Broader Implications of Defining Standards for the Pluripotency of iPSCs

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Citation Daley, George Q., M. William Lensch, Rudolf Jaenisch, Alex Meissner, Kathrin Plath, and Shinya Yamanaka. “Broader Implications of Defining Standards for the Pluripotency of iPSCs.” Cell Stem Cell 4, no. 3 (March 2009): 200–201. © 2009 Elsevier Inc.

As Published http://dx.doi.org/10.1016/j.stem.2009.02.009

Publisher Elsevier B.V.

Version Final published version

Citable link http://hdl.handle.net/1721.1/96298

Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Cell Stem Cell Letter

Broader Implications of Defining Standards for the Pluripotency of iPSCs

George Q. Daley,1,2,* M. William Lensch,1,2 Rudolf Jaenisch,3 Alex Meissner,2,4 Kathrin Plath,5 and Shinya Yamanaka6,7 1Children’s Hospital Boston, Division of Hematology/Oncology, 300 Longwood Avenue, Boston, MA 02115, USA 2Harvard Stem Cell Institute, 42 Church Street, Cambridge, MA 02138, USA 3Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, 9 Cambridge Center, Cambridge, MA 02142, USA 4Department of Stem Cell and Regenerative Biology, Harvard University, 42 Church Street, Cambridge, MA 02138, USA 5University of California, School of Medicine, Department of Biological Chemistry, The Broad Center for Regenerative Medicine and Stem Cell Research, 615 Charles E. Young Drive South, Los Angeles, CA 90095, USA 6Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan 7Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158, USA *Correspondence: [email protected] DOI 10.1016/j.stem.2009.02.009

We read with great interest the excellent whose progeny can reconstitute an entire teratomas but failing the criterion of blasto- review by Maherali and Hochedlinger organism, and is measured most strin- cyst chimerism and germline transmission. (2008) that recommends standards for gently in the mouse using tetraploid Choosing to ignore the need to clearly characterization of pluripotent stem cell embryo complementation (a standard establish the behavior of these and other lines, especially the many new lines being achieved by only a limited subset of novel classes of stem cells has both scien- generated using factor-based reprogram- ESCs). To date, murine iPSCs have not tific (and we might point out, political) ming techniques (induced pluripotent yielded live pups in the tetraploid comple- consequences. MAPCs and AFS cells stem cells, or iPSCs). Of note was the mentation assay, and thus the standard have been touted as viable alternatives to suggestion that iPSCs should be assessed routinely applied is transmission of cells human ESCs. While these cells may be for ‘‘functional differentiation through the through the germline of chimeric animals valuable for generating differentiated cell highest-stringency test acceptable.’’ For to yield live pups (and not just gametes). types in vitro, they meet very different murine iPSCs, this means germline trans- Given practical and ethical limitations on criteria for pluripotency. For nearly a mission following blastocyst chimerism, testing of human embryonic stem cells, decade after human ESCs were first and for human iPSCs this means assess- the gold standard for assessing pluripo- isolated, stem cell scientists operated ment of teratoma pathology. Given the tency is the capacity to generate well- under the presumption that human ESCs fast pace of discovery in the field, the value differentiated teratomas following injec- were equivalent to mouse ESCs. Recent and relevance of time-consuming charac- tion into immunodeficient mice (Brivanlou evidence gleaned from a careful compar- terization of cell lines are bound to be et al., 2003). Although nonquantitative ison of growth factor requirements and debated. We’d like to highlight what’s at and subjective, teratoma histology in the behavior in various assays of differentiation risk when the pressure for rapid publica- hands of a skilled pathologist can distin- now indicates that human ESCs are most tion erodes the imperative for applying guish tumors composed predominantly similar to EpiSCs. How much do we know rigorous and uniform standards before of poorly differentiated neuroectodermal about iPSCs given that factor-based re- assigning the label ‘‘iPSC’’ to novel cell elements from cystic masses composed programming is barely more than two years lines. of well-differentiated tissue from all three old? The initial iPSCs isolated by Takahashi The term ‘‘pluripotency’’ can be as- embryonic germ layers. The former and Yamanaka (2006) behaved quite differ- signed according to lax or stringent behave as malignancies and are akin to ently from the subsequently cultured lines; criteria. A diverse array of stem cell types teratocarcinomas, while the latter behave the initial iPSC lines chimerized embryos have been labeled pluripotent: multipo- as encapsulated, benign masses and are but didn’t yield live pups or chimerize the tential adult progenitor cells (MAPCs); true teratomas that arise from pluripotent germline. Partially reprogrammed colonies amniotic fluid-derived stem cells (AFS); stem cells (Lensch et al., 2007). can be identified and pushed toward full marrow-isolated adult multilineage induc- Importantly, in the mouse there are pluripotency with subsequent chemical ible cells (MIAMI); testes-derived stem several types of embryo-derived stem cells treatment (Meissner et al., 2008), and cells; and a variety of embryonic stem cells that all share the most basic capacity for human colonies that might be mistaken derived by parthenogenesis, blastomere differentiation into all three germ layers: for faithfully reprogrammed iPSCs indeed culture, and somatic cell nuclear transfer. classical ESCs, epiblast-derived stem cells fail a number of criteria for pluripotency, In the loosest sense, a pluripotent cell (EpiSCs), and Fibroblast Growth Factor/ including marker expression and formation includes in its progeny elements of all Activin/Bio cultivated stem cells (FAB- of highly differentiated teratomas. In fact, three embryonic germ layers (ectoderm, SCs), among them. These different types the mere detection of cell-type-specific endoderm, and mesoderm), regardless of embryo-derived stem cells behave markers on cells grown in culture is of experimental context. In the strictest differently when subjected to specific a less stringent criterion for functional sense, pluripotency pertains to cells in vivo assays, in some instances forming differentiation than the presence of

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well-differentiated cells in teratomas, as lab comparisons will be significantly of pluripotency available. We need to assessed by histological criteria. compromised. Applications in regenera- understand the behavior of iPSCs in Practitioners of reprogramming appre- tive medicine may favor one type of plurip- a standard set of assays to enable cross- ciate that the process produces a range otent cell type over another; indeed, lab comparisons and to move toward of colony morphologies, and some that because of safety concerns, lines that a deeper understanding of the molecular appear morphologically similar to ESCs don’t form teratomas may be preferable basis of pluripotency and lineage restric- do not share essential molecular features as sources of cell products for clinical tion. and behave quite differently in culture. applications. However, if we accept lax Variability in epigenetic remodeling, the rather than strict criteria before assigning extent of methylation, and the persistence the ‘‘iPSC’’ label, we deprive the label of REFERENCES of expression of integrated proviruses all its integrity and risk muddying the litera- might alter the differentiation potential of ture with data from a disparate array of Brivanlou, A.H., Gage, F.H., Jaenisch, R., Jessell, iPSC lines. Our concern is not whether diverse cell lines. T., Melton, D., and Rossant, J. (2003). Science 300, 913–916. a cell line fails certain pluripotency criteria; The field of factor-based reprogram- the essential scientific imperative is that ming is in its infancy. For the foreseeable Lensch, M.W., Schlaeger, T.M., Zon, L.I., and we know as much about the nature of the future, as we are learning more about Daley, G.Q. (2007). Cell Stem Cell 1, 253–258. cell line as possible before we label it. what signaling pathways and epigenetic Maherali, N., and Hochedlinger, K. (2008). Cell When one lab produces a pluripotent cell modifications distinguish one particular Stem Cell 3, 595–605. using a particular protocol, and another pluripotent stem cell state from another, Meissner, A., Mikkelsen, T.S., Gu, H., Wernig, M., lab produces pluripotent cells with and until such time as reliable molecular Hanna, J., Sivachenko, A., Zhang, X., Bernstein, a different protocol, we need to know surrogates of the reprogrammed state B.E., Nusbaum, C., Jaffe, D.B., et al. (2008). Nature whether the two different labs are can be validated, we believe it is best to 454, 766–770. producing comparable cell lines. Other- encourage everyone to characterize their Takahashi, K., and Yamanaka, S. (2006). Cell 126, wise, our collective ability to make cross- cells according to the most stringent test 663–676.

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