Les cellules cancéreuses utilisent une glycolyse anaérobie pour générer l'ATP au lieu de la phosphorylation oxydative. Cette spécificité métabolique offre certains avantages aux cellules cancéreuses: une prolifération rapide et une évasion immune qui implique la sous-régulation de l'expression du CMH-I à la surface des cellules, phénomène lié au changement métabolique. Dans nos expériences, nous forçons les cellules leucémiques à produire de l'énergie par phosphorylation oxydative en les incubant avec de la glutamine comme source d'énergie en absence de glucose. La respiration ainsi forcée induit une augmentation de la transcription et de l'expression du CMH-I. Ce changement de métabolisme induit aussi une augmentation de l'expression de MAPK ERK5 et son accumulation dans les mitochondries. ERK5 intervient dans les changements de l'expression du CMH-I et du métabolisme. La sur-régulation du CMH-I induite par la respiration est bloquée dans les cellules leucémiques exprimant le shRNA shERK5. ERK5 régule la transcription de l'histone désacétylase de classe III Sirtuin 1 par l'activation de sa cible MEF2, ayant pour conséquence la liaison de MEF2 au promoteur de SIRT1. La régulation transcriptionnelle de SIRT1 induite par ERK5 intervient dans la réponse antioxydante des cellules leucémiques, et la sous-régulation d'ERK5 affecte cette réponse antioxydante. L'augmentation du métabolisme de la glutamine observée dans les cellules leucémiques est initiée par la glutaminase (GLS), enzyme qui est le facteur limitant de la vitesse du métabolisme de la glutamine. miR-23a cible l'ARN messager de GLS et inhibe l'expression de GLS. Le milieu glutamine induit la translocation de p65 dans le noyau, qui mène à une augmentation de l'activité transcriptionnelle de p65. NF-KB p65 inhibe l'expression de miR-23a en amenant HDAC4 sur le promoteur de miR-23a. Cela permet aux cellules leucémiques d'augmenter l'utilisation de la glutamine en tant que source alternative de carbone. Ainsi, la respiration forcée dans les cellules leucémiques contrôle l'expression du CMH-I, la réponse antioxydante et facilite la prolifération tumorale. / Cancer cells have anaerobic-like glycolysis to generate ATPs instead of oxidative phosphorylation. This specific metabolism provides advantages to cancer cells: rapid growth and immune evasion, which involves downregulation of MHC-I at the cell surface and it is linked to metabolic change. In our experiments, we force leukemic cells to produce energy by oxidative phosphorylation by incubating them with glutamine as an energy source in the absence of glucose. The forced respiration increases MHC-I transcription and protein level. This change of metabolism also leads to increase MAPK ERK5 expression and accumulation in mitochondria. ERK5 mediates changes in both MHC-I and metabolism. The respiration-induced upregulation of MHC-I is blocked in leukemic cells stably expressing short hairpin ERK5 (shERK5). ERK5 transcriptionally regulates the class III histone deacetylase Sirtuin 1 through activation of its target MEF2 and subsequently MEF2 binding to SIRT1 promoter. The ERK5-induced transcriptional regulation of SIRT1 mediates the antioxidant response in leukemic cells and downregulation of ERK5 impairs the antioxidant response. The increased glutamine metabolism found in leukemic cells is initiated by glutaminase (GLS), a rate limiting enzyme for glutamine metabolism. miR-23a targets GLS mRNA and inhibits GLS expression. The glutamine medium induces p65 translocation to the nucleus that leads to increase p65 transcriptional activity. NF-KB p65 inhibits miR-23a expression by bringing HDAC4 to the miR-23a promoter. This allows leukemic cells to increase the use of glutamine as an alternative source of carbon. Thus, forcing respiration in leukemic cells controls MHC-I expression, antioxidant response and facilitate tumor growth.

Cell migration is essential for homeostasis and the development of metastases. We hypothesize that cell migration is regulated by mitochondria and endoplasmic reticulum morphology. Using live cell microscopy, we found that mitochondria specifically migrate into the biochemically dense leading edge of the cell interacting with focal adhesions as well. At the leading edge the mitochondria are visibly shorter and less tubular than the perinuclear area. This is related to the elevated levels of fission events per minute in the leading edge and elevated levels of fusion events per minute in the trailing edge. We observe that mitochondria migrate along microtubules and simultaneously interact with the ER. When the ER is sheet-like the mitochondria are longer and tubular and when the ER is tubular the mitochondria are shorter and punctate. This change in ER and mitochondria morphology changes the cell’s ability to migrate. CLIMP63 cells have more sporadic turns, take longer to make turns, have shorter distances travelled and shorter displacements. To determine whether mitochondria dynamics play a role we examined these cell migration parameters in the presence of OPA1 and Drp1. This allowed us to conclude that the ER morphology is responsible for the distance and displacement the cell travels while the mitochondria is responsible for the angles the cell turns. When the ER is sheet-like the cells will be travel shorter total distances and displacements and when the cell has longer mitochondria it will be sporadic turns and take longer to make these turns.

No description available.

Mitochondrial calcium (Ca2+) uptake has been studied for over five decades, with crucial insights into its underlying mechanisms enabled by development of the chemi-osmotic hypothesis and appreciation of the considerable voltage present across the inner mitochondrial membrane (ΔΨm) generated by proton pumping by the respiratory chain (Carafoli, 1987; Nicholls, 2005). However, the molecules that regulate mitochondrial Ca2+ uptake have only recently been identified (Jiang et. al., 2009; Perocchi et. al., 2010) and further work was needed to clarify how these molecules regulate mitochondrial Ca2+ uptake. Leucine Zipper EF hand containing Transmembrane Protein 1 (LETM1) acts as a regulator of mitochondrial Ca2+ uptake distinct from the mitochondrial Ca2+ uniporter (MCU) pathway (Jiang et. al., 2009). However, a controversy exists regarding the function of LETM1 (Nowikovsky et. al., 2004). Therefore, I asked if LETM1 played a role in mitochondrial Ca2+ uptake and if LETM1 regulated cellular bioenergetics and basal autophagy. To further characterize mitochondrial calcium uptake, we asked how Mitochondrial Calcium Uptake 1 (MICU1) regulates MCU activity by quantifying basal mitochondrial Ca2+ and MCU uptake rates in MICU1 ablated cells. The following work characterizes the molecules that regulate mitochondrial Ca2+ uptake and their mechanistic function on decoding calcium signals. Since LETM1 is the Ca2+/H+ antiporter, I hypothesize that alterations in LETM1 expression and activity will decrease mitochondrial Ca2+ uptake and will result in impaired mitochondrial bioenergetics. As a regulator of free intracellular Ca2+, mitochondrial Ca2+ uptake and the orchestra of its regulatory molecules have been implicated in many human diseases. Mitochondria act both upstream by regulating cytosolic Ca2+ concentration and as downstream effectors that respond to Ca2+ signals. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter (Jiang et. al., 2009); however characterization of the functional role of LETM1-mediated Ca2+ transfer remained unstudied. Therefore the specific aims of this project were to determine how LETM1 regulates Ca2+ homeostasis and bioenergetics under physiological settings. Secondly, this project aimed to characterize how LETM1-dependent Ca2+ signaling regulates ROS production and autophagy. The data presented here confirmed that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake. Furthermore, in-depth approaches including either deletion of EF-hand or mutation of critical EF-hand residues (D676A D688KLETM1) impaired histamine (GPCR agonist)-induced mitochondrial Ca2+ uptake. Knockdown of LETM1 resulted in bioenergetic collapse and promoted LC3-positive multilamellar vesicle formation, indicative of autophagy induction. Interestingly, knockdown of LETM1 significantly reduced complex IV but not complex I and complex II-mediated oxygen consumption rate (OCR). In contrast, cellular NADH and mitochondrial membrane potential (ΔΨm) were unaltered in both control and LETM1 knockdown cells. LETM1 has been implicated in formation of the supercomplexes of the electron transport chain (Tamai et. al., 2008). In support, these studies show that LETM1 knockdown results in increased reactive oxygen species (ROS) production. These results for the first time demonstrate that LETM1 controls cellular bioenergetics through regulation of mitochondrial Ca2+ and ROS. MICU1 was identified as an essential regulator of the mitochondrial Ca2+ uniporter (Perocchi et. al., 2010). Therefore, this project specifically aimed to determine how MICU1 regulates the mitochondrial Ca2+ uniporter. Interestingly, the data presented here suggest that MICU1 is not necessary for uniporter activity. Instead, loss of MICU1 caused mitochondria to constitutively load Ca2+ at rest which resulted in a host of cellular phenotypes. This result led to further questions on how MICU1 knockdown affects cellular bioenergetics and if MICU1 is essential for cell survival under stress. MICU1 ablation influenced pyruvate dehydrogenase activity and ROS production. Subsequent investigations demonstrated that increased basal ROS left cells poised to ceramide-induced cell death thereby suggesting the role of MICU1 in cell survival. Collectively, the data presented here show that MICU1 is necessary to control constitutive mitochondrial Ca2+ uptake during rest. This work demonstrates that LETM1 regulates a distinct mode of mitochondrial Ca2+ uptake pathway whereas MICU1 controls mitochondrial Ca2+ uniporter activity. Further studies are required to uncover the potential role of these two mitochondrial-resident Ca2+ regulators in health and disease. / Biochemistry

Researchers have identified several methods for treating acute myocardial infarction (AMI) patients affected by ischemia and reperfusion injury. Some of these therapies include thrombolysis, balloon angioplasty, and coronary arterial bypass graft (CAGB). This lab has previously demonstrated that transplantation of mitochondria into the ischemic zone of a rabbit heart during reperfusion significantly improved recovery as compared to current techniques. In order for this therapy to be translated into the clinic a rapid isolation method for producing highly pure and functional mitochondria will be required. Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 minutes to complete. Herein, a method for rapid isolation of mitochondria from mammalian tissue biopsies is described. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters which eliminates repetitive centrifugation steps. Mitochondrial isolation time is less than 30 minutes compared to 60-100 minutes using alternative methods. This isolation protocol yields approximately 2 x 10^10 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

Strap is a DNA damage responsive p53 cofactor, reflecting its control by the DNA damage signalling pathway. This study identified Strap at the mitochondria where it is damage regulated and can augment p53-dependent cytochrome c release leading to apoptosis. Moreover, p53 and Strap facilitate each other’s localisation from the mitochondria to the nucleus during the DNA damage response. Two ATM/ATR phosphorylation consensus sites in Strap were identified by mass spectrometry and phosphorylation of all the ATM/ATR consensus sites resulted in mitochondrial localisation of Strap during DNA damage. Targeting Strap to the mitochondria depletes cellular ATP when cells favour energy production through oxidative phosphorylation and sensitises cells to p53-dependent damage-induced apoptosis. These results thus imply that Strap co-ordinates different arms of the p53 response, and might be responsible for integrating its mitochondrial and nuclear functions.

<p>Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.</p><p>To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.</p><p>I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.</p><p>Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.</p> / Dissertation

Alzheimer’s disease (AD) is a multifactorial, progressive, age-related neurodegenerative disease. Oxidative stress hypothesis is most prevalent and is gaining significant support. Inspite of the progress achieved on oxidative stress related damages in AD brain; the modification occurring on the various cellular antioxidant enzymes antioxidant has not been identified. Tyrosine nitration, a marker for peroxynitrite induced oxidative damage to protein is widespread in AD brain and Manganese superoxide dismutase (MnSOD), primary mitochondrial antioxidant enzyme is prone to peroxynitrite induced nitration and inactivation. Nitration of proteins involved in energy metabolism has been demonstrated in AD brain, which may explain the altered glucose metabolic status existing in AD brain. In the present study, we investigated the effect of tyrosine nitration of MnSOD on energy metabolism by the use of AD mouse model and cultured neuronal cells. The AD mouse model was generated from a double homozygous knock-in mouse, designated as APP/PS-1 mice, by incorporating the Swedish familial AD mutations in APP and P264L familial AD mutation in PS – 1. These animals develop age dependent increase in Aβ deposition beginning at 6 months along with an increase in insoluble Aβ1-40/Aβ1-42 levels. Genotype and age associated increase in nitration of MnSOD without any change in protein levels was also observed. MnSOD activity and mitochondrial respiration was decreased in APP/PS-1 mice. There was also concomitant increase in levels of lactate, an index of glycolytic activity in APP/PS-1 mice. To directly investigate the role of MnSOD inactivation in mitochondrial function and subsequent alteration in glycolytic activity, SH-SY5Y neuroblastoma cells line was used and treated with peroxynitrite. Enhanced nitration and reduction in the activity of MnSOD was observed upon peroxynitrite treatment. Peroxynitrite treatment also induced mitochondrial dysfunction, but MnSOD was inactivated at a concentration of peroxynitrite 10 times lower than that required to inhibit mitochondrial respiration. Mitochondrial dysfunction was alleviated by SOD mimetic and reproduced by MnSOD siRNA. The decline in mitochondrial function did not result in decreased ATP levels but was accompanied by an up-regulated glycolysis signified by high levels of lactate and lactate dehydrogenase activity but decreased activity of pyruvate dehydrogenase. These changes were prevented by SOD mimetic and were promoted by MnSOD siRNA. Specific reduction of MnSOD in MnSOD heterozygous knock-out mice resulted in decreased RCR and complex I activity with increased lactate levels. Taken together, these data demonstrate a critical role of MnSOD in influencing the mitochondrial function and thereby the switch in the energy metabolism switch that might occur under the pathological condition of MnSOD deficiency.

UVB radiation is a known environmental carcinogen that causes DNA damage and increase ROS generation in mitochondria. Accumulating evidence suggests that mtDNA damage and increased ROS generation trigger mitochondrial translocation of p53. Within mitochondria, p53 interacts with nucleoid macromolecular complexes such as mitochondrial antioxidant MnSOD, mitochondrial DNA polymerase Polγ, and mtDNA. Mitochondria are considered to be a potential source for damage-associated molecular patterns (DAMPs) such as mtDNA, cytochrome C, ATP, and formyl peptides. Intracytoplasmic release of DAMPs can trigger inflammasome formation and programmed cell death processes. Autophagic clearance of mitochondria with compromised integrity can inhibit inflammatory and cell death processes. In this study we investigated whether and how MnSOD plays a protective role in UVB-induced mitochondrial damage. The possibility of MnSOD participating in the mtDNA repair process was addressed in vivo using transgenic and pharmacological approaches. The results demonstrate that MnSOD functions as a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ and that MnSOD coordinates with p53 to prevent mtDNA damage. We also investigated whether autophagy is an adaptive response mechanism by which skin cells respond to mitochondrial injury, using mouse keratinocytes (JB6 cells) and C57/BL6 mice as in vitro and in vivo models. The results demonstrate that UVB induces autophagy initiation in murine skin tissues and that down regulation of AKTmTOR levels triggers initiation of autophagy processes. These results suggest that autophagy may play a role in scavenging damaged mitochondria. Taken together, the results from these studies suggest that MnSOD plays a protective role against UVB-induced mitochondria injury beyond its known antioxidant function. Within the mitochondrial matrix, MnSOD acts as an antioxidant and fidelity protein by prevention of UVB-induced nitration of Polγ. The functions of MnSOD may be to enhance mitochondrial membrane integrity and to prevent the genesis of oxidatively damaged mitochondrial components and subsequent intracytoplasmic spillage. Activation of autophagy serves as an additional response that scavenges damaged mitochondria.

La transplantation d’hépatocytes est une nouvelle approche thérapeutique pour le traitement des maladies métaboliques. Elle peut être proposée en alternative à la transplantation de foie entier ou, à tout le moins, en attente de celle-ci chez les patients instables, à risque de décompensation métabolique. Les essais cliniques effectués chez 9 patients aux cliniques St Luc ainsi que ceux publiés dans la littérature démontrent l’intérêt de la transplantation de cellules hépatiques à court et moyen terme. La qualité de la suspension cellulaire transplantée reste le premier facteur limitant pour le développement clinique de la technique. La cryopréservation reste le moyen le plus approprié pour la conservation à long terme des cellules. Elle permet de constituer une banque de cellules pouvant être utilisées à tout moment. Nous avons d’abord analysé les protocoles de cryopréservation décrits dans la litérature, ainsi que leurs limites tant au niveau de la préservation de la qualité cellulaire après décongélation in vitro qu’après transplantation in vivo. Dans ce travail, nous avons démontré l’intérêt d’utiliser des cellules cryopréservées/décongelées, afin de stabiliser des patients atteints de maladies du cycle de l’urée, avant la greffe de foie entier. Les tests de contrôle de qualité effectués sur ces cellules ont cependant montré une altération aux niveaux biochimique et cellulaire, après décongélation. Nous avons ainsi démontré une chute des concentrations intracellulaires d’ATP, signe d’une atteinte mitochondriale. Nos travaux ont également permis de mettre en évidence une diminution de la consommation d’oxygène des hépatocytes en suspension, due plus particulièrement à une atteinte du complexe 1 de la chaîne respiratoire. Cette atteinte mitochondriale peut déjà être observée après l’incubation de la suspension cellulaire à –20°C. Aux alentours de cette température critique se fait le passage de l’état aqueux à l’état cristallin suggérant que les dégâts mitochondriaux observés sont dès lors vraisembablement dus à la formation de glace intracellulaire durant le processus de cryopréservation ou de décongélation. Diverses tentatives visant à améliorer les paramètres mitochondriaux affectés par le processus de congélation/décongélation par l’addition d’agents protecteurs du complexe 1 (Bilobalide), d’ inhibiteurs du pore de transition de perméabilité (Ciclosporine A), d’ anti-oxydants ou encore de solutions hyperosmotiques à la solution de cryopréservation, n’ont pas permis d’améliorer la qualité cellulaire. Le tri de sous-types de populations hépatocytaires ou l’isolement de foies hépatectomisés n’ont pas permis de révéler de différences de capacité de résistance à la cryopréservation. Toujours dans le but d’améliorer le rendement de la transplantation d’hépatocytes et d’augmenter l’efficacité d’implantation dans le parenchyme receveur, nous avons démontré dans la deuxième partie de la thèse la capacité des hépatocytes isolés (fraîchement isolés ou cryopréservés/décongelés) à induire un phénomène de coagulation dépendant du facteur tissulaire. Cette activité pro-coagulante, inhibée in vitro par lea N-acetyl-L-cystéine, pourrait être le point de départ d’une réaction inflammatoire aspécifique influençant ainsi la réussite de la transplantation cellulaire. En conclusion, nous proposons dans ce travail différentes stratégies en vue de l’amélioration du rendement de la thérapie cellulaire. La vitrification, autre technique de cryopréservation, permettrait d’éviter la formation d’eau intracellulaire. Enfin la modulation de l’activité pro-coagulante par la N-acetyl-L-cystéine, due à la transplantation cellulaire, constitue une piste intéressante pour essayer d’améliorer l’implantation des cellules transplantées et ainsi le rendement de la greffe. / Liver cell transplantation provides clinical benefit to patients with congenital metabolic abnormalities and currently represents an alternative to orthotopic liver transplantation or at least an interim measure for unstable patients awaiting transplantation. Our team and others have already demonstrated that transplanted hepatocytes can achieve metabolic control in the short or medium term. The quality of transplanted cells remains the first limiting factor for the success of liver cell transplantation. Because the use of freshly isolated cells is restricted by contemporary organ donation, cryopreservation remains necessary for long-term storage and permanent availability of the cells. In this thesis, we have first reviewed and discussed established hepatocyte cryopreservation protocols, especially the cooling procedure, and have focussed on the in vitro and in vivo assays used for the evaluation of post-thawing hepatocyte quality. Amongst 9 cell transplanted patients in our center, several received exclusively or predominantly cryopreserved/thawed hepatocytes. We demonstrated post-transplantation benefits of using these cells in control patients with congentital abnormalities in the urea cycle, particularly with respect to clear evidence of cell engraftment and de novo appearance of enzyme activity. However, despite these clinical benefits, we found an in vitro relationship between the low post-thawing quality of cryopreserved /thawed hepatocytes and an alteration in their mitochondrial function. This post-thawing mitochondrial damage was already evident after the first −20°C cryopreservation step of our protocol, suggesting it occurrs early in the process, around the nucleation point, by intracellular ice formation. Cellular impairment could therefore be possibly explained by mechanical alteration of mitochondria due to water crystallisation during the cryopreservation process or thawing procedure. We also observed a poor efficacy of cryopreserved/thawed hepatocytes (as compared to freshly isolated cells) when used liver engraftment in two mice transplantation models. The marked reductions in intracellular ATP concentrations and the decreases in oxygen consumption by hepatocytes were therefore used as markers for the evaluation of the effects of several compounds such as bilobalide, hyperosmotic or anti-oxidant molecules, pore transition permeability inhibitors, and for the evaluation of the resistance of selected hepatocyte subtypes to cryopreservation protocols. We also demonstrated that isolated hepatocytes exert tissue factor-dependent pro-coagulant activity, which may contribute to the early loss of infused cells. We observed that the addition of N-acetyl-L-cysteine to hepatocyte suspensions inhibits coagulation activation. In conclusion, this work has identified several ways to improve the clinical benefit of liver cell transplantation, including new cryopreservation strategies, such as vitrification. In addition, modulation of the pro-coagulant activity induced by cell infusion with N-acetyl-L-cysteine might beneficially enhance cell engraftment.