山中伸弥Cell Stem Cell倡议建立干细胞新标准

　　

生物通报道，iPS创建人之一山中伸弥（Shinya Yamanaka），哈佛大学干细胞研究中心的George Q.Daley以及麻省理工，白头医学研究所的科研人员在最近一期的Cell Stem Cell上公布一篇关于iPS细胞多能性判定的文章（标题：Broader Implications of Defining Standards for the Pluripotency of iPSCs）

 

2008年，Maherali 和Hochedlinger发表了一篇iPS细胞制备的指导文章，里面详细介绍了诱导多能性干细胞制备的标准程序。文章中尤其强调，要对诱导多能干细胞的多能性进行严格的检测。对人类的iPS细胞而言不仅要检测其多能性尤其要检测其致癌性。因此，研究界需要制定一定的标准来评估多能性。

 

多能性具有多种界定标准，有些标准十分严格，有些标准十分松，比如说，多种人体成体干细胞被认为具有多能性，羊膜衍生的干细胞也被认为具有多能性，骨髓成体干细胞也具有多能性，睾丸祖细胞也被认为有多能性。不同类型的干细胞具有不同的评判标准，这些结构导致对多能性的判定不稳定，无据可循。

 

因此，山中伸弥等人呼吁各研究者要更全面地研究iPS细胞，尤其对iPS细胞后期分化的信号转导，以及表观遗传修饰因子以及如何评估多能性的安全性做深入的调查。希望大家能携手一致建立一套评判的标准，全面鉴定评定干细胞的多能性。

（生物通 小茜）

链接地址：http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(09)00064-2

生物通推荐原文阅读

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We read with great interest the excellent review by Maherali and Hochedlinger, 2008 that recommends standards for characterization of pluripotent stem cell lines, especially the many new lines being generated using factor-based reprogramming techniques (induced pluripotent stem cells, or iPSCs). Of note was the suggestion that iPSCs should be assessed for functional differentiation through the highest-stringency test acceptable. For murine iPSCs, this means germline transmission following blastocyst chimerism, and for human iPSCs this means assessment of teratoma pathology. Given the fast pace of discovery in the field, the value and relevance of time-consuming characterization of cell lines are bound to be debated. We'd like to highlight what's at risk when the pressure for rapid publication erodes the imperative for applying rigorous and uniform standards before assigning the label iPSC to novel cell lines.

 

The term pluripotency can be assigned according to lax or stringent criteria. A diverse array of stem cell types have been labeled pluripotent: multipotential adult progenitor cells (MAPCs); amniotic fluid-derived stem cells (AFS); marrow-isolated adult multilineage inducible cells (MIAMI); testes-derived stem cells; and a variety of embryonic stem cells derived by parthenogenesis, blastomere culture, and somatic cell nuclear transfer. In the loosest sense, a pluripotent cell includes in its progeny elements of all three embryonic germ layers (ectoderm, endoderm, and mesoderm), regardless of experimental context. In the strictest sense, pluripotency pertains to cells whose progeny can reconstitute an entire organism, and is measured most stringently in the mouse using tetraploid embryo complementation (a standard achieved by only a limited subset of ESCs). To date, murine iPSCs have not yielded live pups in the tetraploid complementation assay, and thus the standard routinely applied is transmission of cells through the germline of chimeric animals to yield live pups (and not just gametes). Given practical and ethical limitations on testing of human embryonic stem cells, the gold standard for assessing pluripotency is the capacity to generate well-differentiated teratomas following injection into immunodeficient mice (Brivanlou etal., 2003). Although nonquantitative and subjective, teratoma histology in the hands of a skilled pathologist can distinguish tumors composed predominantly of poorly differentiated neuroectodermal elements from cystic masses composed of well-differentiated tissue from all three embryonic germ layers. The former behave as malignancies and are akin to teratocarcinomas, while the latter behave as encapsulated, benign masses and are true teratomas that arise from pluripotent stem cells (Lensch etal., 2007).

 

Importantly, in the mouse there are several types of embryo-derived stem cells that all share the most basic capacity for differentiation into all three germ layers: classical ESCs, epiblast-derived stem cells (EpiSCs), and Fibroblast Growth Factor/Activin/Bio cultivated stem cells (FAB-SCs), among them. These different types of embryo-derived stem cells behave differently when subjected to specific invivo assays, in some instances forming teratomas but failing the criterion of blastocyst chimerism and germline transmission. Choosing to ignore the need to clearly establish the behavior of these and other novel classes of stem cells has both scientific (and we might point out, political) consequences. MAPCs and AFS cells have been touted as viable alternatives to human ESCs. While these cells may be valuable for generating differentiated cell types invitro, they meet very different criteria for pluripotency. For nearlya decade after human ESCs were first isolated, stem cell scientists operated under the presumption that human ESCs were equivalent to mouse ESCs. Recent evidence gleaned from a careful comparison of growth factor requirements and behavior in various assays of differentiation now indicates that human ESCs are most similar to EpiSCs. How much do we know about iPSCs given that factor-based reprogramming is barely more than two years old? The initial iPSCs isolated by Takahashi and Yamanaka, 2006 behaved quite differently from the subsequently cultured lines; the initial iPSC lines chimerized embryos but didn't yield live pups or chimerize the germline. Partially reprogrammed colonies can be identified and pushed toward full pluripotency with subsequent chemical treatment (Meissner etal., 2008), and human colonies that might be mistaken for faithfully reprogrammed iPSCs indeed fail a number of criteria for pluripotency, including marker expression and formation of highly differentiated teratomas. In fact, the mere detection of cell-type-specific markers on cells grown in culture is a less stringent criterion for functional differentiation than the presence of well-differentiated cells in teratomas, as assessed by histological criteria.

 

Practitioners of reprogramming appreciate that the process produces a range of colony morphologies, and some that appear morphologically similar to ESCs do not share essential molecular features and behave quite differently in culture. Variability in epigenetic remodeling, the extent of methylation, and the persistence of expression of integrated proviruses all might alter the differentiation potential of iPSC lines. Our concern is not whether a cell line fails certain pluripotency criteria; the essential scientific imperative is that we know as much about the nature of the cell line as possible before we label it. When one lab produces a pluripotent cell using a particular protocol, and another lab produces pluripotent cells with a different protocol, we need to know whether the two different labs are producing comparable cell lines. Otherwise, our collective ability to make cross-lab comparisons will be significantly compromised. Applications in regenerative medicine may favor one type of pluripotent cell type over another; indeed, because of safety concerns, lines that don't form teratomas may be preferable as sources of cell products for clinical applications. However, if we accept lax rather than strict criteria before assigning the iPSC label, we deprive the label of its integrity and risk muddying the literature with data from a disparate array of diverse cell lines.

 

The field of factor-based reprogramming is in its infancy. For the foreseeable future, as we are learning more about what signaling pathways and epigenetic modifications distinguish one particular pluripotent stem cell state from another, and until such time as reliable molecular surrogates of the reprogrammed state can be validated, we believe it is best to encourage everyone to characterize their cells according to the most stringent test of pluripotency available. We need to understand the behavior of iPSCs in a standard set of assays to enable cross-lab comparisons and to move toward a deeper understanding of the molecular basis of pluripotency and lineage restriction.