Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency

The therapeutic potential of embryonic stem cells (ESc) is a key scientific interest, but the ethical implications of stem cell research have been controversial and are often a major detriment to the advancement of our knowledge. Recently, several landmark studies both in mouse and human have defined a set of four transcription factors (Klf4, c-Myc, Oct4 and Sox2) whose ectopic expression enables a somatic nucleus to shift back to a pluripotent state. These cells, referred to as induced pluripotent stem (iPS) cells, not only express markers characteristic of pluripotent cells such as alkaline phosphatase, SSEA1, Nanog and Oct4, but both their epigenetic state and their biological potency have been shown to be indistinguishable from those of ES cells. Although these results have been a great breakthrough in stem cell research and have brought hope for an alternative to embryo-derived ES cells, skeptics argue that these pluripotent cells derive from a small subset of remaining multipotent cells or cells that are more amenable to direct reprogramming. Following this approach, a fundamental question remains unsolved: namely, whether this process holds true in terminally differentiated cells. Also, clarifying whether cells of a distinct lineage are equally amenable to direct reprogramming throughout their developmental stages would determine if distinct prerequisites are needed and would be an important step towards understanding the biological processes involved in this transformation. Here, we used transgenic and inducible expression vectors of the previously described factors to reprogram mouse B lymphocytes. Alone, the four factors were able to convert non-terminally differentiated pro-B cells into a pluripotent state but failed to do so in cells that had undergone full B cell receptor rearrangements. Instead, expression of a myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα), known to disrupt the epigenetic faith of B cells, was shown to be required. Multiple iPS lines were derived and characterized using various methods. Ultimately, they were shown to be fully pluripotent by their contribution to germline transmission. Our study provides definitive proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

Andrea Corsinotti

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development.

EPFL2012

Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency

Gabriela Ecco, Marc Samuel Friedli, Adamantia Kapopoulou, Bastien Mangeat, Evaristo Jose Planet Letschert, Benjamin Rauwel, Helen Mary Rowe, Didier Trono, Priscilla Turelli, Carmen Maria Unzu Ezquerro

Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes. At the iPSC stage, EREs of some classes were repressed, whereas others remained highly expressed, yielding a pattern somewhat reminiscent of that recorded in embryonic stem cells. However, variability persisted between individual iPSC clones in the control of specific ERE integrants. Both during reprogramming and in iPS cells, the up-regulation of specific EREs significantly impacted on the transcription of nearby cellular genes. While transcription triggered by specific ERE integrants at highly precise developmental stages may be an essential step toward obtaining pluripotent cells, the broad and unspecific unleashing of the repetitive genome observed here may contribute to the inefficiency of the reprogramming process and to the phenotypic heterogeneity of iPSCs.