Do you remember the Monopoly card that says “Go to jail. Go directly to jail. Do not pass Go, Do not collect $200?” Well, imagine that there is a cell reprogramming card that says, say when you land on skin cell, “Go to nerve cell, go directly to nerve cell. Do not pass iPSC status. Do not collect pluripotent reprogramming factors.” Very recent research shows that that card exists, and its existence is giving us a broad new perspective on epigenetic regulation of cell fates. By modifying epigenetic factors, it appears that one type of body cell can be changed, very likely, into any other type of body cell, directly and without a need for reversion into induced pluripotent stem cell (iPSC) status. And the process is efficient. This post reviews background on cell reprogramming, the new research in context, and speculates on the implications.
Background on cell reprogramming
Research on reprogramming cells from one type to another goes back to the 1980s, long before the first iPSC was produced. The first work in this area involved fusing two different kinds of cells together to form heterokaryons. A hetrokaryon is “A cell with two separate nuclei formed by the experimental fusion of two genetically different cells(ref).” A 1986 publication reports Rapid reprogramming of globin gene expression in transient heterokaryons, where “Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types.” A series of other publications based on studies of heterokaryons followed. A 1993 publication Reversibility of the differentiated state in somatic cells reported “Analysis of de novo gene activation in multinucleated heterokaryons has shown that the differentiated state, although stable, is not irreversible, and can be reprogrammed in the presence of appropriate combinations of trans-acting regulatory molecules.”
A 1999 publication Use of somatic cell fusion to reprogram globin genes reports “Experiments with heterokaryons demonstrate that the reprogramming is due to trans-acting factors that are developmental-stage-specific. These results suggest the feasibility of using fusisome-carried sets of nuclear factors to reprogram somatic cells.” A relatively recent January 2009 study Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional reports “Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days.”
Another thread of research related to cell reprogramming was cloning. Dolly, the world’s most famous sheep, was cloned in 1996. “The production of Dolly showed that genes in the nucleus of such a mature differentiated somatic cell are still capable of reverting back to an embryonic totipotent state, creating a cell that can then go on to develop into any part of an animal(ref).”
Another chain of studies in the mid 2000s related to cell reprogramming involved the impact of microenvironment on cell fate. It was found that when cloned liver stem cells were placed into a cardiac microenvironment, they transformed themselves to acquire a cardiac phenotype and function(ref)(ref)(ref). “Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function(ref).”
The hetrokaryon studies, the cloning work and the studies related to the effect of microenvironment indicate that cells of one kind can be directly reprogrammed into cells of another kind and that there is some kind of molecular signaling process involved. The big more-recent cell reprogramming news of course was the ability to revert any cell to embryonic stem cell-like pluripotency, the creation of induced pluripotent stem cells (iPSCs) starting in 2006. The first comprehensive discussion of iPSCs in this blog was the March 2009 post Rebooting cells and longevity, and iPSCs have been mentioned or discussed in many subsequent blog posts. The first studies described the use of four transcription factor proteins to create iPSCs: Oct4, Sox2, Klf4, and c-Myc. Much progress in creating iPSCs in the last year including use of other transcription factor combinations, safer less-oncogenic vectors for insertion of the transcription factors, induction of stem cell expression without using transgenes, and, most recently, the use of vitamin C to improve the efficiency of reprogramming(ref)(ref).
The 2008 publication Reprogramming of somatic cell identity summarized the situation as of the time “Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the reexpression of all developmental programs. Recent breakthroughs highlight the importance of transcription factors as well as epigenetic modifiers in the establishment, maintenance, and rewiring of cell identity.”
Nonetheless, the excitement about iPSCs led many researchers to forget or ignore the earlier research on cell reprogramming and assume that if one wants to start with, say, skin cells and end up with nerve (or heart or liver) cells, just about the only practical approach is a two-step one: 1. Take some skin cells and revert them to being iPSCs, an inefficient process even when using vitamin C, and then 2. Somehow convince those iPSCs to progressively differentiate to become nerve cells, possibly a quite tricky thing to do in-vivo. The new research finding suggests that with the right transcription factors it might be possible to start out with any kind of cell and end up with any other kind of cell without going through an intermediate stage.
Direct cell reprogramming
2008 saw the publication of a breakthrough study In vivo reprogramming of adult pancreatic exocrine cells to beta cells. “Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble
The new January 2010 research study report Direct conversion of fibroblasts to functional neurons by defined factors reports “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.”
The transcription factors used in both studies are different than those used to create iPSCs and the cell-type conversion process is much more efficient than that of reverting cells to iPSC status. The 2008 study was exciting because it described direct cell reprogramming in-vivo in a way that addresses a disease, albeit in a mouse model. Regarding the 2010 study, the “neurons could integrate into pre-existing neural networks and form independent synapses with each other. — This system bypasses production of tumorigenic pluripotent cells, a main barrier to using iPSCs in regenerative medicine, and may provide a platform for more efficient disease modeling and drug discovery(ref).” “They also tested the procedure on skin cells from the tails of adult mice. They found that about 20 percent of the former skin cells transformed into neural cells in less than a week. That may not, at first, sound like a quick change, but it is vast improvement over iPS cells, which can take weeks. What’s more, the iPS process is very inefficient: Usually only about 1 to 2 percent of the original cells become pluripotent(ref).”
· hESCs, iPSCs and other stem cell types are likely to turn out to be extremely important, but are not the only games-in-town for producing desired cells where and as needed.
· Cloning taught us that all body cells encompass the same genes and that any one cell encompasses the possibilities in all other cells. The differences among cells are ones of epigenetic gene expression. The latest research indicates it may be possible freely to change one cell type to another via introducing highly specific transcription factors.
· It may turn out to be practical to convert many cell types to other cell types in-vitro, in-vivo or both, allowing the development of many new regenerative medicine applications. The challenge is discovering the transcription factors and other epigenetic modifiers needed and how to introduce them so as safely get a desired result.
· The new work probably makes addressing cell senescence even more critical. I suspect that transforming an old-near-senescent skin cell into a nerve cell will produce an old near-senescent nerve cell unless issues like telomere lengths are also addressed.
· The new work is likely to contribute to an acceleration in research relating to the discovery and isolation of gene transcription factors(ref), micro-RNAs(ref), HATs and HDACs(ref), DNA demethylases(ref) and other gene regulatory factors. Unraveling all of those may well take decades.
I am not worried about running out of work here.