development in stem cell cultures are very similar to the process of motor neuron formation during fetal development. However, stem cell-derived motor neurons appear to be more homogeneous than their fetal counterparts and lack several defining characteristics of mature cells. We are currently investigating the basis of these differences and whether there are any consequences on the function of the stem cell-derived neurons. We have also developed methods for evaluating the communication of stem cell-derived motor neurons with muscle cells. We anticipate that this assay platform will be valuable for modeling the pathology of neurodegenerative diseases that affect motor function. Lastly, we have obtained evidence that the forced expression of genes associated with specific motor neuron groups can strongly influence their trajectory and rate of motor axon growth, and improve innervation of limb muscles.

Year 3

The main goals of our project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the third year of this project, we have assembled a nearly complete documentation of the developmental progression of human stem cell-derived motor neurons in cell culture compared to that seen in normal fetal development. From this analysis we conclude that the process of forming motor neurons in the culture setting faithfully replicates many aspects of their formation in the intact spinal cord. However, the types of motor neurons that are formed in stem cell cultures are more limited in their subtype diversity, which has implications for the utility of these cells as therapeutic agents and models to investigate disease mechanism. We have nevertheless found that we can extend the diversity of stem cell derived motor neurons by programming the cells to express specific proteins that promote the formation of different motor neuron subtypes. These findings suggest a general strategy for creating different functional classes of motor neurons for therapeutic uses and research applications. Lastly, we have developed two simple cell culture systems to measure the communication between motor neurons and muscle cells. Breakdown in this communication is thought to underlie many motor neuron diseases, and we anticipate that this platform will provide a means for studying the underlying pathology of these diseases, and facilitate the discovery of novel therapeutic agents.

Year 4

The main goals of our project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the final period of this project, we have completed our documentation of the developmental progression of human stem cell-derived motor neurons in cell culture compared to that seen in normal fetal development. From this analysis we conclude that the process of forming motor neurons in the culture setting faithfully replicates many aspects of their formation in the intact spinal cord. However, the types of motor neurons that are formed in stem cell cultures are more limited in their subtype diversity, which has implications for the utility of these cells as therapeutic agents and models to investigate disease mechanism. We have nevertheless found that we can extend the diversity of stem cell derived motor neurons by programming the cells to express specific proteins that promote the formation of different motor neuron subtypes. These findings suggest a general strategy for creating different functional classes of motor neurons for therapeutic uses and research applications. Lastly, we have developed a novel cell culture system to measure the communication between motor neurons and muscle cells. Breakdown in this communication is thought to underlie many motor neuron diseases, and we anticipate that this platform will provide a means for studying the underlying pathology of these diseases, and facilitate the discovery of novel therapeutic agents.

© 2013 California Institute for Regenerative Medicine