Reprogramming and iPSC Formation - Then and Now

Personalized disease treatment, tailored to an individual’s genetic background, is the future of medicine. Stem cells hold the promise of that objective becoming a reality. A decade ago, the discovery that pluripotent stem cells need not come from embryos caused a paradigm shift in the field of stem cell research.

Embryonic stem cells (ESCs) brought as much scientific promise as they did ethical controversy. The ability to create stem cells from non-embryonic cells, through the process of reprogramming, lifted the ethical hindrance and is driving stem cell research in a new direction at a rapid pace.

A history of reprogramming

Reprogramming is the process of reverting differentiated, tissue specific cells into induced pluripotent stem cells (iPSCs).

The first iPSCs were generated in 2006 from mouse embryonic fibroblasts in the lab of Dr. Shinya Yamanaka, work that earned him the Nobel Prize in Physiology or Medicine in 2012 (along with Sir John B. Gurdon) "for the discovery that mature cells can be reprogrammed to become pluripotent".

Yamanaka’s lab identified 24 proteins that were important in differentiation, none of which induced pluripotency individually. However, a marathon runner by hobby, Yamanaka took a long, systematic approach to reveal a combination of four of them, OCT4, SOX2, KLF4 and cMYC, (otherwise known as OSKM) that together reprogrammed the cells to a stem cell state. Reprogramming in this way, often referred to as the Yamanaka method, creates immortal, pluripotent cells. 

How is reprogramming evolving?

The method of reprogramming has changed significantly in the last decade. One of the biggest changes has been in how the factors such as OSKM are delivered to the cells. Up until recently, iPSCs were generated by infection of somatic cells with viral vectors, either retroviruses or lentiviruses. This method results in transgenes that integrate into the genome of the cell. The flexibility of the expression of the genes is lost upon integration which is problematic once the reprogramming process is complete. More recently, genes have been added to the cells directly as RNAs or through the use of viruses that do not integrate . These new methods limit the genes’ expression to the duration of the reprogramming process. Because the genes are inactive when the cells re-differentiate, there is less interference in the process of expressing tissue specific factors. 

Although many stem cell researchers dream of iPSCs being used to treat disease directly, that path has not been clear. With only one clinical trial to date , Yamanaka is instead focusing efforts on establishing an iPSC cell bank. But, the clinical utility of iPSCs, for the moment, is not as exciting as their role in research, and their usefulness in modeling disease.

Dr. Charles P. Emerson is the Director at the University of Massachusetts Medical School Wellstone Muscular Dystrophy Cooperative Research Center for facioscapulohumeral muscular dystrophy (FSHD). He has been utilizing iPSCs to model muscular dystrophy.

“The amount of potential held in iPSCs is truly remarkable,” says Dr. Emerson.

Regarding the impact that iPSCs have had on advancing the field of muscular dystrophy, Dr. Emerson explains, “iPSCs have given us the ability to look at disease in a very meaningful way, in large part because we can study the disease on an individual level, in the context of the patient's unique genetic makeup. Now, when a patient comes in with a disease, we have the ability to make iPSCs to study their particular disease process. This personalized look at disease is incredibly powerful and will certainly inform the development of new and exciting treatments in the future.”

If iPSCs can be used in the lab to study correcting diseases, how will that translate into therapies?

The answer lies, in part, by obtaining iPSCs that are both corrected of genetic mutations and engraftable into tissue. The advent of CRISPR-Cas9 gene editing technology will certainly catapult the field of iPSCs in the area of correcting mutated genes in the cells. But, grafting them without rejection or other complications such as teratoma (tumor) formation is not well understood.

This particular area of iPSC research seems to be a priority of the National Institutes of Health (NIH.) A recent Funding Opportunity Announcement (FOA) entitled “Improvement of Animal Models for Stem Cell-Based Regenerative Medicine (R24)” will fund research “aimed at characterizing animal stem cells and improving existing, and creating new, animal models for human disease conditions. The intent of this initiative is to facilitate the use of stem cell-based therapies for regenerative medicine.”

The biggest challenges

While there are many challenges to this complex matter, a few stand out as the most pertinent.

The biggest question remains, how similar are iPSCs to ESCs and are the differences between iPSCs and ESCs meaningful?

Dr. Jared Churko, an Instructor at the Cardiovascular Institute at Stanford University explains, “A lot of the studies that look at the transcriptomics show that the two cell types are very similar, with 99% of the same genes expressed. That small difference could mean something, but, it depends on what the difference is. What we need to do is to figure out how important that difference is and if it effects the resulting cell line downstream.” He adds, “In a lot of cases, it won’t matter.” 

To take it one step further, Dr. Churko, Dr. Joseph Wu and colleagues analyzed how different methods of delivery of the transcription factors affect the resulting iPSCs. They performed systematic analysis of six distinct methods of delivery (minicircle plasmid, episomal plasmid, direct messenger RNA transfection, Sendai virus, direct microRNA transfection, and a lentiviral system) of the reprogramming factors. The iPSCs showed significant gene-expression changes, with up to 500 differentially expressed genes, when compared with ESCs.

Among the genetic differences between ESCs and iPSCs, epigenetic genes rise to the top – indicating the large role that epigenetic memory plays in this process.

The epigenetic landscape – mostly referring to histone modifications (methylation and acetylation) and DNA methylation – are reset when a cell is reprogrammed. This process can be incomplete, and how much of the old epigenetic marks remain is still an unknown. This is a fundamental part of the reprogramming process and can have implications on pluripotency and differentiation capacity. The role of the epigenome in the process of reprogramming is only recently being fully appreciated. Recent research suggests that reprogramming is, indeed, being thought of largely as a process determined by epigenetic remodeling.

Churko says that most epigenetic differences observed have not prevented the potential in using iPSCs. “Even with these epigenetic differences, iPSCs maintain their pluripotent state and are capable in differentiating to various cell types. Also, if there are differences in epigenetic marks in genomic regions not being expressed, these differences may not impact the function of the cells.”

Churko explains that there are other factors at play, too, for example “continual culturing and passaging of hiPSCs over time can result in genetic modifications.”

Despite the challenges, he and others feel that the first places that may see clinical advancements are in diseases of the eye, blood diseases and Duchenne muscular dystrophy or other muscle diseases.

There are still many questions to be answered regarding the process of reprogramming and the resulting iPSCs. However, over the last decade, iPSCs have risen to the top of the list of the most powerful research tools.

As Churko says, with enthusiasm, “The field is wide open and we are just beginning to figure out the full potential of iPSCs. It is just a matter of time before more uses come out of them.”