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Mechanisms regulating cell fate specification during embryonic development and tissue homeostasis

Our lab is interested in understanding how multipotent progenitors are specified during embryonic development, what are the mechanisms that control the differentiation of these progenitors into the different cell lineages that composed adult tissue and what are the mechanisms regulating the maintenance of adult tissue.

 

 

Mechanisms mediating multipotent cardiovascular progenitor specification

The heart is composed by different cell types, including contractile cardiac cells, vascular cells, smooth muscle cells as well as pacemaker cells. During embryonic development as well as during embryonic stem cell (ESC) differentiation, the different cardiovascular cell types arise from the differentiation of multipotent cardiovascular progenitors. The mechanism that promotes multipotent cardiovascular progenitor specification from undifferentiated mesoderm cell remains largely unknown.

Using ESCs, in which gene expression can be temporally regulated, we showed that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cell autonomous mechanism.

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Control Video

Mesp1 Induction Video

 

Genome-wide transcriptional analysis and chromatin immunoprecipitation experiments indicate that Mesp1 rapidly activates and represses a discrete set of genes. Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes in the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly and strongly represses the expression of key genes regulating other early mesoderm and endoderm cell fates, ensuring the unidirectionality and specificity of cardiovascular cell fate specification induced by Mesp1.

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Our study demonstrates that Mesp1 acts as a key molecular switch during specification of MCPs from undifferentiated mesoderm, residing at the top of the hierarchy of the cardiovascular transcriptional network and stimulating the coordinated expression of the main transcription factors necessary for cardiovascular development. In addition, our study provides a novel and robust method for generating cardiovascular cells during ESC differentiation and opens new avenues for cardiac cellular therapy in humans.

Bondue, A., Lapouge, G., Paulissen, C., Semeraro, C., Iacovino, M., Kyba, M. and Blanpain, C.
Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification.
Cell Stem Cell 3(1):69-84
PubMed | PDF

Mesp1 at the heart of mesoderm lineage specification. (PREVIEW)
Wu SM
Cell Stem Cell. 2008, Jul 3; 3(1):1-2.

While progresses have been recently accomplished in characterizing these two type of MCPs, little is known about their specification. Do these MCPs arise from a common progenitor? If so, do these earliest MCPs represent a homogenous or a heterogeneous cell population? What are the cell surface markers expressed by the early MCPs, allowing their prospective isolation? What are the transcription factors expressed by the early MCPs that act alone or in combination with Mesp1 to promote MCP specification and cardiovascular lineage differentiation?

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To address these questions, we engineered Mesp1-GFP reporter ESCs allowing tracking and isolation of the earliest Mesp1 expressing cells during ESC differentiation. We showed that these early Mesp1 expressing cells are strongly enriched for MCPs of both heart fields, which give rise upon differentiation to all cardiovascular cell lineages both in vitro and in vivo. By transcriptionally profiling the early Mesp1 expressing cells, we uncovered cell surface markers allowing their prospective isolation, cellular and molecular characterization. Using gain and loss of Mesp1 function during ESC differentiation, we demonstrated that Mesp1 is required to promote the specification of MCPs and the expression of cardiovascular transcription factors in MCPs. We found that Isl1 is expressed in a subpopulation of Mesp1 expressing cells and stimulates cardiovascular commitment in these early MCPs. Our study identifies the early MCPs residing at the top of the cellular hierarchy of the different cardiovascular lineages during ESC differentiation and provides novel insights into the cellular and transcriptional hierarchy acting during the early step of cardiovascular differentiation.

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Bondue A, Tännler S, Chiapparo G, Chabab S, Ramialison M, Paulissen C, Beck B, Harvey R, Blanpain C.
Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation.
J Cell Biol. 2011 Mar 7;192(5):751-65.
PubMed | PDF

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Mechanisms regulating cell fate decision during epithelial development

Our lab is interested in understanding how the different epithelial cell lineages of the skin epidermis and the mammary glands are specified during embryonic development.

Mechanisms mediating touch cell specification

Merkel cells are neuroendocrine cells located in the skin epidermis that mediate touch sensation. Since their first identification by Frederich Merkel in 1875, the embryonic origin of Merkel cells remain matters of intense controversy. Merkel cells present many characteristics of neuronal cells such as factors involved in neuronal cell fate determination factors, neuro-peptides and many components of the synaptic machinery suggesting they arise from neuronal cells. On the other hand, Merkel cells also express charcteristics of epitelial cells suggesting that they may arise from the epithelial cells of the skin.

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We used genetic approach in mice to determine the embryonic origin of Merkel cells, the mechanisms that specify these cells during embryonic development and that maintain these cells during adult life. We found that Merkel cells originate from embryonic epidermal progenitors and not from neural crest cells as it has been previously suggested. We also show that Merkel cells undergo a slow but significant turnover during adult life that is ensured by epidermal stem cells. We showed that a proneural factor named Atoh1/Math1 is required in embryonic epidermal progenitors for Merkel cell specification. The ablation of this proneural factor in epidermal cells provides the first animal model completely deficient for Merkel cells.

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Our study offers new ways to define the role of Merkel cells in sensory perception and open new avenues to define the cell at the origin and mechanisms leading to the formation of Merkel cell carcinoma, a poorly understood and extremely aggressive cancer in humans with rising incidence especially in aging and immunocompromised patients. We are collaborating with Dr Hassan, VIB, KUL, to generate animal model of this devastating cancer. This work also provide new cues to understand how it is possible to generate neuron like cells from skin cells, which may be useful in the future to generate bona fide neurons from skin cells, a highly accessible tissue.

This study has been published and makes the cover of the Journal Cell Biology of the October 5th issue and is accompanied by an editoral highlight.

Van Keymeulen A., Mascre G., Kass Youseff K., Harel I., Michaux C., De Geest N., Spalski C., Achouri Y., Bloch W., Hassan B.A. and Blanpain C.
Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis.
Journal of Cell Biology, 2009, 187:91-100
PubMed | Preview | PDF | F1000 Biology | F1000 Medicine

Ben Short « Merkel bear the touch of epidermis » Journal of Cell Biology, 2009, 187

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Mechanisms mediating mammary cell specification

The mammary gland expands considerably during puberty and pregnancy, during which it differentiated into milk-producing cells. Two different cell types formed the mammary gland: the myoepithelial cells and luminal cells, which can differentiate either into ductal cells or milk-producing cells. Whereas ductal and milk-producing cells secrete the water and nutriments essential for the survival of young mammalian offspring, the myoepithelial cells, through their contraction, guide the circulation of the milk throughout the ductal tree toward the nipple.

Transplantation of a single FACS isolated mammary epithelial cell into the mammary fad pad can reconstitute, although at low frequency, a normal mammary gland, suggesting that rare multipotent stem cells reside at the top of the cellular hierarchy of the mammary gland homeostasis (Stingl et al. and Shackleton et al. Nature 2006).

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To precisely define the cellular hierarchy of mammary gland during physiological conditions, we used a novel state of the art genetic lineage tracing approach to fluorescently mark the different cell types of the mammary gland and follow the fate of fluorescent marked cells overtime. While during embryonic development, both luminal and myoepithelial cells arise from multipotent progenitors, during puberty and pregnancy, the luminal and myoepithelial lineages contain long lived unipotent stem cells which present extensive renewing capacities, as demonstrated by their ability to expand during morphogenesis and undergo massive expansion during several cycles of pregnancy.

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Transplantation studies of genetically YFP marked cells reveal that both types of unipotent stem cells conserve their lineage-restricted potential when these stem cells are transplanted together into the mammary fat pad at non limiting dilution, whereas myoepithelial stem cells but not luminal stem cells can expand their differentiation potential and reform mammary gland when transplanted alone or at limiting dilution with a low ratio of luminal cells, suggesting that myoepithelial stem cells are indeed mul tipotent SC in transplantation assays, that restrain their luminal differentiation potential during physiological conditions.

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Altogether, these new data demonstrate that the different cell types of the mammary epithelium initially develops from multipotent embryonic progenitors that are rapidly replaced after birth by two types of lineage restricted unipotent SCs, able to differentiate into either myoepithelial or luminal lineages that ensure the massive expansion of mammary cells during puberty and pregnancy.

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This study has been published in the online early edition of Nature 2011

Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G, Rock J, Sharma N, Dekoninck S, Blanpain C.
Distinct stem cells contribute to mammary gland development and maintenance.
Nature. 2011 Oct 9. doi: 10.1038/nature10573. [Epub ahead of print]
PubMed | PDF | PDF (Supplementary Info)

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Multipotent and unipotent progenitors contribute to prostate postnatal development

The prostate is a secretory gland surrounding the urethra at the base of the bladder producing the seminal fluid providing nutrients, ions and enzymes necessary for the survival of the spermatozoids during their journey through the female reproductive tract. The adult prostate is a pseudo-stratified epithelium composed of three main cell lineages: the basal cells, the luminal cells and the neuroendocrine cells.

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Androgen deprivation through castration induces the regression of the adult prostate, which can generate a new fully developed prostate on androgen re-administration, suggesting the presence of castration-resistant stem cells within the adult prostate epithelium. However, little is known about the cellular mechanisms leading to prostate postnatal development and its lineage segregation during tissue morphogenesis.

To define the cellular hierarchy that control postnatal prostate development, we used state of the art genetic lineage tracing approach to fluorescently mark the different cell types of the prostate epithelium and follow the fate of their progeny overtime. We found that prostate postnatal development is mediated by basal multipotent stem cells that differentiate into basal, luminal and neuroendocrine cells, as well as by unipotent basal and luminal progenitors. Clonal analysis of basal cells revealed the existence of bipotent and unipotent basal progenitors as well as basal cells already committed to the luminal lineage. Using mathematical modeling, we proposed that the apparent cellular heterogeneity of basal progenitors can be potentially explained by stochastic cell fate decision of a single multipotent progenitor.

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Our results demonstrate the existence of multipotent basal progenitors during prostate postnatal development and unravel the cellular hierarchy acting during this period.

This study has been published in Nature Cell Biology online as AOP October 14 2012 and in the November 2012 issue.

Ousset M, Van Keymeulen A, Bouvencourt G, Sharma N, Achouri Y, Simons BD, Blanpain C.
Multipotent and unipotent progenitors contribute to prostate postnatal development.
Nat Cell Biol. 2012 Oct 14. doi: 10.1038/ncb2600. [Epub ahead of print]
PubMed | PDF

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Mechanisms regulating adult tissue homeostasis and repair

Our lab is interested in understanding how the different epithelial cell lineages of the skin epidermis and the mammary glands are maintained during adult homeostasis and repaired following injuries.

Studying genome maintenance and DNA damage response in stem cells

The maintenance of the stem cell genomic integrity is essential for tissue homeostasis. Mutations in stem cells may result in precancerous condition if the mutations provide a survival advantage, or induce stem cell depletion resulting in tissue atrophy or ageing if the mutations is detrimental to the stem cell fitness.

We are studying the response how epidermal stem cells maintain their genomic integrity and response to exogenous and endogenous DNA damage during morphogenesis and tissue homeostasis.

Studying the mode of chromosome segregation in multipotent hair folicle stem cells

In tissues with high cellular turnover such as the skin epidermis, SCs divide many times during the life of the organism, thus being at high risk of accumulating errors during DNA replications. Consequently, SCs may have acquired during evolution specialized mechanisms to ensure the maintenance of their genome integrity.

One putative mechanism, by which SCs may protect their genome from accumulating mutations arising during DNA replication, is known as the immortal strand hypothesis, in which SC always retain the older DNA strand (the “immortal" strand), while the other daughter cell committed to terminal differentiation inherits the newer DNA strand, which may contain de novo mutations.

Here, we used different in vivo approaches to determine how hair follicle SCs segregate their DNA strand during cell division. Double labeling studies showed that HF SCs incorporate two sequentially two different nucleotide analogs, contradictory to the immortal strand hypothesis. The co-segregation of DNA and chromatin labeling during pulse-chase experiments demonstrated that label retention in SCs is rather a mark of relative quiescence. Our results demonstrate that DNA strand segregation occurs randomly in the majority of HF SCs during development, tissue homeostasis and upon activation and that immortal strand segregation proabably does not represent a genome protection mechanism in adult bulge SCs. However, we cannot always exclude the possibility that there is a small minority of stem cells that segregate their chromosome strands in a biased mode, or a minority of chromosomes that follow the asymmetric segregation.

Possible outcome of double labeling studies according to the asymmetrical and random strand segregation models.

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Our results have been published as featured article in Stem Cells

Sotiropoulou, P., Candi, A. and Blanpain, C. (2008).
The Majority of Multipotent Epidermal Stem Cells Do Not Protect Their Genome By Asymmetrical Chromosome Segregation.
Stem Cells 26(11):2964-73
PubMed | PDF

Studying DNA damage response in epithelial stem cells

Little is known about how adult stem cells sense and respond to DNA damage within their natural niche.

Here, we used skin epidermis as a model to dissect the response of multipotent epithelial stem to DNA damage within their in vivo niche. Using a variety of techniques including functional analysis, biochemical characterization and transcriptional profiling of isolated stem cells before and after DNA damage, we demonstrated that multipotent hair follicle bulge stem cells are extremely resistant to DNA damage-induced cell death compared to their more mature counterparts. Two important molecular mechanisms contribute to the higher resistance of bulge stem cells to DNA damage induced cell death. Bulge stem cells express higher levels of the anti-apoptotic factor Bcl-2, which counter-balance the effect of the pro-apoptotic genes induced by the DNA damage. Moreover, the stabilization and activation of p53 is more rapidly attenuated in bulge stem cells, due to faster DNA repair through more effective non-homologous end joining mechanisms.

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Resistance of haïr follicle stem cells to DNA damage induced cell death. Green show apoptoic cells (active caspase 3) and Red show hair follicle stem cells (CD34) one day after DNA damage.   Accelerated DNA repair activity of hair follicle stem cells as shown by the lower amount of phosphorylated H2AX (green) in bulge SC.

This study indicates that the resistance to DNA damage and accelerated DNA repair represent novel characteristics of adult stem cells. Further studies will be required to determine whether this characteristic represents a more general mechanism shared by different types of adult stem cells as it has been subsequently shown for murine hematopoietic stem cells. These results also show that hair follicle stem cells are poised to repair DNA through non-homologous end joining, an error prone DNA repair mechanism, possibly allowing long-term accumulation of mutations in stem cells, and suggesting that this mechanism could be at double edge for adult stem cells, promoting the short-termed survival after DNA damage at the expense of long-term maintenance of their genomic integrity. These results may also have important implications for understanding the increased susceptibility of certain tissues to DNA damage-induced tumorigenesis and ageing.

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The results of our study has been published in the June issue of Nature Cell Biology 2010 and accompanied by a research highlighted in Nature Review Molecular and Cellular Biology

Sotiropoulou PA, Candi A, Mascré G, De Clercq S, Kass Youseff K, Lapouge G, Dahl E, Semeraro C, Denecker G, Marine JC and Blanpain C (2010).
Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death.
Nat Cell Biol. 2010 Jun;12(6):572-82. Epub 2010 May 16.
PubMed | PDF

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Distinct contribution of stem and progenitor cells to epidermal maintenance

The skin, which is an essential barrier that protects our body against the external environment, undergoes constant turnover throughout life to replace dead cells that are constantly sloughed off from the skin surface. During adult life, the number of cells produced must exactly compensate the number of cells lost. Different theories have been proposed to explain how this delicate balance is achieved.

Using a novel genetic lineage tracing experiments to fluorescently mark two distinct epidermal cell populations, and follow their survival and contribution to the maintenance of the epidermis overtime, we uncover the existence of two types of dividing cells. One population of proliferative cells presented a very long term survival potential while the other population is progessively lost overtime.

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In collaboration with Pr. Benjamin D. Simons (University of Cambridge), we developed a mathematical model of our clonal data that proposes that the skin epidermis is hierarchically organized with slow cycling stem cells residing on the top of the cellular hierarchy that give rise to more rapidly cycling progenitor cells that ensure the daily maintenance of the skin epidermis. Analysis of cell proliferation confirms the existence of slow cycling stems cells and gene-profiling experiments demonstrate that the stem and the progenitors cells are characterized by distinct gene expression.

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Importantly, upon wounding only stem cells are capable of extensive tissue regeneration and undergo major expansion during this repair process, while the progenitors did not expand significantly, and only provide a short-lived contribution to the wound healing response.

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This work demonstrates the existence of slow-cycling stem cells that promote tissue repair and more rapidly cycling progenitors that ensure the daily maintenance of the epidermis.

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The results of our study has been published in the September issue of Nature 2012.

Mascré G, Dekoninck S, Drogat B, Youssef KK, Broheé S, Sotiropoulou PA, Simons BD, Blanpain C.
Distinct contribution of stem and progenitor cells to epidermal maintenance.
Nature. 2012 Sep 13;489(7415):257-62.
PubMed | PDF | PDF (Supplementary Info)

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