Franzi Chiba

Ryan George

John Blair

John Boyle

Lana Kevin

Lana Bosanac

Maja Petkovic

Katlin Silm

Damien Jullié

Milos S Simic

Robert T Schinzel

Erica Moehle

Jonathan J Halloran

Kartoosh Heydari

Melissa Sanchez

Dirk Hockemeyer

Andrew Dillin
email: dillin@berkeley.edu

The activation of the UPR ER is an essential step in the acquisition of pluripotency during reprogramming

Somatic cells can be reprogrammed into a pluripotent stem cells state and is

achieved by the forced expression of 4 transcription factors: OCT4, SOX2, KLF4 and c-MYC. This process theoretically requires a global remodeling of the organelles and a drastic change in metabolism. Furthermore, reprogramming has an inherent property of stochastic variation that is limiting and largely unknown. We hypothesize that this variation is due, in part, by variable regulation of the protein homeostasis network. We therefore postulated that the early steps of reprogramming would result in the activation of a variety of stress pathways that regulate the protein homeostasis network, which might in turn impact the efficiency of reprogramming. We focused in particular on the endoplasmic reticulum unfolded protein response (UPR ER ).

We find that the UPR ER is activated during reprogramming and that its activation can increase the efficiency of this process. We find that stochastic activation of the UPR ER can predict reprogramming efficiency. These results suggest that the low efficiency of cellular reprogramming is partly the result of the cell's inability to initiate a proper stress response to cope with the newly expressed load of proteins that will eventually change the fate of this cell.

Preface

This manuscript documents my doctoral work in Andrew Dillin's laboratory, UC Berkeley, USA, started in January 2013.

My doctoral experience was the occasion to show my ability to construct an innovative scientific project literally from the ground. Our laboratory's main focus is the study of aging mostly in C. elegans and mice as well as the study of cellular stresses. Even though the laboratory's expertise was not in stem cell biology, with Andrew Dillin's support, I decided to explore the role of stress pathways during cellular reprogramming. It was therefore very challenging to build all the protocols and techniques in cellular reprogramming by myself. But the outcome is very promising. For the first time we were able to bridge two fields and offered a new way to look at cellular reprogramming through the prism of protein quality control. The present manuscript will elaborate exclusively on this work.

Besides this exciting project, I had the chance to build another project to study the tissuespecific requirement of RPN-6, a subunit of the 19S proteasome, in C. elegans. Very stimulating results came from this project but we decided to focus on the role of the endoplasmic reticulum stress during cellular reprogramming. Therefore, no further mention of this project will follow.

The time I spent in the Dillin lab was also a great opportunity to establish collaborations and keep bridging fields. This also provided me with a platform to explore other model organisms such as C. elegans and techniques like genome-wide CRISPR-Cas9 screens.

These collaborations are still ongoing and very promising. Some of this work already gave rise to publications in very high profile journals: the assumption that because germ cells mediate inheritance, there must be a deletion or inactivation of the unnecessary genetic codes in somatic cells. This is known as the Weismann barrier [START_REF] Weismann | The germ-plasm; a theory of heredity[END_REF]. Later on, in the mid-20 th century, Conrad Waddington used the image of a ball rolling downhill to describe embryonic development. The ball starts from the top of Waddington's mountain symbolizing the immature stem cells and rolls down in valleys representing the mature differentiated states [START_REF] Waddington | The strategy of the genes: a discussion of some aspects of theoretical biology[END_REF]. In this model the ball is "trapped" in the valley with no possibility to come back or move to another valley because of gravity. Therefore, the cells are committed to one lineage that will result in a permanent cell state (Figure 1, p.13).

Résumé

The first evidence that this theory was not accurate was discovered by Sir John [START_REF] Gurdon | The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles[END_REF]. He reported that cells could be reprogrammed to a different state using somatic cell nuclear transfer (SCNT) [START_REF] Gurdon | The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles[END_REF]. Gurdon transferred the nucleus of the intestinal epithelium cells into an enucleated egg (Figure 1,p.13). This artificial chimera started to divide and generated an embryo identical to the donor of the somatic cell. This established that the somatic cell nucleus possesses all the genetic information that is present in the embryonic stem cells. Thus, a somatic nucleus can be reprogrammed to an embryonic state capable of generating an entire individual. Later during the 20 th century, other groups expanded SCNT to mammals with the charismatic cloning of the sheep Dolly [START_REF] Wilmut | Viable offspring derived from fetal and adult mammalian cells[END_REF]. Furthermore, mice were successfully derived by SCNT using B cells, which had undergone VDJ-recombination (the mechanism responsible for the high diversity of antibodies and T cell receptors found on B and T cells respectively) [START_REF] Hochedlinger | Monoclonal mice generated by nuclear transfer from mature B and T donor cells[END_REF]. Therefore, terminally differentiated cells were able to reprogram, breaking down the idea of irreversibility of the differentiation process.

The generation of heterokaryons, fusion of two different cell types that then contain two different nuclei, showed that it was possible to reprogram the gene expression profile of the cell (Figure 1, p.13). Genes that were usually silenced in one cell type could be reactivated by the fusion with another cell type that expresses them [START_REF] Blau | Cytoplasmic activation of human nuclear genes in stable heterocaryons[END_REF][START_REF] Takagi | Reversal of Xinactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro[END_REF]. Very interestingly, this observation was expanded to the fusion of somatic cells such as fibroblast with pluripotent cells, cells that have the potential to produce any embryonic tissue. Pluripotency genes expressed predominantly by stem cells such as octamer-binding protein 3/4 (OCT-3/4; OCT4) were then expressed in fibroblasts opening the avenue to cell rejuvenation [START_REF] Cowan | Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells[END_REF][START_REF] Tada | Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells[END_REF]. This discovery implied the existence of factors coming from the stem cell that are able to reprogram the somatic cell into a more "stem-like" cell state and more generally, that key factors could change the fate of a cell. The forced expression of key transcription factors known to mediate the cell identity was used to rewire the gene expression of different cell types and turn them into another. This process is known as transdifferentiation or direct cell conversion. For example, the forced expression of solely MYOD (myoblast determination protein), a muscle differentiation protein, in mouse fibroblasts is sufficient to turn the cells into myoblast-like [START_REF] Davis | Expression of a single transfected cDNA converts fibroblasts to myoblasts[END_REF]. This finding was expanded to other cell type transdifferentiation.

b) Induction of pluripotency

The idea that factors defining a particular cell state could be used to change the fate of other cell types gave rise to the discovery of induced pluripotent stem cells (IPSCs) by the group of Shinya Yamanaka [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF] (Figure 1, p.13).

i) Embryonic stem cells

ESCs are characterized by their ability to indefinitely self-renew and form all the embryonic tissues. This property is named pluripotency. It is noteworthy that totipotency defines cells that contribute to the formation of all the tissues from a developing organism such as extra-embryonic and placental tissues and obviously embryonic tissue. Only the zygote and the two first cleavage division cells possess this property.

The study of pluripotency was made easy by the derivation of ESCs lines form the inner cell mass of blastocyst first in mouse [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[END_REF], and then in human [START_REF] Thomson | Embryonic Stem Cell Lines Derived from Human Blastocysts[END_REF]. Of interest, the culture conditions between mouse and human ESCs were distinct maybe due to species differences and stage of the inner cell mass cells from which they are derived from [START_REF] Nichols | Naive and Primed Pluripotent States[END_REF].

ii) Identifying ES cell-associated transcripts (ECATs)

Several groups, including the Yamanaka group, developed tools to identify key factors required for pluripotency and infinite proliferation, key characteristics of ESCs. This was accomplished mainly by transcriptional profiling of mouse ES cells. These ES cellspecific genes were termed ES cell-associated transcripts (ECATs).

OCT4 and SOX2 (SRY box-containing factor 2) were among the first well-described core transcription factors of pluripotency networks. They regulate the expression of other pluripotency-associated genes [START_REF] Tokuzawa | Fbx15 Is a Novel Target of Oct3/4 but Is Dispensable for Embryonic Stem Cell Self-Renewal and Mouse Development[END_REF][START_REF] Tomioka | Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex[END_REF].

By using the transcriptional profile of mouse ES cells, the Yamanaka group identified NANOG homeobox as an ECAT showing its crucial role in the maintenance of pluripotency [START_REF] Mitsui | The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells[END_REF]. NANOG overexpression was able to overcome the absence of LIF (Leukemia inhibitory factor, an essential cytokine for mouse cell pluripotency used in serum-containing media [START_REF] Smith | Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides[END_REF]). LIF stimulates the STAT3 (signal transducer and activator of transcription 3) pathway preventing ES cell differentiation in culture [START_REF] Matsuda | STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells[END_REF][START_REF] Niwa | Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3[END_REF]. By comparing the expression profile of NANOG overexpression in ES cells with and without LIF (Mitsui et al., 2003;[START_REF] Smith | Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides[END_REF], the Yamanaka group showed that Krüppel-like factor 4 (KLF4) was a target downstream of the LIF-STAT3 signaling pathway.

KLF4 overexpression was able to sustain ES cells self-renewal in a LIF-independent manner indicating that KLF4 is a core component of the pluripotency network [START_REF] Niwa | A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells[END_REF]. With numerous other studies, this helped dissect and establish the core pluripotency circuitry in ESCs (Figure 2,p.16).

Other molecules such as c-MYC (a proto-oncogene promoting cellular proliferation and survival) [START_REF] Cartwright | LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism[END_REF]), β-catenin (a WNT signaling pathway regulator) [START_REF] Sato | Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor[END_REF], TCL1 (T-cell leukemia/lymphoma protein 1, an activator of the PI3K pathway), and the dominant-negative form of GRB2 (growth factor receptor-bound protein 2) (Burdon et al., 1999a(Burdon et al., , 1999b;;[START_REF] Cheng | Mammalian Grb2 Regulates Multiple Steps in Embryonic Development and Malignant Transformation[END_REF] were reported to be necessary for the maintenance and/or specific to ES cells. Based on these observations and others, the Yamanaka group established a list of 24 potential candidates for mediating cellular reprogramming (Figure 2, p.16).

iii) Looking for the reprogramming factors

Takahashi and colleagues [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF] tested the 24 candidates for their ability to transform mouse fibroblasts into embryonic stem cell-like cells. None of them on its own was able to support survival in their experimental design. Interestingly, the combination of the 24 factors gave rise to colonies resembling those of embryonic stem cells. By removing single factors of the 24-pool, they were able to narrow down the list to ten factors that were able to reprogram fibroblasts. Further removal of a particular combination of 4 factors showed to inhibit the formation of the colonies. Conversely, the expression of those 4 factors was able to give rise to colonies. Those factors referred as the Yamanaka factors consist of OCT4, SOX2, KLF4 and c-MYC, also know as OSKM.

The Yamanaka group using the same combination of transcription factors then expanded this discovery to human cells [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. Interestingly, almost at the same time, James Thomson's group at University of Wisconsin, Madison, generated human IPSCs using an alternative combination of transcription factors keeping OCT4 and SOX2 but differing by including NANOG and LIN28 (a cytoplasmic RNA-binding protein) [START_REF] Yu | Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells[END_REF] instead of KLF4 and c-MYC.

c) The power of IPSCs and their limits i) Applications for regenerative medicine and disease research

ESCs are an infinite source of cells that could be used for regenerative medicine and a powerful tool to study the steps of development and differentiation. Yet, ethical concerns were raised due to the use of embryos and the likely immune rejection obstructed the potential use of ESCs.

The possibility to derive IPSCs lines from patient's cells removes these roadblocks.

Autologous patient-specific stem cells can be derived avoiding the complications due to immune rejection. The derivation of these cells comes from the patient somatic tissue therefore escaping the use of embryos.

In order to use IPSCs for clinical applications, it was necessary to achieve reprogramming without changing the genome of the somatic cell by integrating the reprograming factors. Several techniques were developed such as Cre/Lox [START_REF] Soldner | Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors[END_REF] or piggyback [START_REF] Kaji | Virus-free induction of pluripotency and subsequent excision of reprogramming factors[END_REF] system, non-integrating viruses [START_REF] Fusaki | Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome[END_REF], episomal vectors [START_REF] Yu | Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences[END_REF], and direct mRNA [START_REF] Warren | Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA[END_REF] or protein [START_REF] Kim | Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins[END_REF] delivery of the reprogramming factors.

Of great interest is the possibility to study diseases using patient-derived cells with all the genetic alterations. It is therefore possible to establish an in vitro system to investigate a particular disease and to potentially establish therapies [START_REF] Robinton | The promise of induced pluripotent stem cells in research and therapy[END_REF].

Moreover, the combination with the newly developed genome editing strategies combined with IPSCs technologies open astonishing avenues for tackling those issues [START_REF] Hockemeyer | Induced Pluripotent Stem Cells Meet Genome Editing[END_REF].

ii) IPSCs quality and their resemblance to ESCs

IPSCs and ESCs share many similarities such as morphology, overall gene expression, telomeres and mitochondria biology [START_REF] Suhr | Mitochondrial Rejuvenation After Induced Pluripotency[END_REF][START_REF] Van Haute | Human embryonic stem cells commonly display large mitochondrial DNA deletions[END_REF].

Nevertheless, studies pointed out differences in the genome, epigenome, transcriptome and proteome raising concerns about their use for therapeutic applications [START_REF] Gore | Somatic coding mutations in human induced pluripotent stem cells[END_REF][START_REF] Hussein | Copy number variation and selection during reprogramming to pluripotency[END_REF][START_REF] Laurent | Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture[END_REF]. The observed genetic abnormalities could be the result of oncogenic stress induced by the reprogramming factors [START_REF] González | Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State[END_REF]. Indeed, cells exposed to OSKM or OSK show higher levels of phosphorylated histone H2A.X (an early response to double strand breaks). Other studies failed to find genetic and epigenetic abnormalities that would distinguish IPSCs from

ESCs [START_REF] Bock | Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines[END_REF][START_REF] Cheng | Low Incidence of DNA Sequence Variation in Human Induced Pluripotent Stem Cells Generated by Nonintegrating Plasmid Expression[END_REF][START_REF] Gore | Somatic coding mutations in human induced pluripotent stem cells[END_REF][START_REF] Guenther | Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells[END_REF][START_REF] Newman | Lab-Specific Gene Expression Signatures in Pluripotent Stem Cells[END_REF]. Interestingly, these data showed that the extent of variations between IPSCs and ESCs were similar to those seen within different IPSCs and ECSs [START_REF] Vitale | Variability in the Generation of Induced Pluripotent Stem Cells: Importance for Disease Modeling[END_REF].

Embryonic stem cells Induced pluripotent stem cells

Express stemness markers YES YES

Pluripotent

YES YES

Study development and differentiation

YES YES

Use for disease models Some YES, cells are patient-derived

Immune rejection for clinical applications

Very likely, cells are allogeneic NO, cells are autologous patient-specific

Requires embryos or oocytes YES NO

d) Mechanisms of reprogramming: a two-step route

The exact way cellular reprogramming is achieved still remains unknown. The cells have to overcome a series of roadblocks such as apoptosis, cell-cycle arrest and senescence [START_REF] Banito | Senescence impairs successful reprogramming to pluripotent stem cells[END_REF][START_REF] Marión | A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity[END_REF][START_REF] Utikal | Immortalization eliminates a roadblock during cellular reprogramming into iPS cells[END_REF], oxidative burst [START_REF] Ji | Antioxidant Supplementation Reduces Genomic Aberrations in Human Induced Pluripotent Stem Cells[END_REF], and DNA damage [START_REF] Ruiz | Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells[END_REF] in order to successfully become IPSCs. It also includes the silencing of somatic cell genes, switch from an oxidative to a glycolytic metabolism [START_REF] Panopoulos | The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming[END_REF][START_REF] Zhang | Metabolic Regulation in Pluripotent Stem Cells during Reprogramming and Self-Renewal[END_REF] and requires a mesenchymal-toepithelial transition [START_REF] Li | A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts[END_REF][START_REF] Samavarchi-Tehrani | Functional Genomics Reveals a BMP-Driven Mesenchymal-to-Epithelial Transition in the Initiation of Somatic Cell Reprogramming[END_REF]. Population and single-cell based studies suggest a two-step process for cellular reprogramming. These results mainly rely on observations from mouse reprogramming, but seem to be conserved during human cell reprogramming (Figure 3,p.20). In the first step, OSKM bind to various loci not restricted to the ones they would usually bind to in ES cells [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF]. For example, c-MYC binds to methylated H3K4

regions, marking open chromatin, which includes enhancers and promoters of the somatic genes leading to their silencing [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF][START_REF] Sridharan | Role of the Murine Reprogramming Factors in the Induction of Pluripotency[END_REF]. This first wave of gene activation is also characterized by the expression of genes implicated in cytoskeleton organization, metabolism, chromatin organization, cell cycle, mitochondria, DNA repair, RNA processing and proliferation [START_REF] Hansson | Highly Coordinated Proteome Dynamics during Reprogramming of Somatic Cells to Pluripotency[END_REF]Polo et al., 2012a;[START_REF] Zhang | Metabolic Regulation in Pluripotent Stem Cells during Reprogramming and Self-Renewal[END_REF]. At the same time, OSKM bind to promoters and enhancers of early pluripotency-associated genes leading to their expression [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF]. The nature of this early step is rather stochastic and inefficient [START_REF] Buganim | Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase[END_REF] due in particular to repressive methyl histone marks. These marks cover genes required for pluripotency induction and are responsible for closed chromatin conformations [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF].

In a second step, OSKM accesses loci of late pluripotency genes in a more hierarchical and predictable way [START_REF] Buganim | Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase[END_REF]. The access to these late pluripotency gene loci can only occur after the first step. This enables the core pluripotency network to be stably activated.

e) Enhancing the efficiency of reprogramming

Cellular reprogramming is a very inefficient process, depending on the technique and the cell type used; it ranges from 0.001% to 0.1%. Additional factors and molecules were proposed to facilitate reprogramming and increase its efficiency. They are usually referred as "reprogramming enhancers".

i) Genes associated with pluripotency

The expression of other pluripotency-associated genes can increase and in some cases even replace one of the 4 reprogramming factors.

Together with OSKM, TBX3 (T-box transcription factor 3) in mouse and UTF1

(undifferentiated embryonic cell transcription factor 1) or SALL4 (Sal-like protein4) in human, can increase the reprogramming efficiency [START_REF] Han | Tbx3 improves the germ-line competency of induced pluripotent stem cells[END_REF][START_REF] Tsubooka | Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and fibroblasts[END_REF][START_REF] Zhao | Two Supporting Factors Greatly Improve the Efficiency of Human iPSC Generation[END_REF]. KLF4 can be replaced by ESRRβ (Oestrogen-related receptor β)

in mouse and NANOG in humans [START_REF] Feng | Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb[END_REF][START_REF] Picanço-Castro | Pluripotent Reprogramming of Fibroblasts by Lentiviralmediated Insertion of SOX2, C-MYC, and TCL-1A[END_REF].

Similarly, NR5A2 (nuclear receptor subfamily 5 group 2) and TCL1A can substitute OCT4 [START_REF] Heng | The Nuclear Receptor Nr5a2 Can Replace Oct4 in the Reprogramming of Murine Somatic Cells to Pluripotent Cells[END_REF][START_REF] Picanço-Castro | Pluripotent Reprogramming of Fibroblasts by Lentiviralmediated Insertion of SOX2, C-MYC, and TCL-1A[END_REF]. Enhancers of reprogramming or substitutes for OSKM can also be predicted by their role in the maintenance of ES cells pluripotency. Therefore, it is not surprising to find that ESRRβ is an enhancer. Indeed, it is a direct target of NANOG that can rescue pluripotency in NANOG deficient ES cells [START_REF] Festuccia | Esrrb Is a Direct Nanog Target Gene that Can Substitute for Nanog Function in Pluripotent Cells[END_REF].

ii) Genes involved in cell cycle-regulation

Infinite self-renewal is another characteristic of PSCs. c-MYC promotes cell proliferation. Cell proliferation is required to achieve cellular reprogramming. Tumor suppressor p53 inhibits proliferation and thus its inactivation increases cellular reprogramming probably by overcoming DNA damage and senescence [START_REF] Banito | Senescence impairs successful reprogramming to pluripotent stem cells[END_REF][START_REF] Hong | Suppression of induced pluripotent stem cell generation by the p53-p21 pathway[END_REF][START_REF] Kawamura | Linking the p53 tumour suppressor pathway to somatic cell reprogramming[END_REF][START_REF] Li | The Ink4/Arf locus is a barrier for iPS cell reprogramming[END_REF][START_REF] Marión | A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity[END_REF][START_REF] Utikal | Immortalization eliminates a roadblock during cellular reprogramming into iPS cells[END_REF]. In line with this observation, CIP1, INK4A and ARF (all cell cycledependent kinase inhibitors) block cellular reprogramming [START_REF] Banito | Senescence impairs successful reprogramming to pluripotent stem cells[END_REF][START_REF] Li | The Ink4/Arf locus is a barrier for iPS cell reprogramming[END_REF][START_REF] Utikal | Immortalization eliminates a roadblock during cellular reprogramming into iPS cells[END_REF]. Expression of REM2 or cyclin D1 (two cell-cycle enhancers GTP-binding proteins) increases cellular reprogramming [START_REF] Edel | Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1[END_REF].

iii) Epigenetic modifiers

The passage from a differentiated to a pluripotent state requires a dramatic change in the gene expression profile implying a wide range of epigenetic changes. Therefore, by either promoting the expression of pluripotency genes or lowering the expression of somatic genes can increase the reprogramming efficiency. This altered gene expression profile is dependent on epigenetic marks and the roles they have on the transcriptional regulation of the genes. For example, WDR5 (WD repeat-containing protein) [START_REF] Ang | Wdr5 Mediates Self-Renewal and Reprogramming via the Embryonic Stem Cell Core Transcriptional Network[END_REF], DOT1L, SETDB1 and SUV39H1 [START_REF] Onder | Chromatin-modifying enzymes as modulators of reprogramming[END_REF] can positively or negatively affect the efficiency of reprogramming. Vitamin C, by enhancing the activity of JHDM1A and JHDM1B (histone demethylases), increases the efficiency of reprogramming (Wang et al., 2011).

II. Alternative routes for increasing the reprogramming efficiency and understanding its mechanism a) Protein quality control as a necessity for ESCs maintenance

So far, in order to increase the efficiency of reprogramming, studies focused on mainly trying to improve the resemblance of cells to be reprogrammed to ESCs at a transcriptional level and by helping erase the epigenetic memory of the somatic cells in order to speed up the process and hopefully restore the expression of the core pluripotency genes network. This has been proven to be very efficient. We hypothesized that helping somatic cells obtain additional characteristics of ESCs could improve the efficiency of reprogramming. Because of their ability to indefinitely self-renew it is important for the ESCs to protect their protein homeostasis (proteostasis) over many cell divisions. This characteristic has to be shared with other stem cells such as adult stem cells.

In order to find key regulators of the reprogramming process we decided to study the features harbored by SCs to maintain their stemness through the prism of protein quality assurance. By doing that we hoped to find potential cellular pathways that we could modulate in somatic cells in order to increase the efficiency of reprogramming.

The following review is an effort to highlight characteristics of ESCs and also adult SCs This review summarizes how our laboratory relates protein quality control and stemness.

We greatly encourage you to read it to have a broader view of our working hypothesis but it is not necessary for understanding the rest of the dissertation: While we were writing this review, we were excited to see the publication of studies that linked mechanisms ensuring the quality of proteostasis and the efficiency of reprogramming. Interestingly, two major pathways ensuring the protein quality control in the cells were also playing an important role during cellular reprogramming: autophagy [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF][START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF] and the ubiquitin-proteasome system (UPS) (Buckley et al., 2012a;[START_REF] Qin | Systematic Identification of Barriers to Human iPSC Generation[END_REF]. Inhibition of the proteasome activity by either drug treatment with MG132 or genetically by knocking down PSDM14 (a deubiquinating enzyme), decreased significantly the efficiency of reprogramming (Buckley et al., 2012a). Conversely, the knockdown of FBXW7 (F E3 box ligase) increased the reprogramming efficiency [START_REF] Buckley | Regulation of Pluripotency and Cellular Reprogramming by the Ubiquitin-Proteasome System[END_REF]. Recently, in a genome-wide RNAi screen, Qin and colleagues [START_REF] Qin | Systematic Identification of Barriers to Human iPSC Generation[END_REF]) also identified the UPS as a potent barrier for reprogramming.

Even though controversial results have been published, it appears that autophagy also plays an important role in reprogramming. Autophagy is transiently induced during the early stages of reprogramming [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF][START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF]. Whether it plays a positive or negative role is up to debate. Wang and colleagues [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF] reported that knockdown of ATG5, a key player in the autophagosome formation, resulted in an impaired reprogramming. Wu and colleagues [START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF] reported the opposite; they knocked-down additional player in autophagy such as BECN1 and VSP34

and observed a higher efficiency of reprogramming suggesting a negative role of autophagy during reprogramming.

Introduction-Alternative routes for increasing the reprogramming efficiency and understanding its mechanism

25

Based on these observations, we reasoned that not only the quality of the proteostasis has to be high in ESCs but also the path towards the acquisition of pluripotency during reprogramming has to enable the renewal and ensure the quality of the proteaome. This would explain why autophagy is transiently activated during reprogramming [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF][START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF]. Mechanisms important for the maintenance of pluripotency are intrinsically important during the loss of pluripotency that happens during development and differentiation. Because reprogramming seems to be to some extent the reversal of development and differentiation we hypothesized that cellular pathways ensuring the quality of the proteome during normal development and differentiation should also be required for reprogramming.

b) Insights from developmental biology

In order to narrow down which pathways could be the most important for cellular reprogramming, we turned to development. Indeed, reprogramming can be comprehended as a reversal of development. Using a mouse secondary reprogramming system, Cacchiarelli and colleagues [START_REF] Cacchiarelli | Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency[END_REF] observed distinct waves of gene network activation corresponding to developmental genes characteristic of early embryonic patterning genes and followed by a pre-implantation gene signature, such as miR371, DPPA3 (developmental pluripotency-associated 3) and DNMT3L (DNA methyltransferase 3-like). This being the case, we hypothesized that key cellular processes required during normal development and differentiation could be potential candidates to study reprogramming and increases its efficiency. Besides the obvious epigenetic remodeling that is required to change cell fate, we propose that the maintenance of the cell proteostasis should be of great benefit to reprogram efficiently and successfully.

c) The role of the UPR ER during development and differentiation

Cellular reprogramming by its nature requires a wide morphological change of the somatic cell. Remodeling of organelles such as mitochondria has been shown to take place during reprogramming [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF][START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF]. To our knowledge, no data have been published regarding the remodeling of the ER. This was surprising because the ER homeostasis can be disrupted during tissue development, cell differentiation, senescence [START_REF] Pluquet | The unfolded protein response and cellular senescence. A Review in the Theme: Cellular Mechanisms of Endoplasmic Reticulum Stress Signaling in Health and Disease[END_REF], by altered redox status [START_REF] Merksamer | Real-Time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions[END_REF], DNA damage [START_REF] Fornace | DNA damage-inducible transcripts in mammalian cells[END_REF] or during an increase of protein synthesis [START_REF] Kozutsumi | The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins[END_REF]. All these changes also happen during cellular reprogramming.

Interestingly, evidence suggests that the ER stress and UPR effectors are required during development. Indeed, the homozygous deletion of either Hspa5 [START_REF] Luo | GRP78/BiP Is Required for Cell Proliferation and Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic Development[END_REF], Grp94 [START_REF] Wanderling | GRP94 Is Essential for Mesoderm Induction and Muscle Development Because It Regulates Insulin-like Growth Factor Secretion[END_REF], Grp58 [START_REF] Garbi | Impaired assembly of the major histocompatibility complex class I peptide-loading complex in mice deficient in the oxidoreductase ERp57[END_REF], Ire1a [START_REF] Iwawaki | Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability[END_REF], Xbp1 [START_REF] Reimold | An essential role in liver development for transcription factor XBP-1[END_REF], Calreticulin [START_REF] Mesaeli | Calreticulin Is Essential for Cardiac Development[END_REF], or deletion of both Atf6a and Atf6b [START_REF] Yamamoto | Transcriptional Induction of Mammalian ER Quality Control Proteins Is Mediated by Single or Combined Action of ATF6α and XBP1[END_REF] leads to embryonic lethality in mice. This is particularly interesting if reprogramming has reversal features of development; it would not be surprising that the ER stress and UPR modulators would be beneficial for reprogramming. To further support this idea, several components of the endoplasmic reticulum unfolded protein response (UPR ER ) have been shown to have an important role during differentiation. IRE1 increases lymphopoiesis of B cells [START_REF] Zhang | The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis[END_REF], XBP1 induces osteogenic and plasma differentiations [START_REF] Iwakoshi | The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response[END_REF], and CHOP plays an important role in the differentiation of B cells, erythrocytes, osteocytes and chondrocytes [START_REF] Cui | Novel Interaction between the Transcription Factor CHOP (GADD153) and the Ribosomal Protein FTE/S3a Modulates Erythropoiesis[END_REF][START_REF] Pereira | CCAAT/Enhancer Binding Protein Homologous Protein (DDIT3) Induces Osteoblastic Cell Differentiation[END_REF][START_REF] Skalet | Rapid B Cell Receptor-induced Unfolded Protein Response in Nonsecretory B Cells Correlates with Pro-Versus Antiapoptotic Cell Fate[END_REF][START_REF] Yang | Multiple Signals Induce Endoplasmic Reticulum Stress in Both Primary and Immortalized Chondrocytes Resulting in Loss of Differentiation, Impaired Cell Growth, and Apoptosis[END_REF].

The UPR ER , as a stress-coordinated pathway, is important in the regulation of differentiation of the mouse intestinal epithelial stem cell [START_REF] Heijmans | ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response[END_REF].

III. The importance of the ER and UPR ER a) The integrative role of the ER

The ER is the main organelle responsible for the synthesis, maturation and posttranslational modification of secreted and membrane proteins. It is involved in the synthesis of 1/3 of the cell proteome, the biogenesis of membranes structures and metabolic process such as ion storage [START_REF] Kleizen | Protein folding and quality control in the endoplasmic reticulum[END_REF]. Fatty acid desaturation and other lipogenic reactions such as those involved in ceramides, sterols, triacylglycerols and most phospholipids synthesis, occur on the cytosolic face of the ER membrane. The ER houses enzymes involved in fatty acid oxidation and gluconeogenesis. Its membrane forms the nuclear envelope and contributes to the biogenesis of autophagic membranes, peroxisomes and lipid droplets. The transfer of various molecules, lipids and calcium are facilitated by its numerous contacts with other membranes structures of the cell [START_REF] Rutkowski | Regulation of basal cellular physiology by the homeostatic unfolded protein response[END_REF].

Hence, the ER integrates these various aspects of cellular and organismal homeostasis into a unique molecular response: the unfolded protein response (UPR ER ). The UPR ER is an important pathway as shown by its conservation among various species from yeast to mammals [START_REF] Ruberti | Conserved and plant-unique strategies for overcoming endoplasmic reticulum stress[END_REF]. This integration has to take into account the nature of the perturbation, intensity and duration in order to properly maintain homeostasis.

b) The molecular mechanism of the UPR ER

Kozutsumi and colleagues [START_REF] Kozutsumi | The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins[END_REF], and later Dorner and colleagues [START_REF] Dorner | The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion[END_REF] observed that the impairment of ER protein folding in consequence to toxin exposure can lead to the induction of ER chaperones. This set the path to look for signaling mechanisms from the ER to the nucleus and eventually uncover this more general pathway that is the UPR ER .

Upon ER stress, three main responses take place [START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF]. First, a transient adaptation occurs by lowering protein synthesis and translocation in the ER.

Second, UPR ER targets are transcriptionally activated in order to increase the capacity to handle the unfolded proteins, in particular chaperones. Lastly, if the ER isn't able to restore its homeostasis, cell death is triggered as a response to protect the organism.

Yeast has a simple UPR ER , a single arm compared to vertebrates. Ire1p (the ER-resident transmembrane kinase) upon ER stress activates Hac1p, a transcription factor responsible for the activation of numerous genes [START_REF] Sidrauski | The unfolded protein response: an intracellular signalling pathway with many surprising features[END_REF].

In vertebrates, the UPR ER involves three ER-resident transmembrane proteins: IRE1

(inositol-requiring protein-1), PERK (protein kinase RNA (PRK)-like ER kinase) and ATF6 (activating transcription factor 6). Upon acute stress, these three branches reduce the import of proteins into the ER by specific mechanisms.

These three ER-resident transmembrane proteins have a luminal portion able to sense protein-folding environment in the ER, and a cytoplasmic portion transducing the state of the ER to the rest of the cell via transcriptional and translational means. i) IRE1 pathway IRE1 (inositol-requiring protein-1) was discovered in yeast screen aiming at identifying blockers of the UPR ER activation. IRE1 is type 1 ER-resident transmembrane protein. Its cytoplasmic domain contains a kinase domain [START_REF] Cox | Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase[END_REF]Morl et al., 1993).

During stress, IRE1 oligomerizes and trans-autophosphorylates the juxtaposed cytoplasmic kinase domain (Figure 4A,p.29). Interestingly, IRE1 is its own and only substrate unlike classic cascade of kinase activation [START_REF] Shamu | Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus[END_REF]. This

A B C

results in the activation of its endoribonucleolytic activity [START_REF] Sidrauski | The Transmembrane Kinase Ire1p Is a Site-Specific Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response[END_REF].

IRE1 cuts twice the only precursor mRNA Hac1 in yeast [START_REF] Cox | A Novel Mechanism for Regulating Activity of a Transcription Factor That Controls the Unfolded Protein Response[END_REF]Mori et al., 1996) and XBP1, x-box binding protein 1, in metazoans [START_REF] Calfon | IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA[END_REF][START_REF] Yoshida | XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor[END_REF]. A spliced version is then generated after the ligation of the 5' and 3' mRNA ends that encodes an activator of UPR ER target genes. Interestingly, in metazoans, both the precursor and the spliced forms are translated [START_REF] Calfon | IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA[END_REF]. The spliced form of XBP1 is more stable and is a more potent activator of UPR target genes while the precursor encodes a protein that represses UPR target genes [START_REF] Calfon | IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA[END_REF][START_REF] Yoshida | XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor[END_REF]. Among those genes are HSPA5 (also known as BIP or GRP78) and p58 IPK [START_REF] Lee | XBP-1 Regulates a Subset of Endoplasmic Reticulum Resident Chaperone Genes in the Unfolded Protein Response[END_REF].

Levels of XBP1 mRNA will continue to rise even when the ER stress decreases and IRE1

is inactivated [START_REF] Yoshida | pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response[END_REF]. This potentially serves to terminate the activation of the UPR ER since the precursor XBP1 mRNA encodes for a repressor of the UPR ER target genes that could compete with the spliced form for binding sites.

In parallel, IRE1 is able to cleave diverse mRNA at the ER membrane, therefore reducing the load of proteins in the ER by a mechanism called RIDD (regulated IRE1-dependent decay) [START_REF] Hollien | Decay of Endoplasmic Reticulum-Localized mRNAs During the Unfolded Protein Response[END_REF].

ii) ATF6 pathway

Haze and colleagues [START_REF] Haze | Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress[END_REF] searched for proteins able to bind UPR-activated promoters and found ATF6 (activating transcription factor 6). Synthesized as an inactive precursor, ATF6 is tethered to the ER membrane and has a stress-sensing portion in the lumen. Upon ER stress, ATF6 is shuffled from the ER to the Golgi where two Golgiresident proteases will cleave it (Figure 4B,p.29). The first is S1P (site 1 protease) and the second S2P (site 2 protease), which cleave ATF6 in an intramembrane region releasing its cytosolic DNA-binding domain that can in turn go to the nucleus and activate target genes [START_REF] Haze | Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress[END_REF]. This binding domain is a basic leucine zipper (bZIP) domain; it binds to ER stress response element in the promoter of genes such as HSPA5, CHOP, XBP1, GRP94 (glucose-regulated protein 94, an HSP90 chaperone family member) [START_REF] Yoshida | ATF6 Activated by Proteolysis Binds in the Presence of NF-Y (CBF) Directly to the cis-Acting Element Responsible for the Mammalian Unfolded Protein Response[END_REF]. subunit of eIF2α (eukaryotic initiation factor-2) at Ser51. This inhibits the pentameric guanine nucleotide exchange factor eIF2B and prevents the recycling of eIF2α to its active GTP-bound form. Less active eIF2α are available which results in less translation initiation, reducing the load of proteins in the ER [START_REF] Harding | Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase[END_REF]. Parallel to its role in reducing the global translation, phosphorylation of eIF2α at Ser51 leads to the transcriptional activation of genes involved in the UPR ER [START_REF] Harding | An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress[END_REF][START_REF] Lu | Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2[END_REF]. In mammalians, phosphorylated eIF2α results in the translation of ATF4, a transcription factor responsible for the activation of a wide variety of UPR ER genes. The 5'-untransltated region of ATF4 (uORF1, upstream open reading frame 1) facilitates the scanning and reinitiation of ribosomes at downstream coding regions. Under unstressed conditions, ribosomes scan downstream of uORF1 and reinitiate at the next coding region: uORF2, an inhibitory element, which inhibits ATF4 translation. Under stress, levels of the active GTP-bound eIF2α form decrease and results in a delayed reinititation of the ribosomes at the uORF2. This allows the ribosomes to scan through uORF2 and to reinitiate at the coding region of ATF4 [START_REF] Vattem | Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells[END_REF]. Two key target genes of the PERK pathway are CHOP (transcription factor C/EBP homologous protein) and GADD34 (growth arrest and DNA damage-induced 34). CHOP is induced by ATF4 and controls the expression of genes involved in apoptosis [START_REF] Marciniak | CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum[END_REF].

Therefore, the PERK signaling pathway can be very protective under low activation and initiate apoptosis when the stress is prolonged and stronger. CHOP induces the expression of GADD34 which restores the protein translation by dephosphorylating eIF2α [START_REF] Gorman | Stress management at the ER: Regulators of ER stress-induced apoptosis[END_REF]. In order to fine tune the activity of CHOP and prevent the premature activation of apoptosis, it has been observed that p58 IPK (protein 58 inhibitor protein kinase) binds and inhibits the PERK kinase domain, which stalls its activity [START_REF] Yan | Control of PERK eIF2α kinase activity by the endoplasmic reticulum stressinduced molecular chaperone P58IPK[END_REF].

It is noteworthy that other signaling pathways such as amino-acid deprivation or doublestranded RNA accumulation lead to the phosphorylation of eIF2α and the activation of common target genes with the UPR ER . For this reason, the signaling pathway downstream of the eIF2α phosphorylation was called integrated stress response (ISR) [START_REF] Harding | An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress[END_REF]. ATF4, in mammalian cells, accounts for about half of the PERK-dependent UPR genes induction, suggesting the existence of other effectors downstream of phosphorylated eIF2α [START_REF] Harding | An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress[END_REF].

c) Stress recognition

The exact mechanism of how these three transmembrane proteins sense stress and misfolded proteins is still unclear. A titration type hypothesis was proposed to explain the activation of the UPR ER . Under normal conditions, the ATP-dependent ER chaperone HSPA5 maintains these sensors in an inactive state by binding to their luminal domain.

HSPA5 is a member of the HSP70 family of heat-shock proteins; it is the most abundant protein in the ER lumen. Under conditions of stress, HSPA5 binds to nascent peptides and unfolded proteins and promotes proper folding in an ATP-dependent manner preventing protein aggregation. Thus, when excessive amounts of misfolded proteins occur, HSPA5 is titrated away from the three stress-sensing transmembrane proteins.

Consistent with this idea, HSPA5 overexpression attenuates PERK and IRE1 activities and limits the UPR ER [START_REF] Okamura | Dissociation of Kar2p/BiP from an ER Sensory Molecule, Ire1p, Triggers the Unfolded Protein Response in Yeast[END_REF].

A direct binding of the unfolded protein to one of these stress-sensors has also been proposed based on crystal structures studies. The yeast IRE1 luminal domain has a major histocompatibility complex-like domain architecture compatible with peptide binding [START_REF] Credle | On the mechanism of sensing unfolded protein in the endoplasmic reticulum[END_REF].

d) The ER shape and its contribution to the ER function

The ER is comprised of different domains that expand throughout the entire cell. The nuclear envelope is a particular part of the ER composed of two flat and large membrane bilayers (the outer and inner nuclear membranes) punctually connected by nuclear pores [START_REF] Hetzer | PUSHING THE ENVELOPE: Structure, Function, and Dynamics of the Nuclear Periphery[END_REF]. Branching out of the outer nuclear membrane, the peripheral ER is a wide network of tubules and cisternae structures through the entire cytoplasm to the plasma membrane. Importantly, the perinuclear space and the peripheral ER lumen are continuous. Tubules are characterized by their high membrane curvature while cisternae are regions of piled parallel flat bilayer membranes [START_REF] Friedman | The ER in 3D: a multifunctional dynamic membrane network[END_REF]. Key proteins shape the ER and are associated to either tubules or cisternae structures.

Reticulon proteins, such as Reticulon 4 also called Nogo, are responsible for the tubules high curvature and are required for their formation [START_REF] Shibata | The Reticulon and Dp1/Yop1p Proteins Form Immobile Oligomers in the Tubular Endoplasmic Reticulum[END_REF]. CLIMP-63 is responsible for the proper intraluminal spacing of the cisternae [START_REF] Shibata | Mechanisms Determining the Morphology of the Peripheral ER[END_REF].

The shape of the ER is highly correlated to its function. Indeed, muscle cell ER is enriched in tubules devoid of ribosomes. This could help to quickly control calcium levels during contractions. Whereas secretory cells, which require abundant secretion of proteins, have abundant cisternae densely covered with ribosomes [START_REF] Friedman | The ER in 3D: a multifunctional dynamic membrane network[END_REF]. Interestingly, the shape of the ER can be modulated in response to UPR ER activation in order to alleviate the stress [START_REF] Schuck | Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response[END_REF].

Introduction-Working hypothesis 34

IV. Working hypothesis

During normal development and differentiation, cells can dramatically change their morphology and remodel their organelles such as the ER. The UPR ER plays a crucial role during those events. We reasoned that because cellular reprogramming can be comprehended as a reversal of reprogramming, the UPR ER should have an essential function in this process. The ER homeostasis is disrupted under conditions of senescence [START_REF] Pluquet | The unfolded protein response and cellular senescence. A Review in the Theme: Cellular Mechanisms of Endoplasmic Reticulum Stress Signaling in Health and Disease[END_REF], by altered redox status [START_REF] Merksamer | Real-Time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions[END_REF], DNA damage [START_REF] Fornace | DNA damage-inducible transcripts in mammalian cells[END_REF] or during an increase of protein synthesis [START_REF] Kozutsumi | The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins[END_REF].

These events happen during cellular reprogramming. We therefore hypothesized that the UPR ER should be activated and play an active role during reprogramming.

Genes responsible for pluripotency and self-renewal, two of the key characteristics of ESCs, greatly improve the reprogramming efficiency. Because SCs can indefinitely divide, they must maintain an extremely "healthy" proteostasis, another key characteristic of SCs. We therefore hypothesized that cellular pathways insuring this task could enhance cellular reprogramming. We postulated that the proteotoxic-protective role of the UPR ER could be beneficial for cellular reprogramming.

The following chapter summarizes our main findings regarding the contribution of the UPR ER during cellular reprogramming.

Results

This part summarizes the main results achieved during my doctoral work to study the role of the UPR ER during cellular reprogramming. At the time of the writing, this work is almost ready to be submitted, we decided to present our results in a paper manuscript format. Due to formatting requirements for the manuscript, the discussion section of the manuscript is very condensed and doesn't go into details. Therefore, an extended discussion section of the results follows. To avoid multiple identical citations, all the references are summarized in one section at the end of the dissertation.

Introduction

Cellular reprogramming of somatic cells into induced pluripotent stem cells (IPSCs)

through the forced expression of a set of factors (OCT4, SOX2, KLF4 and c-MYC for example) [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF] highlights the remarkable plasticity found within cells and provides an incredible potential for regenerative medicine applications [START_REF] Polo | A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells[END_REF]. However, the quality and the high variability in efficiencies are problematic [START_REF] González | Methods for making induced pluripotent stem cells: reprogramming à la carte[END_REF]. Evidence of DNA damage and genomic instability in IPSCs raises concerns for their use in patients [START_REF] Ruiz | Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells[END_REF]. It is therefore important to better understand the mechanisms underlying reprogramming to improve this method [START_REF] Vierbuchen | Molecular Roadblocks for Cellular Reprogramming[END_REF]. The early phases of reprogramming is hypothesized to be stochastic and responsible for its low efficiency [START_REF] Takahashi | A decade of transcription factor-mediated reprogramming to pluripotency[END_REF].

What drives this early stochastic variation is unknown and it remains the major hurdle in the reprogramming process.

The endoplasmic reticulum (ER) is the site where secreted and membrane-bound proteins are synthesized and represents 1/3 of the proteome. The ER machinery integrates various signals such as growth, differentiation and inflammation. When ER homeostasis is disrupted by increased protein synthesis, cell differentiation, tissue development, senescence, DNA damage and many other stressors, a complex signaling process is activated: the unfolded protein response (UPR ER ) [START_REF] Walter | The Unfolded Protein Response: From Stress Pathway to Homeostatic Regulation[END_REF]. The UPR ER is composed of three branches. They operate in parallel and use distinctive signal transduction mechanism. Each branch senses the protein folding state in the ER lumen using three transmembrane proteins: ATF6 (activating transcription factor 6), PERK (double-stranded RNA-activated protein kinase (PRK)-like ER kinase) and IRE1 (inositol requiring enzyme 1) [START_REF] Walter | The Unfolded Protein Response: From Stress Pathway to Homeostatic Regulation[END_REF]. IRE1 converges on the x-box binding protein 1 transcription factor, XBP1, causing its splicing to create the XBP1s mRNA that can be translated and incorporated into the nucleus to regulate hundreds of genes required for ER protein folding and morphology.

Cellular reprogramming causes a dramatic change in cell morphology and imposes the remodeling of many organelles such as mitochondria [START_REF] Wang | Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency[END_REF]. We therefore hypothesized that cellular reprogramming would restructure the ER and could potentially activate the UPR ER .

Results

The UPR ER is activated during reprogramming

Organelles such as mitochondria, ER and Golgi are less abundant and less mature in embryonic stem cells (ESCs) compared to their differentiated counterparts [START_REF] Sathananthan | The fine structure of human embryonic stem cells[END_REF]. Therefore, cellular reprogramming involves a wide remodeling of these organelles and a dramatic change in gene expression [START_REF] Koche | Reprogramming Factor Expression Initiates Widespread Targeted Chromatin Remodeling[END_REF][START_REF] Mikkelsen | Dissecting direct reprogramming through integrative genomic analysis[END_REF]. Synthesis of new proteins and proteins characteristic of the somatic state coexist for a brief time [START_REF] Koche | Reprogramming Factor Expression Initiates Widespread Targeted Chromatin Remodeling[END_REF][START_REF] Mikkelsen | Dissecting direct reprogramming through integrative genomic analysis[END_REF] possibly creating an imbalance in protein homeostasis. Thus, we hypothesized that cellular reprogramming could activate particular stress pathways regulating protein homeostasis. We focused upon the UPR ER , which integrates intra-and extra-cellular signals for its role in cell differentiation and development.

During ER stress, the transcription of central regulators of the UPR ER stress response are increased as well as their downstream targets. We analyzed the mRNA levels of the major transcription factors representing the three braches involved in the UPR ER : ATF6, ATF4 and XBP1s [START_REF] Walter | The Unfolded Protein Response: From Stress Pathway to Homeostatic Regulation[END_REF], during reprogramming of human somatic cells. All the three factors showed significantly higher levels than the control at day 6 after reprogramming suggesting an activation of the UPR ER (Figure 1A, pp.54-55). We analyzed the canonical downstream transcriptional targets of the UPR ER , such as HSPA5 and GRP94 [START_REF] Walter | The Unfolded Protein Response: From Stress Pathway to Homeostatic Regulation[END_REF]) and found that both had higher levels than cells not undergoing reprogramming (Figure 1A Tubular ER morphology is associated with impaired secretory capacity of the ER. We tested the secretion capacity of cells undergoing reprogramming by following the secretion of the exogenously expressed humanized Gaussia luciferase protein (Gluc) [START_REF] Badr | A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress[END_REF]. We collected the supernatant of cells expressing the reprogramming factors and observed a reduction in secreted Gluc as measured by luciferase activity. The reduced Gluc was not due to reduced expression of during the reprogramming process (Figure 1D, pp.54-55). On the contrary, Gluc is unable to be secreted and stays in the ER.

Because basal levels of ER stress are observed during the early phase of reprogramming, we reasoned that this could be protective against an additional ER stress such as the addition of tunicamycin. We established dose-survival curve to calculate the EC50 and found that cells undergoing reprogramming were more protected than control cells (Figure 1E,. Taken together, ER stress and morphology are dramatically altered during the reprogramming process.

Activation of the UPR ER increases the efficiency of cellular reprogramming

To better understand the role of the UPR ER during reprogramming and test if it could be a limiting factor for successful reprogramming, we followed induction of the endogenous During the process of reprogramming the use of cell surface markers allows an accurate assessment of the reprogramming efficiency. Previous studies showed that the fibroblast surface marker CD13 is downregulated during successful reprogramming while the pluripotency markers such as SSEA-4 and TRA-1-60 are upregulated [START_REF] Chan | Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells[END_REF].

Interestingly, SSEA-4 appears earlier than TRA-1-60, the latter serving as a marker of more mature IPSCs [START_REF] Chan | Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells[END_REF]. Therefore, the simultaneous presence of both SSEA-4 and TRA-1-60 is an indication of cells further along in the reprogramming process (Figure 2A, I, pp.56-57), while cells only positive for SSEA-4 and lacking TRA-1-60 would be less far progressed (Figure 2A, II, pp.56-57). Finally, cells with none of these markers are the furthest from achieving the reprogrammed state (Figure 2A, III, pp.56-57). Based on the distinction of the different reprogramming states using these makers, we analyzed the levels of HSPA5-GFP at different time points of reprogramming (Figure 2B, pp.56-57). Consistently we observed higher levels of HSPA5-GFP in the cells that had progressed the furthest in the reprogramming process (SSEA-4 and TRA-1-60 positive, I).

To validate the GFP reporter, we sorted the three populations (I, II, and III) at day 7 of reprogramming and assessed their UPR ER levels by mRNA levels. As expected, we found higher levels of induction in the SSEA-4+/TRA-1-60+ cells (Figure 2C, pp.56-57).

Additionally, we confirmed the reactivation of the endogenous stemness genes in the SSEA-4+/TRA-1-60+ population (supplementary Figure 3, pp.66-67).

Because of the correlation between increased HSPA5 levels and progression towards the reprogrammed state, we asked what role, if any, did the UPR ER play in the reprogramming process. To address this question, we modulated the UPR ER during reprogramming either pharmacologically or genetically. Pharmacologically, we either activated the UPR ER using APY29, a drug that activates the RNAse activity of IRE1 [START_REF] Hetz | Targeting the unfolded protein response in disease[END_REF] , or inactivated the UPR ER using GSK2656157, a compound that inhibits both PERK and eIF2α phosphorylation (Atkins et al., 2013) (supplementary Figure 4A, pp.68-69). In all cases cell proliferation rates were unaffected (supplementary Figure 4B, pp.68-69). Strikingly, activation of the UPR ER with APY29 increased the percentage of cells expressing the SSEA-4 and TRA-1-60 while limiting the UPR ER with GSK2656157 decreased this population (Figure 2D, pp.56-57).

Intrigued by the pharmacological manipulation of the UPR ER upon reprogramming, we investigated whether overexpression of XBP1s could increase the reprogramming efficiency in keratinocytes. Consistent with the previous results, XBP1s increased the reprogramming efficiency and this activity was dependent upon the transcriptional activity of XBP1s since overexpression of a mutant version of XBP1s that lacked its DNA binding domain was unable to promote reprogramming (Figure 2E, pp.56-57). We confirmed that the increase in reprogramming efficiency was not the result of a higher proliferation rate due to XBP1s overexpression (supplementary Figure 5A, pp.70-71) and also followed cells to full IPSCs formation verifying the expression of stemness genes (supplementary Figure 5B, pp.70-71). We were able to expand these observations by reprogramming fibroblast using an episomal method (supplementary Figure 6, pp.72-73).

Taken together, UPR ER activation is necessary and sufficient to promote reprogramming of fibroblast-like cells. On the basis of these results we concluded that activation of the UPR ER increases reprogramming efficiency.

Activation of the UPR ER must be transient during reprogramming

Interestingly, we observed qualitatively that the success of IPSCs clonal expansion was lower when cells overexpressed XBP1s driven by the EF1α promoter with retroviral reprogramming. On the contrary, in the episomal reprogramming method, IPSCs clonal derivation was very similar between the GFP control and XBP1s overexpression driven by a CMV promoter. EF1α promoter is notoriously used in embryonic stem cells because it is rarely silenced contrary to CMV [START_REF] Xia | Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner[END_REF]. This led us to postulate that high levels of XBP1s in IPSCs would be detrimental and that the UPR ER is required transiently Therefore, activation of the UPR ER must be transient during reprogramming.

HSPA5-GFP levels predict the efficiency of reprogramming

Because reprogramming efficiency could be increased by the activation of the UPR ER , we postulated that the levels of HSPA5-GFP might predict the efficiency of reprogramming in populations of cells undergoing the process of reprogramming. Therefore, we hypothesized that variations in the levels of HSPA5 could be a driving factor for successful reprogramming. During the early phase of reprogramming using our HSPA5-GFP reporter we observed a Gaussian distribution of GFP fluorescence amongst the cell population (Figure 3A, pp.58-59). We subdivided the GFP positive population into 3 equal subpopulations according to their levels of HSPA5-GFP expression (low, medium and high) at day 8 of reprogramming. The percentage of SSEA-4+/TRA-1-60+ cells was the highest in cells with the higher levels of HSPA5-GFP and lowest in the cells with the lower levels of HSPA5-GFP expression (Figure 3A, pp.58-59). We expanded this observation to multiple time points during reprogramming and observed the same result:

higher HSPA5-GFP correlated with increased SSEA-4/TRA-1-60+ cells (Figure 3B, pp.58-59). This finding suggests that levels of HSPA-5 could serve as a good predictor of reprogramming efficiency.

To test this idea, we sorted cells at day 7 of reprogramming based on their levels of HSPA5-GFP into two populations: high and low levels. Cells were plated onto MEFs and we assessed IPS colony formation. After 10 days in culture, cells were stained for TRA-1-60. As postulated, cells with higher levels of HSPA5-GFP at day 7 gave rise to more IPS colonies (Figure 3C,. Taken together, HSPA5-GFP levels appear to be predictive of IPSC formation during the reprogramming process.

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Cellular internal complexity predicts the efficiency of reprogramming

During FACS analysis of the high HSPA5-GFP fibroblast-like cells we noticed that the Side SCatter (SSC) and Forward Scatter (FSC) parameters of these cells were distinct from low HSPA5-GFP cell populations under normal growth conditions (Figure 4A Intrigued by the correlation of increased HSPA5-GFP expression, ER stress, SSC/FSC increases, we hypothesized that SSC and FSC might predict the efficiency of reprogramming, much like increased HSPA5-GFP did. On day 8 of reprogramming we gated cells with high and low SSC/FSC. Interestingly, cells with higher SSC and FSC had a higher percentage of SSEA-4+/TRA-1-60+ cells than their counterparts (Figure 4D, pp.60-61).

Because these results were obtained from fibroblast-like cells derived from ESCs, we further tested the predictive reprogramming efficiency of high SSC/FSC values of primary human dermal fibroblasts (HDFs). We found that much like the fibroblast-like derived ESCs, HDFs with high SSC and FSC values produced a greater proportion of SSEA-4+/TRA-1-60+ cells (Figure 4E, pp.60-61).

We tested the high SSC/FSC and low SSC/FSC populations for their ability to form IPSCs. Surprisingly, cells exhibiting high SSC and FSC at day 7 of reprogramming gave rise to less IPSCs than cells with lower ones (Figure 4F,. This unexpected result will be discussed further down.

Taken together, a strong correlation between the cellular internal complexity and the efficiency to reprogram exist that might be linked to ER stress.

Discussion

Cellular reprogramming is a poorly understood process with really low efficiency. Most of the current knowledge on reprogramming relies on the Yamanaka factors which ensure pluripotency and cell proliferation, and therefore contribute to the identity of ESCs [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF]. The extremely low efficiency of reprogramming can be enhanced by the addition of supplementary factors such as other pluripotencyassociated genes, cell cycle-regulating genes and epigenetic modifiers [START_REF] Takahashi | A decade of transcription factor-mediated reprogramming to pluripotency[END_REF]. However, the lack of other reprogramming enhancers remains a critical issue to advance IPS research. Here we demonstrate that an early ER stress is an essential step for a cell's ability to reprogram. Accordingly, the dramatic morphological changes and organelles remodeling that occur during reprogramming require the activation of potent cellular pathways such as the UPR ER . Our work not only documents this early stress for the first time but also provides strategies to increase the reprogramming efficiency by modulating the UPR ER .

During their discovery of IPSCs, the Yamanaka group hypothesized that the potential reprogramming factors should contribute to the identity of ESCs [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF]. The identification of these ES-cell specific genes was based on their transcriptional profile [START_REF] Mitsui | The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells[END_REF]. It is therefore surprising that XBP1s can robustly increase the reprogramming efficiency. Indeed, XBP1 is not a pluripotent gene and its levels are low in ESCs. Moreover, the UPR ER is less active than in differentiated counterparts. We propose an alternative and novel approach in increasing the reprogramming efficiency based on the theory establishing reprogramming as a process which reverses cellular development [START_REF] Cacchiarelli | Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency[END_REF]. Thus, utilizing genes required for normal development and differentiation could help reprogram better by enabling a successful transition between the two cell states. In line with this theory, XBP1s, among other UPR ER effectors, is required during development and differentiation and therefore expected to regulate reprogramming. Indeed, the homozygous deletion of either Hspa5 [START_REF] Luo | GRP78/BiP Is Required for Cell Proliferation and Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic Development[END_REF], Grp94 [START_REF] Wanderling | GRP94 Is Essential for Mesoderm Induction and Muscle Development Because It Regulates Insulin-like Growth Factor Secretion[END_REF], Grp58 [START_REF] Garbi | Impaired assembly of the major histocompatibility complex class I peptide-loading complex in mice deficient in the oxidoreductase ERp57[END_REF], Ire1a [START_REF] Iwawaki | Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability[END_REF], Xbp1 [START_REF] Reimold | An essential role in liver development for transcription factor XBP-1[END_REF], Calreticulin [START_REF] Mesaeli | Calreticulin Is Essential for Cardiac Development[END_REF], or deletion of both Atf6a and Atf6b [START_REF] Yamamoto | Transcriptional Induction of Mammalian ER Quality Control Proteins Is Mediated by Single or Combined Action of ATF6α and XBP1[END_REF] leads to embryonic lethality in mice. IRE1 increases lymphopoiesis of B cells [START_REF] Zhang | The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis[END_REF], XBP1 induces osteogenic and plasma differentiations [START_REF] Iwakoshi | The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response[END_REF], and CHOP plays an important role in the differentiation of B cells, erythrocytes, osteocytes and chondrocytes [START_REF] Cui | Novel Interaction between the Transcription Factor CHOP (GADD153) and the Ribosomal Protein FTE/S3a Modulates Erythropoiesis[END_REF][START_REF] Pereira | CCAAT/Enhancer Binding Protein Homologous Protein (DDIT3) Induces Osteoblastic Cell Differentiation[END_REF][START_REF] Skalet | Rapid B Cell Receptor-induced Unfolded Protein Response in Nonsecretory B Cells Correlates with Pro-Versus Antiapoptotic Cell Fate[END_REF][START_REF] Yang | Multiple Signals Induce Endoplasmic Reticulum Stress in Both Primary and Immortalized Chondrocytes Resulting in Loss of Differentiation, Impaired Cell Growth, and Apoptosis[END_REF].

The mechanism through which the activation of the UPR ER increases reprogramming efficiency remains to be elucidated. The UPR ER activation leads to a global reduction of protein synthesis [START_REF] Harding | Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase[END_REF] and the degradation of mRNA associated to the ER membrane [START_REF] Hollien | Decay of Endoplasmic Reticulum-Localized mRNAs During the Unfolded Protein Response[END_REF]. A possibility is that the somatic ER associated proteome is cleared from a substantial part of its somatic signature giving room to the new proteome to be set. Therefore, the activation of the UPR ER must be transient, which is suggested by our results. It is also tempting to speculate that the UPR ER activation may lower levels of secreted factors that could inhibit cellular reprogramming. A more comprehensive analysis of the secretome would be interesting to pursue. The ectopic activation of the UPR ER could provide a buffer and a bigger reservoir for the cell to explore different states and consequently reach pluripotency without inducing apoptosis along the way.

We also reported that the number of IPS colonies could be enriched based on the levels of fluorescently tagged endogenous HSPA5 gene, which integrates the global state of the

Results-Discussion

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UPR ER . We propose that technologies that enable tracking UPR ER activity such as the HSPA5-GFP reporter line or live staining will be great tools to increase the number of IPS colonies.

Interestingly, we found a positive correlation between SSC and FSC parameters and HSPA5-GFP levels. Consistent with that, cells with high SSC/FSC values had more SSEA-4+/TRA-1-60+ cells. Very surprisingly, we found that cells with low SSC/FSC sorted at day 7 of reprogramming gave rise to more IPSCs than cells with high SSC/FSC values. We should indeed expect to find more fully reprogrammed cells in the low SSC/FSC population since ESCs exhibit low SSC/FSC compared to derived fibroblastlike cells (supplementary Figure 9A, pp.78-79). While the positive correlation between SSC and FSC parameters and HSPA5-GFP levels holds for a homogenous population such as fibroblast-like cells or ESCs (Figure 4A, pp.60-61 and supplementary Figure 9B, pp.78-79), we think that the correlation is not fully applicable on a population that is going through reprogramming. Our hypothesis is that during the course of reprogramming, cells going through intense remodeling will activate the UPR ER , the ones that exhibit higher levels of HSPA5-GFP are the most likely to fully reprogram, they also have a higher percentage of cells that are SSEA-4+/TRA-1-60+. Successful cells change their morphology to resemble an ESCs and acquire low SSC/FSC values, while still under reprogramming stress and keeping their HSPA5-GFP levels high. The correlation between high HSP5-GFP levels and high FSC/SSC values, true on the population level, is lost for these few cells. This happens for a very small subset of cells while most of the other cells still remain with high SSC/FSC values, high HSPA5-GFP and are SSEA-4+/TRA-1-60+.

We predict that studying the pathways required to transit from one cell state to another can identify potent facilitators of reprogramming such as effectors ensuring protein quality control. Previous work in our lab [START_REF] Vilchez | Increased proteasome activity in human embryonic stem cells is regulated by PSMD11[END_REF] and others [START_REF] Buckley | Regulation of Pluripotency and Cellular Reprogramming by the Ubiquitin-Proteasome System[END_REF] has already linked protein quality control through the ubiquitin-proteasome system with stem cell maintenance and differentiation. We showed that high levels of proteasome activity are required for hESCs maintenance [START_REF] Vilchez | Increased proteasome activity in human embryonic stem cells is regulated by PSMD11[END_REF].

Furthermore, knockdown of the ubiquitin E3 ligase FBXW7 increased the Results-Material and methods

48

reprogramming efficiency. Conversely, knockdown of the deubiquinating enzyme PSMD14 failed to reprogram and generate IPSCs from mouse embryonic fibroblasts [START_REF] Buckley | Regulation of Pluripotency and Cellular Reprogramming by the Ubiquitin-Proteasome System[END_REF]. The role of other regulatory elements of protein quality control such as the mitochondrial unfolded protein response (UPR mt ), and molecular chaperones involved in the heat shock response remain largely unexplored in the regulation of stem cell differentiation or reprogramming. How these processes are involved in reprogramming, as well as their potential cross-play with the UPR ER will need to be explored. We believe that our observations can be expended to transdifferentiation paradigms, an extremely promising field for regenerative therapies.

Material and methods

Cell culture. Human dermal fibroblasts (Lonza CC-2511 and CC-2509), HEK293FT

(ThermoFisher, R70007), BJ human fibroblasts (ATCC, CRL-2522), fibroblast-like cells and irradiated CF-1 mouse embryonic fibroblasts (GlobalStem) were grown in DMEM, 10% FBS, 1x Pen/Strep, 1x glutamax and 1X non-essential amino acids (NEAA) (all from Invitrogen).

The hESC line H9 (WA09, WiCell Research Institute) and the other hIPS generated lines were cultured with mTeSR1 media (Stem Cell Technologies) on Geltrex (Invitrogen).

Human keratinocytes (Lonza 192907) were cultured with KGM-Gold media (Lonza).

Plasmids. A list of the plasmids and the cloning strategy can be found in supplementary Viral production. Lentiviral and moloney-based retroviral pMX-derived vectors were co-transfected with their respective packaging vectors in 293FT cells using JetPrime transfection reagent to generate viral particles as previously described [START_REF] Ruiz | Generation of a Drug-inducible Reporter System to Study Cell Reprogramming in Human Cells[END_REF].

The viral supernatant was filtered through a 0.45 uM filter. bFGF (all from Invitrogen), and 55 uM β-mercaptoethanol (Sigma). To evaluate reprogramming efficiency, the same number of infected cells was plated, after 2-3 weeks cells were fixed with 4% PFA and stained for TRA-1-60 as previously described [START_REF] Onder | Chromatin-modifying enzymes as modulators of reprogramming[END_REF] and scored. Briefly, fixed cells were blocked for 1 hour at room temperature in 1xPBS, 3% FBS, 0.3% Triton X-100, then incubated with biotin-anti-Tra-1-60 (eBioscience13-8863-82, 1:250) over night at 4C and the next day streptavidin horseradish peroxidase (Biolegend 405210, 1:500) for 2 hours at room temperature.

Staining was developed with the sigmaFast DAB kit (D0426). Alternatively, an alkaline phosphatase (AP) staining was performed for episomal reprogramming experiments as instructed by the Millipore detection kit (SCR004). Briefly, cells were fixed in 4% PFA for less than a minute to avoid losing the AP activity. Cells were rinsed with TBS-T and covered with Fast Red Violet Solution/water/Naphthol (2:1:1) for 20 min followed by a wash with PBS. AP positive colonies were then counted.

For time course studies, imaging and flow cytometry, cells were plated on geltrex coated plates instead of MEFs.

Where indicated, after plating on geltrex, cells were incubated with APY29 (Chem Scene, CS-2552) or GSK 2656157 (Chem Scene, CS-3262) for 3 days.

Alternatively, cells were also reprogrammed using an episomal electroporation system [START_REF] Okita | A more efficient method to generate integration-free human iPS cells[END_REF]. Briefly, cells were first selected with the appropriate factor. 500,000 cells were then electroporated with the episomal constructs using the nucleofector kit (Lonza, VPD-1001). Cells were plated and kept in their original media. After 6 days, cells were dissociated and plated on freshly plated MEFs. Cells were switched to IPS media the next day.

Derivation of fibroblast-like cells. Stem cells were differentiated into fibroblast-like cells using an embryoid body (EB)-mediated protocol. Stem cells grown on Geltrex were detached using dispase, resuspended in DMEM/F12, 20% FBS, 1x glutamax, 1x NEAA, 1x Pen/Strep and 55 µM β-mercaptoethanol and grown on low adhesion plates for 4 days with media change. EBs were plated on gelatin-coated plates and cultured with the same media. When EBs spread and cells appeared fibroblast looking, the culture was dissociated using TrypLE and replated using a regular fibroblast media. This was serially done until the whole population became uniform.

RNA isolation and real-time PCR. Cells were collected in Trizol®. A classic chloroform extraction followed by a 70% ethanol precipitation was performed. The mixture was then processed through column using the RNeasy quiagen kit as described by the manufacturer. Quantitect reverse transcription kit (Quiagen) was used to synthesize complementary DNA. Real-time PCR was performed using Sybr select mix (Life Technologies). GAPDH expression was used to normalize gene expression values.

Primer sequences can be found in the supplementary Table 3, p.83.

Western blot analysis. Cells were washed with PBS and RIPA buffer was added to the plates on ice. Cells were scraped, collected and stored at -20C. The RIPA buffer was always supplemented with Roche cOmplete mini, and phosSTOP when needed. 20 µg of protein was loaded per lane and actin or tubulin was used as a loading control in pre-cast 4-12% Bis-Tris NuPage gels (Invitrogen). Proteins were blotted on nitrocellulose membranes using the NuPage reagents according to the manufacturer instructions.

Membranes were prepared for imaging using Odyssey® CLx Imaging System-LI-COR Biosciences with the appropriate reagents. Briefly, membranes were incubated in the proprietary blocking buffer for 1 hour at room temperature. Overnight primary antibody incubation at 4C was done using the blocking buffer and 0.1% Tween-20. Membranes were washed in TBS-T then incubated with secondary antibody for 1 hour at room temperature. Membranes were then washed in TBS-T with a final wash in TBS. For the list of antibodies and concentrations refer to supplementary Flow cytometry. For cell analysis, cells were dissociated with TrypLE and pelleted. 100 µL of a fluorescent-conjugated antibodies cocktail (5 µL of SSEA-4 330408, 5 µL of TRA-1-60 330610 Biolegend) in staining media (1xPBS, 2% FBS) was used to resuspend the pellet and incubated 30min on ice. Cells were then resuspended in excess of staining media, span down and resuspended in staining media, filtered through a cell strainer and kept on ice. Cells were analyzed using the BD Bioscience LSR Fortessa. The analysis was done using the FlowJo software.

For cell sorting, a similar procedure was followed. Cells were eventually resupsended in their media supplemented with rock inhibitor and sorted accordingly using the BD Bioscience Influx Sorter. Cells were then transferred to appropriate dishes for culture and kept on rock inhibitor during the next 24 hours.

ER secretion assay.

Transduced cells with Gluc-CFP were incubated 24 hours with fresh media and the supernatant was collected for analysis. An equal volume of Gluc Glow buffer (nanolight) was added to the supernatant in a 96-well plate format. The luminescence was measured by a TECAN plate reader and integrated over 50 ms.

Cell health/survival assay. Cells were plated on 96-well plates and treated with the appropriate condition. After the desired incubation time, cell titer glow buffer (Promega)

was added to the wells (1:5 volume) and incubated for 12min on a shaker. The luminescence was measured with the TECAN plate reader and integrated over 1s.

Electron microscopy. Cells were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 5 min. Samples were rinsed with 0.1M sodium cacodylate Buffer (3x5 min)

followed by the addition of 1% osmium tet, 1.5% ferrocyanide in 0.1M cacodylate buffer (5min). After washing with water (3x5min), 2% uranyl acetate was added for 5min followed by a water rinse. A dehydration series of ethanol was then completed: 35%, 50% 75%, 100%, 100% (5 min each). A 1:1 ethanol/resin (3x10min) incubation followed

Results-Acknowledgements 52 by 100% resin (3x10min) was done. The samples were cured over 48hrs, sectioned at 50nm with a microtome using a Diatome. Sections were placed on a coated copper mesh grid. They were then stained with uranyl acetate for 5 min, and then stained with lead citrate for 5 min before imaging.

Genome editing and southern blot. Transcription activator-like effector nuclease (TALENs) technology was used to create a fusion HSPA5-GFP by insertion of eGFP at the 3' end of the HSPA5 locus. We followed the protocol described in [START_REF] Hockemeyer | Genetic engineering of human pluripotent cells using TALE nucleases[END_REF]. TALENs were cloned to bind ACAGCAGAAAAAGATGA and ATTACAGCACTAGCA sequences and generate a double-stranded break around the STOP codon. The donor plasmid OCT4-eGFP-PGK-Puro, published in [START_REF] Hockemeyer | Genetic engineering of human pluripotent cells using TALE nucleases[END_REF], was adapted to target HSPA5 by changing the homology arms. H9 cells were electroporated and clonal expansion after puromycin selection was done. Successful targeting was confirmed by southern blot using the GFP probe published in [START_REF] Hockemeyer | Genetic engineering of human pluripotent cells using TALE nucleases[END_REF].

Statistical analysis.

The software Prism was used to perform the statistical tests. The corresponding statistical tests and the number of biological repeats, denoted as n, are indicated in the figure legends. For drug dose response assays, a log(drug) vs normalized response with viable slope model was used to determine the EC50.

Figures and their Legends

Stemness genes Endo OCT4

TGTACTCCTCGGTCCCTTTC TCCAGGTTTTCTTTCCCTAGC [START_REF] Maherali | A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells[END_REF] Endo SOX2

GCTAGTCTCCAAGCGACGAA GCAAGAAGCCTCTCCTTGAA [START_REF] Maherali | A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells[END_REF] Endo Nanog

Discussion

Our research shows that the UPR ER is transiently activated during reprogramming. This is in line with the expectations, as this process results in dramatic morphological changes and organellar remodeling. Our work not only documents an early stress that upregulates the UPR ER but also provides strategies to increase the reprogramming efficiency by modulating the UPR ER .

I. Main results

a. Possible roles of single reprogramming factors in inducing the

UPR ER during cellular reprogramming

The UPR ER is activated during cellular reprogramming. Whether the induction is the result of the cellular reprogramming process itself or due to the direct action of a single reprogramming factor is unclear. Soufi and colleagues [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF] performed a chromatin immunoprecipitation sequencing (ChiP-seq) to map the early protein-DNA interactions of OCT4, SOX2, KLF4, and c-MYC with the human genome using a doxycycline-inducible system. The authors used a 48 hr dox induction point on the basis that OSKM expression was maximal at that time and that most of the transcriptional changes are not happening yet. Therefore, only the primary effects of the reprogramming factors are studied. Based on their genome-wide ChiP-seq data we picked seven UPR ER related genes of interest and summarized their results in Table 2, p.83. Strikingly, all the genes involved in the UPR ER were bound by one or multiple reprogramming factors with the exception of ATF4 and GRP94. This suggests that activation of the UPR ER , at least of some of its components, could potentially be the result of the overexpression of any single reprogramming factor independently; therefore questioning the idea that the process of cellular reprogramming triggers the activation of the UPR ER . We think that while it is possible that each reprogramming factor can bind the DNA at UPR ER promoter genes and induce their expression at a low level, it is more likely that the wide remodeling and transcriptional changes occurring during reprogramming induce an excessive load on the ER and result in a potent activation of the UPR ER . Key points should be raised regarding the study in order to draw the proper conclusions. The possible cooperation between the different factors is not addressed by this experiment. Indeed, transcriptional activation of certain target genes involves OCT4 and SOX2 to co-bind on their promoters. There is a sequential binding step that is required in order to induce the expression of these target genes [START_REF] Chen | Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells[END_REF].

Discussion-Main results

88

Additionally, the physical presence of a transcription factor on a promoter does not necessitate transcriptional activity. Also, in a context where high overexpression of the reprogramming factors is observed, the specificity of binding must be questioned.

Though the direct contribution of the reprogramming factors directly binding the UPR ER promoters resulting in the induction of the UPR ER cannot be excluded, we believe that it only plays a minor role. The major cellular and organellar remodeling that occurs during reprogramming appears to have a more potent responsibility as detailed further below.

b. The temporal requirement of the UPR ER

Our data suggest a temporal requirement of the UPR ER during cellular reprogramming.

This observation comes from the fact that embryonic stem cells have lower UPR ER activity than some of their differentiated counterparts (supplementary Table 1,

89

UPR ER with the drug GSK2656157, a compound that inhibits both PERK and eIF2α phosphorylation [START_REF] Atkins | Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity[END_REF], for 3 days during the beginning of cellular reprogramming decreased the percentage of the SSEA-4+/TRA-1-60+ population. It would be interesting to vary the exposure time to the drugs and/or change the time window during which the drugs are applied during cellular reprogramming to further support our statement.

An indirect validation that the induction of the UPR ER was required transiently came from our experiments that use the EF1α promoter to drive XBP1s. We initially decided to choose the EF1α promoter because the overexpression is mild preventing initiation of cell death while maintaining the UPR ER activated and because the promoter is not completely silenced in ESCs [START_REF] Xia | Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner[END_REF]. Therefore, the expression of the transgenes will be sustained during most of the reprogramming process. We assumed this would be beneficial for reprogramming. We observed that despite substantially increasing the reprogramming efficiency, IPSCs clones derived from the EF1α promoter driving XBP1s cells had poor survival. The picked colonies would round up and not spread evenly leading to the formation of an embryoid body (EB)-like structure. A major difference observed at the time of picking was that the entire colony would detach preventing us from effectively being able to dissociate it. The extracellular matrix was stickier when the colonies came from XBP1s overexpression cells. Considering the role of the ER for secretion and the synthesis of transmembrane proteins we speculated that XBP1s was not completely silenced and could be responsible for this phenotype. It is established that the EF1α promoter is not silenced in ESCs while others like CMV are [START_REF] Xia | Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner[END_REF]. We observed a similar "rounding up" phenotype when we overexpressed XBP1s in ESCs with the EF1α promoter. In line with these observations, all the IPSCs lines successfully derived from XBP1s overexpression showed silencing of the transgene while silencing of the transcriptional inactive XBP1s-DBD was not required for successful derivation.

Additionally, when we derived IPSCs using an episomal reprogramming protocol together with the CMV promoter driving either GFP or XBP1s, we did not observe any differences in deriving IPSCs between the two conditions. As anticipated, the expression of XBP1s was similar between the derived lines and the controls suggesting a proper silencing of the transgene promoter.

To confirm the transient role of the UPR ER , it would be important to use an inducible system to express XBP1s. This would also allow testing the time window of the UPR ER activation requirement during cellular reprogramming.

c. Reconciling the granularity and shape with IPSCs formation prediction

We also reported that the number of IPS colonies could be enriched by FACS sorting based on the fluorescently tagged endogenous HSPA5 gene. High levels of HSPA5-GFP correlated with a higher efficiency of reprogramming. High levels of HSPA5-GFP are an indicator of cells remodeling their ER, and by extension, those going through reprogramming. The high HSPA5-GFP population had a higher percentage of SSEA-4+/TRA-1-60+ cells, suggestive of being more advanced toward full pluripotency and gave rise to more IPSC colonies. It would be interesting to extend this finding to other genes involved in the UPR ER . The utility of this method is limited by the fact that it involves the creation of a reporter line. Other methods such as live staining that could report the status of the UPR ER would be perhaps more versatile tools to increase the number of IPS colonies.

Remarkably, we found a correlation between the basal levels of HSPA5-GFP and the Side SCatter (SSC) and Forward Scatter (FSC) parameters in fibroblast-like cells (Figure 4A,. SSC reflects the internal cellular complexity and membrane texture while FSC is an indicator of the cell size. Indeed, high levels of HSPA5-GFP correlated with higher SSC/FSC values. Even more exciting, during cellular reprogramming, cells exhibiting high SSC/FSC values showed a higher population of SSEA-4+/TRA-1-60+ like high HSPA5-GFP cells which we know to be more efficiently reprogrammed (Figure 3C,. It was therefore tempting to hypothesize that high SSC/FSC would be predictive of higher number of IPSC colonies. Very surprisingly, the opposite was true (Figure 4F,. This observation raises interesting questions regarding our results.

While it is true that potential IPSCs have to stain positive for SSEA-4 and TRA-1-60, the reciprocal is not true. Indeed, all SSEA-4+/TRA-1-60+ cells are not going to give rise to Discussion-Main results

91

completely reprogrammed cells [START_REF] Kahler | Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting[END_REF] would explain why high HSPA5-GFP correlates better with more IPSC formation. Our hypothesis is that during the course of reprogramming, cells going through intense remodeling will activate the UPR ER , the ones that exhibit higher levels of HSPA5-GFP are the most likely to fully reprogram, they also have a higher percentage of cells that are SSEA-4+/TRA-1-60+. Successful cells change their morphology to resemble an ESCs and acquire low SSC/FSC values, while still under reprogramming stress and keeping their HSPA5-GFP levels high. This is when the correlation between high HSP5-GFP levels and high FSC/SSC values, true on the population level, is lost for these few cells.

This happens for a very small subset of cells while most of the other cells still remain with high SSC/FSC values, high HSPA5-GFP and are SSEA-4+/TRA-1-60+.

An interesting experiment to do would be to sort high and low SSC/FSC in the high and low HSPA5-GFP; this could help to further enrich in reprogrammed cells. It would be exciting to compare this method to the SSEA-4/TRA-1-60 enrichment strategy in term of reprogramming efficiency. [START_REF] Banito | Senescence impairs successful reprogramming to pluripotent stem cells[END_REF][START_REF] Marión | A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity[END_REF][START_REF] Utikal | Immortalization eliminates a roadblock during cellular reprogramming into iPS cells[END_REF], oxidative burst [START_REF] Ji | Antioxidant Supplementation Reduces Genomic Aberrations in Human Induced Pluripotent Stem Cells[END_REF], and DNA damage [START_REF] Ruiz | Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells[END_REF].

II. How

All these reprogramming-associated consequences are known in other contexts to disrupt the ER homeostasis. Therefore, the beneficial role of the UPR ER activation during reprogramming can be explained by a better capacity to integrate and respond to these cues and alleviate more efficiently the ER stress during reprogramming.

Cellular reprogramming requires a dramatic remodeling of the cell structure and in particular of its organelles. A very powerful remodeling process in the cell is autophagy.

It is a self-catabolic mechanism through which dysfunctional and unnecessary components of the cell are degraded such as organelles and proteins [START_REF] Bento | Mammalian Autophagy: How Does It Work?[END_REF].

It has been recently reported that robust induction of autophagy happens during reprogramming in mouse fibroblasts [START_REF] Wu | Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming[END_REF]. The authors limit the role of autophagy to the degradation of p62 whose accumulation in autophagy-deficient cells facilitates reprogramming. The cell reshaping, such as the cell size and the mitochondrial remodeling, is achieved by the inhibition of mTORC1. Remarkably, ER stress is capable at the same time to activate mTORC1 [START_REF] Kato | mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway[END_REF] and autophagy [START_REF] Ogata | Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress[END_REF] suggesting it could be an early step in the process. It is noteworthy that p62 is activated by TRIM-13, an ER resident ubiquitin E3 ligase, during tunicamycin-induced ER stress [START_REF] Tomar | TRIM13 regulates caspase-8 ubiquitination, translocation to autophagosomes and activation during ER stress induced cell death[END_REF]. The role of TRIM-13 during reprogramming would be interesting to investigate since another E3 ligase, FBXW7, was shown to regulate cellular reprogramming and stemness in mice (Buckley et al., 2012b). Interestingly, knockdown of FBXW7 increased the reprogramming efficiency. Conversely, knockdown of the deubiquinating enzyme PSMD14 failed to reprogram and generate IPSCs from mouse embryonic fibroblasts (Buckley et al., 2012a).

Previous work in our lab has already linked protein quality control with stem cell maintenance. A high level of proteasome activity is observed in hESCs and is required for their pluripotency. PSMD11 levels, a 19S proteasome subunit, was shown to play an important role in increasing the proteasome activity in hESCs [START_REF] Vilchez | Increased proteasome activity in human embryonic stem cells is regulated by PSMD11[END_REF]. We anticipate that increased levels of proteasome activity through the overexpression of PSMD11 would increase the reprogramming efficiency.

Besides the induction of ER chaperones, the UPR ER activates key players of the ERassociated protein degradation (ERAD) pathway [START_REF] Travers | Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation[END_REF]. This process mediates the delivery of unfolded proteins from the ER to the cytosol for proteasomal degradation. When the proteasome machinery is unable to degrade the unfolded proteins either because of their size of because of their aggregation status, another more potent mechanism is activated: autophagy. Not only are proteins and aggregates degraded, but entire organelles are recycled. Key members of the autophagy pathway are UPR ER target genes [START_REF] Bernales | Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response[END_REF].

Interestingly, in some cases UPR ER activation can induce the expression of c-MYC [START_REF] Shi | Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress[END_REF] and KLF4 [START_REF] Sugiura | The Unfolded Protein Response Is Activated in Differentiating Epidermal Keratinocytes[END_REF]. This activates the expression of the endogenous c-MYC and KLF4 possibly speeding up the process of reprogramming.

The role of other regulatory elements of protein quality control such as the mitochondrial UPR (UPR mt ), and molecular chaperones involved in the heat shock response remain largely unexplored in the regulation of stem cell differentiation or reprogramming. How these processes are involved in reprogramming, as well as their potential cross-play with the UPR ER will need to be explored.

Mitochondria have been shown to go through remodeling during reprogramming [START_REF] Folmes | Somatic Oxidative Bioenergetics Transitions into Pluripotency-Dependent Glycolysis to Facilitate Nuclear Reprogramming[END_REF], thus, it is tempting to speculate that the UPR mt might be turned on during the early stages of cellular reprogramming. Links between regulatory components of the Discussion-How does the UPRER activation increase cellular reprogramming efficiency?

94

UPR ER with mitochondrial regulation and function exist. For example, ATF4 can control the expression of Parkin, a ubiquitin ligase crucial for mitochondria function and dynamics [START_REF] Bouman | Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress[END_REF]. Parkin, in turn, is able to enhance branches of the UPR ER through the activation of XBP1s [START_REF] Duplan | ER-stress-associated functional link between Parkin and DJ-1 via a transcriptional cascade involving the tumor suppressor p53 and the spliced X-box binding protein XBP-1[END_REF]. The activity of PGC1α, a master regulator of mitochondrial biogenesis, was associated with ATF6 [START_REF] Arensdorf | Temporal clustering of gene expression links the metabolic transcription factor HNF4α to the ER stress-dependent gene regulatory network[END_REF].

Evidence tends to support the presence of cross talk between UPR, autophagy and mitochondria underlying the UPR ER activation [START_REF] Senft | UPR, autophagy, and mitochondria crosstalk underlies the ER stress response[END_REF] offering new avenues to study and understand cellular reprogramming through the prism of protein control quality.

b. Resetting the ER towards pluripotency

Acquisition of pluripotency implies a major remodeling of the organelles. ER morphology, for instance, changes drastically. The ER size correlates with the UPR ER activation in order to respond to physiological demand. The UPR ER can modulate the secretory capacity of the ER. During reprogramming, it is important to erase the somatic proteome and establish one of an embryonic stem cell. The UPR ER activation could reset the ER associated proteome and contribute to reprogramming through two major pathways: translation and translocation.

The phosphorylation of eIF2α by PERK reduces translation initiation. This not only reduces the load of potentially misfolded proteins, which increases the quality of the ER proteome, but also stops the expression of the somatic genes. In parallel, the activation of IRE1 leads to the degradation of ER-bound mRNA through the RIDD pathway [START_REF] Hollien | Decay of Endoplasmic Reticulum-Localized mRNAs During the Unfolded Protein Response[END_REF]. Thus, a global reset of the somatic ER associated proteome occurs.

The GADD34 restoration of translation initiation through eIF2α dephosphorylation could enable the translation of the newly activated genes by the reprogramming factors. Indeed, it is very likely that the reprogramming factors induce a high expression of genes that are ensuring stemness and therefore their mRNA would outcompete the ones that ensure the somatic cell identity. This could lead to the reprogramming of the ER proteome.

A complementary mechanism occurs at the translocons. Translocons are channels through which nascent peptides enter the ER lumen. ER chaperones can assist during translocation of the newly synthesized peptide into the lumen [START_REF] Brodsky | BiP and Sec63p are required for both co-and posttranslational protein translocation into the yeast endoplasmic reticulum[END_REF].

When the UPR ER is activated, the chaperones are titrated away from this task and as a result the translocation efficiency is reduced. Thus, fewer proteins are loaded into the ER reducing the global load. This could also contribute to reset the previous somatic ER proteome. Interestingly, Kang and colleagues [START_REF] Kang | Substrate-Specific Translocational Attenuation during ER Stress Defines a Pre-Emptive Quality Control Pathway[END_REF] observed a preferential translocation of proteins such as HSPA5 into the ER under stressful conditions. This mechanism is based on the "strength" of the signal peptide harbored by the secreted proteins. Besides increasing the quality of the ER proteome, it would be interesting to investigate if stem cell specific secreted proteins present a "strong" signal peptide that could play a role in the establishment of pluripotency. Conversely, some secreted proteins or receptors reminiscent of the somatic state could be negative regulators of stemness. A more comprehensive analysis of the secretome could give very interesting hints into this hypothesis. Consistent with this hypothesis, the mesenchymal-to-epithelial transition relies on signaling pathways and is an essential step during cellular reprogramming of mouse embryonic fibroblasts [START_REF] Li | A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts[END_REF]. Li and colleagues proposed that SOX2, OCT4 and c-MYC inhibit the TGF-β (transforming growth factor β) signaling pathway while KLF4 induced E-cadherin, an epithelial gene. While there might be speciesspecific differences, this observation highlights the role of signaling molecules during cellular reprogramming and potentially a connection with the modulation of ER secretion capacity by the activation of the UPR ER .

c. Between life and death, a second chance for reprogramming

The different UPR ER branches play important roles in the decision whether the cell has to activate apoptosis or not. Apoptosis is a major barrier for cellular reprogramming [START_REF] Marión | A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity[END_REF]. A notable gene involved in pro-apoptotic signal is CHOP. As mentioned in the introduction, CHOP is induced by ATF4 [START_REF] Marciniak | CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum[END_REF] and ATF6 [START_REF] Yoshida | ATF6 Activated by Proteolysis Binds in the Presence of NF-Y (CBF) Directly to the cis-Acting Element Responsible for the Mammalian Unfolded Protein Response[END_REF]. Sustained and/or strong activation of the UPR ER leads to the expression of CHOP and initiates apoptosis. Interestingly, sustained activation of the IRE1 pathway via the splicing of XBP1 promotes cell survival [START_REF] Lin | Divergent Effects of PERK and IRE1 Signaling on Cell Viability[END_REF].

Therefore overexpression of XBP1s or addition of APY29 (promoting the splicing of XBP1) during reprogramming could protect the cells from the stress imposed by reprogramming. This could offer a wider range of possibilities for the cell to explore different cell states and maybe find a path towards pluripotency. The protection conferred by higher levels of UPR ER activity increases the cell state plasticity.

d. Reprogramming as a reversal of development: lessons from the role of the UPR ER during normal development and differentiation

Reprogramming can be comprehended as a reversal of development. Using a mouse secondary reprogramming system, Cacchiarelli and colleagues [START_REF] Cacchiarelli | Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency[END_REF] observed distinct waves of gene network activation corresponding to developmental genes characteristic of early embryonic patterning genes and followed by a preimplantation gene signature. We reported in the introduction that numerous UPR ER effectors are required during development. For example the homozygous deletion of either Hspa5 [START_REF] Luo | GRP78/BiP Is Required for Cell Proliferation and Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic Development[END_REF], Grp94 [START_REF] Wanderling | GRP94 Is Essential for Mesoderm Induction and Muscle Development Because It Regulates Insulin-like Growth Factor Secretion[END_REF], Grp58 [START_REF] Garbi | Impaired assembly of the major histocompatibility complex class I peptide-loading complex in mice deficient in the oxidoreductase ERp57[END_REF], Ire1a [START_REF] Iwawaki | Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability[END_REF], Xbp1 [START_REF] Reimold | An essential role in liver development for transcription factor XBP-1[END_REF], Calreticulin [START_REF] Mesaeli | Calreticulin Is Essential for Cardiac Development[END_REF], or deletion of both Atf6a and Atf6b [START_REF] Yamamoto | Transcriptional Induction of Mammalian ER Quality Control Proteins Is Mediated by Single or Combined Action of ATF6α and XBP1[END_REF] leads to embryonic lethality in mice. Furthermore, several components of the UPR ER have an important role during differentiation. IRE1 increases lymphopoiesis of B cells [START_REF] Zhang | The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis[END_REF], XBP1 induces osteogenic and plasma differentiations [START_REF] Iwakoshi | The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response[END_REF], and CHOP plays an important role in the differentiation of B cells, erythrocytes, osteocytes and chondrocytes [START_REF] Cui | Novel Interaction between the Transcription Factor CHOP (GADD153) and the Ribosomal Protein FTE/S3a Modulates Erythropoiesis[END_REF][START_REF] Pereira | CCAAT/Enhancer Binding Protein Homologous Protein (DDIT3) Induces Osteoblastic Cell Differentiation[END_REF][START_REF] Skalet | Rapid B Cell Receptor-induced Unfolded Protein Response in Nonsecretory B Cells Correlates with Pro-Versus Antiapoptotic Cell Fate[END_REF][START_REF] Yang | Multiple Signals Induce Endoplasmic Reticulum Stress in Both Primary and Immortalized Chondrocytes Resulting in Loss of Differentiation, Impaired Cell Growth, and Apoptosis[END_REF]. These observations demonstrate the pleiotropic mode of action of the UPR ER in different differentiation contexts and during normal development. It is therefore not surprising that the UPR ER plays such an important role during cellular reprogramming.

The role of the UPR ER during normal development can be comprehended as a need to deal with the newly synthesized and the remodeling of the ER. Pieces of evidence suggest a more physiological role where the UPR ER is activated even when the proteinprocessing capacity of the ER is not exceeded. During development, some cells require a drastic expansion of their ER. The UPR ER has an important proactive role in this context.

For instance, in the context of B cells differentiation into antibody-producing plasma cells the UPR ER is activated even before any Ig synthesis occurs, suggesting a possible proactive role of the UPR ER (van Anken et al., 2003). Consistently, B cells lacking the ability to produce Ig still activate XBP1 and differentiate normally [START_REF] Hu | XBP-1 regulates signal transduction, transcription factors and bone marrow colonization in B cells[END_REF].

These observations suggest that the UPR ER can be activated in preparation for the upcoming load of proteins. This proactive role suggests that the UPR ER is a driving force for cellular state changes and not just a consequence. We believe that the proactive Transdifferentiation occurs naturally. The newt, a type of salamander, is able to regenerate its lens after lentectomy. Pigmented epithelial cells change their morphology and lose their pigments and after a proliferation phase, cells switch to the new lineage and differentiate into mature lens cells [START_REF] Tsonis | A newt's eye view of lens regeneration[END_REF]. Experimental transdifferentiation, also called direct conversion, can be achieved by the forced expression of different transcription factors. For example, fibroblast can be transdifferentiated into functional neurons by the overexpression of ASCL1 (achaete-scute homologue 1), BRN2 (brainspecific homeobox and POU domain 2) and MYT1L (myelin transcription factor 1-like) [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF]. Likewise, fibroblasts can be converted to functional cardiomyocytes with MEF2C (myocyte-specific enhancer factor 2C) and TBX5 (T-box 5) [START_REF] Ieda | Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors[END_REF].

Together with cellular reprogramming, these mechanisms can help uncover cellular and molecular pathways leading to the development of regenerative strategies.

Transdifferentitaiton involves the reprogramming of a cell's fate making it similar to what happens during cellular reprogramming with the Yamanaka factors. It would be fascinating to study the contribution of the UPR ER during these processes.

b. A platform to study aging and rejuvenation

In our review, Vilchez and colleagues [START_REF] Vilchez | Proteostasis and aging of stem cells[END_REF], we highlighted the crucial role of protein quality control in the control of stemness maintenance. We also draw a parallel with cellular pathways required for organismal lifespan and health extension or "healthspan". It appears that the same cellular mechanisms at play in stem cell maintenance and lifespan extension operate. Extrapolating this hypothesis, the study of mechanisms involved in either stem cell maintenance or lifespan extension could benefit from each other. In a very striking publication, Chen and colleagues [START_REF] Chen | Rapamycin and other longevity-promoting compounds enhance the generation of mouse induced pluripotent stem cells[END_REF] showed that pro-longevity compounds such as rapamycin (an mTOR inhibitor), resveratrol, fisetin (two sirtuin activators), curcumin (an antioxidant) or spermidine (an autophagy inducer) were able to increase the reprogramming efficiency. Consistent with this observation, activation of the UPR ER is a potent pro-longevity pathway as published by our laboratory [START_REF] Taylor | XBP-1 Is a Cell-Nonautonomous Regulator of Stress Resistance and Longevity[END_REF]. We therefore hypothesize that we could use the knowledge from studies that identified barriers to cellular reprogramming and test these pathways for their ability to extend lifespan and healthspan and vice versa.

IV. General conclusion: Key points

• The UPR ER is transiently activated during cellular reprogramming Mori, K., Kawahara, T., Yoshida, H., Yanagi, H., and Yura, T. (1996). Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells 1, 803-817.

•
Morl, K., Ma, W., Gething, M.-J., and Sambrook, J. (1993). A transmembrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell 74, 743-756.

  Les cellules somatiques peuvent être reprogrammées en cellules pluripotent en surexprimant 4 facteurs de transcriptions: OCT4, SOX2, KLF4 et c-MYC. Ce processus nécessite en théorie un remodelage des organelles et un changement drastique du métabolisme. De plus, la reprogrammation cellulaire possède une composante stochastique qui est peu comprise et conduit à une faible efficacité. Nous avons fait l'hypothèse que cette variabilité est en partie due aux variations de la régulation de l'homéostasie protéique. Nous nous attendons à ce que la première phase de reprogrammation active les voies de stress qui régulent l'homéostasie protéique, ce qui impacterait l'efficacité de reprogrammation. Notre attention s'est dirigée vers le rôle de la réponse aux protéines dépliées du réticulum endoplasmique. Nous avons découvert que cette voie est active pendant la reprogrammation cellulaire et que son activation peut augmenter l'efficacité de ce processus. Par ailleurs le niveau d'activation de cette voie peut prédire l'efficacité de reprogrammation. Ces résultats suggèrent que la faible efficacité de reprogrammation cellulaire est en partie due à l'incapacité des cellules à activer cette voie de stress afin de pouvoir correctement répondre à la nouvelle charge The notion of cell fate dates back to the late 19 th century when August Weismann made

Figure 1 :

 1 Figure 1: The plasticity of the cell fate. Cell fate acquisition was believed to be unidirectional, starting from an immature pluripotent to a mature differentiated state. Waddington described this process as a ball rolling from the top of the Waddington "mountain" to "valleys" where it will be "trapped". Somatic cell nuclear transfer and somatic cell fusion with pluripotent stem cell indicated that this hypothesis was wrong and established the first indication of cellular fate plasticity. The epigenetic memory of the somatic cell can be erased. It was later shown that ectopic expression of key transcription factors was able to convert a cell type to another. Overexpression of MYOD (myoblast determination protein) in fibroblast converted them into myoblasts. The most sticking evidence of cell fate plasticity came from the Yamanaka group showing that somatic cells were able to be reprogrammed into a pluripotent stem cells state called induced pluripotent stem cell (IPSC) by the ectopic expression of solely four factors: OCT4, SOX2, KLF4 and c-MYC (OSKM). PSC: pluripotent stem cell; ES: embryonic stem; SCNT: somatic cell nuclear transfer. This figure is adapted from (Takahashi and Yamanaka, 2016).

Figure 2 :

 2 Figure 2: The stem cell core circuitry and potential reprogramming factors. A complex set of signaling controls embryonic stem cell (ESC) pluripotency and selfrenewal. This circuitry is mostly base on mice data but seems to be conserved in humans. OCT4 (also known as POUF5F1), SOX2 and NANOG form a transcriptional module essential for ESC maintenance. Both SOX2 and NANOG (not shown) interact with OCT4 and positively regulate their three transcripts. These genes activate the expression of other pluripotency genes (left of the core circuitry) and at the same time repress lineage commitment genes (right of the core circuitry). The list of the 24 candidate genes for reprogramming selected by the Yamanaka group are enumerated in the table on the right. The core circuitry figure was adapted from (MacArthur et al., 2009) and the list of the 24 candidate genes from (Takahashi and Yamanaka, 2016).

Figure 3 :

 3 Figure 3: Mechanisms of reprogramming in two steps. During the first stochastic phase, OSKM (OCT4, SOX2, KLF4 and c-MYC) act as pioneer factors and bind many different regions of the genome that are not OSKM targets in embryonic stem cells. This generates a dynamic state of the chromatin. Among the early genes OSKM binds to are: identity genes of the somatic cell (i.e. mouse embryonic fibroblast: MEF genes) such as epithelialto-mesenchymal transition identity genes (EMT) and mesenchymal-to-epithelial transition identity genes (MET) (orange box); genes involved in cellular proliferation, apoptosis and metabolism (red box); unknown target thought to facilitate the genomic fluidity (light grey); and distal regions of early pluripotency genes (dark grey). The light blue box represents the late pluripotency genes that are at this time refractory to be bound by OSKM. A second phase that is more hierarchical occurs. The first part of it is very speculative; in rare cells the earlyactivated pluripotency genes can start a more deterministic activation of core pluripotency genes such as Sox2 through a direct or hierarchical fashion. Sox2 is part of pluripotency initiating factors (PIFs) indispensable for the initiation of the core pluripotency circuitry. The endogenous pluripotency proteins OCT4, SOX2 and Nanog (OSN) bind their target genes (Boyer et al., 2005) and maintain the pluripotency of the induced pluripotent stem cells (IPSCs) in the absence of the exogenous targets. This figure was taken from (Buganim et al., 2013).

  that could be important for cellular reprogramming. The mechanisms in play in both ESCs and adult SCs to ensure their self-renewal and maintenance could be used to increase the efficiency of reprogramming. It underlines the need to maintain a healthy proteostasis in SCs in order to ensure their function during development and throughout the course of life. How SCs are able to maintain their function through aging and development are important characteristic of stemness.

  Figure 4, p.29, summarizes the mechanism of the UPR ER ; a more detailed description of the three pathways involved follows.

Figure 4 :

 4 Figure 4: The molecular mechanisms of the UPR ER . A: In stressed cells, IRE1 oligomerizes and trans-autophosphorylates, which unmasks its dormant endoribonucleolytic activity. Active IRE1 excises a small RNA fragment in XBP1 mRNA. This spliced version encodes a potent transcription factor, XBP1s, which activates a variety of genes including chaperones. This helps to deal with the unfolded proteins. In parallel, active IRE1 degrades specific mRNAs, which results in a reduced protein load on the ER. B: Upon stress, ATF6 is delivered to the Golgi apparatus where it is cleaved by SP1 and SP2. The cytosolic portion of ATF6 is then imported into the nucleus where it activates UPR target genes. C: Similarly to IRE1, PERK oligomerizes and is activated by transautophosphorylation during stress. Subsequent phosphorylation of eIF2α at Ser51 leads to a global decrease of translation. Through a particular mechanism, ATF4 translation is increased. ATF4 activates CHOP and GADD34. The later is important to dephosphorylate eIF2α and stop the activation of the PERK pathway. The figure was adapted from Walter and Ron (Walter and Ron, 2011).

  kinase RNA (PKR)-like ER kinase) is another ER-localized type I transmembrane protein. It has a stress-sensing luminal domain and a cytoplasmic portion that contains a protein kinase domain. Upon stress, PERK is able to transautophosphorylate after oligomerization (Figure 4C, p.29). PERK phosphorylates the α-

  Figure1A, pp.62-63). To corroborate the RNA levels, we analyzed HSPA5 protein levels and found that it too was increased(Figure 1B,. To further characterize the activation of the UPR ER we analyzed the phosphorylated state of IRE1 and PERK and found that both were highly phosphorylated during the reprogramming process (Figure1B, pp.54-55 and supplementary Figure 1C, pp.62-63). During the reprogramming process, the 4 reprogramming factors are delivered by viral infection, to rule out the possibility that the UPR ER is induced by the use of a viral delivery system, we used an episomal delivery by electroporation of the reprogramming factors and observed UPR ER activation (supplementary Figure 1B, pp.62-63).

  HSPA5 fused to eGFP. Using transcription activator-like effector nuclease (TALENs) genome editing, we placed the eGFP encoding sequence into the 3' end of one allele of the HSPA5 locus in H9 ESCs. Successful targeting was confirmed by southern blotting (supplementary Figure2A, pp.64-65). The HSPA5-GFP cell line was then differentiated into fibroblast-like cells to use for cellular reprogramming using an embryoid mediated differentiation protocol[START_REF] Ruiz | Generation of a Drug-inducible Reporter System to Study Cell Reprogramming in Human Cells[END_REF]. HSPA5-GFP fibroblast cells responded faithfully to ER stress caused by tunicamycin, showing robust induction under the stress condition. Likewise, after removal of the tunicamycin, GFP levels dropped from these cell lines (supplementary Figure2B,C, pp.64-65).

  during reprogramming. Consistent with this observation, EF1α driving XBP1s IPSC successfully derived clones showed silencing to levels similar to EF1α driving emGFP derived clones while the XBP1s-DBD (coding for the transcriptionally inactive XBP1s) IPSC derived clones failed to do so (supplementary Figure7A, pp.74-75). Remarkably, overexpression of XBP1s using the EF1α promoter in H9 ESCs prevented their proper spreading (supplementary Figure7B, pp.74-75). Notably, basal levels of UPR ER activity are low in embryonic stem cells compared to their differentiated counterparts as shown by transcriptome analysis from a published data set[START_REF] Lowry | Generation of human induced pluripotent stem cells from dermal fibroblasts[END_REF][START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF] (supplementary Table1, p.80), the HSPA5-GFP levels (supplementary Figure7C, pp.74-75) and western blot ofXBP1s and ATF6 (supplementary Figure 7D,.

  , pp.60-61). SSC reflects the internal cellular complexity and membrane texture while FSC measures the size of the cells(Figure 4A,, suggesting that high HSP5-GFP cells might have more complex internal granularity and possibly be larger. Under normal conditions, the top 33% HSPA5-GFP fibroblast-like cells had high internal cellular complexity and size; conversely the lowest 33% HSPA5-GFP cells had a lower internal cellular complexity and size. Therefore, there appears to be a gradient that positively correlates the levels of HSPA5-GFP with SSC and FSC. To exclude the possibility that those are two distinct populations resulting from a heterogeneous differentiation, we sorted these two populations. Seven days later the HSP5-GFP medians were similar (Figure4B, pp.60-61). In addition, when the UPR ER was ectopically induced, by expression of XBP1s, the SSC and FSC parameters were increased(Figure 4C,. Interestingly, the addition of tunicamycin to naïve cells also changed the SSC and FSC measurements to match those found with ectopic XBP1s expression. Addition of tunicamycin to the XBP1s cells did not further change the SSC and FSC(Figure 4C,. Lastly, knockdown of XBP1 decreased the population of cells with highSSC and FSC values (supplementary Figure 8B and C,.

iPSC generation .

 generation Primary cells were spinfected with the viral supernatant containing the reprogramming factors and other factors during 1hour at 1000g in presence of 5ug/mL of polybrene (Millipore) twice, 24 hours apart. The regular media was replaced after each round. Selection was started the next day of the last transfection, 48 hours later cells were dissociated with TrypLE (Invotrogen) and plated on top of irradiated MEFs in their regular media. The next day cells were switched to IPS media containing DMEM/F12, 20% knockout serum replacement, 1X Pen/Strep, 1X glutamax, 1X NEAA, 10ng/mL

Figure 1 :

 1 Figure 1: The UPR ER is activated during reprogramming. A: Relative mRNA levels of the three UPR ER branches effectors relative to GAPDH determined by qRT-PCR (n=3). D3 GFP control was set to 1. B: Time course reprogramming western blot analysis of HSPA5, P-IRE1 and IRE1. C: Electron microscopy of day 3 reprogramming fibroblasts and GFP control, scale bar = 0.2 µm. Pseudo-colors blue and red mark respectively the nucleus and the ER. D: Secretion capability of the ER measured by luciferase activity secreted in the media (n=12) and western blot analysis of the Gaussia luciferase. E: Sensitivity to tunicamycin treatment determined by EC50 measurement at day 4 of reprogramming of fibroblast-like cells (n=3). * indicates statistical difference (p-value<0.05) using an unpaired two-tailed t-test, n.s. indicates statistical non significance. Error bars indicate the standard deviation.

  55

  Figure 1

Figure 2 :Figure 3 :

 23 Figure 2: The reprogramming efficiency is improved upon UPR ER activation. A: Flow cytometry analysis of fibroblast-like HSPA5-GFP cells at day 8 of reprogramming stained with SSEA-4 and TRA-1-60 surface markers. I,II,III represent the different cell states of reprogramming. B: Median HSPA5-GFP of the different cell states (I,II,III) during reprogramming (n=3). C: Relative mRNA levels of the UPR ER effectors relative to GAPDH determined by qRT-PCR (n=3). Values for SSEA-4-/TRA-1-60-were set to 1. D: Percentage of SSEA-4+/TRA-1-60+ cells at day 14 of reporgramming after drug treatment with APY29, an inducer of the UPR ER , and GSK2656157, an inhibitor of the UPR ER from day 2 to day 5 of reprogramming (n=3). E: Relative reprogramming efficiency of keratinocytes measured by colony TRA-1-60 staining after 3 weeks in culture upon overexpression of emGFP, XBP1s and XBP1s-DBD (missing its DNA binding domain) with the EF1α promoter, shown are two biological replicates done in duplicate. Conditions with different letter denote a statistical significant difference between them (p-value<0.05, Tukey's multiple comparison test). Error bars indicate the standard deviation.

Figure 4 :

 4 Figure 4: SSC and FSC parameters predict the reprogramming efficiency. A: Left panel explains the meaning of the Side Scatter and Forward Scatter parameters on the cellular levels. Right panel represents the distribution of the top high (red) and low (blue) 1/3 HSPA5-GFP regarding their SSC and FSC values. B: Median HSPA5-GFP values after sorting fibroblast-like HSPA5-GFP on day 0 and after 7 days in culture (n=3). C: Top, respectively bottom, panel show the median Side SCatter, respectively Forward SCatter, after overexpression of luciferase (LUC), XBP1s and XBP1s-DBD with and without 1µg/mL tunicamycin treatment for 24 hours. D: SSEA-4 and TRA-1-60 parameters are shown based on the SSC and FSC selected population. Shown is the percentage within these populations at day 8 of reprogramming of the double positive cells (n=3). E: Upper panel shows the percentage of SSEA-4+/TRA-1-60+ neonatal fibroblasts at day 10 of reprogramming within the population. Bottom panel shows the relative reprogramming efficiency of sorted neonatal fibroblasts cells at day 10 based on their FSC/SSC values (n=3). F: Relative reprogramming efficiency of sorted fibroblastlike HSPA5-GFP cells at day 7 based on their FSC/SSC values (n=3). Conditions with different letter denote a statistical significant difference between them (p-value<0.05, Tukey's multiple comparison test). * indicates statistical difference (p-value<0.05) using an unpaired two-tailed t-test, n.s. indicates statistical non significance, only statistical comparisons of importance were performed. Error bars indicate the standard deviation.

  p.80 and supplementary Figure 6, pp.72-73). As observed either by transcriptome analysis, protein levels or HSPA5-GFP levels, UPR ER effectors are lower in hESCs compared to the derived fibroblast-like cells or normal fibroblasts. The fact that UPR ER activation was beneficial for cellular reprogramming (Figure 2, pp.56-57) was surprising and lead us to hypothesize that perhaps transient UPR ER activation is needed during a specific time during reprogramming, after which UPR ER activity levels are decreased to a low basal state. Our data are consistent with this hypothesis. The time course experiments in Figure 1B, pp.54-55 and supplementary Figure 1C, pp.62-63 show a transient activation of the PERK and IRE1 branches of the UPR ER during the early phase of cellular reprogramming. Activation of the UPR ER by pharmacological means using APY-29, an activator of the IRE1 ribonuclease activity (Hetz et al., 2013), increased the percentage of the SSEA-4+/TRA-1-60+ cells in the population during reprogramming. This effect was achieved by only exposing the cells to the drug during 3 days at the beginning of the reprogramming process. Therefore, a short and transient ectopic induction of the UPR ER was sufficient to increase the efficiency of reprogramming. Conversely, inhibiting the Discussion-Main results

  function of the UPR ER could be at play during cellular reprogramming. By further activating the UPR ER , genetically or pharmacologically, the cells are better primed for cellular reprogramming. The ER could better cope with the load of proteins generated by the addition of the Yamanaka factors and also with the results of the morphological changes. This is line with our data suggesting a transient role of the UPR ER . Activation of the UPR ER by XBP1s overexpression or addition of APY29 for only 3 days during the early step of reprogramming, when most of transcriptional changes happen, was able to prime the cells to reprogram with a higher efficiency. Conversely, inhibiting the UPR ER with GSK2656157 for only 3 days during the early step of reprogramming had potent effect in reducing the efficiency of reprogramming. We predict that ectopic activation of the UPR ER later during cellular reprogramming will have less effect on the efficiency of cellular reprogramming.Discussion-Extrapolation of the results to other paradigms98III. Extrapolation of the results to other paradigms a. Extending the findings to other cell state switch paradigmsWe believe that the transient UPR ER activation is a more general process that enables cells to transit between two different states by insuring the integrity of the ER proteome but also other biological pathways in the cell, thus helping cells overcome this barrier of changing cellular states. We predict that this process should operate in a very similar fashion during cases of transdifferentiation happening naturally or induced experimentally; during this process cells revert to a point where they are able to change lineages. Cells can then differentiate into another cell type. This processes involves the reprogramming of the cell's fate.

  Ectopic activation of the UPR ER increases cellular reprogramming• Levels of the UPR ER activity, as determined by HSPA5-GFP intensity,

Table 1

 1

, p.19 briefly summarizes the similarities between ESCs and IPSc and their potential for diverse applications.

Table 1 : Comparison of embryonic stem cells and induced pluripotent stem cells. Both cells are pluripotent stem cells suitable for the study of stemness, development and differentiation. They differ in their potential to study disease specific models, their applicability for clinical applications and their origin.

 1

Table 2 ,

 2 pp.81-82.

  Table 4, pp.84-85. Primary antibody incubation was done overnight. After PBS washes, secondary antibody was added for 1 hour at room temperature. After PBS washes, slides were mounted with mounting media containing DAPI. For the list of antibodies and concentrations refer to supplementary Table 4, pp.84-85.

Fluorescent immunostaining. Cells on slides were fixed with 4% PFA for 15min and washed with PBS. 2% donkey-serum blocking buffer in PBS was used for 1 hour at room Results-Material and methods 51 temperature.

Table 2 : Occupancy of OCT4, SOX2, KLF4, c-MYC at 48h of reprogramming. These data were adapted from Soufi and colleagues (Soufi et al., 2012). TSS: transcription start site; MACS: Model-based analysis of ChIP-seq; FDR: false discovery rate.

 2

			Chromatin occupancy within 20 kb upstream
	RefSeq annotation	TSS and gene body (peaks called with MACS
				at 0.005 FDR)
	transcript ID	gene name	c-MYC	KLF4	OCT4	SOX2
	NM_007348 ATF6	bound	bound	bound	bound
	NM_182810 ATF4	not bound not bound not bound not bound
	NM_005080 XBP1	bound	bound	not bound not bound
	NM_005347 HSPA5	bound	not bound not bound not bound
	NM_003299 HSP90B1/GRP94	not bound not bound not bound not bound
	NM_004083 DDIT3/CHOP	bound	not bound not bound not bound
	NM_014330 PPP1R15A/GADD34 bound	bound	not bound not bound

  . Approximately 3.6% of the SSEA-4+/TRA-1-60+ cells gave rise to IPSC colonies in the Kahler and colleagues study starting with human fibroblasts. It is important to consider that the actual IPSCs are merely a small fraction of cells, diluted in a larger population that can exhibit some characteristics that would mislead us. In the previous study, this means that 96.4% of the cells are blocked in an intermediate state of reprogramming. From our results, high SSC/FSC values have more SSEA-4+/TRA-1-60+ cells. Importantly, this does not imply low SSC/FSC have none. We would, in fact, expect to find more mature IPSCs among the low SSC/FSC population because ESCs have low SSC/FSC when compared to their differentiated counterparts such as fibroblast-like cells (supplementary Figure8, pp.76-77). It is noteworthy that the correlation between high HSPA5-GFP and high SSC/FSC was established under unstressed conditions in fibroblast-like cells and ESCs(Figure 4A,. During the process of reprogramming,

the cell state dramatically changes and does not resemble a fibroblast anymore. We believe that the correlation between the cell SSC/FSC parameters and HSPA5-GFP levels is lost for the fully reprogrammed cells, because diluted in a larger population, while still valid for most of the cells in a transient state of reprogramming. Cells with low SSC/FSC (that include fully reprogrammed cells) can exhibit high HSPA5-GFP levels, which

does the UPR ER activation increase cellular reprogramming efficiency? a. Cytoprotective role of the UPR ER during reprogramming and its interplay with other cytoprotective pathways

  Modulation of the UPR ER can be seen as a cytoprotective response to protect the cells undergoing reprogramming. It has been shown that reprogramming can result in apoptosis, cell-cycle arrest and senescence

Acknowledgments

Acknowledgments

I would like to express my gratitude to everyone in Pr Andrew Dillin's laboratory for making me feel like part of a family. I wish to express my deepest and sincere gratitude to my supervisor Andrew Dillin for his endless optimism, enthusiasm, support in my work and inspiration. Also, an enormous thanks to my other thesis co-director Olivier Bensaude for his invaluable advice and flexibility throughout my PhD. I extend my gratitude to Dirk Hockemeyer and Xavier Darzacq for external tutorship and helpful discussions. I would like to thank my reading committee and part of my jury, Valérie Lallemand-Mezger and Eric Chevet. I sincerely hope you will enjoy your summer reading. I would like to extend my thanks to the rest of the jury, Jonathan Weitzman, Charles Durand, Han Li and Andrew Dillin for kindly accepting to be present at my thesis defense.

I thank my colleagues and friends at the lab, for providing a positive and stimulating work environment: Celine Riera, Carlos Daniel De Magalhaes Filho, Carsten Merkwirth, Jenni Durieux, Kristan Steffen, David Vilchez, Nate Baird, Pete Douglas, Ye Tian, Kristen Berendzen, Erik Kapernick, Thomas Heimbucher, Larry Joe, Sarah Uhlein Tronnes, Melissa Sanchez, Jonathan Halloran, Gilbert Garcia, Hope Henderson, Joseph Daniele, Ashley Frakes, Ryo Higuchi-Sanabria, Sabine Jordan, Hyun-Eui Kim, Erica Moehle, Vidhya Ramachandran, Robert Schinzel, Ophir Shalem, Brant Webster, Suzanne Wolff and Nan Xin.

Especially to Celine, Casi, Daniel and Jenni for their unselfish help.

Acknowledgements

Funding was provided by HHMI and CIRM grants. We thank Dr B. Tannous who generously provided the Gluc-CFP, the Tjian lab for the episomal reprogramming vectors, Dr A. Panopoulos and Dr S. Ruiz for the pMX reprogramming vectors and the reprogramming protocols.

Supplementary Table 1: Transcriptome analysis of ER UPR genes in fibroblasts,

IPSCs and ESCs. The data analysis of Lowry and colleague data set [START_REF] Lowry | Generation of human induced pluripotent stem cells from dermal fibroblasts[END_REF] was done by Soufi and colleague [START_REF] Soufi | Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome[END_REF]. We picked seven UPR ER related genes of interest and summarized their results. As control, we picked Nanog a stemness marker and COL1A1 a fibroblast marker.

RefSeq annotation