Large scale production of stem cells requires an intimate knowledge of the genetic networks that convert adult cells into induced pluripotent stem cells (iPSCs). The original protocol established by Shinya Yamanaka and his colleagues used four genes all clustered on a retrovirus vector, but there are safer, more technically subtle ways to make iPSCs.
Because iPSCs are made from a patient’s own cells, they are less likely to be rejected by the patient’s immune system. They also show tremendous developmental flexibility, they can potentially be differentiated into any adult cell type in the body. The problem with iPSCs comes from the difficulty of making large quantities of them in a reasonable amount of time. However, a new research publication from scientists at the University of Toronto, the University for Sick Children and Mount Sinai Hospital, in collaboration with colleagues from the United States and Portugal, identifies specific proteins that play central roles in controlling pluripotency that may mean a potential breakthrough in producing iPSCs.
Researchers discovered these proteins by using something called the “splicing code.” Benjamin Blencowe discovered the splicing code a few years ago. “The mechanisms that control embryonic stem cell pluripotency have remained a mystery for some time. However, what Dr. Blencowe and the research team found is that the proteins identified by our splicing code can activate or deactivate stem cell pluripotency,” said Brendan J. Frey, from the University of Toronto Departments of Electrical Engineering and Medicine, who published with Benjamin Blencowe the paper that deciphered this splicing code (see Nature 2010 465: 53-59). While a complete recipe for producing iPSCs may not be available yet, it is beginning to look more likely, according to Frey.
In this paper, Blencowe and his collaborators identified two proteins known as muscleblind-like RNA binding proteins, or MBNL1 and MBNL2. These proteins are conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between embryonic stem cells and other cell types.
RNA splicing occurs in plant, animal, fungal, and protist cells (only very, very rarely in bacteria), and involves the removal of segments of primary RNA transcripts. When RNA molecules are transcribed in eukaryotic cells, they are engaged by cellular machinery called the RNA spliceosome. The RNA spliceosome removes segments known as “introns” and the excised introns are degraded and the remaining RNA segments, which are known as “exons, are ligated together to form a mature messenger RNA.
Some introns are removed from primary RNA transcripts by all cells, but others are removed in some cells but not others. This phenomenon is known “alternative splicing” and it is responsible for the differential regulation of particular genes.
Alternative splicing is mediated by sequences called splicing enhancers and splicing silencers that are six to either nucleotides long and bind proteins that either induce or repress alternative splicing in those cells that express the proteins that bind these splicing enhancers or silencers.
MBNL is one of these proteins that bind to RNA splicing silencers. If the quantity of MBNL proteins in differentiated cells is decreased, then these cells switch to an embryonic stem cell-like alternative splicing pattern for approximately half of their genes. Conversely, overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in a protein-coding gene called the forkhead family transcription factor FOXP1. FOXP1 controls pluripotency, and consistent with a central and negative regulatory role for MBNL proteins in pluripotency, knockdown of MBNL significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.
Thus MBNL proteins should be one of the main targets for the mass production of iPSCs.