Direct induction of functional neurons from fibroblasts by three transcription factors; what’s the limit of reprogramming?

At the end of 2007, the Japanese group demonstrated the feasibility to reprogram mouse fibroblasts into multipotent stem cell-like cells, which they called iPS cells. Later, people showed that human fibroblasts can be reprogrammed! Also, any terminal differentiated cells can be induced to the group state – pluripotent stem cell stage.

However, until now, there is no way directly reprogram fibroblasts into neurons – what people was doing was reprogramming fibroblasts into iPS cells, then differentiated into neuronal cells.

The following paper demonstrated the feasibility of direct conversion of fibroblasts to functional neurons! Now, the hot question is NOT which cell types cannot be reprogrammed, but is which cell types cannot be reprogrammed into … the sky is the limit

Nature advance online publication 27 January 2010
DOI: 10.1038/nature08797

Direct conversion of fibroblasts to functional neurons by defined factors

Thomas Vierbuchen 1,2, Austin Ostermeier 1,2, Zhiping P. Pang 3, Yuko Kokubu 1, Thomas C. Südhof 3,4 & Marius Wernig 1,2

1 Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology,
2 Program in Cancer Biology,
3 Department of Molecular and Cellular Physiology,
4 Howard Hughes Medical Institute, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA

Correspondence and requests for materials should be addressed to M.W. (Email: wernig@stanford.edu).

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

Couple key methods from the paper

• We had three criteria for identifying candidates with neuron-inducing activity: (1) we reasoned that cell-fate-inducing factors should be enriched in the gene category of transcriptional regulators. (2) We included factors previously involved in reprogramming to pluripotency (Klf4, c-Myc and Sox2). (3) We searched for genes specifically expressed in neural tissues. Those were selected based on published expression arrays of MEFs, embryonic stem cells and neural progenitor cells retrieved from the Gene Expression Omnibus database (GSE8024, http://www.ncbi.nlm.nih.gov/gds) and the EST Profile function of NCBI’s Unigene database (http://www.ncbi.nlm.nih.gov/unigene).
• After 16–20 h in media containing lentivirus, the cells were switched into fresh MEF media containing doxycycline (2 mg/ml 21) to activate expression of the trans-duced genes. After 48 h in MEF media with doxycycline (Sigma), the media was replaced with N3 media 22 containing doxycycline. The media was changed every 2–3 days for the duration of the culture period.
• The following method was used to calculate the efficiency of neuronal induction. The total number of Tuj11 cells with a neuronal morphology, defined as cells having a circular, three-dimensional appearance that extend a thin process at least three times longer than their cell body, were quantified 12 days after infection. This estimate was based on the average number of iN cells present in 30 randomly selected x20 visual fields. The area of a x20 visual field was then measured, and we used this estimated density of iN cells to determine the total number of neurons present in the entire dish. We then divided this number by the number of cells plated before infection to get a percentage of the starting population of cells that adopted neuron-like characteristics.
• For RT–PCR analysis, RNA was isolated using Trizol (Invitrogen) following the manufacturer’s instructions, treated with DNase (NEB) and 1.5 mg was reverse-transcribed with Superscript II (Invitrogen). PCR products were analysed on a 1% gel.
• For details about cortical culture, glia cell isolation, and electrophysiology, please check the original article.

At the end of the paper, the authors speculated that “We assume that high expression levels of strong neural cell-fate-determining transcription factors can activate salient features of the neuronal transcriptional program. Auto-regulatory feedback and feed forward activation of downstream transcriptional regulators could then reinforce the expression of important cell-fate-determining genes and help to further stabilize the induced transcriptional program. Robust changes in transcriptional activity could also lead to genome-wide adjustments of repressive and active epigenetic features such as DNA methylation, histone modifications and changes of chromatin remodelling complexes that further stabilize the new transcriptional network 12,33. It is possible that certain subpopulations of cells are ‘primed’ to respond to these factors, depending on their pre-existing transcriptional or epigenetic states 34.“