The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34+CD45RA+ precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells

Human embryonic stem cells, a model to study the early steps of lymphopoiesis

Les cellules souches embryonnaires humaines (CSEh) sont des outils puissants pour explorer la genèse des différents tissus de l’organisme, notamment le tissu hématopoïétique. Dans le but d’obtenir des types cellulaires cliniquement utiles, la majorité des travaux se sont concentrés sur l’obtention des cellules hématopoïétiques terminales, notamment des cellules lymphoïdes (lymphocytes B, lymphocyte T et cellules NK), à partir des cellules souches pluripotentes humaines. En revanche, le rendement des cellules hématopoïétiques obtenues dans ce modèle reste faible. D’autre part, les étapes précoces de l’hématopoïèse, notamment l’identification de la cellule souche hématopoïétique (CSH), des progéniteurs myéloïdes et lymphoïdes à partir des cellules souches pluripotentes, sont encore très peu définies. Nous nous sommes intéressés aux étapes précoces de la lymphopoïèse dans le modèle des CSEh. Dans un premier temps, nous avons étudié le rôle de l’homéoprotéine HOXB4 dans l’expansion des progéniteurs NK dérivés des CSEh. Nous avons montré que l’exposition des cellules des corps embryonnaires (EB pour Embryoid Body), dérivées de la différenciation des CSEh, à la lignée MS-5/SP-HOXB4, une lignée modifiée qui exprime constitutivement HOXB4, induit une expansion des progéniteurs NK dérivées des CSEh. De plus, les cellules NK qui en dérivent sont matures et fonctionnelles, de part leur activité cytolytique vis-à-vis d’une lignée érythro-leucémique (K562). Outre l’effet de HOXB4 sur l’expansion des progéniteurs NK, cette étude a permis de démontrer en particulier le rôle de la lignée stromale MS-5 dans l’induction de la spécification lymphoïde à partir des CSEh. Dans un deuxième temps, nous avons analysé plus précisément les étapes précoces de la lymphopoïèse humaine à partir des CSEh. En effet, nous avons montré, au cours de la première partie, que la coculture des cellules dérivées des EB avec MS-5 induit l’expression en surface du CD45RA, un marqueur de spécification lymphoïde, au sein des progéniteurs hématopoïétiques CD34+. Ainsi, sur la base de ces données et des données antérieurs concernant les étapes précoces de la lymphopoïèse humaine fœtale et adulte, nous avons identifié et caractérisé in vitro à partir des CSEh deux populations originales de progéniteurs lymphoïdes précoces multipotents (MELP pour Myeloid Early Lymphoid Progenitor): La progéniteur CD34+CD45RA+CD7+ dont le potentiel de différenciation est biaisé vers le lignage T et NK ; et le progéniteur CD34+CD45RA+CD7- a un potentiel de différenciation biaisé vers les lymphocytes B. Cette étude a un intérêt dans la compréhension du processus de la lymphopoïèse humaine dans le modèle des cellules souches pluripotentes. En perspective, ces données pourraient avoir également un intérêt dans la modélisation de maladie de défauts génétiques de développement du système lymphoïde.Human embryonic stem cells (hESC) are powerful tools to explore tissue genesis of the organism, especially hematopoietic tissue. In order to obtain cellular types clinically useful, the majority of works have been focalised on final output of hematopoietic cells, especially lymphoid cells (lymphocyte B, lymphocyte T and NK cells), from human pluripotent stem cells. However, the obtained hematopoietic cells yield is very poor. In the other hand, initial steps of hematopoiesis, especially the identification of the hematopoietic stem cell, myeloid and lymphoid progenitors, from pluripotent stem cells, are poorly defined. We were interested to early steps of lymphopoisis in the hESC model. Initially, we studied the role of HOXB4 homeprotein on CSEh-derived NK progenitor. We showed that exposure of embryoid body (EB), derived from hESC, to the modified line that express constitutively HOXB4 “MS-5/SP-HOXB4”, induce hESC-derived NK progenitor expansion. Furthermore, the derived NK cells are mature and fonctionnal, by cytolytic activity on erythro-leucemic line K562. Furthermore the effect of HOXB4 on NK progenitor expansion, this study demonstrated, particularly the role of MS-5 line on the lymphoid specification from hESC.Secondly, we analysed more precisely the early steps of human lymphopoiesis from hESC. We showed, in the first part, that MS-5 coculture of the EB-derived cells induce surface expression of CD45RA (marker of lymphoid specification) on hematopoietic progenitor CD34+. Thus, on the basis of these data and previous data concerning the initial steps of fetal and adult lymphopoiesis, we identified and characterized in vitro from hESC, two populations of multipotent early lymphoid progenitor (MELP): the CD34+CD45RA+CD7+ progenitor whose the differentiation potential is biased to T and NK lineage, and the CD34+CD45RA+CD7- progenitor has differentiation potential biased to B lineage. This study is essential in understanding of normal and pathological lymphopoisis process in pluripotent stem cells model. Additionally, this study paves the way for the modeling of genetic disorders of lymphoid system

Modulation de la perception olfactive par l'endothéline

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Endothelin as a neuroprotective factor in the olfactory epithelium

International audienceIn mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ET(B) receptors, while ET(A) receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ET(A) and ET(B) receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ET(B)-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ET(B) and indirectly by limiting non neuronal cells death through ET(A)

Coherence and Stochastic Dominance

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Endothelin modulates both short-term detection kinetics of odors and long-term cellular population dynamics in olfactory mucosa

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Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

International audienceHematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process

NK-cell culture procedure of hESC-derived hematopoietic precursor cells.

<p>Schematic representation of the three steps of NK-cells differentiation from hESCs. hEBs derived from the H1 hESC cell line were dissociated then co-cultured with MS-5/SP-HOXB4 cells or MS-5/EGFP cells as control during 2 weeks. Then, the cells derived from the first step of co-culture were submitted to a second step of 3-week co-culture with unmodified MS-5 cells, in permissive conditions for NK-cell differentiation in presence of SCF, IL-2 and IL-15. NK-cell culture differentiation was conducted directly with un-co-cultured hEB-derived cells as control. (B) Analysis of the presence of the HOXB4 protein within hEB-derived cells co-cultured with either MS-5/SP-HOXB4 (dark line) or MS-5/EGFP (dotted line) stromal cell lines. Data are from one experiment out of two. Gray histogram corresponds to isotypic control. Abbreviations : cy, cytoplasmic.</p

Functional activity of NK cells derived from co-culture with MS-5/SP-HOXB4.

<p>(A) The functional activity of cells was assessed in the presence of IL-12 and against K562 target cells; the NK-cell activity was evaluated by the mobilisation of the CD107a antigen on the cell surface and the intra-cytoplasmic expression of IFNγ. Cells were gated on CD56⁺ cells (not shown). (B) Cytotoxic activity of NK effector cells against target K562 cells. a) Target K562 cells were labelled by CFSE. b) Percentage of 7AAD⁺ death cells among the CFSE⁺ target cells at various Effector-Target (E/T) ratios (one experiment out of two). c) Percentage of cytotoxicity (<i>i.e</i> percentage of 7AAD⁺ death cells among the CFSE⁺ target cells) according to the ratio E/T (dotted line and bold line represent two independent experiments).</p

Analysis of NK differentiation potential and NK progenitor cell expansion mediated by HOXB4.

<p>(A) Percentages of NK cells (CD56⁺CD3⁻CD19⁻) among nucleated cells collected at the end of 5 weeks of co-culture. Cells derived from the 2 weeks primary co-cultures with MS-5/SP-HOXB4 or MS-5/EGFP were then plated on unmodified MS-5 cells in conditions known to promote NK-cell differentiation and maintained during three weeks. At the end of the culture period, cells were analyzed by FACS for the expression of CD56 and CD3/CD19 markers. Un-co-cultured hEB cells were directly cultured under NK-cell differentiation conditions for 3 weeks (n = 5, *<i>p</i><0.05, **<i>p</i><0.01). (B) Fold increase of total NK cells. NK cells were derived from total cells isolated from the primary 2-week co-cultures of hEB-derived cells with either MS-5/SP-HOXB4 or MS-5/EGFP control and then cultured under NK-cell differentiation condition for three weeks. NK cells were then numbered. Bars represent fold amplifications relative to day-0 control (un-co-cultured hEBs) (designated as 100%) (n = 5, *<i>p</i><0,05).</p