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

The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors

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Temperature Effect on Ionic Current and ssDNA Transport through Nanopores

International audienceWe have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15kBT and the translocation barrier is ∼35kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient

DNA Unzipping and Protein Unfolding Using Nanopores

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HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4

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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

Biomédicaments dans le psoriasis de l’enfant de moins de 12 ans. Cohorte rétrospective Franco-Italienne

International audienceIntroductionL’utilisation des biomédicaments est indiquée dans les formes modérées à sévères du psoriasis chez l’adulte et l’enfant. Jusqu’en 2020, 2 biomédicaments étaient autorisés avant 12 ans, l’adalimumab (ADA) et l’étanercept (ETC), 3 autres molécules devraient être disponibles en 2021 (AMM européenne en 2020) : l’ustékinumab (UST), le sécukinumab et l’ixékizumab. Cette population des enfants de moins de 12 ans est sous-représentée dans les essais thérapeutiques, sans analyse dédiée le plus souvent, ainsi que dans les cohortes rétrospectives. Une première étude, l’étude « BiPe » menée en France sur 134 enfants et 184 lignes de traitements étudiait tous les enfants d’âge inférieur à 18 ans. Moins d’un tiers des enfants avait moins de 12 ans. L’objectif de l’étude « BiPe Jr » étaient d’évaluer le taux de maintien et la tolérance des biomédicaments dans le psoriasis des enfants de moins de 12 ans.Matériel et méthodesÉtude multicentrique franco-italienne, rétrospective, par appel à cas (SFDP, GrPso, SIDerP) d’enfants atteints de psoriasis ayant reçu au moins une injection de biothérapies, ayant ou non l’AMM, avant l’âge de 12 ans. Etaient exclus les enfants traités dans le cadre d’un essai thérapeutique et l’indication rhumatisme psoriasique. Les données sociodémographiques, cliniques et thérapeutiques ont été recueillies.RésultatsSoixante-quinze enfants (filles, n = 45 (58,1 %)) ont été inclus pour un total de 97 lignes de biomédicaments (ADA (n = 46), ETC (n = 35), UST (n = 11), anakinra (n = 2), infliximab (n = 2) et sécukinumab (n = 1), avec 1 à 6 lignes initiées avant 12 ans. Quinze enfants (25,8 %) étaient en surpoids, 25 (33,8 %) avaient un antécédent familial de psoriasis. La forme « psoriasis en plaques » était la plus fréquente (n = 43, 57,3 %), suivie par la forme palmoplantaire (n = 16, 21,3 %) ; 5 (7,6 %) avaient un rhumatisme et 24 (36,4 %) une onychopathie psoriasique. Le PGA moyen à l’initiation était de 3,8 ± 0,9 et le PASI de 13,7 ± 9,4. L’âge moyen au début de la biothérapie était de 9,0 ± 0,6 ans. Une tendance à un meilleur maintien de l’UST et de l’ADA versus l’ETC était observée (p = 0,12) ; trente-quatre enfants ont arrêté leur biomédicament pour perte d’efficacité (n = 19), inefficacité primaire (n = 8), choix de la famille (n = 4) et effets indésirables (n = 3). Six événements indésirables graves (EIG) ont été rapportés et sont détaillés.DiscussionCette 1re étude de large ampleur, en vie courante, menée chez l’enfant de moins de 12 ans complète les résultats de l’étude BiPe menée chez les enfants et adolescents psoriasiques. La comparaison des taux de maintien des biomédicaments n’a pas mis en évidence de différence significative malgré une discrète tendance à un meilleur maintien de l’UST et de l’ADA, comme déjà observé chez les plus grands. Les EIG mis en évidence étaient rares mais rappellent la nécessité d’une vigilance accrue du risque infectieux

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