Large Screening and Analyses of Established Induced Pluripotent Stem Cell Lines Finds Rogue Lines


Induced pluripotent stem cells (iPSCs) have come a long way since the first lines were made by Shinya Yamanaka and his colleagues in 2006. Initial successes of iPSCs in animal models generated a good deal of hope that iPSCs might find a place in the annals of regenerative medicine. However, since that time, further work has created doubts about the safety of these cells, since some, though admittedly not all, iPSC lines show some genetic abnormalities. However, as screening techniques have become better and have increased in sensitivity, the possibility of accurately ascertaining the quality of iPSC lines draws closer and closer.

A new paper that appeared in the June 9 edition of the journal Stem Cell Reports by Carolyn Lutzko and others from a multi-institutional research group known as the Progenitor Cell Biology Consortium, have used these new screening technologies to screen large numbers of established iPSC lines. The results were somewhat sobering; about 30 percent of iPSC lines analyzed from 10 research institutions were genetically unstable and not safe for clinical use.

This work comprehensively characterized of a large collection of iPSC lines. The technology to produce safe and effective iPSCs exists. Nevertheless, this does not mean that all iPSC lines were produced safely and effectively. In this paper, Lutzko and her colleagues discovered that some iPSC lines that were made with inferior protocols. Some iPSC lines were contaminated with bacteria or carried mutations associated with cancer.

“It was very surprising to us the high number of unstable cell lines identified in the study, which highlights the importance of setting safety standards for stem cell therapies,” said Carolyn Lutzko, PhD, senior author and director of translational development in the Translational Core Laboratories at Cincinnati Children’s Hospital Medical Center. “A good number of the cell lines we studied met quality standards, although the unexpected number of lines that did not meet these standards could not be used for clinical therapies.”

In this paper, Lutzko and her collaborators compared 58 different iPSC lines that had been submitted by various research institutions. The cells were generated with a variety of genes, methods and cells of origin that ranged from skin fibroblasts to infant cord blood cells. All iPSC lines were analyzed for genetic stability, degree of pluripotency, and several other scientific criteria.

In order for an iPSC line to be considered for clinical work, they must exhibit a high degree of genetic stability. Genetically unstable iPSC lines run the risk of form derivatives that can become cancerous, show poor survival, or differentiate into unwanted cell types upon transplantation. It also is essential that iPSC lines exhibit the ability to continuously renew and expand without losing pluripotency or introducing new genetic mutations.

All iPSC lines were also compared to human embryonic stem cell lines in order to compare them to an outside standard.

How did these 58 iPSC lines fare in this rather exacting gauntlet of tests? It depended on several factors. First of all the cell of origin was very important. Skin fibroblasts tended to make rather low-quality iPSC lines, on the average, but cord blood stem cells usually made rather high-quality iPSC lines. Additionally, the specific reprogramming method employed also made a difference. Some of the iPSC lines included in the test were reprogrammed by means of viruses that integrate into the genome of the host cell (24%). Others were reprogrammed with plasmids (64%), which do not integrate into the host cell genome and are lost soon after reprogramming and growth occurs. Others were reprogrammed with modified RNAs (7%), and a few others (5%) were reprogrammed with other types of viruses that do not integrate into the genome of the host cell (Sendai virus). In all cases, the iPSC lines were made by introducing genes into a mature cell that drove that cell to de-differentiate and grow. Slightly different cocktails of genes were used, but the results were largely the same – the induction of pluripotency.  On the average, non-integrating methods of introducing reprogramming genes into cells resulted in higher-quality iPSC lines, with a few notable exceptions.

Pluripotency for each iPSC line was tested by means of implanting undifferentiated iPSCs into nude mice and observing the cells form differentiated tumors called “teratomas.” Teratomas contain tissues derived from all three primary germ layers; endoderm (gut region), ectoderm (epidermis, nerve tissue, etc.) and mesoderm (muscles, blood cells, etc.).

Prior to this study, the prevailing view was that low-quality iPSC lines were not pluripotent and could not form proper teratomas. This hypothesis had not been tested because of the expense of implanting all these iPSC lines into nude mice. To test this hypothesis, Lutzko and her colleagues tested if all iPSC lines, both high and low quality lines, could generate teratomas. Their tests showed that both genetically stable and unstable iPSC lines formed teratomas with cells from all three germ layers. Although genetically unstable iPSC lines demonstrated pluripotency, the concern in a clinical context would be that they also could result in cancer – again emphasizing the need for safe reprogramming methods, according to study authors.

The enormous amount of data generated by these experiments required sophisticated computing for high-level computational analyses. First author, Nathan Salomonis, PhD, a researcher in the Division of Biomedical Informatics at Cincinnati Children’s. Salomonis used computational approaches to collate, examine, and analyze the data and produce large data sets that can compare the different methods of cell programming, the differences in gene regulation between lines, and the functional quality of each iPSC line.

According to Salomonis, his robust data sets uncovered those iPSC lines that had lost their ability to differentiate into particular adult cell types. This massive collection of raw processed data is available through the online web database.

Salomonis said that, in the future, members of this research consortium will test the ability of each iPSCs line to differentiate into specific cell types – such as brain, heart, lung and other cells in the human body. After these data are verified and published, this information will be added to the online database as a public resource.

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mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).