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Keeping things simpleMahendra Rao
Improved methods for human pluripotent stem cell culture and for reprogramming of human somatic cells to pluripotency may bring us closer to the routine generation of personalized pluripotent stem cells.
It has been a little more than a decade since Thomson and colleagues first described the successful isolation and propagation of human embryonic stem cells (ESCs)1. Much has changed since then. Pluripotent cell lines have been derived from morula-stage embryos, from single isolated blastomeres, from par-thenotes and from late-stage embryos2. Lines have been derived from embryos generated by somatic-cell nuclear transfer and more recently by reprogramming adult cells from a variety of different tissues, using a process discovered by Yamanaka and colleagues and modified by a variety of researchers3. In two papers in this issue of Nature Methods, researchers report improvements to pluripo-tent stem cell culture4 and to the reprogram-ming of human cells5.
The derivation and culture of human pluri-potent stem cells initially required propaga-tion on feeder cultures and in most cases used serum-containing medium. With the need for larger numbers of cells and the need to grow pure populations of cells for analysis, screen-ing or drug discovery, investigators began to experiment with various ways of at least prop-agating cells in more defined conditions.
Initial attempts to replace serum with a serum-replacement solution and to use con-ditioned medium from fibroblasts rather than the fibroblasts themselves, together with the use of a substrate such as Matrigel or Geltrex (which themselves are far from defined) resulted in successful stable long-term culture of pluripotent cells. Building on this success, investigators determined the role of key growth factors secreted by feeder cells that could replace conditioned medium and identified the relevant attachment receptors on stem cells as well as key regulators of the cell cycle.
Over the past five years several second-generation stem cell culture media have been described6. These medium optimization efforts examined basal medium components, looked at novel pathways for self-renewal and
investigated the utility of adding antiapoptotic agents, altering cell cycle time or inhibiting dif-ferentiation using small molecules or defined factors. The use of such media made it pos-sible to standardize manufacturing processes; furthermore, it has led to better comparability across laboratories and to several key discover-ies about the basic biology of stem cells and the pluripotent state.
The International Stem Cell Initiative led a study7 that evaluated a variety of stem cell culture media and determined that at least two commercially available media8,9 ade-quately supported the feeder-free, serum-free propagation of a variety of pluripotent stem cell lines. These media work well, are robust, support a variety of lines, can be used with synthetic substrates and can be used for the initial generation and establishment of blas-tocyst- or somatic cell–derived pluripotent lines. Nevertheless these commercial media are expensive and cumbersome to use, and it is often difficult to adapt cells grown on feed-ers to growth in these media. There is thus an ongoing need to further simplify ESC cul-ture medium, with a notable previous attempt being that of Ding and colleagues, who had suggested that a basal medium with N2 and B27 supplements along with growth factors would be sufficient10.
In this issue of Nature Methods, Chen et al.4 report the systematic evaluation of candidate components in pluripotent stem cell culture media (Fig. 1). They develop a chemically defined medium that, coupled with a defined substrate, provides a complete solution for the culture of ESCs and for the derivation and propagation of integration-free induced pluripotent stem cells (iPSCs). The medium contains eight critical components for pluri-potent cell culture, making it far simpler than current formulations. Their systematic approach showed that many components pre-viously added to culture media are unneces-sary and that still other components are then
needed to mitigate their unwanted effects on pluripotent stem cell growth.
Perhaps the most intriguing result was the finding that serum albumin is neither required in stem cell culture medium nor is it necessary to substitute for this protein with an alternative. Addition of albumin or a substitute protein to culture medium in which serum is eliminated has been a dogma in the media world, and the data showing that cells grew better without this component were unexpected. It is intriguing that the overall formulation is very similar to previously reported media, but much simpler, suggesting that previous media were over-engineered. The authors noted that albumin was critical only to counteract the toxic effect of beta-mercaptoethanol, an antioxidant4. Antioxidants are widely used in cell culture, and this basic idea of simplification and albu-min removal may have widespread applicabil-ity to developing defined media formulations for the growth of other cell types as well.
Mahendra Rao is at Stem Cells and Regenerative Medicine, Life Technologies, Frederick, Maryland, USA. e-mail: [email protected]
iPSC
Neuralcells
Research Therapy?
Cardiaccells Hepatocytes
Cell from an individual
Chen et al.4
Simplified, definedculture medium
More efficientreprogramming
Okita et al.5
Figure 1 | Improvements at different steps of cellular reprogramming could be combined to efficiently generate iPSCs for basic and applied research or for cell therapy.
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390 | VOL.8 NO.5 | MAY 2011 | nature methods
In the same issue, Okita et al.5 report integration-free generation of iPSC lines, at higher efficiency than previously reported (Fig. 1). The authors showed that a three-vector EBNA-based system modified by using non-transforming L-MYC instead of c-MYC and by modulating the cell cycle with knockdown of TP53 (also known as p53) allowed them to obtain multiple iPSC lines. The authors used this approach to obtain integration-free iPSC lines from two human leukocyte anti-gen (HLA)-homozygous individuals5; as such these lines could serve as donors for 20% of the Japanese population.
It is exciting to speculate that one can now derive iPSCs from an identified individual using improved-efficiency, integration-free methods, in a chemically defined medium that improves the stability and propagation charac-teristics of these lines, to yield a master bank of cells for therapy in a large fraction of indi-viduals, with little or no immune suppression,
or for use in basic or applied research. These results may bring us one step closer to mak-ing regenerative medicine cost-effective for the average individual.
ComPetinG FinanCiaL interests The author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemethods/.
1. Thomson, J.A. et al. Science 282, 1145–1147 (1998).
2. Condic, M.L. & Rao, M. Stem Cells Dev. 19, 1121–1129 (2010).
3. Takahashi, K. et al. Cell 131, 861–872 (2007).4. Chen, G. et al. Nat. Methods 8, 424–429 (2011).5. Okita, K. et al. Nat. Methods 8, 409–411 (2011).6. Rajala, K. et al. Hum. Reprod. 22, 1231–1238
(2007). 7. International Stem Cell Initiative Consortium et al.
In Vitro Cell Dev. Biol. Anim. 46, 247–258 (2010).
8. Ludwig, T.E. et al. Nat. Methods 3, 637–646 (2006).
9. Wang, L. et al. Blood 110, 4111–4119 (2007).10. Yao, S. et al. Proc. Natl. Acad. Sci. USA 103,
6907–6912 (2006).
high-content screening: getting more from lessJ Philip McCoy Jr
A parallel microfluidic cytometer combines low-pixel-count, one-dimensional images with parallel-channel flow cytometry for high-speed, high-throughput screening of cells.
We often hear the adage ‘he who dies with the most [toys], wins’, but in recent times the opposite saying ‘he who needs the least, wins’ has become popular. These sayings summarize two very different approaches to life based on either maximizing or minimizing acquisition. Likewise, our approaches to science tend to follow divergent paths. On one hand, instru-ments and technologies are developed to cap-ture as much data as possible with the need for complex data analysis and/or subsequent data reduction to identify the salient data that can be used to address the pertinent questions. On the other hand, formulating focused, minimalistic approaches to gather only the most pertinent data for specific questions can be a power-ful approach as well. In this issue of Nature Methods, Daniel Ehrlich and colleagues provide an example of the latter by using low-pixel-count,
one-dimensional (1D) imaging (Fig. 1) combined with parallel microfluidics1. With less information acquired from each cell, data files are more manageable, analysis is easier, and throughput can be increased beyond tradi-tional high-content screening (HCS) analysis.
Contemporary approaches to drug dis-covery frequently use a combination of high-throughput screening and HCS or both. High-throughput screening measures the fluorescence of cellular features tagged with fluorochrome-conjugated antibodies or other fluorescent labels in a very rapid manner and is quite often performed by flow cytometry, a technique particularly well suited to this endeavor. Traditional flow cytometry can eas-ily capture emissions from ten or more differ-ent fluorochromes on a cell at rates exceeding 25,000 cells per second. Although this is
J. Philip McCoy Jr. is at the Flow Cytometry Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. e-mail: [email protected]
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