Rôle d'une boucle autocrine impliquant l'IL-6R dans la détermination des cellules souches hématopoïétiques

L'hématopoïèse est un processus complexe au cours duquel des cellules souches hématopoïétiques (CSH) pluripotentes prolifèrent et se différencient pour donner un effectif complet de cellules sanguines matures et fonctionnelles. Le maintien de la population de CSH est assuré par un mécanisme d'autorenouvellement. De nombreuses cytokines sont impliquées dans la régulation des phases précoces de l'hématopoïèse. Leur production est assurée par les cellules du microenvironnement, mais aussi par les CSH elles-mêmes. Le but de ce travail repose sur le rôle de l'IL-6 et de ses récepteurs dans la détermination des CSH. L'expression de l'IL-6Rm sur les CSH augmente suite à une stimulation de la gp130 par une molécule fusionnant l'IL-6 à l'IL-6Rs (l'Hyper-Interleukine-6-HIL-6). Cette stimulation augmente la production d'IL-6Rs uniquement dans une population restreinte de CSH résistantes au 5-FU. D'autres cytokines de la famille de l'IL-6, tels que le LIF ou l'IL-11, ne permettent pas de reproduire ces réponses. La production d'IL-6Rs est stimulée par le PMA et est inhibée par le TAPI, indiquant qu'une ADAM clive l'IL-6Rm. Des stimulations transitoires par l'HIL-6 permettent également d'augmenter la production d'IL-6Rs par clivage de l'IL-6Rm. L'activation de l'ADAM induite par la gp130 est régulée en partie par la voie PI-3K. Ces événements plaident donc pour l'existence d'une boucle autocrine basée sur l'expression des formes solubles et membranaires de l'IL-6R. La fonctionnalité de la boucle a été testée en stimulant la gp130 de CSH en culture. Cependant, aucune augmentation du nombre de progéniteurs issus de ces CSH stimulées n'a été observée. Cette observation a été corroborée par la simulation de modèles informatiques RPHF. En revanche, la fréquence des LTC-IC augmente dans des cultures à long terme de CSH exposées à l'HIL-6 en présence de cellules stromales, suggérant ainsi le rôle important du microenvironnement dans la fonctionnalité du circuit de la régulation auto-paracrine. L'effet de l'HIL-6 est inhibé par le TAPI, soulignant ainsi l'importance de la trans-signalisation dans le maintien des CSH. Une analyse du cycle cellulaire des CSH révèle que l'HIL-6 n'augmente pas l'entrée en cycle, privilégiant ainsi une action de l'HIL-6 sur la survie cellulaire plutôt que sur la prolifération.The hematopoisis is a complex process where the proliferation and the differenciation of pluripotent hematopoietic stem cells (HSCs) lead to the formation of mature and functional blood cells. The maintenance of HSCs population is sustained by a mechanism of renewal. Numerous cytokines are involved in the regulation of early phases of hematopoiesis. These cytokines are secreted by the cells of the microenvironment, and the HSCs themselves. The role of IL-6 and its receptors in the maintenance of the HSCs is studied herein. The stimulation of gp130 by the fusion molecule linking IL-6 to sIL-6R (the hyper-interleukin-6, HIL-6) enhanced the expression of mIL-6R on HSCs. This stimulation increases the production of sIL-6R only on the population of 5-FU-resistant HSCs. However, same responses were not obtained by stimulation with others IL-6 family members, such as LIF or IL-11. production of sIL-6R is stimulated by PMA and is inhibited by TAPI, demonstrating that an ADAM is involved in shedding of mIL-6R. The production of sIL-6R by shedding of mIL-6R was also increased by transient stimulations with HIL-6. Gp-induced-activation of ADAM is partially mediated by PI-3K pathway. Thus, all these data pleading to the existence of a mIL-6R and sIL-6R based autocrine loop. The functionality of this loop has been tested in culture of gp130-stimulated HSCs. Nevertheless, there was no increase in the number of progenitor derived from stimulated HSCs. This observation has been confirmed using the simulation of informatic HFPN models. In contrast, the LTC-IC frequency increases in long-term culture of HSCs in presence of stromal cells and HIL-6, suggesting the important role of the microenvironment in the functionality of the auto-paracrine circuit of regulation. The effect of HIL-6 is inhibited by TAPI, underlining the importance of the trans-signalling in the maintenance of HSCs. The cell-cycle analysis of HSCs shown that HIL-6 has no effect in the entry in cell-cycle, suggesting preferential role of HIL-6 in the cell survival rather than in the proliferation.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells.

BACKGROUND & AIMS: Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. METHODS: We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. RESULTS: In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. CONCLUSIONS: We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies

Modélisation et simulation de la régulation de l'hématopoïèse précoce grâce aux Réseaux de Petri Hybrides Fonctionnels

National audienceThe regulation of haematopoiesis is a complex biological system with numerous interdependent processes. In vivo, Haematopoietic Stem Cells (HSCs) self-renew so as to maintain a constant pool of these cells. It would be very interesting to maintain these cells in vitro, in view of their therapeutical importance. Unfortunately, there is currently no known process to activate HSCs self-renewal in vitro. Since the difficulties related to in vitro experiments, modeling and simulating this process is indispensable. Moreover, the complexity of haematopoiesis makes it necessary to integrate various functionalities: both discrete and continuous models as well as consumption and production of resources. Hence, we focussed on the use of Hybrid Functional Petri Nets, which offer a number of features and flexibility. We started with modelling and simulation of the role of a cytokine, interleukin-6, in the regulation of early haematopoiesis

Modélisation et simulation de la régulation de l'hématopoïèse précoce grâce aux Réseaux de Petri Hybrides Fonctionnels

National audienceThe regulation of haematopoiesis is a complex biological system with numerous interdependent processes. In vivo, Haematopoietic Stem Cells (HSCs) self-renew so as to maintain a constant pool of these cells. It would be very interesting to maintain these cells in vitro, in view of their therapeutical importance. Unfortunately, there is currently no known process to activate HSCs self-renewal in vitro. Since the difficulties related to in vitro experiments, modeling and simulating this process is indispensable. Moreover, the complexity of haematopoiesis makes it necessary to integrate various functionalities: both discrete and continuous models as well as consumption and production of resources. Hence, we focussed on the use of Hybrid Functional Petri Nets, which offer a number of features and flexibility. We started with modelling and simulation of the role of a cytokine, interleukin-6, in the regulation of early haematopoiesis

Modeling and Simulation with Hybrid Functional Petri Nets of the role of interleukin-6 in human early haematopoiesis

International audienceThe regulation of human haematopoiesis is a complex biological system with numerous interdependent processes. In vivo Haematopoietic Stem Cells (HSCs) self-renew so as to maintain a constant pool of these cells. It would be very interesting to maintain these cells in vitro, in view of their therapeutical importance. Unfortunately, there is currently no known process to activate HSCs self-renewal in vitro. Since the difficulties related to in vitro experiments, modeling and simulating this process is indispensable. Moreover, the complexity of haematopoiesis makes it necessary to integrate various functionalities: both discrete and continuous models as well as consumption and production of resources. We thus focus on the use of Hybrid Functional Petri Nets, which offer a number of features and flexibility. We begin by modeling and simulating the role of a specific cytokine, interleukin-6, in the regulation of early haematopoiesis. Results obtained in silico lead to the disappearence of HSCs, which is in agreement with in vitro results

Stem cells for liver tissue repair: Current knowledge and perspectives

Stem cells from extra- or intrahepatic sources have been recently characterized and their usefulness for the generation of hepatocyte-like lineages has been demonstrated. Therefore, they are being increasingly considered for future applications in liver cell therapy. In that field, liver cell transplantation is currently regarded as a possible alternative to whole organ transplantation, while stem cells possess theoretical advantages on hepatocytes as they display higher in vitro culture performances and could be used in autologous transplant procedures. However, the current research on the hepatic fate of stem cells is still facing difficulties to demonstrate the acquisition of a full mature hepatocyte phenotype, both in vitro and in vivo. Furthermore, the lack of obvious demonstration of in vivo hepatocyte-like cell functionality remains associated to low repopulation rates obtained after current transplantation procedures. The present review focuses on the current knowledge of the stem cell potential for liver therapy. We discuss the characteristics of the principal cell candidates and the methods to demonstrate their hepatic potential in vitro and in vivo. We finally address the question of the future clinical applications of stem cells for liver tissue repair and the technical aspects that remain to be investigated

Modeling and Simulation with Hybrid Functional Petri Nets of the role of interleukin-6 in human early haematopoiesis

International audienceThe regulation of human haematopoiesis is a complex biological system with numerous interdependent processes. In vivo Haematopoietic Stem Cells (HSCs) self-renew so as to maintain a constant pool of these cells. It would be very interesting to maintain these cells in vitro, in view of their therapeutical importance. Unfortunately, there is currently no known process to activate HSCs self-renewal in vitro. Since the difficulties related to in vitro experiments, modeling and simulating this process is indispensable. Moreover, the complexity of haematopoiesis makes it necessary to integrate various functionalities: both discrete and continuous models as well as consumption and production of resources. We thus focus on the use of Hybrid Functional Petri Nets, which offer a number of features and flexibility. We begin by modeling and simulating the role of a specific cytokine, interleukin-6, in the regulation of early haematopoiesis. Results obtained in silico lead to the disappearence of HSCs, which is in agreement with in vitro results

Towards the modelling of the regulation of early haematopoiesis

International audienceHaematopoietic stem cells (HSCs) can either remain quiescent, with a constant and low rate of self renewal, or become committed to differentiation into haematopoietic cell lineages. The switch between those two fates can be seen as the switch from one stationary state to another in a dynamic process (epigenetic switch). The control of early haematopoiesis is quite complex, involving several factors and feedback circuits. Its study needs modelling, which was not straightforward, since it requires to take into account both consumption and production of resources as well as discrete and continuous time. This was performed using Hybrid Functional Petri Nets (HFPN). The model presented simulates the results obtained in vitro, where the quiescent stem cells are rapidly lost, and shows the potentiality of HFPN to model complex systems such as the regulation of haematopoiesis. In silico simulations can then be performed to study the mechanism responsible for the epigenetic switch