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Background aims Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). Methods cGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. Results The authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. Conclusions The authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum‐free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA‐CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA‐treated cultures were able to engraft in immune‐deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA‐mediated ex vivo HSC expansion for gene modification of adult HSCs., Valproic acid (VPA)‐mediated ex vivo expansion of adult bone marrow and mobilized peripheral blood CD34+ cells resulted in a cellular product characterized by high viability, enrichment with CD34+CD45RA‐CD90+ cells, increased aldehyde dehydrogenase (ALDH) activity, robust multipotent clonogenic potential, and decreased mitochondrial potential. VPA‐expanded grafts were able to establish unbiased multilineage human hematopoietic‐cell chimerism in NSG mice at 16 weeks post‐transplantation.

Understanding mechanisms that determine the behavior of human hematopoietic stem cells (HSCs) is essential for developing novel strategies to expand ex vivo the number of fully functional HSCs. In this review, we focus on the complex interplay between intrinsic mechanisms regulated by transcriptional and mitochondrial networks and extrinsic signals imposed by the bone marrow microenvironment, which in concert regulate the balance between HSC self‐renewal and differentiation. Such integrated signaling mechanisms that dictate the fate of HSCs in vivo must be recapitulated ex vivo to achieve successful expansion of clinically relevant HSCs. We also highlight some of the most recent ex vivo HSC expansion strategies that have currently entered clinical development. Finally, based on the evidence reviewed here and lessons learned from ex vivo HSC expansion, we raise some critical questions regarding HSC fate and the cellular plasticity of hematopoietic cells that challenge the unidirectional model of human hematopoiesis., In this review, we focus on the complex interplay between intrinsic mechanisms regulated by transcriptional and mitochondrial networks and extrinsic signals imposed by the bone marrow microenvironment, which in concert regulate the balance between HSC self‐renewal and differentiation. We also highlight some of the most recent ex vivo HSC expansion strategies that have currently entered clinical development and raise some critical questions regarding HSC fate and the cellular plasticity of hematopoietic cells that challenge the unidirectional model of human hematopoiesis.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis., Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment. Recent evidence, however, highlights that alterations in mitochondrial metabolism and activity are not simply passive consequences but active drivers of HSC fate decisions. Modulation of mitochondrial activity and metabolism is therefore critical for maintaining the self-renewal potential of primitive HSCs and might be beneficial for ex vivo expansion of transplantable HSCs. In this review, we emphasize recent advances in the emerging role of mitochondria in hematopoiesis, cellular reprograming, and HSC fate decisions.

This study investigated the relationship of oxytocin (OT) to chondrogenesis and osteoarthritis (OA). Human bone marrow and multipotent adipose-derived stem cells were cultured in vitro in the absence or presence of OT and assayed for mRNA transcript expression along with histological and immunohistochemical analyses. To study the effects of OT in OA in vivo, a rat model and a human cohort of 63 men and 19 women with hand OA and healthy controls, respectively, were used. The baseline circulating OT, interleukin-6, leptin, and oestradiol levels were measured, and hand X-ray examinations were performed for each subject. OT induced increased aggrecan, collagen (Col) X, and cartilage oligomeric matrix protein mRNA transcript levels in vitro, and the immunolabelling experiments revealed a normalization of Sox9 and Col II protein expression levels. No histological differences in lesion severity were observed between rat OA groups. In the clinical study, a multivariate analysis adjusted for age, body mass index, and leptin levels revealed a significant association between OA and lower levels of OT (odds ratio = 0.77, p = 0.012). Serum OT levels are reduced in patients with hand OA, and OT showed a stimulatory effect on chondrogenesis. Thus, OT may contribute to the pathophysiology of OA.

The limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34+ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34+CD90− cells into CD34+CD90+ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.

Objective: Brite adipocytes are inducible energy-dissipating cells expressing UCP1 which appear within white adipose tissue of healthy adult individuals. Recruitment of these cells represents a potential strategy to fight obesity and associated diseases. Methods/Results: Using human Multipotent Adipose-Derived Stem cells, able to convert into brite adipocytes, we show that arachidonic acid strongly inhibits brite adipocyte formation via a cyclooxygenase pathway leading to secretion of PGE2 and PGF2a. Both prostaglandins induce an oscillatory Ca þþ signaling coupled to ERK pathway and trigger a decrease in UCP1 expression and in oxygen consumption without altering mitochondriogenesis. In mice fed a standard diet supplemented with u6 arachidonic acid, PGF2a and PGE2 amounts are increased in subcutaneous white adipose tissue and associated with a decrease in the recruitment of brite adipocytes. Conclusion: Our results suggest that dietary excess of u6 polyunsaturated fatty acids present in Western diets, may also favor obesity by preventing the “browning” process to take place.

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSCs) for patients that require allogeneic stem cell transplantation. The use of UCB in adults is restricted due to the limited number of HSCs within a single unit. This limitation can be overcome by ex-vivo expansion. Several strategies have successfully expanded the numbers of clinically functional HSCs. Such strategies, including the aryl hydrocarbon receptor antagonist, SR1, pyrimidoindole derivative, UM171, nicotinamide, NAM and more recently, valproic acid (VPA), have entered clinical development. HSC grafts expanded by SR1 and UM171 have been reported to establish rapid and sustained hematological reconstitution in allogeneic transplant recipients [1-3]. Our group has started a clinical trial using VPA-expanded allogeneic UCB grafts (NCT03885947). Initial data have shown multi-lineage donor cell reconstitution with especially rapid T cell and platelet engraftment in patients with treatment refractory acute leukemias. To gain insight into the mechanisms underlying these clinical HSC expansion procedures, we recreated the different approaches to compare the various expanded grafts. Although there is successful clinical data with the NAM approach, we were unable to recreate these conditions in our laboratory and therefore, we could not compare these grafts to those expanded with SR1, UM171 and VPA [4-7]. These comparative studies indicated that each strategy increased to varying degrees both the percentage and absolute numbers of CD34+CD133+CD90+EPCR+ as opposed to cultures treated with their respective cytokine cocktail alone. Although SR1 treatment resulted in the generation of greater numbers of total nucleated cells from purified 1x106 UCB-CD34+ cells (16x107 in SR1 vs 8x107 in UM171 and 7x107 in VPA), the numbers of CD34+CD133+CD90+EPCR+ cells (4x105 in SR1 vs, 7x105 in UM171 and 8.7x105 in VPA) as well as of other CD34+ subpopulations with different degrees of stem cell activity were lower than those observed in VPA and UM171-expanded cultures. In order to understand the underlying mechanism by which these agents act, we isolated CD34+CD90-EPCR- cells and treated with VPA, SR1 or UM171. VPA treatment for 24 hrs resulted in acquisition of CD90 and EPCR expression (80% and 25% of cells acquired CD90 and EPCR, respectively). UM171 treatment led to a modest increase in cells expressing EPCR and CD90. By contrast, SR1 treatment did not induce a phenotypic switch. Comparison of RNA-seq data indicated a strong induction of CD90, EPCR and CD133 in the CD34+ grafts expanded with VPA and UM171, but not in grafts generated with SR1. Twenty five out of 53 and 5 out of 9 genes upregulated more than 2 fold by UM171 and SR1, respectively, were also upregulated in VPA-expanded grafts. Eight out of 39 and 6 out of 77 genes downregulated more than 1.5 fold by UM171 and SR1, respectively, were also downregulated in VPA-expanded grafts. However, grafts expanded with each of these agents shared only three upregulated and three downregulated genes suggesting that different mechanisms underlie the actions of each of these agents. In fact, enhanced histone acetylation induced by VPA was not observed in SR1 and UM171 expanded cells. Both UM171 and VPA did not signal through the AHR pathway, which is required for SR1-mediated expansion. These data suggest that these agents target distinct pathways leading to ex vivo HSC expansion. Since mitochondrial activity is an additional critical regulator of the HSC fate, we examined the mitochondrial profile. VPA-expanded HSCs exhibited a remodeled mitochondrial network associated with low levels of mitochondrial ROS and mass. HSCs expanded with UM171 unlike SR1 displayed low ROS levels. Neither UM171 nor SR1 impacted mitochondrial mass. In vivo transplantation of UM171 and VPA expanded grafts in NSG mice showed similar degrees of human cell chimerism (~25%). Whereas both expanded grafts were capable of establishing multilineage hematopoiesis, we found a greater degree of CD34+ and T cell chimerism in NSG mice engrafted with VPA-expanded grafts. Studies of the engraftment potential of SR1 expanded grafts are ongoing. In conclusion, our findings suggest that although VPA, UM171 and SR1 expand greater numbers of human HSCs that are useful for allogeneic stem cell transplantation, their underlying mechanisms of action and pathways involved are different. Disclosures Schaniel: Summer Road: Research Funding. Hoffman:Merus: Research Funding.

Adult hematopoietic stem cells (HSCs) provide an attractive target for gene modification strategies for the correction of genetic blood disorders. A significant barrier to the translation of these strategies to the clinic has been the decline in numbers of fully functional HSCs associated with ex vivo culture. In order to create optimal conditions for gene modification, HSCs are typically cultured with cytokines to trigger their proliferation. Cytokine stimulation, however, induces stem cell differentiation, resulting in loss of long-term marrow repopulating potential. To overcome this barrier, we treated two sources of adult HSCs, G-CSF mobilized peripheral blood (mPB) and bone marrow (BM) CD34+ cells with valproic acid (VPA), a histone deacetylase inhibitor. This approach has previously been shown to promote the expansion of fully functional cord blood HSCs (Chaurasia et al. JCI, 2014). Cryopreserved mPB or BM CD34+ cells were thawed and cultured in serum-free media supplemented with stem cell factor, FLT-3 ligand, thrombopoietin and interleukin 3. The cells were primed with cytokines for 16 hours followed by addition of VPA (1mM) and incubation in the presence of VPA and cytokines for 7 days. The viability, number of total nucleated cells (TNC), CD34+ cells and frequency of various HSC subpopulations were determined shortly after thawing the primary cells (PCs) and at daily time points, in both VPA-treated cultures and cultures containing cytokines alone (control). The viability of mPB and BM cells treated with VPA for 7 days, each exceeded 90%. While not significantly affecting the expansion of TNC, VPA treatment maintained a higher percentage of CD34+ cells throughout the culture period, and led to a higher expansion of CD34+ cells compared to that observed in control cultures (29 vs 5.5-fold in mPB and 21 vs 5-fold in BM, p Since the phenotype of cells expanded ex vivo does not always correlate with function, we used aldehyde dehydrogenase (ALDH) activity as a functional marker of HSCs. VPA-expanded mPB CD34+ cells showed significantly higher ALDH activity compared to PCs and controls, after both 3 and 7 days of incubation (Table 1). We have previously shown that epigenetic reprogramming induced by VPA is linked to mitochondrial remodeling and suppression of ROS (Papa et al. ASH 2016). We used the mitochondrial membrane potential dye TMRM to assess mitochondrial activity, and found a significant decrease in TMRM in VPA-treated cultures from mPB, as compared to control cultures, at 3 and 7 days (Table 1). This reduction in mitochondrial membrane potential is associated with primitive HSCs. To test their functional capacity, we transplanted the progeny of 2X105 mPB and BM CD34+ cells cultured with or without VPA, into sub lethally irradiated NSG mice. The degree of human cell chimerism in mouse bone marrows was determined 16 weeks later by human CD45 expression. While mice transplanted with grafts from the control cultures had limited or no human chimerism (range 0-1.4%), those transplanted with VPA-treated mPB grafts had up to 50% human chimerism (range 2-50%) (Table 1). The engrafted cells belonged to both myeloid and lymphoid lineages. Comparable results were observed using BM. Several groups have demonstrated the application of expansion methods adapted from cord blood to adult HSCs. These efforts met with successful albeit modest results, perhaps owing to a limited effect of the evaluated agents on adultHSCs. To our knowledge, the above studies represent the most robust demonstration of expansion of HSCs from adult sources, evident as early as after 3 days of culture. These studies strongly support the evaluation of VPA treatment in HSC gene modification strategies. Disclosures No relevant conflicts of interest to declare.

International audience; Osteoporosis and overweight/obesity constitute major worldwide public health burdens that are associated with aging. A high proportion of women develop osteoporosis and increased intraabdominal adiposity after menopause. which leads to bone fractures and metabolic disorders. There is no efficient treatment without major side effects for these 2 diseases. We previously showed that the administration of oxytocin (OT) normalizes ovariectomy-induced osteopenia and bone marrow adiposity in mice. Ovariectomized mice, used as an animal model mimicking menopause, were treated with OT or vehicle. Trabecular bone parameters and fat mass were analyzed using micro-computed tomography. Herein, we show that this effect on trabecular bone parameters was mediated through the restoration of osteoblast/osteoclast cross talk via the receptor activator of nuclear factor-κB ligand /osteoprotegerin axis. Moreover, the daily administration of OT normalized body weight and intraabdominal fat depots in ovariectomized mice. Intraabdominal fat mass is more sensitive to OT that sc fat depots, and this inhibitory effect is mediated through inhibition of adipocyte precursor's differentiation with a tendency to lower adipocyte size. OT treatment did not affect food intake, locomotors activity, or energy expenditure, but it did promote a shift in fuel utilization favoring lipid oxidation. In addition, the decrease in fat mass resulted from the inhibition of the adipose precursor's differentiation. Thus, OT constitutes an effective strategy for targeting osteopenia, overweight, and fat mass redistribution without any detrimental effects in a mouse model mimicking the menopause.

International audience; : Chondrogenesis has been widely investigated in vitro using bone marrow-derived mesenchymal stromal cells (BM-MSC) or primary chondrocytes. However, their use raises some issues partially circumvented by the availability of Adipose tissue-derived MSCs. Herein; we characterized the chondrogenic potential of human Multipotent Adipose-Derived Stem (hMADS) cells, and their potential use as pharmacological tool. hMADS cells are able to synthesize matrix proteins including COMP, Aggrecan and type II Collagen. Furthermore, hMADS cells express BMP receptors in a similar manner to BM-MSC, and BMP6 treatment of differentiated cells prevents expression of the hypertrophic marker type X Collagen. We tested whether IL-1β and nicotine could impact chondrocyte differentiation. As expected, IL-1β induced ADAMTS-4 gene expression and modulated negatively chondrogenesis while these effects were reverted in the presence of the IL-1 receptor antagonist. Nicotine, at concentrations similar to those observed in blood of smokers, exhibited a dose dependent increase of aggrecan expression, suggesting an unexpected protective effect of the drug under these conditions. Therefore, hMADS cells represent a valuable tool for the analysis of in vitro chondrocyte differentiation and to screen for potentially interesting pharmacological drugs.

Umbical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs) for allogeneic transplantation of patients suffering from hematological malignancies and genetic disorders. Its use, however, as an HSC graft for adults has been restricted due to the limited numbers of HSCs within a single UCB unit. We have previously reported that UCB CD34+ cell numbers can be expanded over a seven-day period of incubation by ex vivo treatment with a cytokine combination and the histone deacetylase inhibitor, valproic acid (VPA) (Chaurasia et al. J Clin Invest. 2014:124(6): 2378-2395). In this study, we addressed the mechanism underling this ex-vivo expansion of UCB-derived CD34+cells. We found that VPA triggered two distinct phases of HSC behavior. The first phase (days 1-4) was characterized by a prompt elevation of the expression of stem cell phenotypic markers (CD90 and CD49f), which was associated with acetylation of histone H3, and expression of pluripotency genes including OCT4, NANOG, and SOX2. During this period, the cells underwent a limited number of cell divisions. The second phase (days 4-7) was characterized by a greater number of cell divisions that resulted in a drastic increase in the absolute number of phenotypically defined HSCs. Both acetylation of H3 and expression of the pluripotency genes were transient events since they were no longer evident during the second phase. During the entire incubation period, VPA-induced HSC cells maintained a distinct mitochondrial metabolic profile characterized by low mitochondrial potential, ROS levels and mass. Remarkably, the p38 activity, a downstream target of ROS, was also suppressed in VPA-expanded HSCs. Given that both ROS and p38 activity lead to exhaustion and limit the lifespan of the pool of HSCs, our findings suggest that the VPA-expanded HSCs are equipped with mechanisms that monitor and ensure their fitness.In addition, removal of VPA from the culture after 24, 48 and 72hrs of incubation reversed the acetylation of H3 and the expression of the pluripotency genes. These events were further accompanied by the rapid loss of stem cell marker expression (24hrs after removal) leading to decreased HSC numbers. Importantly, upon VPA removal, the expanded cells exhibited an increase in mitochondrial ROS, mass and potential suggesting that the epigenetic reprograming and mitochondrial remodeling triggered by VPA were linked and were both reversible events. Both transcript and protein levels of p53 were also increased during the first phase but not during the second phase of the ex-vivo expansion of CD34+cell. Importantly, removal of VPA diminished the up-regulation of p53 during the first phase. We further assessed the role of p53 in VPA stem cell expansion with pifithrin-α, a small molecule inhibitor of p53 function. P53 inhibition led to a significant elevation of the mitochondrial ROS without affecting cellular viability. Moreover, treatment with pifithrin-α reduced both the percentage and the absolute number of HSCs induced by VPA. Conversely, co-treatment with VPA and a low concentration of nutlin (1μM), an inhibitor of p53 degradation decreased further mitochondrial ROS levels and augmented the percentage of HSCs induced by VPA. By contrast, treatment with higher concentrations of nutlin (10μM) led to HSC cell death. These findings raise the possibility that VPA induces a moderate up-regulation of p53 as a stress-response mechanism to cope with oxidative stress. It seems plausible that p53 along with alterations in mitochondrial mass and potential are among the mechanisms required to repress oxidative stress and thus, are implicated in the HSC expansion by VPA. Collectively, our data establishes that VPA expands ex-vivo the pool of HSCs from CD34+ CB cells by triggering epigenetic modifications that are tightly linked to mitochondrial remodeling. Notably, these events are reversible and do not result in the malignant transformation of the CD34+ cells. Moreover, these data indicate that VPA modulates and maintains a critical threshold of p53 that is required for HSCs expansion but is not sufficient to trigger the exhaustion and death of HSCs. In this context, VPA also represses p38 activity that if up-regulated can limit the lifespan of HSCs. These studies demonstrate that effective ex vivo HSC expansion requires the coordination and retention of a number of critical biological events that define an HSC Disclosures No relevant conflicts of interest to declare.

It is well established now that adult humans possess active brown adipose tissue which represents a potential pharmacological target to combat obesity and associated diseases. We had shown previously that human multipotent adipose-derived stem (hMADS) cells are able to differentiate into cells which exhibit the key properties of human white adipocytes, and to convert into functional brown adipocytes upon PPARγ activation that could explain UCP1-expressing cells within islets surrounded by white adipocytes. Herein we further characterize hMADS cells differentiation into brown adipocytes that behave like mouse brite adipocytes previously described. We analyzed the expression of gene markers known to be associated with mouse white and brown adipocytes. When shifting from a white to a brown fat cell phenotype, the striking enhancement of uncoupling activity appears mainly due, if not all, to an increase in UCP1 expression whereas induction of UCP2 is weak and UCP3 expression is unchanged. Conversion of white hMADS adipocytes is dependent on PPARγ activation with rosiglitazone as the most potent agonist and is inhibited by a PPARγ antagonist. Furthermore our data show that, in contrast to mouse cellular models, hMADS cells conversion into brown adipocytes is not induced by BMP7 treatment and not modulated by activation of the Hedgehog pathway. No primary or clonal precursor cells of human brown adipocytes have been obtained so far that can be used as a tool to develop therapeutic drugs and to gain further insights into the molecular mechanisms of brown adipogenesis in humans. Thus hMADS cells represent a suitable cell model to delineate the formation and/or the uncoupling capacity of human brown/brite adipocytes that could help to dissipate caloric excess intake among individuals.

Introduction L’expression de la GTPase Rab4b est diminuee dans le tissu adipeux (TA) de souris et de patients obeses. Rab4b est principalement exprime dans les cellules immunitaires dont les lymphocytesT qui participent au developpement de la resistance a l’insuline dans l’obesite. Nous etudions l’impact de l’absence de Rab4b dans les lymphocytesT sur l’homeostasie gluco-lipidique et lymphocytaire. Materiels et methodes L’invalidation de Rab4b dans les lymphocytesT est obtenue par croisement des souris Rab4bflox/flox et exprimant la Cre sous dependance du promoteur Lck des lymphocytesT (LymphT-Rab4bKO). Leur phenotype est determine par exploration metabolique (glycemie, insulinemie, GTT, ITT, cages metaboliques) et par analyse du TA et du foie (histologie, genomique, proteique). Les lymphocytesT thymiques et peripheriques (sang, TA…) sont caracterises par cytometrie en flux et leurs fonctions endocytiques par imagerie quantitative multiparametrique. Resultats Sous regime normal, les souris LymphT-Rab4bKO presentent une hyperglycemie a jeun, un QR nocturne diminue, mais une tolerance au glucose et a l’insuline normale. Nourries, elles sont normoglycemiques et hyperinsulinemiques. Leur masse de TA epididymaire est identique avec une hypertrophie adipocytaire, une infiltration de macrophages pro-inflammatoires, et une diminution des lymphocytesT dont les Treg. L’expression d’IRS1 et Glut4 est diminuee, signe d’une insulinoresitance adipocytaire. Sous regime hyper-lipidique, leur poids est identique mais elles ont une intolerance au glucose et a l’insuline accrue et une steatose hepatique aggravee. L’invalidation de Rab4b augmente l’apoptose des lymphocytesT lors de la selection thymique positive causant une lymphopenie. Elle perturbe la morphologie et la fonction des endosomes et modifie la quantite de recepteurs a la membrane plasmique avec une diminution du TCR. Conclusion La perte de Rab4b dans les lymphocytesT affecte leur biogenese entrainant un remodelage cellulaire du TA pouvant participer au developpement d’une insulinoresistance adipocytaire. Ce defaut du TA conduit a une aggravation des perturbations gluco-lipidiques lorsque les souris sont confrontees a une alimentation hyperlipidique.

International audience; HIV-protease inhibitors (PIs) markedly decreased mortality of HIV-infected patients. However, their use has been associated with occurence of metabolic abnormalities the causes of which are not well understood. We report here that lopinavir, one of the most prescribed PI, dose-dependently co-induced insulin resistance and ER stress in human adipocytes obtained from differentiation of precursor cells. Insulin resistance was subsequent to IRS1 phosphorylation defects and resulted in a concentration-dependent decrease of glucose uptake. The major ER stress pathway involved was the phosphorylation of eIF2-alpha. Salubrinal, a selective eIF2-alpha dephosphorylation inhibitor, induced insulin resistance by targeting IRS1 phosphorylation at serine 312 and acted synergistically with LPV when both drugs were used in combination. This study points out the key role of eIF2-alpha phosphorylation in the development of PI-associated insulin resistance and ER stress. Thus, this protein represents a promising therapeutic target for development of new PIs devoid of adverse metabolic effects.

Introduction L'obesite se caracterise par une inflammation chronique du tissu adipeux. L'inflammation contribue au developpement de pathologies comme la resistance a l'insuline et le diabete de type 2. Le but de cette etude est de determiner le role de REDD1, un regulateur de mTORC1, dans le developpement de l'inflammation du tissu adipeux. Materiels et methodes Des souris ont ete soumises a un regime riche en graisse pendant 17 semaines. Des adipocytes 3T3-L1 et des macrophages J774, dans lesquels l'expression de REDD1 a ete inhibee par des ARN interferant, des MEF et des BMDM (macrophages issus de la moelle osseuse) isoles a partir de souris sauvages ou invalidees pour REDD1 (REDD1 KO) ont ete traites par de l'interleukine IL1β ou du LPS/ATP et l'expression des proteines a ete analysee par western blot. Des souris sauvages ou REDD1 KO ont ete injectees avec du LPS et l'expression de marqueurs inflammatoires a ete analysee dans le tissu adipeux par RT-PCR quantitative. Resultats L'expression de REDD1 est augmentee dans le tissu adipeux de souris soumises a un regime riche en graisse, et en reponse a l'IL1β et au LPS dans des adipocytes et des macrophages (J774 et BMDM). Le LPS/ATP stimule l'inflammasome de facon dependante de REDD1 puisque l'absence de REDD1 (a l'aide d'ARN interferant ou dans des BMDM issus de souris REDD1 KO) inhibe l'expression de NLRP3 et le clivage de caspase-1. De meme la phosphorylation de p65-NF?B en reponse a l'IL1β est inhibee en absence de REDD1. In vivo , l'injection de LPS dans des souris augmente l'expression de marqueurs de l'inflammation comme l'IL1β, le TNFα et l'IL6. Cette augmentation est perdue lorsque des souris REDD1 KO sont injectees avec du LPS. Conclusion En conclusion, nos resultats suggerent que REDD1 pourrait participer au developpement de la resistance a l'insuline en modulant la reponse inflammatoire. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.