Mélanie Bilodeau¹, and Guy Sauvageau ^{1, 2}

¹ Laboratory of Molecular Genetics of Stem Cells, Institut de Recherche en Immunologie et Cancérologie (IRIC), C.P. 6128 succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.

² Department of Medicine and Division of Hematology and Leukemia Cell Bank of Quebec Maisonneuve-Rosemont Hospital, Montréal, Québec, Canada.

E-mail:  guy.sauvageau@umontreal.ca

NetworkEditor's Perspective: "Gene Transcription yields both proteins and non-coding RNAs".
Abstract:
Introduction:
Constraints:
Sufficiency:
mRNA knockdown:
Noncoding RNA:
Fig.1: Candidate transcription factors that determine stem-cell identity.
References:
Additional References:
Further Topics:
Other Links:
Further Information:

Understanding how self-renewal and pluripotency — two key characteristics of stem cells — are controlled may allow generation of stem cell lines from somatic tissues, thus avoiding the ethically contentious need to derive them from embryos. A step forward in this understanding was recently taken by two teams, who exploited recombinant retroviruses in gain-and-loss of function experiments to characterize candidate transcription factors with the potential to regulate 'stemness'.

For years, many predicted that understanding the properties of murine and human embryonic stem cells (mES and hES cells, respectively) could lead to the design of cell replacement therapies [1]. Using a list of candidate factors, Takahashi and Yamanaka [2] show that a specific combination of only four factors allows the generation of pluripotent stem cells from mouse adult fibroblast cultures, avoiding the controversial need for deriving them from an embryo. Ivanova et al. [3] chose a RNA interference (RNAi) strategy to identify known and novel transcriptional regulators from two distinct pathways that control mES cell self-renewal [3].

Both cell extrinsic and intrinsic factors regulate mES cell self-renewal (indefinite cell division maintaining stem-cell characteristics) and pluripotency (the ability of a single stem cell to form all lineages). In terms of cell extrinsic factors, mES cell maintenance in vitro requires the presence of the leukaemia inhibitory factor (LIF) and bone morphogenic protein (BMP), which signal through Stat [3] and Smad proteins, respectively [4]. Evidence indicates that Wnt signalling is also involved [4]. The cell intrinsic machinery includes the transcription factors Oct4, Nanog and Sox2 (ref. 5). The pluripotent state may also be regulated by epigenetic mechanisms involving members of the Polycomb group proteins [5].

Until now, only two relatively inefficient methods have allowed somatic-cell reprogramming to a pluripotent state: nuclear transfer and cell fusion. Interestingly, reprogramming efficiency is influenced by the developmental and/or epigenetic stage of the donor cell [6] and may be enhanced by ectopic expression of Nanog [7]. Importantly, the exact blend of factors necessary to reprogramme a cell remained unknown until recently [2]. Takahashi and Yamanaka selected 24 candidate genes, either with documented roles in ES self-renewal and pluripotency, or that were specifically expressed in these cells. These factors were expressed alone, or in combination, using retroviral gene transfer in mouse embryonic fibroblasts (MEFs) or adult tail-tip fibroblasts (TTFs). The cells used in these experiments were derived from a transgenic mouse bearing a bgeo gene integrated into the Fbx15 locus, which is active in mESCs. The newly generated pluripotent clones engineered to express a given subgroup of transcription factors were then selected with G418 for bgeo expression. By testing increasingly narrow combinations of candidates, the authors identified a minimal set of genes necessary to obtain G418^R pluripotent stem cells derived from MEFs (iPS-MEF) and TTFs (iPS-TTF) cultures. Only four factors turned out to be essential: Oct4, Sox2, c-Myc and Klf4 (ref. 2). The iPS clones isolated resembled mES cells both in terms of morphological criteria and transcriptional signatures (as evaluated by RT–PCR and microarray analyses), as well as by their capacity to differentiate into the three germ layers during in vitro embryonic body differentiation and in vivo by teratoma formation. Interestingly, the selected stem-cell lines (iPSs) strictly required LIF and feeder cells to remain undifferentiated, suggesting that the four transduced factors were not sufficient to confer cell autonomous properties. Although Nanog was not required for their generation, it may overcome the need for LIF and BMP (provided by the serum) and accentuate the ES cell-like properties of the reported iPSs. When introduced in mouse blastocyst, iPS-TTF could contribute to derivatives of the three germ layers up to day E13.5; however, no chimeric mice where born [2].

It seems that the formation of iPS is constrained in some manner. Using the data provided, we estimated the efficiency of reprogramming at between approximately one per 2,500 to approximately one per 30,000 infected embryonic and adult fibroblasts, respectively. Because of the impact of these findings and the low frequency of the observed phenomenon, two critical issues arose: which cell type(s) was reprogrammed?; is the reported combination of factors sufficient for reprogramming? Although the authors reasonably argued that the low frequency of iPS derivation is due to selective pressure for the rare cells that express appropriate levels of the factors, it will be necessary to experimentally demonstrate this in subsequent studies. As recently reported by Hochedlinger et al. [8], definitive proof of mature cell nuclear reprogramming will emerge when similar studies are performed with differentiated cells that are genetically marked.

The issue of whether these factors are sufficient for reprogramming remains open, especially considering the notable range in frequency of iPS generation and the inability of these cells to behave as normal mES cells. The interesting possibility that insertional mutagenesis has contributed must be considered because approximately 20 integrations are reported per clone. The identification of common insertion sites in the various clones could thus reveal novel reprogramming factors. As the diversity of the retroviral particles used in this study was seemingly generated by transfection of plasmid pools, the possibility that unexpected recombinant viruses have been generated remains. Other caveats not related to retroviral infection have already been discussed by Rodolfa and Eggan and will not be repeated here [9]. Nevertheless, even if some points need to be clarified concerning the iPS and the genes involved, the reported somatic-cell reprogramming with just a handful of factors is of major interest and represents a breakthrough in the stem-cell field.

Ivanova et al. [3] used a complementary approach to identify mES cell self-renewal genes. They report a list of around 65 candidate transcription factors from a selection of 901 genes down-regulated during retinoic acid-induced mES cell differentiation and generated shRNA-based lentiviral vectors for these genes. The efficiency of mRNA knockdown of this approach compares favourably to most other methods reported to date [10]. To evaluate the effect on self-renewal capacity, they designed a competitive in vitro assay between transduced and untransduced cells which resulted in a final list of ten candidate genes. Two of these genes were eliminated because they induce cell loss. The others were tested with an elegant rescue experiment using a lentiviral vector containing both the shRNA and its corresponding cDNA expressed under the control of an inducible promoter. Only one candidate gene failed in the rescue experiment. The functions of all the other candidates were characterized by knocking down their expression in ES cells or forcing their expression during differentiation. Analyses were then performed by RT–PCR for expression markers corresponding to the three germ layers and the trophoectoderm, or by the contribution of these modified ES cells to chimeric mice. The effect of knockdown experiments was also evaluated by microarray analysis. These results showed, for the first time, that two separate pathways seem to regulate self-renewal: one including Nanog, Oct4 and Sox2; and the other Esrrb, Tbx3, Tcl1 and Dppa4. One hundred and sixty candidates for positive regulators of differentiation were selected from the microarray data and overexpressed in ES cells and eighteen induced differentiation. The direct transcriptional link between these genes and the seven candidate self-renewal factors will need to be demonstrated. Nevertheless, these results represent an enormous endeavour in understanding the transcriptional complexity underlying mES cell self-renewal.

Together, these studies confirm the involvement of Oct4 and Sox2 in the maintenance of ES cell identity and further underscore their ability to induce nuclear reprogramming (Fig. 1). Based on this, it should be of interest to test the reprogramming potential of the other factors identified by Ivanova et al. [3] and also the target genes shared by Oct4 and Sox2 (ref. 11). Moreover, we anticipate that these results will become even more significant when epistatic and synthetic interactions are uncovered, as may be achieved through high-throughput ES cell experiments. Thus, it can now be concluded that a small set of transcription factors are key components regulating ES cell fate decisions. Understanding their post-translational modification in self-renewing cells will become necessary. Non-coding microRNA are also expected to be implicated in the regulatory network [12]. In addition, accumulating evidence indicates that up to 2% of the genome involves non-coding regions kept under active evolutionary selection, with more than 5000 sequences of over 100 bp that are absolutely conserved between human and mouse [13]. Interestingly, some of these conserved elements, referred as "bivalent domains", may silence developmental genes in ES cells but also keep them poised for activation [14]. It could be envisioned that chromosome engineering technology will become part of the growing list of complementary approaches to decipher the function of these conserved regions that are likely to control stemness [15].

Figure 1. Candidate transcription factors that determine stem-cell identity.

Figure 1. Candidate transcription factors that determine stem-cell identity.

Oct4 and Sox2 (blue) contribute to reprogramming fibroblasts into a pluripotent state (green) [2]. Interestingly, these genes (red) are also required for maintenance of mES cell self-renewal [3].

Added Reference:

16. Zhang J, Tam W-L, Tong GQ, Wu Q, Chan H-Y, Soh B-S, Lou Y, Yang J, Ma Y, Chai L, Ng H-H, Lufkin T, Robson P, and  Lim B,
"Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1".
Nature Cell Biology - vol. 8, no. 10, pp. 1114 - 1123 (October, 2006)
Published online: 17 September 2006; | doi:10.1038/ncb1481
http://www.nature.com/ncb/journal/v8/n10/full/ncb1481.html

NetworkEditor's Perspective: "Gene Transcription yields both proteins and non-coding RNAs".

This interesting review by Mélanie Bilodeau and Guy Sauvageau raises the point that current research on
reprogramming differentiated cells to become stem cells may need to consider noncoding activator RNA species as well as proteins when gene expression is assessed.

1. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".

2. Frenster JH, "Yeast RNA Re-Programming of Already-Active Mammalian Chromatin".

3. Frenster JH, and Hovsepian JA, "Overshoot in Late Telophase for RNA Re-Programming of Mitotic Chromatin".

4. Byrne JA, Simonsson S, Western PS, and Gurdon JB, "Nuclei of Adult Mammalian Somatic Cells are Directly Reprogrammed to oct-4 Stem Cell Gene Expression by Amphibian Oocytes".

5. Katoh M, Shaw C, Xu Q, Van Driessche N, Morio T, Kuwayama H, Obara S, Urushihara H, Tanaka Y, and Shaulsky G, "An orderly retreat: Dedifferentiation is a regulated process".

6. Xie H , Ye M , Feng R , and Graf T, "Stepwise Reprogramming of B Cells into Macrophages".

7. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, and Takahashi T, "Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival".

8. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming".

9. Hochedlinger K, Blelloch R, Brennan C, Yamada Y, Kim M, Chin L, and Jaenisch R, "Reprogramming of a melanoma genome by nuclear transplantation".

10. Hovsepian JA, and Frenster JH, "Reprogramming as an Approach to Neoplasms".

11. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, and Slack FJ, "RAS is regulated by the let-7 microRNA family". Cell 2005;120:635-647.

12. Eder M, and Scherr M, "MicroRNA and Lung Cancer".

Links to RNA and Biological Causality:

A Brief History of  Activator RNA:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".