Plasticité de l’identité cellulaire / Plasticity of cellular identity

Séminaire FSER organisé par Sophie Jarriault (IGBMC, France)et Oliver Hobert (Columbia University, USA) du 23 au 28 mai 2016

Participants

Paola Arlotta, Yann Barrandon, Cédric Blanpain, Helen Blau, Patrick Collombat, Magdalena Götz (Goetz), Thomas Graf, Oliver Hobert, Sophie Jarriault, Kim Bak Jensen, Leanne Jones, Lucie Laplane, Sri Teja Mullapudi, Freddy Radtke, Joel Rothman, Austin Smith, Shahragim Tajbakhsh, Marius Wernig, David Zarkower

Introduction

La conférence « Plasticité de l’identité cellulaire » a réuni, en mai 2016, dix-huit biologistes et une philosophe des sciences venant du monde entier. Cette conférence a eu lieu au Domaine des Treilles, un cadre splendide favorisant les interactions informelles. Le format de cette rencontre intime a été clé pour permettre ces débats animés et échanges riches qui ont fait le succès de cette rencontre.

Résumé

L’objet de cette conférence était de débattre de la façon dont l’identité cellulaire est acquise, fixée ou changée. L’acquisition puis le maintien d’une identité cellulaire au fil du temps sont des propriétés critiques pour le fonctionnement au long cours de nos organes et tissus. Comment une identité cellulaire est-elle acquise, a quel point et combien de temps est-elle labile, et comment peut-elle être échangée pour une autre dans certaines circonstances ? En effet, des expériences précurseurs ont montré que l’identité cellulaire spécialisée n’est pas écrite dans la pierre, et peut être reprogrammée, que ce soit induit de façon expérimentale, ou lors des phénomènes naturels de conversion entre types cellulaires. Ce séminaire a donc réuni de jeunes chercheurs et des scientifiques de renom qui, à l’aide d’un éventail de modèles cellulaires et animaux, travaillent sur différents aspects de la plasticité et de la reprogrammation cellulaire, et en sondent les principes généraux. Ces interventions ont été organisées en quatre sessions, examinant comment une identité cellulaire est acquise ; maintenue ; reprogrammée, revenant à un état non-engagé type cellule souche ou adoptant une autre identité spécialisée ; ou comment l’identité cellulaire peut osciller entre deux états (i.e. pluripotent et plus engagé). La réunion s’est conclue par un débat général sur les thèmes émergents et les défis futurs clôturant une semaine de discussions fructueuses.

Compte-rendu (en anglais)

Below is a summary of the original contributions of the participants, arranged in 4 main topics :

Lability and reversals of commitment

Austin Smith discussed his latest findings on the principles of pluripotency. Pluripotency means plasticity of single cells to engender all lineages of the mature organism. In mammals a pluripotent tissue, the epiblast, is generated during pre-implantation development. The epiblast subsequently constitutes the substrate for germ cell induction, neural induction and gastrulation. Different pluripotent stem cell types can be established, representing the pre-implantation and gastrulation stages respectively. Observations of heterogeneous gene expression and phenotypic variability in stem cell cultures have given rise to the notion of pluripotency as a metastable state or “precarious balance”, sustained by competition between lineage-specifying transcription factors. However, this concept is not readily compatible with the dynamic and directional progression of pluripotency in utero. Austin Smith proposed an alternative framework in which the continuum of pluripotency is parsed into three sequential phases; naïve, formative and primed. He hypothesised that competence for multi-lineage specification requires wholesale remodelling of transcriptional control circuitry and chromatin in order to programme the tabula rasa of naïve pluripotency. This formative transition may be illuminated by interrogation of pluripotent stem cell entry into differentiation in defined conditions in vitro.

Leanne Jones presented her work on the mechanisms regulating the stem cells-niche interaction and the transitions from one to the other, using both the Drosophila germline and the intestinal stem cells as models systems. Her talk focused on possible common mechanisms found across different stem cell types and the role of escargot in particular. Escargot was found to be necessary to restrict the conversion of niche cells (Hub cells) to somatic cyst stem cells (CySC) in the germline. In the intestine, escargot was found to promote self-renewal of the intestinal stem cells. She also elaborated on how escargot activity evolves during ageing when increased ISC proliferation and a block in terminal differentiation are observed.

Lucie Laplane, a philosopher of science, questions what a stem cell is. This is both a biological and philosophical issue as it raises the question of the identity of a particular category of biological objects. In her presentation, she first reviewed debates about the identity of stem cells as found in the scientific literature. Based on these data, she suggested a classification that distinguishes four possible identities for stem cells: categorical (stemness is an intrinsic property of stem cells), dispositional (stemness is an intrinsic property whose expression relies on extrinsic stimuli), relational (stemness is an extrinsic property induced by the micro-environment), or systemic (stemness is an extrinsic property that is maintained and control at the cell population level). Lucie Laplane then investigated the consequences of this classification for biomedical sciences. She argued that clarifying the true identity of each kind of stem cell is of major importance at least for oncology, as the efficiency of some therapeutic strategies (cancer stem cell-targeting therapies, niche-targeting therapies, and differentiation therapies) directly relies on whether stemness is a categorical, a dispositional, a relational, or a systemic property.

Kim Jensen discussed how cellular identity is controlled and dynamically regulated during tissue morphogenesis and in response to tissue remodelling following injury. He described how adult stem cells in the intestinal epithelium appears rather late during morphogenesis, a phenomena conserved amongst species. Interestingly, a precursor to the adult stem cell state can be trapped in its foetal attractor state, and cell with similar properties are most often produced with current directed differentiation protocols for pluripotent stem cells. He presented recent work where he is trying to understand the relationship between the foetal and adult stem cells, how the transition is regulated at the mechanistic level, and how insight into the process of tissue maturation is of interest during tissue repair and directed differentiation.

Helen Blau discussed her resuscitation of the heterokaryon cell fusion system to analyse regulators of the plasticity of cell fate, a system she developed and used 25 years ago to show that ‘terminally’ differentiated human cells could be reprogrammed, providing evidence for an unexpected plasticity of cell fate. Recent work in her lab has entailed fusing mouse embryonic stem cells with human fibroblasts in non-dividing syncytia in a quest to identify novel regulators at the onset of reprogramming to pluripotency. A time course within hours of cell fusion allowed analyses of chromatin accessibility by ATAC Seq which predicted gene expression by RNA Seq at subsequent time points. Using this highly efficient and near-synchronous reprogramming system, Blau’s group identified a cascade of previously unrecognized regulators that act prior to and induce OCT 4 expression. The role of these regulators was validated in Blau’s laboratory by showing that they are required and sufficient for reprogramming to induced pluripotency (iPS), findings excitingly corroborated at the Les Treilles meeting in the lymphoid to iPS reprogramming database of Thomas Graf, exemplifying the value and true spirit of these meetings.

Natural dedifferentiation or transdifferentiation

Sophie Jarriault discussed the latest findings of her team on the mechanisms endowing specific cells with the ability to naturally change their identity. Using a single cell event of natural transdifferentiation in C. elegans, she showed that Notch signal naturally contributes to establish a cellular context favourable to transdifferentiation and is sufficient to create a cell competent to undergo transdifferentiation. She further emphasised how the signalling dynamic is key to Notch action, leading the Notch pathway to exert dichotomic effects on transdifferentiation, when altered. Altogether, her data show that both an extrinsic signal from the micro-environment and the intrinsic cellular context combine to impact on the ability of a cell to change its identity.

Yann Barrandon presented his work on the plasticity of thymic epithelial cells. The thymus is an organ of endodermal origin that contains epithelial cells (TECs) that are critical for proper thymopoiesis. In the rat, some TECs are clonogenic and can be extensively cultured. When transplanted in embryonic skin, they cross germ layer boundaries and become bona fide hair follicle multipotent stem cells. He found that in human, clonogenic TECs balance between a thymic program, a partial EMT and a partial epidermal program.

Cédric Blanpain discussed the importance of the cancer cell in regulating tumour heterogeneity. He showed using genetically engineered mouse models allowing lineage tracing together with oncogenic activation in different cell lineages, how the cancer cell of origin controls tumour initiation and heterogeneity in different models of skin epidermis and mammary gland tumours. He presented evidence that in basal cell carcinoma initiation, adult stem cells are reprogrammed into an embryonic hair follicle progenitor fate before progressing into invasive tumours and showed that targeting this reprograming step by Wnt inhibitors blocks tumour progression. Finally, he showed the importance of the cancer cell of origin in controlling EMT, and discussed the molecular mechanisms and epigenetic landscape that prime oncogene targeted cells to undergo EMT.

Induced direct reprogramming

Patrick Collombat presented his work on pancreatic cell type conversion for therapeutic approaches to diabetes. The mature pancreas includes three main cell subtypes: acinar, duct and endocrine cells. Acinar cells produce and secrete digestive enzymes, which are routed to the intestine by a branched ductal network. Endocrine cells are involved in regulating nutrient metabolism and glucose homeostasis. Specifically, these are organized into cell clusters termed islets of Langerhans that are composed of four cell subtypes, each secreting a specific endocrine hormone: alpha-, beta-, delta-, and PP-cells producing glucagon, insulin, somatostatin, and pancreatic polypeptide (PP), respectively. Both type 1 and 2 diabetes (T1D and T2D, respectively) ultimately result in pancreatic beta-cell loss and chronic hyperglycemia. Importantly, despite the most recent advances in diabetes care, patients suffering from T1D still display, on average, a shortened life expectancy and a worsened quality of life as compared to healthy individuals (WHO, 2014).

The recent discovery that genetically-modified pancreatic alpha-cells can regenerate and convert into beta-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. P. Collombat reported the identification of G8 as an inducer of alpha-to-beta-like cell conversion in vivo. Such conversion induces alpha-cell replacement mechanisms through the mobilisation of duct-lining precursor cells that adopt an alpha-cell identity prior to being converted into beta-like cells, solely upon sustained G8 exposure. Importantly, these neo-generated beta-like cells are functional and can repeatedly reverse chemically-induced diabetes in vivo. Similarly, the treatment of transplanted human islets with G8 results in a loss of alpha-cells and a concomitant increase in beta-like cell counts, suggestive of alpha-to-beta-like cell conversion processes also in humans. This newly discovered G8-induced alpha-cell-mediated beta-like cell neogenesis could therefore represent an unprecedented hope towards improved therapies for diabetes.

Joel Rothman has identified a molecular switch in the model animal C. elegans that can robustly convert and remodel fully mature cells of one type (the heart-like pharynx and the gonad) into those another organ type, the intestine, revealing substantial plasticity in the phenotypes of fully differentiated cells. With this system, his team has identified genes that are necessary for this transdifferentiation process, including transcriptional regulators and those involved in protein turnover. While the pathways used during transdifferentiation appear to be similar to those during de novo generation of the bona fide intestine during embryogenesis, they are deployed over quite different time courses.

Thomas Graf presented work on how C/EBPa can induce both the transdifferentiation of B cells into macrophages when continuously expressed and enhance the reprogramming of B cells into iPS cells when transiently expressed before activation of the Yamanaka factors. He showed evidence that the two activities can be dissociated, and that the C/EBPa pulse causes major changes in genome topology. Based on these findings he discussed the provocative idea that C/EBPa acts like a conventional transcription factor during induced transdifferentiation but might exert a different role when acting as a ‘gate opener’ during iPS cell reprogramming.

Oliver Hobert summarized his labs work on understanding how individual neuron types in the nervous system of the nematode C. elegans acquire their terminally differentiation state. One specific type of transcription factor, called “terminal selectors” appear to play a dominant role in this terminal differentiation process. He then discussed the question whether these terminal selectors are not only required to execute specific differentiation programs within their normal context, but whether they can also be used to reprogram the identity of other cells upon ectopic mis-expression. Experiments that test this prediction showed a relatively limited capacity of these transcription factors, but this capacity can be greatly enhanced through the removal of specific chromatin modifications.

Marius Wernig described his lab’s molecular and mechanistic dissection of transcription factor-induced cellular reprogramming events. He dissected the contribution of individual factors that are involved in reprogramming to neurons, including a bHLH-type transcription and a novel, little characterized Zn finger transcription factor, Myt1. One intriguing and unanticipated insight was the finding that during reprogramming from fibroblast to neurons, one intervening transcriptional state which appeared muscle-like was transiently observed and required to involve the Myt1 transcription factor.

Magdalena Götz reported new unpublished data from reprogramming of glial cells into neurons. Identification of reprogramming hurdles in astrocytes in vitro allowed improving direct reprogramming of glial cells into neurons after brain injury. In particular easing metabolic conversion by promoting cell survival and protecting cells during the conversion process from oxidative stress hugely improved reprogramming to a 90% efficiency in vivo. This now leads to the fascinating question of functional integration of these cells. To determine if this is at all possible, transplantation of embryonic cerebral cortex cells was performed and their brain wide-connectivity was determined. This revealed strikingly appropriate integration into the injured visual cortex where transplanted cells not only acquired their appropriate input patterns with the correct topographic mapping but also acquisition of orientation and direction selective visual receptive field properties. Thus, new neurons can perfectly integrate into a brain region that never does so normally in adulthood.

Cellular ID programming, control and maintenance

Paola Arlotta‘s talk first focused on pyramidal neuron diversity in the mammalian neocortex, and her work to understand the molecular mechanisms that establish neuronal diversity during corticogenesis. She described a new methodology to purify and profile by RNAseq distinct classes of pyramidal neurons and described the identification of signature of genes that identify specific neuron types, dynamically, through early development. She then described the role and functional logic of one transcription factor, Fezf2. Fezf2 is a selector gene able to instruct the identity of one class of pyramidal neurons, corticospinal motor neurons, when expressed in a plastic cellular context, including young post mitotic neurons of a different lineage, where it can instruct direct lineage reprogramming into corticospinal motor neuron in vivo. Finally, she touched upon recent efforts to model the process of human cortical development in the dish through the generation of long-term cultures of 3D human whole-brain organoids. The value of the models to understand human cortical development and disease were discussed as well as the ethical implications associated with generation of human brain tissue in vitro.

David Zarkower In mammals sex is determined in the foetal gonad when either Sertoli (male) or granulosa (female) cells are specified. Recent work from his lab has found that these cell types, once specified, must be maintained in each sex. In males the DMRT1 transcription factor actively maintains Sertoli cells fate by activating male-promoting and repressing female-promoting genes. DMRT1 prevents retinoic acid signalling from stimulating transdifferentiation and allows retinoic acid to be used to control spermatogenesis.

Freddy Radtke discussed the origin of chronic inflammation, associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem cell function is a contributing factor in inflammation-related diseases, although the underlying cellular and molecular mechanisms remain to be fully elucidated. Freddy Radtke and his team delineated the effect of chronic inflammation on epithelial stem cells using the corneal epithelium as a model tissue. Using a combination of mouse genetics, pharmacological approaches and in vitro assays, they demonstrate that chronic inflammation indirectly regulates stem cell fate choice by altering the mechanical properties of the surrounding microenvironment. Subsequently, aberrant mechano-transduction in corneal epithelial stem/progenitor cells induces epidermal differentiation via elevated b-catenin signalling. Corneal differentiation can be restored using small molecule inhibitors of mechano-transduction. Collectively, this study demonstrates that chronic inflammation and mechano-transduction are linked and act to elicit pathological responses in epithelial stem cells. This therefore establishes a new mechanism by which chronic inflammation can contribute to disease.

Shahragim Tajbakhsh discussed the intriguing nature of heterogeneities among skeletal muscle founder stem cells in different anatomical locations. Although cell fate is established by Myf5, MyoD or Mrf4 throughout the body, the upstream cells giving rise to these progenitors use distinct transcription factor regulatory networks (eg. Pax3, Tbx1, Pitx2) to specify stem cells in somitic versus cranial mesoderm derived muscle masses. Diversity is also seen during homeostasis in the adult, where muscle stem cells are quiescent, yet this state is not uniform as deeper states of quiescence with distinct molecular signatures have been identified in normal tissues, post-mortem, and aged mice. These states appear to be in part associated with the stress incurred, thus pointing to the adaptable and flexible nature of this stem cell population.

Sri Teja Mullapudi presented his approaches to identify small molecules that can impact on the generation or amplification of pancreatic β cells, with the aim to provide new therapeutic leads for diabetes. Given the socioeconomic significance of diabetes, understanding the mechanisms governing pancreatic β cell differentiation as well as systemic metabolism would open new avenues to preventing, managing and treating the disease. Using transcriptional reporters in the zebrafish model, he reported on the design and execution of high throughput in vivo chemical screens aimed at identifying small molecules which can direct differentiation and proliferation of pancreatic β cells. Additionally, using an array of metabolic sensors, he presented screening strategies for small molecules modulating processes such as peripheral glucose uptake, lowering glucose levels and improving lipid profiles in diabetic disease models. The hop is to ultimately unravel novel small molecules and signaling pathways which can potentially be manipulated to treat diabetes, including cases where insulin resistance has already set in.

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Additional event

In addition to the scientific presentation and discussions on the control of the cellular identity, an outreach event was organised by Héloïse Dufour, from the “Cercle FSER” (Fondation Schlumberger pour l’Education et la Recherche). During this event, several classes of highschool students from the area came to the Foundation des Treilles to meet with the participants of this Treilles Meeting, and visit the Domaine des Treilles. Up to 3 students met with 13 scientists, one at a time in a « speed dating » format, and asked them questions related to their research or sought career planning advices. The aim of this session is to encourage informal exchanges between scientists and the public to help bridge the gap between science and society and foster interest in fundamental science in the new generation. Students, teachers and researchers were all enthralled by the event, and the feedback collected by Heloïse Dufour using formal feedback forms were excellent and showed a positive impact on how young people view science.