Generating pluripotent cell lines from neurons.

Generating pluripotent cell lines from neurons.

Funding Type: 
New Faculty I
Grant Number: 
RN1-00584
Award Value: 
$2,786,560
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The goal of our research is to generate cell lines from neurons with the long term goal to understand how to generate the best possible stem-cell derived models for human neurological diseases such as Alzheimer's, Parkinson's and Huntington's diseases. These studies might also reveal basic mechanisms of disease that could inspire new drug development efforts. We proposed to use two cutting edge methods in stem cell biology to accomplish this: cloning and direct reprogramming (generation of induced pluripotent stem cells with reprogramming factors). At the time, methods for direct reprogramming were in their infancy and it was not possible to generate mice entirely from these cells. Because reprogramming neurons is likely to be inefficient and difficult, we proposed to generate entire mice derived from reprogrammed cells. Al cell types, including neurons in these mice would carry special genes to allow us to then turn their neurons into stem cell lines. We have successfully generate such mice and in doing so, we became the first to show that fertile adult mice could be generated from skin cells using induced pluripotent stem cells. This result helps to validate the utility of stem cells and induced pluripotent stem cells and represents the achievement of a critical milestone in our project timeline. Present efforts will use these mice as a unique resource to achieve the goal of the application.

Year 2

The goal of this grant is to generate cell lines from mouse neurons in order to learn how to produce the best possible models of neurological disease. Neurons do not divide and neurons from adults cannot be grown in a dish so very little is known about the genomes of neurons. We have developed reprogramming methods to generate cell lines from neurons and we have established methods to perform whole genome sequencing of pluripotent cell lines that establish whether mutations derive from donor cells or arose during reprogramming. By combining these methods we will be able to survey the genomes of neurons and determine whether it is important to use neurons as sources to generate cell lines for disease modeling.

Year 3

The goal of this grant is to generate stem cell lines from neurons. This is important because neurons are affected in many diseases yet they cannot be made into dividing cell lines using standard methods and therefore it is difficult to study human neurologic disease. A second goal is to use neuron-derived cell lines to determine whether neurons harbor genomic mutations or stable DNA marks that are relevant to brain development and/or human neurologic disease. We have successfully generated the first mouse pluripotent cell lines that are derived from adult cortical neurons and are in the process of using whole genome sequencing to identify any stable DNA marks that are unique to neurons. Results of this research will help to establish the best methods for modeling human neurological disease using stem cell technologies and establish the extent and identity of stable DNA changes in cortical neurons.

Year 4

A major goal of regenerative medicine is to generate in vitro models and sources of cell replacements for diseases of the brain and nervous system. These diseases have been particularly difficult to study because it has not been feasible to culture and propagate human neurons from affected individuals. Over the five year course of this grant, many ground breaking discoveries have emerged that will allow researchers to generate human neurons in vitro fro a wide variety of individuals who differ in their disease state or genetic propensity to disease. While this is clearly an exciting prospect, we still do not understand two key features of neurons. First, it is not know why neurons cannot be induced to divide in vitro. Second, neuronal genomes have never been sequenced to determine whether they maintain their integrity over the lifetime of the organism. Here we proposed to test whether neuronal genomes are maintained similarly to other cell types by using somatic cell nuclear transfer to produce clones mice and clones ES cell lines from adult post mitotic neurons. We succeeded at this and have now shown that the genomes of adult most mitotic cortical neurons can produce fertile and viable mice. Further, we performed high resolution genome sequencing of the cell lines derived from these neurons. Remarkably these studies showed that each of three neurons taken from the same adult organism differs from its neighbors in ~80 locations. These data suggest that it will be important to continue these studies on human neurons to ensure that we are developing the most effective models of human disease in a dish.

© 2013 California Institute for Regenerative Medicine