Mechanisms of human induced neuronal cell reprogramming

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Grant Award Details

Grant Type:

Basic Biology V

Grant Number:

RB5-07466

Investigator(s):

Name:

Dr. Marius Wernig MD, PhD

Institution:

Stanford University

Type:

Human Stem Cell Use:

Directly Reprogrammed Cell

Award Value:

$1,178,370

Status:

Closed

Progress Reports

Reporting Period:

Year 1

Reporting Period:

Year 2

Reporting Period:

Year 3

For successful lineage reprogramming the target cell program needs to be activated and the donor cell program needs to be silenced. Typically, the same factors are used to reprogram many different donor cell types. This raised the puzzling question how the identical combination of transcription factors can silence so many different donor programs because most transcriptional repressors are considered lineage-specific such as REST which blocks neuronal genes and Groucho which blocks hepatic genes. Therefore, a different set of lineage repressors would have to be induced in different donor cell types.

Here we found that the reprogramming factor Myt1-like (Myt1l) is a pro-neuronal “master repressor” which inhibits scores of lineage programs except the neuronal lineage. Its repressive function is mediated by direct interaction and recruitment of the Sin3/HDAC chromatin complex through a novel protein domain. Myt1l is the only pan-neuronal transcription factor known that is also specifically expressed in neurons. Surprisingly, its physiological targets are almost identical in neurons as in fibroblasts when ectopically expressed suggesting that unrelated lineage programs (such as the fibroblast program) must be silenced by active recruitment of transcriptional repressors rather then by passive epigenetic mechanisms to maintain the neuronal identity.

In this project we also defined the chromatin accessibility changes induced by the neuronal reprogramming factors throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hours after reprogramming. Many changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs. Integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during reprogramming,andidentifiedseveralkeylineagefactorsthatmediatethereprogrammingprocess.

Grant Application Details

Application Title:

Mechanisms of human induced neuronal cell reprogramming

Public Abstract:

We and other groups have recently shown that it is possible to convert skin cells from foreskin of newborns into nerve cells that closely resemble nerve cells of the brain in terms of both shape and functional properties. If it were possible to also reprogram human adult skin cells into similarly functional nerve cells, we would be able to generate functional nerve cells from patients that are suffering from a variety of brain diseases. These cells could be used to study the processes underlying these diseases. Furthermore, new skin-derived nerve cells could be used for novel transplantation-based therapies for neurodegenerative diseases such as Parkinson’s disease. It is also conceivable that brain cells other than nerve cells could be converted in the living brain into specific nerve cells that are lost due to the disease process.
However, all these potential translational applications rely on our ability to generate functional nerve cells not only from newborns but also from adult skin cells. Unfortunately, all efforts to convert human adult fibroblasts into mature functional nerve cells have failed thus far. We reason that there must be molecular differences between newborn and adult cells that are responsible for the lack of reprogramming. In this proposal we suggest to identify these molecular blockers of nerve cell reprogramming from adult skin cells, which would enable us to devise methods to overcome these blockers.

Statement of Benefit to California:

The goal of our proposal is to enable the reprogramming of adult human skin cells into fully functional and mature nerve cells. If successful, these reprogrammed nerve cells could be used for a variety of clinical applications. First, such cells could be derived from patients suffering from a variety of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease but also neurodevelopmental diseases such as Autism, Schizophrenia, or bipolar disease. Such nerve cells could then be used to capture processes that underlie those diseases which would enable us to test ways to block these disease processes such as the testing of small molecule drugs. Another potential use of these cells would be cell transplantation. In particular neurodegenerative diseases (i.e. diseases where neurons are lost) would be good candidate diseases for such an approach as one could attempt to generate nerve cells in the dish and use them to replace those nerve cells that are lost in the brain. Finally, this nerve cell reprogramming method could be used directly in the brain without any cell transplantation. It is conceivable that the delivery of just a handful of reprogramming factors directly into the brain can convert non-nerve cells into new nerve cells that again could take over function of previously lost cells. Our proposal is an important first step towards the realization of these applications that aim to improve the life of people that suffer from incurable disease.

Publications

Nature (2016): Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. (PubMed: 27281220)
Nature (2017): Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. (PubMed: 28379941)