By Vince Giuliano
The last seven months have seen a great deal of research related to and induced pluripotent stem cells (iPSCs) and cell reprogramming. This blog post is devoted to some important perspectives that have emerged resulting from that research. A subsequent blog entry will be devoted to the topic of longevity of stem cells and the roles stem cells play in human longevity.
The February 2011 publication Genomic instability in iPS: time for a break (Blasco et al) reports “Since their discovery, manuscripts characterizing properties of induced Pluripotent Stem (iPS) have flooded the literature. Among others, the analysis of the transcriptome and epigenome of iPS is now a recurrent theme that is helping to understand the molecular mechanisms behind reprogramming. Recent works have revealed that transcriptional and epigenetic reprogramming is often incomplete, which has raised some concerns on the nature of iPS. Inevitably, now the genome itself of iPS has been scrutinized; and the reports come with an unexpected twist: the presence of mutations in the genome of iPS. —.”
Continuing: “The term iPS was officially born, and has arguably become one of the fastest moving fields in biomedical research. However, a careful look at the original protocol raised the concern that one of the four factors included in the reprogramming cocktail was a well-known oncogene (Myc). In addition, reprogramming can also be stimulated by the presence of other oncogenes such as SV40 large T antigen (Mali et al, 2008) or by the loss of tumour suppressors like p53 or Arf (Menendez et al, 2010). To further fuel the concerns, developmental problems and tumours were reported in mice derived from iPS (Okita et al, 2007; Zhao et al, 2010). As a consequence, much of the recent works on iPS have been dedicated to the development of safer protocols such as defining an even more minimal set of factors that do not include Myc or the transient delivery of the reprogramming factors by non-integrating methods. Now, four independent works report on genomic analyses of iPS and reveal a worrisome presence of mutations in these cells.”
One of the works mentioned is the March 2011 publication is Somatic coding mutations in human induced pluripotent stem cells. “Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.”
A second relevant work is the May 2011 publication Genomic instability in induced stem cells. The work “looks at the problem from a cancer-angle. Given that oncogenes are known to generate a type of DNA damage known as replicative stress (RS) (Halazonetis et al, 2008), and that some of the reprogramming factors like c-Myc or Klf4 are known proto-oncogenes, they explored whether the reprogramming protocol could generate RS. In fact, a previous report had already shown that cells undergoing reprogramming presented a pan-nuclear phosphorylation pattern of histone H2AX, which is reminiscent of RS (Marion et al, 2009). In addition, DNA repair deficient cells show a poor reprogramming efficiency again, suggesting that some form of DNA damage could be generated during reprogramming. To evaluate this hypothesis, Pasi et al performed comparative genomic hybridization (cGH) analyses of iPS genomes. Their data show a significant number of chromosomal aberrations on iPS, which the authors suggest was in part influenced by the use of Myc. In fact, the authors report that whereas Myc is sufficient for the reprogramming of mammary progenitors into mammary stem cells, this protocol is accompanied by chromosomal abnormalities. Interestingly, this work revealed that the chromosomal rearrangements that occur during reprogramming frequently involved deletions mapping closely to known fragile sites, or to very large genes, supporting the concept that reprogramming could be accompanied by significant amounts of RS(ref).”
A third relevant 2011 paper is Copy number variation and selection during reprogramming to pluripotency. “Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.”
A fourth relevant work is the January 2011 publication Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. This work argues that all pluripotent stem cells exhibit genomic instability. As related by Blasco et al: “By performing a very comprehensive high-resolution SNP analysis of 189 pluripotent (iPS and ES) and 119 non-pluripotent samples, the authors found that the genomes of pluripotent cells are amazingly plastic, with frequent CNVs in pluripotency-related genes and pseudogenes. Noteworthy, the pattern of genomic aberrations was different in iPS or ES, again suggesting some intrinsic changes linked to the reprogramming process. The process of reprogramming led to small deletions, which included tumour suppressors, and which could be consistent with the idea of reprogramming-induced RS. However, time in culture led to the accumulation and selection of novel genomic aberrations in both iPS and ES, which were quantitatively of the same magnitude as those inflicted during reprogramming. This work illustrates the remarkable plasticity of pluripotent genomes and strongly suggests that the use of early passage lines should be an important factor to consider when working with pluripotent cells.” Further. “For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety(ref).”
IPSCs tend to be oncogenic
The problem of mutations in iPSCs poses as serious challenge to the use of such stem cells for human therapeutic purposes. The propensity of iPSCs to generate tumors has been known for some time. For example, see the 2009 review article The tumorigenicity of diploid and aneuploid human pluripotent stem cells and the 2011 article Stem cells: The dark side of induced pluripotency.
Another 2011 review article The tumorigenicity of human embryonic and induced pluripotent stem cells reports “Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity(ref).”
An April 2011 publication Intramyocardial transplantation of undifferentiated rat induced pluripotent stem cells causes tumorigenesis in the heart warns “Our study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease.”
A June 2011 publication Dissecting the Oncogenic Potential of Human Embryonic and Induced Pluripotent Stem Cell Derivatives reports “In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. — Overall, our findings suggest that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be performed before they can be used for regenerative therapy.”
Much is being learned about iPSCs. There is a great deal of variability among iPSCs depending on cell type of origin and reprogramming method and typically, variable pluripotency unequal to that of hESC counterparts.
The May 2011 publication The transcriptional and signalling networks of pluripotency reports “Pluripotency and self-renewal are the hallmarks of embryonic stem cells. This state is maintained by a network of transcription factors and is influenced by specific signalling pathways. Current evidence indicates that multiple pluripotent states can exist in vitro. Here we review the recent advances in studying the transcriptional regulatory networks that define pluripotency, and elaborate on how manipulation of signalling pathways can modulate pluripotent states to varying degrees.” This article gets into some of the exquisite detail involved in reversion to and maintenance of pluripotency. “These studies additionally revealed that in mESCs many of the key pluripotency-associated factors (Oct4, Sox2, Nanog, Esrrb, Sall4, Dax1, Klf2, Klf4, Klf5, Stat3 and Tcf3) may autoregulate their own expression21, 22, 23, 24, 25, 26, 32, 33, 34. It is possible that certain transcription factors directly downregulate the transcription of their own genes to prevent overactivation of gene expression. Overexpression of pluripotency-associated transcription factors has been shown to perturb the homeostasis of mESCs; for example, overexpression of Oct4 and Sox2 triggers differentiation16, 35. Hence, the continual activation of these genes may destabilize the mESC state. — Biological networks consist of highly connected nodes called hubs, which if removed would lead to fragmentation of the network. Some of the genes that constitute hubs receive extensive inputs. For example, the enhancer region of the Oct4 gene is bound by at least 14 transcription factors (Oct4, Sox2, Nanog, Sall4, Tcf3, Smad1, Stat3, Esrrb, Klf4, Klf2, Klf5, E2f1, n-Myc and Zfx), and the enhancer region of the Nanog gene is bound by at least 9 transcription factors (Nanog, Klf4, Klf2, Klf5, Sall4, E2f1, Esrrb, Stat3 and Tcfcp2l1; refs 23, 25, 36). These genomic sites serve as key contact points and represent the most crucial integration nexus within the transcriptional regulatory network. — There is also a correlation between the level of occupancy of gene promoters and transcriptional status. Genes bound by more transcription factors tend to be more actively transcribed, whereas genes with low level of transcription-factor occupancy are silenced in mESCs23, 24.”Increased numbers of transcription-factor-binding datasets coupled with precise measurement of gene expression could provide a more sophisticated and integrated analysis to reveal the underlying rules of ESC-specific gene regulation and the combinatorial nature of transcription factor regulation37, 38, 39.” Also, the article discusses the interface of the pluripotent transcription factor networks with histone modification, microRNAs and non-coding RNAs. A chart only for action of the Oct4 gene is:
The article concludes: “Several stem cell lines with key characteristics of pluripotency have been derived from mammalian embryos. Although these stem cells express transcription factors (Oct4, Sox2 and Nanog) that are classically associated with pluripotency, there are substantial differences in the features of the transcriptional regulatory networks that characterize them. Deciphering these networks is likely to provide new mechanistic insights into the regulation of pluripotent states. It is also evident that transcription factors are powerful modulators of pluripotent states as they can induce the transition between different states. Many of the methodologies at hand to convert or induce pluripotent states involve the use of chemical inhibitors targeting specific signalling pathways, highlighting the importance of understanding the roles of signalling through extrinsic factors. Overall, the combinatorial use of transcription factors and chemical modulators will enable the development of new approaches to shape cellular states, and possibly create novel ones.”
The March 2011 publication Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells reports “Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.”
The November 2011 publication A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity reports identifying factors relating to pluripotency in hESCs that can be looked for also in iPSCs. “The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.”
iPSCs can generate immune reactions even in individuals from which the source cells were taken
It has always been thought that iPSCs that are autologous to an individual (i.e. resulting from reprogramming of cells from an individual and then re-introduced into that same individual) would not initiate an immune system rejection response. Surprisingly, not so! At least, not so in our mouse cousins. The June 2011 publication Immunogenicity of induced pluripotent stem cells reports “Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells1, 2, 3, 4, 5. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.”
Incomplete epigenetic reversal appears to be a characteristic of iPSCs
A major reason why iPSCs generated through most known methods fail to exhibit full hESC-type pluripotency is that epigenetic markers of the source cell types are not completely wiped out. The March 2011 publication Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells relates to this point. “Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chromosome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming.”
Micro RNAs can be used for reprogramming to generate iPSCs
“Reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs) was first achieved by ectopic expression of OCT4, SOX2, KLF4 and c-MYC or OCT4, SOX2, LIN28 and NANOG87, 88, 89. Using the same approach, Ding and colleagues infected human fibroblasts with OCT4, SOX2, NANOG and LIN28 to generate iPSCs90. Instead of using conventional hESC culture conditions, mESC medium with human LIF was then used to select the reprogrammed cells. These cells were maintained with a combination of chemical inhibitors (MEK, ALK5 and GSK3 inhibitors)(ref).” Subsequently a number of alternative approaches have been developed for reprogramming into iPSCs. I reported on some of those approaches a little less than a year ago in the blog entry Induced pluripotent stem cells – developments on the road to big-time utilization. One of the latest approaches involves the use of micro RNAs.
The April 2011 publication Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells reports “The embryonic stem cell–specific cell cycle–regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells1. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle2, 3. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.”
LincRNAs are are direct targets of key pluripotency transcription factors and involved in reprogramming to iPSCs
The December 2010 publication Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells reports: “The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome, resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells, suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches, we found that one such lincRNA (lincRNA-RoR) modulates reprogramming, thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells.”
A few observations
This has been a selective reporting on iPSC research published in the last seven months. Even though I have covered a fair number of publications here, there is much additional ground that I might cover in later blog entries. In particular I would like to provide an update on advances in other forms of cell reprogramming and, as I stated above, I plan soon to generate a subsequent blog entry devoted to the topic of longevity of stem cells and the roles stem cells play in human longevity. I will also do a blog post at some point on progress in directing iPSC and hESC differentiation into other somatic cell types.
The nature of stem cells of all kinds, the broad area of cell reprogramming, and the generation of iPSCs are topics on the cutting edge of research in biology. The rate of research in these areas is accelerating mightily and a lot is being learned.
The complexity in these areas is extraordinary. Involved are a large number of renewal genes, signaling pathways, transcription factors and co-factors, lincRNAs and microRNAs, pluripotency factors, histone acetylation and DNA methylation, other chromatin remodeling, pathways involving mitochondria, cell senescence and repair mechanisms and factors still being discovered.. I don’t think this should be too surprising. We should not be impatient since what is being discovered, finally, is the nature of life itself.
We are probably close to the start of the journey which may take decades before we are reasonably near the end of understanding most what we really need to know to promote health and extend life.
For the present iPSCs are useful for in-vitro and animal studies and for modeling the effects of drugs(ref), but they are still beset with multiple problems identified above that means they cannot yet be used for human therapeutic purposes. My vision of closing the loop in the stem cell supply chain remains as a possibility for the future but many obstacles must be overcome before that possibility matures into reality.