Die Nicht-alkoholische Steatohepatitis (NASH), gehörig zu der Gruppe der chronischen Lebererkrankungen als eine schwere Form der Nicht-alkoholischen Fettleber-erkrankungen (NAFLD), nimmt in ihrer Prävalenz ständig zu. Gründe dafür sind u.a. eine gesteigerte Nahrungsaufnahme sowie Veränderungen der Nahrungszusammen-setzung. Es kommt zur Ausbildung einer Steatose, die sich unter Mitwirkung verschie-dener Einflussfaktoren zur Steatohepatitis weiterentwickeln kann, wobei die Pathoge-nese noch nicht genau verstanden ist. Die Nicht-alkoholische Steatohepatitis geht oft einher mit Insulinresistenz und starkem Übergewicht. Die Folgen für die Leber sind Funktionseinschränkungen und –verlust, hervorgerufen durch eine massive Akkumula-tion von Triglyzeriden in den Hepatozyten, Entzündungsprozesse sowie einem fibro-tischen Umbau der Leber. Im fortgeschritten Stadium wird eine Lebertransplantation unausweichlich, die jedoch aufgrund des zunehmenden Mangels an Spenderorganen oft nicht möglich ist. Eine Alternative bietet die Transplantation mesenchymaler Stammzellen (MSC). MSC können in vitro in leberzellähnliche Zellen differenziert wer-den und weisen dabei essentielle hepatozytäre Eigenschaften auf, wodurch sie als möglicher Ersatz bzw. als Überbrückungstherapie bis zur Lebertransplantation in Frage kommen. Die vorliegende Arbeit beschäftigte sich mit dieser Fragestellung. Dazu wur-de ein Tiermodell der NASH mittels Methionin-Cholin-defizienter Diät (MCD-Diät) etab-liert und die Transplantation von hepatozytär differenzierten MSC durchgeführt. An-hand spezifischer zellulärer und biochemischer Marker der NASH konnte die Wirkung des Zelltransplantats auf die Empfängerleber analysiert werden. Es hat sich gezeigt, dass die MSC einen anti-inflammatorischen, anti-fibrotischen und pro-proliferativen Einfluss auf das Empfängerparenchym hatten und somit zur Verbesserung der Symptomatik der NASH beitrugen.

Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...

Opioids are one of the oldest analgesics used to relieve pain. Besides their therapeutic properties, they also have negative side effects, which include impaired tissue regeneration. Therefore, it can be assumed that opioids also have a negative effect on stem cells which are responsible for tissue healing. One of stem cell populations involved in wound regeneration are mesenchymal stem cells (MSCs). MSCs are undifferentiated, multipotent cells that could be find in almost all tissues. They have immunoregulatory properties and they can migrate to the site of inflammation or injury where they contribute to healing of tissues. Therefore, the purpose of this thesis was to evaluate the effect of morphine and methadone on properties and migration of MSCs. Their effect on the metabolic activity of MSCs and also on the production of cytokines and growth factors was measured. The effect of these opioids on the immunoregulatory properties of MSCs acting on both innate and adaptive immune cells in vitro was studied. The effect of morphine on expression of adhesive molecules on MSCs was also examined. Furthermore, the effect of morphine on migration properties of systemically administered exogenous MSCs in vivo was investigated in mouse models. Distribution of MSCs to individual organs and to the site of...

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

L’AVC représente la première cause d’handicap acquis chez l’adulte. L’AVC ischémique, représentant 87% des AVCs, est une pathologie complexe dont le premier facteur de risque aggravant est l’hypertension artérielle. À l’heure actuelle les seuls traitements disponibles sont la thrombolyse et la thrombectomie. Cependant, ces traitements présentent de nombreuses contre-indications et effets secondaires limitant leurs applications chez les patients. L’objectif des travaux menés dans cette thèse est l’évaluation d’un traitement pharmacologique, le RGTA (ReGeneraTing Agent), combiné ou non à un traitement cellulaire utilisant les cellules souches mésenchymateuses (CSMs), chez des rats normo- et hyper-tendus. Les résultats obtenus dans cette thèse montrent qu’à la suite d’une ischémie cérébrale, les traitements évalués offrent une neuroprotection et une récupération fonctionnelle persistantes, chez les animaux noromo- et hyper-tendus. Cette récupération est expliquée par la réduction du volume lésionnel, par une meilleure plasticité cérébrale (angiogenèse, neurogenèse), ainsi par la potentialisation de l’effet des CSMs par le RGTA. En conclusion, nos études démontrent l’efficacité d’une thérapie robuste de neurorprotection chez le rongeur à la suite d’une ischémie cérébrale. / Stroke is the leading cause worldwide of adult severe disability. The limited available treatments for ischemic stroke, which accounts for 87% of strokes, makes it necessary to develop new therapeutical approaches. Stroke is a complex pathology and chronic hypertension (CAH) represents the first aggravating risk factor for ischemic stroke. At the present time, the only two available treatments for ischemic stroke, thrombolysis and thrombectomy, present several side effects limiting their clinical use. Here we evaluate the effect of a molecular RGTA (ReGeneraTing Agent) based therapy combined or not to a cellular therapy based on the use of mesenchymal stem cells (MSCs) for the treatment of ischemic stroke in normo- and hyper-tensive rats. The results demonstrate that the evaluated therapies confer a long lasting neuroprotection accompanied by animals’ functional recovery. Further analysis suggest that RGTA enhances brain plasticity (angiogenesis, and neurogenesis), protects the extracellular matrix structure, and potentiates MSCs’ beneficial effects. In conclusion, our studies demonstrate the efficacy of a molecular and cellular combined therapy conferring a persistent neuroprotection and functional recovery for the treatment of ischemic stroke.

There are approximately 600,000 cases of delayed or aberrant fracture healing in people each year, with a small subset of these fractures experiencing disunion. Dietary phosphate deficiency has been shown to impair oxidative phosphorylation and decrease BMP-2 mediated chondrogenic differentiation during fracture healing. Prior studies using pre-committed chondro-progenitor ATDC5 cell line grown in phosphate deficient media showed that energy consumption was linked to protein production and collagen hydroxylation but inversely related to matrix mineralization. The goal of this study was to further define the relationship between energy consumption and BMP-2 mediated stem cell chondrogenic differentiation and further examine how dietary phosphate, and promotion of collagen hydroxylation via ascorbate availability effected these processes. C3H10T1/2 murine cells, a multi-potential cell line, were expanded in pre-differentiation growth medium (DMEM with 10% FBS and 1% Pen/Strep). Once cells reached 60% confluence (day 0), they were grown in differentiating media (α-MEM with 5% FBS and 1X insulin-transferrin-selenium) containing either 100% (1mM) or 25% (0.25mM) inorganic phosphate (Pi), ± 200ng/mL BMP-2(BMP), and ±0.2 mM L-ascorbic acid (AA). In total, there were 8 groups with varying combinations of these three substances. Intracellular lipid, total DNA, protein, and hydroxyproline (HP) content were examined. Chondrocyte gene expression (Col2a1, Acan, ColXa1) and adipocyte gene expression (Pparg, Plin1, Ucp1) were measured to check for cell lineage commitment and specific differentiation of the C3H10T1/2. All measurements were acquired at day 8. The +BMP differentiation media groups contained significantly less DNA content and more protein content than the –BMP differentiation media groups (both p<0.0001). There was also a significant interaction between phosphate and ascorbic acid treatment (p=0.0296), with 25% Pi +AA groups producing significantly more protein than 100% Pi +AA groups. Hydroxyproline production was not different in 100% Pi or 25% Pi conditions (p=0.2951). AA presence in culture media led to greater HP production than culture media lacking AA (p=0.0035) There was a trend of an interaction between phosphate content and AA availability (p=0.0744). 100% Pi ±AA groups produced significantly different amounts of HP while 25% Pi ±AA groups did not produce significantly different amount of HP. Col2a1, Acan, and ColXa1 expression were all increased in +BMP groups. Ascorbic acid treatment groups expressed significantly more Col2a1and Acan than –AA groups. 100% Pi media led to greater Acan expression over 25% Pi groups (p=0.0009), whereas 25% Pi media trended to lead to greater ColXa1 expression over 100% Pi groups (p=0.0734). Pparg and Plin1 expression were increased in the 25% Pi condition. There were no significant differences in expression of Ucp1. C3H10T1/2 cells were significantly affected by phosphate concentration, BMP-2 treatment, and ascorbic acid supplementation. Phosphate deficiency hindered maturation of early chondrocytes into proliferating chondrocytes while also promoting MSC differentiation into the adipocyte cell lineage. Hypertrophic chondrocyte expression was decreased in phosphate deficient media, which may coincide with increased protein production observed in low phosphate conditions. BMP-2 promoted chondrogenesis which resulted in increased protein production. Whereas, lack of ascorbic acid in cell culture media led to decreased hydroxyproline production.

Rotator cuff injuries cause pain, disability, and loss of shoulder function in over 17 million individuals in the United States that result in over 500,000 surgeries performed annually, though with alarming failure rates of 20-94% (Colvin et al., 2012; Galatz et al., 2004; Harryman et al., 1991; Jain et al., 2014; Mather et al., 2013; Oh et al., 2007; Vitale et al., 2007; Yamaguchi et al., 2006). These surgeries involve repair or reconstruction of the damaged rotator cuff tendon(s) along with enlargement of the subacromial space by debriding the overlying bone (acromion) and removing the subacromial bursa (Beard et al., 2018; Burkhart et al., 2016; Dines et al., 2006; Lo & Burkhart, 2003; Rossi & Ranalletta, 2020). The subacromial bursa is a synovial-like tissue that is situated between the acromion and the tendons of the rotator cuff. This tissue has been long understood to serve a primarily mechanical role by providing cushioning and protecting from friction-wear from the acromion on the underlying tendons. More recently, the identification of a robust vascular network within the bursa, a resident population of mesenchymal stem cells, and inflammatory responsiveness to rotator cuff pathology have supported the existence of a biological role of this tissue in addition to its mechanical one (Blevins et al., 1997; Gotoh et al., 1998, 2001; Põldoja et al., 2017; Rathbun & Macnab, 1970; Steinert et al., 2015; Yepes et al., 2007). These observations make surgical excision of the bursa problematic, given our current lack of understanding of the implications of removing the bursa on the biological response to tendon injury. Therefore, the goals of this dissertation were three-fold: (1) to determine the role of the subacromial bursa in the rotator cuff tendon response to injury and healing, (2) to interrogate patterns of cellular crosstalk between the subacromial bursa and the rotator cuff following injury, and (3) to demonstrate therapeutic potential of targeting the subacromial bursa for modulating inflammation and improving tendon healing.Motivated by clinically observed phenotypic changes in the subacromial bursa with rotator cuff pathology, the profiles of human bursa and rotator cuff tendon tissues were assessed using histology, proteomics, and transcriptomics. This data set, analyzed in the context of patient demographics and diagnoses, revealed distinct bursa proteomes according to tissue phenotype (i.e., fibrous, vascular, or fatty), patient age, and presence of a tear in the underlying rotator cuff. These results suggested the presence of crosstalk between the rotator cuff and the bursa that had not been previously appreciated. Employing multiple methods of validation, including histology, microcomputed tomography, gene expression, and flow cytometry, the rat bursa was established as an appropriate animal model of the human bursa. Therefore, we used the rat model to investigate the role of the bursa in tendon injury response and healing; tendon injuries were created surgically with or without a subsequent repair to study healing and responses to injury, respectively. The role of the bursa in the response to injury was assessed using gene expression, transcriptomics, and histology. The bursa promoted inflammatory gene expression in the injured supraspinatus but resolved inflammatory gene expression in the intact infraspinatus. The role of the bursa in tendon healing was assessed using gene expression, histology, microcomputed tomography, and tensile mechanical testing of the cuff tendons. Consistent with responses during the inflammatory phase of healing, the bursa promoted expression of genes related to aberrant, scar-mediated healing in the supraspinatus, whereas it promoted tenogenic and tendon extracellular matrix gene expression in the intact infraspinatus. Mechanical testing demonstrated that the bursa protected the infraspinatus from the inflammatory environment caused by the supraspinatus injury but had a limited functional effect on the healing supraspinatus. Microcomputed tomography also indicated bursa-dependence in cortical and trabecular bone remodeling following tendon injury. Cross-talk between the bursa and the tendon was then studied in a novel tissue explant co-culture platform using gene expression and nitric oxide release as outcome measures. These experiments revealed that the activated bursa engaged in immunomodulation of tendon fibroblast responses to inflammatory stimulus. The in vitro platform also established the glucocorticoid dexamethasone as a viable therapeutic candidate for bursa-targeted treatment based on its capacity to regulate the bursa’s response to an inflammatory stimulus and enhance the bursa’s immunomodulatory potential. Therefore, in the final component of this thesis, dexamethasone was delivered via PLGA microspheres in vivo to the bursa to modulate the post-injury inflammatory response in the supraspinatus and the infraspinatus tendons. Results supported the therapeutic potential of this treatment approach to improve rotator cuff healing outcomes. This body of work demonstrated a robust involvement of the bursa in rotator cuff responses to injury, with distinct roles in the underlying injured and intact tendons. This work also established, for the first time, the immunomodulatory capacity of the activated bursa and provided strong evidence against the clinical practice of bursectomy. Finally, use of sustained-release dexamethasone to dampen the inflammatory responses to rotator cuff injury offers a new direction for harnessing the inherent properties of the subacromial bursa therapeutically for improved rotator cuff tendon healing.

Mesenchymale Stammzellen bieten eine interessante Option in der regenerativen Medizin, da sie praktisch unlimitiert verfügbar sind. Um das Verhalten von humanen MSC zu studieren, werden Untersuchungen momentan an immundefizienten Mäusen durchgeführt, deren Verwendung kostenintensiv und aufwendig ist. Fra-gestellung war, ob durch Immunisolation (Alginat, Dialyseschlauch, Diffusionskammer) die Knorpel erhaltenden -, bzw. bildenden Eigenschaften von MSC-Konstrukten ebenso gut in immunkompetenten Mäusen untersucht werden können. Gleichzeitig sollte geprüft werden, ob die mit einer Immunabschirmung einhergehende Reduktion der Zellversorgung und damit die Annäherung an die Gelenksituation ihre Mineralisierung vermindern kann und ob Mauszellen für eine Veränderung der vordifferenzierten Knorpelpellets verantwortlich sind. Hierzu wurden hBMSC chondrogen differenziert. Die Zellpellets wurden mit Alginat, dem Dialyseschlauch oder der Diffusionskammer verkapselt und parallel zu unver-kapselten Kontrollpellets subkutan in immundefiziente SCID-Mäuse sowie in immunkompetente BDF1-Mäuse implantiert. Die Explantate wurden mit Alzianblau-, Alizarinrot-, Kollagen Typ II-Färbungen, sowie einer ALU in-situ Hybridisierung mar-kiert und mittels Histologiescore doppelt blind bewertet (MannWhitneyU). Überra-schenderweise zeigten die unverkapselten Kontrollen in den BDF1-Mäusen weder Zeichen von Inflammation noch von Destruktion und 4/5 der Pellets waren auf Kol-lagen Typ-II und Alzianblau positiv. Gleichzeitig war der Grad der Mineralisierung in den BDF1-Mäusen gegenüber SCID-Mäusen reduziert (p = 0,03). Durch Alginat wurde die Mineralisierung in den BDF1 Mäusen (0/8) völlig verhindert, während in den SCID-Mäusen noch 7/8 der Pellets Kalzifizierung zeigten (p = 0,001). Die Verkapselung mit Alginat verglichen mit der Kontrolle führte in beiden Mausstämmen zu höheren Scores für Kollagen Typ II (SCID: p = 0,013, BDF1: p = 0,042) und zeigte gleichzeitig eine Reduktion der Mineralisierung (SCID: p = 0,018, BDF1: p = 0,031). In SCID-Mäusen war außerdem der Alzianblau-Wert gegenüber den Kontrollen erhöht (p = 0,003). Die Diffusionskammer erwies sich als ungeeignet, da die Pellets ihre knorpeligen Eigenschaften verloren. Durch die Verwendung des Dialyseschlauches konnte lediglich in der SCID-Maus eine Erhöhung der Kollagen Typ II (p = 0,03) und eine Reduktion der Kalzifizierung (p = 0,004) erreicht werden. Sowohl im Alginatbead in der BDF1-Maus (1/3 Spendern), als auch im Dialyseschlauch mit Kollagenmembran (2/3 Spendern) konnte eine erfolgreiche in vivo Chondrogenese durchgeführt werden. Zur Untersuchung der in vivo Stabilität knorpeliger MSC-basierter Konstrukte stellt die BDF1-Maus eine attraktive, kostengünstige Alternative mit einer gegenüber der SCID-Maus verringerten Mineralisierungsrate dar. Die in vitro gebildete knorpelige Extrazellulärmatrix erzeugt dabei bereits eine Immunisolation, welche die Transplantatdestruktion verhindert. Ob ein intaktes lymphozytäres System die Knorpelstabilität gegenüber defizienten Immunsystemen begünstigt, muss durch die Untersuchung weiterer Ansätze belegt werden. Im Gegensatz zur Diffusionskammer bietet Alginat das richtige Maß an Versorgungsreduktion, um die Stabilisierung des Knorpelphänotyps der Konstrukte zu ermöglichen.

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La thérapie cellulaire utilisant une greffe d’hépatocytes est une stratégie prometteuse pour traiter les maladies du foie. Cependant, plusieurs limitations freinent leur transfert pour des applications cliniques, comprenant la production à haut débit d'hépatocytes fonctionnels, leur survie en culture, l’âge du donneur et la source des cellules souches hépatiques (CS). Les avancées scientifiques réalisées à ce jour ont permis d’identifier de nouveaux marqueurs moléculaires et les voies de signalisation impliquées dans la différenciation des CS en hépatocytes fonctionnels. En effet, la voie de signalisation Wnt a montré être importante pour réguler de nombreux processus biologiques des CS permettant de contrôler leur différenciation hépatique dont l’activation des GTPases et les gènes ciblant la voie de signalisation de Lgr5. Récemment, des études ont montré que le marqueur Lgr5 (récepteur 5 couplé à la protéine G contenant une répétition d’acides aminés riche en leucine) est décisif pour maintenir une expansion à long terme des CS hépatiques in vitro. En outre, Lgr5 fonctionne principalement comme un effecteur de la Cdc42 (cycle de division cellulaire 42) qui est un membre de la famille des Rho-GTPase. Une forte expression de la Cdc42 a montré être corrélée avec le vieillissement des SCs hématopoïétiques. Néanmoins, cette corrélation n'a jamais été étudiée à ce jour dans les cellules souches mésenchymateuses dérivées du tissu adipeux humain (hADSCs).Au cours de nos travaux de thèse, nous nous sommes intéressés (i) à proposer une nouvelle technologie de reprogrammation d’hépatocytes matures murins en progéniteurs endodermiques (mEndoPCs) exprimant Lgr5 capables de générer des organoïdes spécifiques du foie et de se différencier en hépatocytes et cholangiocytes in vitro et en des structures biliaires et hépatiques in vivo (ii) à étudier l’activité de Cdc42 dans les hADSCs âgées et l'impact de son inhibition sélective par le ML141 sur leur potentiel de différenciation hépatique in vitro.Nous montrons qu’il a été possible de générer des mEndoPCs et à améliorer la différenciation hépatique des hADSCs âgés. Nous montrons également que Lgr5 et Cdc42 sont régulés de façon distincte par la voie de signalisation Wnt. De plus, nos résultats ont révélé que les voies LIFR/STAT3 et Lgr5/WNT sont essentielles pour l’auto renouvellement des mEndoPCs permettant leur expansion illimitée in vitro en présence d’activateur de STAT3. Les voies MAPK/PKA, WNT/ β-caténine et la production d'exosomes ont montré avoir rôle majeur dans l’âge des hADSCs. Nous montrons qu’une transition mésenchymato-épithéliale était nécessaire pour différencier les hADSCs en hépatocytes fonctionnels. D’autre part, ML141 est proposé comme un nouvel outil pharmacologique permettant de reverser l’âge des hADSC âgés et d’amplifier le taux de prolifération, d’adhésion et de fonctionnalité hépatique à un niveau équivalent aux hADSCs jeunes. Enfin, le transfert de ces méthodologies à l’homme pourrait servir pour la médecine régénératrice du foie, comme outil pour évaluer la toxicité hépatique des médicaments et pour l'ingénierie et la reconstitution d’un foie entier par des approches de « bio printing ». / Hepatocytes cell-based transplantation is a promising strategy for treating liver diseases. However, there are still several limitations for their use in clinical applications among them the generation of highthrouput of functional hepatocytes, their life span in culture, the age of the donor age and the source of hepatic stem cells (SCs). At present, the challenge lies to develop approaches aiming the identification of the new molecular markers signaling pathways involved in the differentiation of SCs toward functional hepatocytes. In fact, Wnt(s) pathways governs multiple biological processes controlling the differentiation fate of SCs into hepatocytes, some of them result in the activation of small GTPase and the Lgr5 pathway regulators. Indeed, Lgr5 (a target gene of Wnt, the Leucine-rich-repeat-containing G protein-coupled Receptor 5) was shown to be crucial for maintaining long-term expansion of hepatic SC in vitro. In addition, Lgr5 primarily functions as an effector of the Cdc42 GTPases (a RhoGTPase protein, the cell division cycle 42). Higher activity of Cdc42 was reported to be correlated to hematopoietic SCs aging. However, this correlation has never been studied before in adipose tissue Mesenchymal Stem Cells (ADSCs) which were proposed recently as a promising tool for liver regeneration.In this study, we were interested (i) to propose a novel method of reprogramming mouse mature hepatocytes into murine endodermic progenitors (mEndoPCs) that express Lgr5, generate liver-specific organoids and can differentiate into hepatocytes and cholangiocytes in vitro and give arise to bile duct structures and into functional hepatocytes in vivo, and (ii) to study the activity of Cdc42 in human aged-derived hADSCs and the impact of its selective inhibition by ML141 on their hepatic differentiation potential in vitro.In our study we succeeded to generate mEndoPCs and to improve the functionality of the aged-hADSCs derived-hepatocytes. We showed that both Lgr5 and Cdc42 are regulated distinctly by Wnt signaling pathways. In addition, our results revealed that LIFR/STAT3 and LGR5/WNT pathways are important to maintain the unlimited expansion of mEndoPCs in vitro when STAT3 pathway is activated. MAPK/PKA, WNT/ β-catenin pathways and the exosome’s production were shown to be deregulated with hADSCs aging. We showed also that a mesenchymal to epithelial transition was crucial to transdifferentiate hADSCs into functional hepatocytes. On the other side, ML141 is proposed as a new pharmacological tool to rejuvenate aged-hADSCs toward functionally younger-like cells thus by promoting cell proliferation, doubling and cell adherence. Finally, the transfer of these methodologies to human could serve the regenerative medicine of the liver as a good tool for hepatocyte-based drug toxicity screening systems and for the liver engineering using a « bio printing » approach.