A few days after I returned from giving a presentation [1] to the Midwestern Stem Cell Conference (hosted by Oakland University), it was announced that John Gurdon [2] and Shinya Yamanaka [3] just won the 2012 Nobel Prize in Physiology and Medicine (Figure 1). The award was given for work in cellular reprogramming, which over the past 30 years has moved from the fringe of biological science to a well-established set of biotechnological protocols with much potential for enabling future scientific breakthroughs (Figure 2).
Figure 1. Headshots of John Gurdon (left) and Shinya Yamanaka (right). COURTESY: Nobel Prize committee website.
Gurdon’s seminal work with frog embryos (1962) helped to establish an approach called nuclear (or indirect) reprogramming [4]. This method ultimately enabled technologies such as animal cloning using somatic cell nuclear transfer (SCNT). The advent of indirect cell reprogramming allowed for the reversion of a mature somatic cell to a stem-like state, which at the time went against prevailing ideas about cellular differentiation. This lead to the creation of Dolly the Sheep, the first mammal reproduced using cloning techniques (Figure 3, top). While a powerful technology in its own right, indirect reprogramming is a low-throughput (e.g. serial) technique that has a rather low overall efficiency. While this is sufficient for reproductive applications, a more flexible approach was needed for other applications.
Figure 2. Intellectual trajectory of Gurdon and Yamanaka’s work. COURTESY: Figure 1 in [5].
These drawbacks provided an impetus for the development of direct reprogramming methods. Direct reprogramming methods involve delivery of a reprogramming agent directly into the cell (for example, see Figure 4), changing gene expression, epigenetics, and ultimately cell pheontype rather than replacing the cell nucleus wholesale [6]. Early experiments with the transcription factor MyoD was found to be sufficient for converting fibroblast cells into skeletal muscle fibers [7]. While this process is also one with relatively low efficiency, it can be administered to large populations of cells simultaneously.
It took another 20 years for Yamanaka (2007) to achieve his winning result (fibroblasts to induced pluripotent, or iPS, cells [8] – see Figure 3, bottom) with four factors (Oct4, Sox2, Klf4, and c-Myc) [9]. However, the field is taking off in a number of promising directions. One is the use of iPS cells for therapeutic applications: fibroblasts taken from a patient donor (e.g. a sufferer of Alzheimer Disease) can be used to create an iPS model of the disease [10]. Another is the role transcription factor cross-antagonisms [11] and other systems-level phenomena play in efficient conversion of somatic cells to a stable iPS state [12]. In addition to induced pluripotent cells, the same basic techniques have been used to generate induced neural cells (iNCs) and induced cardiomyocytes (iCMs) that are fully functional and phenotypically stable [13].
Figure 3. Clonal animals at play (top), and clonal (e.g. iPS) cell lines at work forming a colony (bottom). COURTESY: Stem Cell School.
Figure 4. Examples of cell reprogramming using a variety of transcription factors and input cells. Notice that these methods result in a variety of cell phenotypes. COURTESY: Figure 1 from [6].
More observations on and explanations about this wonderful field of science can be found in the Notes section below. Have fun!
NOTES:
[1] This was a very well-run small regional conference with many excellent talks. My talk was only tangentially related to stem cell biology. It was entitled: “Simulating the Dynamic Regulation of a Cell: Relevance to Cell Reprogramming”. See my talk here.
[2] Gurdon, J.B. and Byrne, J.A. The first half-century of nuclear transplantation. PNAS, 100, 8048-8052 (2003).
[3] Takahashi, K. et.al Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. (2007).Cell, 131(5), 861-872.
[4] Gurdon, J.B. and Melton, D.A. Nuclear reprogramming in cells. Science, 322, 1811-1815 (2008).
[5] Yamanaka, S. Induced Pluripotent Stem Cells: past, present, and future. STEM, 10(6), 678-684 (2012).
[6] Similar to an approach called lineage reprogramming or transdifferentiation. For more information, please see: Thomas, G. and Enver, T. Forcing cells to change lineages. Nature, 462, 587-594 (2009).
[7] While this transcription factor (MyoD) was delivered using cDNA constructs, more recent approaches have used transgenes encoded on a viral vector.
[8] fibroblasts are the main cell type in skin. Here is a generic example of fibroblast cells in culture:
iPS cells exhibit extensive phenotypic diversity (e.g. partial iPS, or piPS, phenotypes), the extent of which has not been fully characterized. Here is an example of this diversity (COURTESY: myself (primary data), Cellular Reprogramming Laboratory, Michigan State University):
[9] For generation of iPS cells, the four factor approach (Oct4, Sox2, Klf4, and c-Myc) is generally used. Oct4 and Sox2 are the most important factors (the main “hub” in the pluripotency gene network), while c-Myc is viewed by some people as dispensable. The following visuals might help:
Abbey Road to pluripotency: relative importance of the four factors?
The four transcription factors used for making iPS cells. COURTESY: Stem Cell School.
Yamanaka arrived at the four factor cocktail by screening a much larger number of factors, and then reducing the list to the smallest number of factors sufficient for creating a reprogrammed phenotype in a significant number of cells. For the skeptics out there, I can only say that this is not yet an exact or predictive science. See my previous post on the Stem Cell School web resource.
[10] iPS disease models can be used either for basic science or for cell therapy. For more information, please see the following citations:
a) Kiskinis, E. and Eggan, K. Progress toward the clinical application of patient-specific pluripotent stem cells. Journal of Clinical Investigations, 120(1), 51–59 (2010)
b) Patel, M. and Yang, S. Advances in Reprogramming Somatic Cells to Induced Pluripotent Stem Cells. Stem Cell Reviews, 6(3), 367–380 (2010)
c) Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K., and Daley, G.Q. Disease-specific induced pluripotent stem cells. Cell, 134, 877-886 (2008).
[11] Visvader, J.E., Elefanty, A.G., Strasser, A., and Adams, J. M. GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO Journal, 11, 4557–4564 (1992).
[12] While the role of each of the four factors is fairly well-established, it is not clear what the cumulative effects of their downstream interactions are.
For more information, please see: Loh, K.M and Lim, B. A Precarious Balance: pluripotency factors as lineage specifiers. Stem Cell, 8(4), 363-369 (2011) AND Iwasaki, H. et al. The order of expression of transcription factors directs hierarchical specification of hematopoietic lineages. Genes and Development, 20, 3010–3021 (2006).
Other genes may play a critical role in establishing pluripotency, especially with respect to the time course of reprogramming. For a recent paper on the topic, please see:
Buganim, Y., Faddah, D.A., Cheng, A.W., Itskovich, E., Markoulaki, S., Ganz, K., Klemm, S.L. van Oudenaarden, A., and Jaenisch, R. Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase. Cell, 150, 1209–1222 (2012).
[13] For more information on iNCs, please see: Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., Citri, A., Sebastiano, V., Marro, S., Sudhof, T.C., Wernig, M. Induction of human neuronal cells by defined transcription factors. Nature, 476, 220-223 (2011).
For more information on iCMs, please see: Srivastava, D. and Ieda, M. Critical Factors for Cardiac Reprogramming. Circulation Research, 111, 5-8 (2012).
For more information on in vivo generation of iCMs in mouse, please see: Song, K., Nam, Y-J., Luo, X., Qi, X., Tan, W., Huang, G.N., Acharya, A., Smith, C.L., Tallquist, M.D., Neilson, E.G., Hill, J.A., Bassel-Duby, R., and Olson, E.N. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature, 485, 599-606 (2012).
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