Induced pluripotent stem cells: Choosing a reprogramming method

options for making induced pluripotent stem cells

Alexander DeVine is a research assistant in the Stem Cell Research Program at Boston Children’s Hospital.

Few discoveries have so transformed human stem cell research as have induced pluripotent stem cells (iPSCs). Like embryonic stem cells (ESCs), iPSCs possess, in principle, the potential to produce any of the cells in the human body—hence the term pluripotent. Because they can be derived by “reprogramming” easily accessible cell types (e.g., blood or skin cells) from any patient, rather than by creating and dissecting an embryo from donated sperm and eggs, iPSCs are more readily available to researchers than ESCs and better poised for clinical application.

In the seven years since Shinya Yamanaka, Jamie Thomson, and Boston Children’s Hospital’s own George Daley independently described the first methods for generating human iPSCs, these versatile cells have taken stem cell laboratories by storm. Today, they are used around the globe to study human development and to model a plethora of common and rare genetic conditions, from Parkinson’s disease to Fanconi anemia to type I diabetes. iPSCs are also starting to enter the clinic: in Japan, patients are already being recruited to a clinical trial to test the safety and efficacy of iPSC-derived therapeutics for the treatment of blindness.

In the early days of iPSC research, the genetic reprogramming factors required to turn adult cells into pluripotent stem cells could only be efficiently delivered by engineered lentiviruses, which introduce DNA into the genomes of their target cells in a process called “genomic integration.” While this delivery approach is effective for creating pluripotent cells, it also introduces unwanted viral sequences. Consequently, lentivirally-derived iPSCs are not considered safe enough for producing human therapeutics, nor precise enough for modeling complex diseases in the laboratory.

Comparing reprogramming methods to create iPSCs
Reprogramming factors can be added to patient cells using one of four different vectors. mRNA reprogramming requires repeated additions, as indicated by the multiple arrows. Over time, the cells convert to iPSCs. Unlike the other methods, lentiviral reprogramming inevitably results in genomic integration. In rare instances, genomic integration may occur in cells reprogrammed by episomes (see the yellow cell in the teal dish). mRNA and episomal reprogramming, which do not use viruses, are currently the best candidates for clinical use. (Courtesy Alexander DeVine)

Fortunately, many groups have devised innovative alternatives to the original iPSC derivation technology. We now have self-extinguishing vectors based on the Sendai virus, which reprogram adult cells to iPSCs without inserting genetic material into the cell’s chromosomes. Another technique circumvents viral vectors altogether: it repeatedly introduces messenger RNA (mRNA) transcripts of the reprogramming factors into cells until they become pluripotent; the transcripts themselves, like the mRNAs cells already produce, are rapidly eliminated. Yet another technique entails one-time introduction of episomes. These small circles of DNA encode the reprogramming factors and associate with adult cells’ genetic material without integrating into the genome. As the cells divide, the episomes are replicated imperfectly, causing them to disappear over several generations of cell division.

Which technology is best for making iPSCs?

With so many reprogramming methods, investigators wanting to use iPSCs but lacking the bandwidth to test them first-hand often find themselves overwhelmed by the options. How difficult is each method to use? How efficient is it? Will it work with the patient cell samples available in my project? How resilient is it to operator error? Can I afford the needed reagents? How likely is it to introduce genetic or epigenetic artifacts or other biases, which could confound future experiments? How readily can the method be adapted for clinical application?

Thorsten Schlaeger, PhD, director of the Human Embryonic Stem Cell Core for the Boston Children’s Hospital’s Stem Cell Program, has repeatedly fielded such questions from researchers seeking to employ iPSCs in their studies. In a recent Nature Biotechnology article, he and colleagues at Boston Children’s, the Harvard Stem Cell Institute, Massachusetts General Hospital, Harvard Medical School, Johns Hopkins, MIT and other institutions offer a comparison of the different non-integrating reprogramming methods.

In brief, there are few differences in the quality of iPSCs derived by the various methods, but each technique has its pros and cons:

  • mRNA reprogramming is very efficient, requires the least amount of patient material, works particularly well with skin cells from very young donors, and never results in retention of the delivery vectors. But it is relatively labor-intensive and cannot reprogram blood cells.
  • Sendai-viral reprogramming is less technically demanding and more reliable than mRNA reprogramming. But it is costly; the vectors are eliminated only slowly; and it is not yet amenable to clinical adaptation.
  • DNA episomes can reprogram many cell types, and are easy and inexpensive to prepare in both research and pharmaceutical settings. Like Sendai-viral reprogramming, episomal reprogramming is minimally labor intensive, and, like mRNA reprogramming, it does not utilize viral derivatives that might harm patients. However, there is risk that cells will misidentify episomes as broken pieces of their own DNA and splice them into their own genomes. These rare genomically-integrated cells are already identified and removed as part of the episomal reprogramming workflow, but in the clinic, where time is often of the essence, this technique may not always be feasible.

Interest in regenerative medicine and disease modeling continues to expand. We hope this side-by-side comparison of techniques will make iPSCs more widely accessible to scientists—and iPSC-based therapeutics more available to patients.

Ed note: Read about a recent “roadmap” comparing iPSC creation with two alternative cell engineering methods: differentiation and direct conversion of one adult cell type to another.