The Stem Cell “Tunnel” Effect
Authors: Thomas P Zwaka
Journal: Molecular Therapy (2010)

In this issue, Kane et al. suggest that induction of pluripotency from terminally differentiated cells may not always require reprogramming factors but might be prompted by integration of an empty retroviral vector alone.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar These findings have potential implications for the safety of induced pluripotent stem (iPS) cells as well as for our understanding of the fundamental molecular principles underlying pluripotency.It almost sounds like a control experiment gone wrong: iPS cells surface in a petri dish treated with empty vector retrovirus. And yet, as unlikely as this may appear, Kane and co-workers report just that. The result they describe is reminiscent of the “tunnel effect” in quantum physics, whereby a particle can “tunnel” through any barrier, but with a very low probability. According to this new report, somatic cells apparently can, on rare occasions, tunnel through the epigenetic barrier that normally prevents dedifferentiation into the primitive blank state of pluripotentiality.Ironically, shortly after the first wave of reprogramming articles,2Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18486) Google Scholar,3Okita K Ichisaka T Yamanaka S Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3499) Google Scholar,4Wernig M Meissner A Foreman R Brambrink T Ku M Hochedlinger K et al.In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.Nature. 2007; 448: 318-324Crossref PubMed Scopus (2204) Google Scholar there was some discussion of the possibility that, in addition to the direct reprogramming activity of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc, positional mutagenesis due to retroviral integration could play at least a supportive role in the reprogramming process. Lending additional credence to this possibility was the fact that the early studies, which all used retrovirus-mediated gene transfer to deliver the reprogramming factors, commonly reported a relatively high number of integration sites.3Okita K Ichisaka T Yamanaka S Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3499) Google Scholar Although subsequent careful and systematic mapping of the retroviral landing spots did not reveal any elite integration sites,5Varas F Stadtfeld M de Andres-Aguayo L Maherali N di Tullio A Pantano L et al.Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.Stem Cells. 2009; 27: 300-306Crossref PubMed Scopus (50) Google Scholar,6Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; 321: 699-702Crossref PubMed Scopus (859) Google Scholar this issue remained a subject of debate.7Hawley RG Does retroviral insertional mutagenesis play a role in the generation of induced pluripotent stem cells.Mol Ther. 2008; 16: 1354-1355Abstract Full Text Full Text PDF PubMed Scopus (27) Google ScholarKane and colleagues now report that a relatively high dose of retrovirus alone'i.e., without reprogramming factors'provoked the emergence of iPS-like cells in three of six human fibroblast cell lines tested.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Follow-up experiments revealed that the cell lines obtained under such circumstances not only resembled human iPS and embryonic stem cells morphologically but were apparently capable of differentiating into the three principal germ layers both in vitro, after embryoid body formation, and in vivo, after injection into immunocompromised mice, in which they formed teratocarcinomas at the injection site. These properties are the hallmarks of pluripotentiality and satisfy the criteria typically applied experimentally. Although such experiments are critical, additional follow-up studies will be needed to fully gauge the pluripotency of these cells. These should include an assessment of the DNA methylation patterns in the promoter regions of the Oct4 and Nanog genes, tests to establish that clonally obtained cell lines retain the potential to differentiate into the three principal germ layers, determination of telomerase activity, and confirmation that physiologically functional cell types (e.g., beating cardiac myocytes and neurons with characteristic action potentials) can be obtained. Finally, future studies would need to carefully and rigorously compare the differentiation potential of these new iPS-like cells to other well-established “conventional” iPS and human embryonic stem cells.Could the retroviral integration process play a functional role in this unorthodox reprogramming process? The authors argue “yes,” citing their identification of certain genes previously associated with pluripotency near the viral genomic integration sites. However, this observation cannot distinguish between a possible epiphenomenon and a truly mechanistic association. As it currently stands, this study provokes a number of questions. First, did the lentivirus indeed play a causal role? The authors argue that published studies using nonintegrative means to deliver reprogramming factors are plagued by extremely low efficiencies; however, a recent study reported extremely high and efficient reprogramming using direct delivery of reprogramming factors via messenger RNA.8Warren L Manos PD Ahfeldt T Loh YH Li H Lau F et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.Cell Stem Cell. 2010; (e-pub ahead of print 29 September 2010)PubMed Google Scholar Second, was the retrovirus needed at all, or would a spontaneous conversion have occurred even in untreated cells, albeit at a much lower rate? Could viral infection potentially activate an extremely small subset of fibroblasts that already expressed some or all of the reprogramming factors? Studies suggest that reprogramming efficiency increases if some of the reprogramming factors are already present in the cell types to be reprogrammed.9Kim JB Greber B Araúzo-Bravo MJ Meyer J Park KI Zaehres H et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-653Crossref PubMed Scopus (557) Google Scholar It will also be important to assess the significance of the massive chromosomal abnormalities observed. Are these mere by-products, or are they necessary contributors to the reprogramming process? Does retroviral mutagenesis provoke such aberrations? A recent report describing an alarmingly high frequency of chromosome abnormalities in iPS cells suggests that it does.10Mayshar Y Ben-David U Lavon N Biancotti J-C Yakir B Clark AT et al.Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (591) Google ScholarAnother important question is whether retrovirus-mediated reprogramming poses additional poorly understood risks. And might it, in and of itself, affect cell fate and phenotype? The answers to these questions could turn out to be relevant not only for the nascent reprogramming field but also for other gene therapy studies in which retroviral gene transfer methods are utilized therapeutically. Although there appears to be a general consensus that integration-free reprogramming methods would be safer in a clinical setting, it has also been acknowledged that, at least in initial clinical trials that utilize iPS cell technology, the ability to specifically trace genetically marked, transplanted cells is extraordinarily useful for assessing safety and efficacy.11Ellis J Baum C Benvenisty N Mostoslavsky G Okano H Stanford WL et al.Benefits of utilizing gene-modified iPSCs for clinical applications.Cell Stem Cell. 2010; 7: 429-430Abstract Full Text Full Text PDF PubMed Scopus (17) Google ScholarThe most worrisome aspect of the study by Kane et al.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar is also the most intriguing: if rigorously confirmed, the described ability to obtain iPS cells without reprogramming factors severely challenges our understanding of the reprogramming process, compromising the current view that sequential stepwise activation of individual reprogramming factors fulfills very specific defined roles. Instead, it implies that stochasticity could play a much greater role than we currently assume. In this issue, Kane et al. suggest that induction of pluripotency from terminally differentiated cells may not always require reprogramming factors but might be prompted by integration of an empty retroviral vector alone.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar These findings have potential implications for the safety of induced pluripotent stem (iPS) cells as well as for our understanding of the fundamental molecular principles underlying pluripotency. It almost sounds like a control experiment gone wrong: iPS cells surface in a petri dish treated with empty vector retrovirus. And yet, as unlikely as this may appear, Kane and co-workers report just that. The result they describe is reminiscent of the “tunnel effect” in quantum physics, whereby a particle can “tunnel” through any barrier, but with a very low probability. According to this new report, somatic cells apparently can, on rare occasions, tunnel through the epigenetic barrier that normally prevents dedifferentiation into the primitive blank state of pluripotentiality. Ironically, shortly after the first wave of reprogramming articles,2Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18486) Google Scholar,3Okita K Ichisaka T Yamanaka S Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3499) Google Scholar,4Wernig M Meissner A Foreman R Brambrink T Ku M Hochedlinger K et al.In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.Nature. 2007; 448: 318-324Crossref PubMed Scopus (2204) Google Scholar there was some discussion of the possibility that, in addition to the direct reprogramming activity of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc, positional mutagenesis due to retroviral integration could play at least a supportive role in the reprogramming process. Lending additional credence to this possibility was the fact that the early studies, which all used retrovirus-mediated gene transfer to deliver the reprogramming factors, commonly reported a relatively high number of integration sites.3Okita K Ichisaka T Yamanaka S Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3499) Google Scholar Although subsequent careful and systematic mapping of the retroviral landing spots did not reveal any elite integration sites,5Varas F Stadtfeld M de Andres-Aguayo L Maherali N di Tullio A Pantano L et al.Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.Stem Cells. 2009; 27: 300-306Crossref PubMed Scopus (50) Google Scholar,6Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; 321: 699-702Crossref PubMed Scopus (859) Google Scholar this issue remained a subject of debate.7Hawley RG Does retroviral insertional mutagenesis play a role in the generation of induced pluripotent stem cells.Mol Ther. 2008; 16: 1354-1355Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Kane and colleagues now report that a relatively high dose of retrovirus alone'i.e., without reprogramming factors'provoked the emergence of iPS-like cells in three of six human fibroblast cell lines tested.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Follow-up experiments revealed that the cell lines obtained under such circumstances not only resembled human iPS and embryonic stem cells morphologically but were apparently capable of differentiating into the three principal germ layers both in vitro, after embryoid body formation, and in vivo, after injection into immunocompromised mice, in which they formed teratocarcinomas at the injection site. These properties are the hallmarks of pluripotentiality and satisfy the criteria typically applied experimentally. Although such experiments are critical, additional follow-up studies will be needed to fully gauge the pluripotency of these cells. These should include an assessment of the DNA methylation patterns in the promoter regions of the Oct4 and Nanog genes, tests to establish that clonally obtained cell lines retain the potential to differentiate into the three principal germ layers, determination of telomerase activity, and confirmation that physiologically functional cell types (e.g., beating cardiac myocytes and neurons with characteristic action potentials) can be obtained. Finally, future studies would need to carefully and rigorously compare the differentiation potential of these new iPS-like cells to other well-established “conventional” iPS and human embryonic stem cells. Could the retroviral integration process play a functional role in this unorthodox reprogramming process? The authors argue “yes,” citing their identification of certain genes previously associated with pluripotency near the viral genomic integration sites. However, this observation cannot distinguish between a possible epiphenomenon and a truly mechanistic association. As it currently stands, this study provokes a number of questions. First, did the lentivirus indeed play a causal role? The authors argue that published studies using nonintegrative means to deliver reprogramming factors are plagued by extremely low efficiencies; however, a recent study reported extremely high and efficient reprogramming using direct delivery of reprogramming factors via messenger RNA.8Warren L Manos PD Ahfeldt T Loh YH Li H Lau F et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.Cell Stem Cell. 2010; (e-pub ahead of print 29 September 2010)PubMed Google Scholar Second, was the retrovirus needed at all, or would a spontaneous conversion have occurred even in untreated cells, albeit at a much lower rate? Could viral infection potentially activate an extremely small subset of fibroblasts that already expressed some or all of the reprogramming factors? Studies suggest that reprogramming efficiency increases if some of the reprogramming factors are already present in the cell types to be reprogrammed.9Kim JB Greber B Araúzo-Bravo MJ Meyer J Park KI Zaehres H et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-653Crossref PubMed Scopus (557) Google Scholar It will also be important to assess the significance of the massive chromosomal abnormalities observed. Are these mere by-products, or are they necessary contributors to the reprogramming process? Does retroviral mutagenesis provoke such aberrations? A recent report describing an alarmingly high frequency of chromosome abnormalities in iPS cells suggests that it does.10Mayshar Y Ben-David U Lavon N Biancotti J-C Yakir B Clark AT et al.Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar Another important question is whether retrovirus-mediated reprogramming poses additional poorly understood risks. And might it, in and of itself, affect cell fate and phenotype? The answers to these questions could turn out to be relevant not only for the nascent reprogramming field but also for other gene therapy studies in which retroviral gene transfer methods are utilized therapeutically. Although there appears to be a general consensus that integration-free reprogramming methods would be safer in a clinical setting, it has also been acknowledged that, at least in initial clinical trials that utilize iPS cell technology, the ability to specifically trace genetically marked, transplanted cells is extraordinarily useful for assessing safety and efficacy.11Ellis J Baum C Benvenisty N Mostoslavsky G Okano H Stanford WL et al.Benefits of utilizing gene-modified iPSCs for clinical applications.Cell Stem Cell. 2010; 7: 429-430Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The most worrisome aspect of the study by Kane et al.1Kane NM Nowrouzi A Mukherjee S Blundell MP Greig JA Lee WK et al.Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.Mol Ther. 2010; 18: 2139-2145Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar is also the most intriguing: if rigorously confirmed, the described ability to obtain iPS cells without reprogramming factors severely challenges our understanding of the reprogramming process, compromising the current view that sequential stepwise activation of individual reprogramming factors fulfills very specific defined roles. Instead, it implies that stochasticity could play a much greater role than we currently assume.