Reprogrammed cells, confirm your pluripotence!
According to the study, the results of which were published on October 11 in the online version of Nature Biotechnology in the article "Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells", scientists should pay more attention to the identification of pluripotency of reprogrammed human cells. The authors formulate strict molecular criteria for pluripotency, separately emphasizing that the use of only one marker in this case is completely unacceptable. In addition, in the article you can find a detailed description of the methods most suitable for the task.
Scientists are increasingly improving in the transformation of adult human cells into stem cells with properties similar to those of embryonic cells – the so-called induced pluripotent stem cells (iPSCs). However, it is often quite difficult to prove that the resulting cells have actually reached the desired level of pluripotency. Experimenters working with mouse cells use molecular tags integrated into pluripotency-associated genes such as Fbx15, Oct4 or Nanog. Unfortunately, no adequate analogues for working with human cells have been found to date. According to one of the authors, George Daley from the Harvard Stem Cell Institute, it is obvious that visual examination of cells under a microscope is completely insufficient; in addition, traditional markers of pluripotency can also be misleading.
Using the methods of visualization of living cells, the authors traced the changes in the expression of various pluripotency markers during the reprogramming process and characterized the cell types they isolated based on their morphology and molecular characteristics. At the beginning of the experiment, tens of thousands of human fibroblasts were placed in each well of the tablet, almost all of which as a result formed colonies morphologically indistinguishable from embryonic stem cells. However, a series of in vitro and in vivo tests showed that only a small number of the resulting cells were truly pluripotent.
Based on the imaging results, the authors divided all colonies morphologically indistinguishable from embryonic stem cell colonies into three types, called types I, II and III. Cells of all three types were injected into immunodeficient mice to test their ability to form teratomas (tumors consisting of all types of human tissue) – this method is one of the "gold standards" for testing the pluripotency of human stem cells. As a result, type II and type III cells formed tumors whose cells differentiated into tissues of different germ layers. The degree of observed differentiation varied, but in any case it is a sign of a certain degree of pluripotency. At the same time, a number of epigenetic factors, including hypomethylation of promoter genes for NANOG and OCT4, as well as histone modifications characteristic of the pluripotent state, were found only in type III cells.
According to one of the authors, a biologist from Boston Children's Hospital Thorsten Schlaeger, the purpose of the work is to demonstrate to all researchers working with induced pluripotent stem cells the need for a thorough multi-stage verification of their pluripotency, including tests for the ability to form teratomas, as well as the study of epigenetic traits and gene expression. This is quite difficult, but it is necessary to avoid problems, misunderstandings and conflicting results.
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of The Scientist: How to ID human pluripotency.
21.10.2009