The ability to make stem cells from other cell types has largely been a recent phenomenon. It took a huge amount of research and testing to get to that point and, even today, it requires a complex cocktail of chemicals to induce the needed genetic and epigenetic changes that revert a cell to its infancy. The process isn’t the most efficient method, but when it involves the creation of cells of universal possibility, any procedure that sees success is desirable.

Even from the beginning however, it was known that there were other options. But we just didn’t know how to properly utilize them. A set of transcription factor proteins were known to work as a treatment to induce pluripotency, but not on the large scale and not without issues. There just wasn’t any good method at the time to overexpress those factors in a reliably repeatable manner.

So the chemical cocktail has been the norm since. Researchers haven’t stopped investigating other possibilities though and now, scientists at the Gladstone Institutes in California may have achieved that alternative. And it is seemingly an obvious one in hindsight. What else is CRISPR for, after all?

Pluripotency Gene Activators

When first starting their experiment, the researchers weren’t entirely sure which genes or how many of them would need to be activated in order to induce the differentiated cells back into stem cell states. They decided to work with mouse embryonic fibroblasts (MEFs), a commonly used limited differentiated cell type used in stem cell research. Because they are limited lines that will quickly go through their generations, reach their senescence cell limits, and die, it is easy to use MEFs to determine if you’ve succeeded at reverting to immortal stem cell lines.

As for genes, the scientists decided on Sox2, a deterministic transcription factor that helps to maintain a pluripotent state. But they were unsure if it would therefore be able to induce a return to said state. They also included the gene Oct4, which is a transcription factor additionally involved in the cellular self-renewal cycle.

Since the purpose of this experiment was to activate these transcription factor genes and not remove or alter their sequence, the researchers chose against using regular CRISPR-Cas9 and instead went with dead Cas9 (dCas9). Its ability to fuse with transactivation domains on the genome was what they were looking for, a mechanism we’ve discussed before.

A Brand New Dead CRISPR

This dCas9 was combined with the SunTag system, a peptide and antibody chain that is able to recognize the target gene, transcriptionally activate it, and then fluorescently tag resulting proteins for tracking and analysis. Combined with the binding capabilities of dCas9, the joint system titled dCas9-SunTag-VP64 was created with enhanced gene targeting and activation capabilities.

The VP64 part of the name refers to a specialized protein domain made for dCas9 that’s composed of four VP16 proteins, which are able to enact the process of chromatin remodeling to make it easier for the entire complex to access the gene of interest. Since the genes are important pluripotency genes contained in a cell that’s no longer in that stage, it is likely that they would be turned off and wrapped up into their chromatin structure. Thus, to access and activate them, the chromatin has to be unwound and the histones deconstructed to get at the DNA.

Several single guide RNAs (sgRNAs) were added to guide the complex to both of the target genes and the entire set of components were delivered to the cells via lentivirus inoculation. The first test actually directly used mouse embryonic stem cells, which were then forced into differentiation using retinoic acid, after which the dCas9 complex was set loose. The sgRNAs were set up so that different sets of them targeted slightly different parts of the genes and the surrounding areas of the genome, to see if certain promoters or enhancers were preferable for increasing gene activation for these particular genes.

Activation and Induction

They found that targeting the promoter seemed to be the better option, with Oct4 seeing a 100-fold increase in transcription and Sox2 seeing a 15-fold increase. With the successful testing of whether the complex could activate the genes, the next step was to see whether the same complex and gene activation could truly revert MEFs back into stem cells. Over the course of a week, the two genes were found to become more and more robust in their activity, with reprogramming clusters forming. After two weeks, colonies of cells were found that had been auspiciously induced to pluripotency.

The last part of their experiment involved determining if all of the genes and activation areas were needed to have that effect or if a smaller change would be just as sufficient. Instead of the host of sgRNAs that were used prior, only a single one was used one by one to find individual gene loci that were required. At the end of it all, they determined that only targeting the promoter of Sox2 was essential.

Finally a stringent test was run to ensure these induced stem cells were authentic or not and whether they would retain their state. They were found to be capable of pluripotent differentiation and were germline competent, expressing the appropriate genes and proteins just like original embryonic stem cells.

However, the scientists chose to run an additional experiment focusing on the Oct4 gene, which they suspected might require simultaneous activation of the promoter and the enhancer sequence in order for the chromatin remodeling to be achieved. Their test showed that doing this and using VP64 for histone acetylation was enough to cause pluripotency induction. Therefore, it was epigenetic remodeling that had to be done in order to get Oct4 to lead to stem cell production.

Rewards To Do More Work

Overall, it appears that the SunTag addition was enough to cause Sox2 to induce reversion, a feat that had been tried with the gene before, but met with failure due to not including SunTag. Similarly, the inclusion of VP64 and its epigenetic remodeling capabilities helped Oct4 to be expressed at the levels needed for induction.

The remodeling and reprogramming of the combined dCas9-SunTag-VP64 complex showcases two separate gene alteration methods that can create mouse embryonic stem cells from fibroblasts. The system will need to be replicated with other cell types and, eventually, in human cell lines before we will see any real use from it, but this is a big step toward having alternatives to the older methods of stem cell production. This new method may also prove capable of mass production of stem cells in the future, giving treatment availability for the many conditions that need them.

Press Article Link

Study Link

Photo CCs: Mice embryonic fibroblasts GFP from Wikimedia Commons

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