The Niche

Gene-free reprogramming in human cells

A new paper in Cell Stem Cell describes how scientists reprogrammed human cells to pluripotency without using any DNA at all. Instead, reprogramming proteins were engineered so that they could enter the nucleus. These proteins were produced in cultures of mammalian cells and secreted into the culture media. When fibroblasts derived from newborns were exposed to those cell extracts, the cells reprogrammed to teratoma-producing induced pluripotent stem (iPS) cells, a big first for human cells.

Nature’s David Cyranoski covered the story for Nature News, and he generously provided some outtakes of quotes from his reporting, although even these are condensed from what he provided. His original notes were over ten pages!

Those quoted include several well-known experts:

The scientists who led the most-recent work: Kwang-Soo Kim of CHA Stem Cell Institute in Seoul, South Korea, and Harvard Medical School in Cambridge, Massachusetts; Robert Lanza, chief scientific officer of Advanced Cell Technology in Santa Monica, California.

The scientists who, among other work, reported protein-only reprogramming in mouse cells in April: Sheng Ding of The Scripps Research Institute in La Jolla, California; Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Münster, Germany.

Outside experts: James Thomson of the University of Wisconsin–Madison, the first to derive human embryonic stem cells and human iPS cells; Shinya Yamanaka of Kyoto University, Japan, and the Gladstone Institute in San Francisco, the first to reprogram both mouse and human cells.

Below, they all discuss the significance of the results and hurdles ahead, the differences between the human and mouse techniques and the need to compare different cells.


Significance of reprogramming human cells without genetic engineering and hurdles to applications

Robert Lanza: This is the ultimate stem cell solution — you just add some proteins to a few skin cells and voila — patient-specific stem cells!

Kwang-Soo Kim: I would say that our work is proof of concept that human somatic cells can be reprogrammed by direct protein delivery without any other manipulations. However, this is the very first of this protein-induced iPS research, and [it] needs much further work. For example, since we used the total cell extracts, we need to purify the CPP [cell-penetrating protein]-reprogramming proteins and use these pure forms.

Shinya Yamanaka: This report is important since it is the first demonstration of human iPS cell generation without gene delivery.

The avoidance of genetic materials is an advantage of this method. We have to confirm the uniformity and completeness of reprogramming in these iPS cells before we can apply the technology to patients.

Hans Schöler: The issue now is that the hard work still is before us. Shinya Yamanaka has laid out the playground and all subsequent studies were more or less predictable. One of the next steps might to be to replace all factors [with] small chemical compounds, which would be good for high-throughput automatic conversion of somatic cells into pluripotent cells.

Need to compare cells

Lanza: Our iPS cells appear to be just as similar to ES [embryonic stem] cells as others. This is based on cell characterization — markers of pluripotency, differentiation both in vitro and in teratomas, global gene-expression patterns and bisulfate sequencing analysis, among others (all of these [parameters] were compared between both standard viral-generated iPS cells and normal human ES cells).

Kim: It will be useful to generate iPS cells using viral, chemical plus viral, protein plus chemical and proteins only and then fully characterize their chromosomal integrity and differentiation potential.

James Thomson: It is not yet clear what approach, or combination of approaches, will most consistently meet these criteria, but protein transduction is certainly a promising approach.

If a normal iPS cell line is obtained, it would differ from an ES cell line in name only and share all of the most difficult downstream challenges for therapeutic applications [immune rejection, neoplastic transformation and mutation accumulation during culture, for example, he says].

Sheng Ding: It is still too early to decide on a standard, and there will be different ways to do this with its own advantages and disadvantages There are and will be different standards/solutions. Certainly, using [a] chemically defined approach would be the future, for example, protein-based methods and/or small molecule–based methods).

Schöler: The hard work begins, as we need to determine what these cells are actually worth with respect to their therapeutic potential. This, however, I think is difficult as long as we have not shown that human ES cells can cure a disease. Until proven wrong I have problems seeing how a cell that has been reprogrammed from a somatic cell can be as good as pluripotential cells from an embryo. Mouse studies indicate that there might be problems that we still need to solve. [Compared to cloned mouse ES cells, he says, mouse iPS cells are not as efficient at generating embryos in the so-called tetraploid complementation assay.] The question is if and how much it matters if iPS cells are not epigenetically identical to human ES cells. Unfortunately, the answer needs proof of long-term functionality of cells in an organism.

Technique details and differences from mouse work

[Note: The big differences between the techniques in the mouse and human reprogramming work were in how the proteins were made and purified as well as the addition of the small molecule valproic acid to boost reprogramming rates. In addition to the quotes below, Kim and Lanza believe that dispensing with the molecule is safer as it can cause mutations. Schöler and Ding question this and argue that a move toward using more small molecules and less protein could lead to safer, easier reprogramming.]

Kim: Initially, we tried these CPP-attached proteins expressed in E. coli, without any success. After these failures, we finally decided to express these CPP-attached reprogramming proteins in mammalian cells and to use the whole cell extracts for reprogramming. These mammalian [cells that] expressed CPP-attached protein [were] amazingly transported into the cell nucleus.

Lanza: Mammalian versus bacteria makes a HUGE difference, especially factors such as protein folding and solubility. There are also significant differences between the human versus murine systems, including protein sequences, timing and laboratory methodologies used — all of which can easily make the difference between success and failure of the procedure.

Ding: There are many different methods of producing proteins in very large quantity for therapeutic uses. Making proteins in E. coli is widely used and has as many advantages (for example, protein refolding) over other expression systems (such as insect or mammalian cells), as it has certain disadvantages. I would not say which method is better at this point.

Technically, [making reprogramming proteins in mammalian cells] may not be an improved/advanced method, simply because they did not use purified proteins rather than using cell extracts (a mixture of undefined materials). Their work is a nice proof of concept in human cells, but using the purified proteins (as we did) would have more advantages over undefined mixtures in practical applications (for example, production, quality control, consistency and avoiding harmful contaminants).

Schöler: You can get huge amounts of proteins from E. coli. It made the point that a protein cocktail can do it; is it the perfect cocktail? Probably not. There are many ways to try to improve the vector-free system, small chemical compounds being one.

Lanza: Efforts are currently underway to purify and scale-up the production of the proteins. It’s impossible at this point to estimate what the cost would be to make one iPS cell line. By the time it’s on the market, my guess is that it will be — or at least should be — extremely reasonable.

We hope to file at least one IND [investigational new drug application] with the FDA by mid to late next year. ACT will be focusing on retinal degenerative diseases, and Stem Cell & Regenerative Medicine International will be focusing on several haematopoietic and vascular diseases.

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