Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia�Creperfusion

During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R.     

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.     

Reducing endoplasmic reticulum stress does not improve steatohepatitis in mice fed a methionine- and choline-deficient diet

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of nonalcoholic steatohepatitis. The ER stress response is activated in the livers of mice fed a methionine- and choline-deficient (MCD) diet, yet the role of ER stress in the pathogenesis of MCD diet-induced steatohepatitis is unknown. Using chemical chaperones on hepatic steatosis and markers of inflammation and fibrosis in mice fed a MCD diet, we aim to determine the effects of reducing ER stress. C57BL/6J mice were fed a MCD diet with or without the ER chemical chaperones 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) for 2 wk. TUDCA and PBA effectively attenuated the ER stress response in MCD diet-fed mice, as evidenced by reduced protein levels of phosphorylated eukaryotic initiation factor 2α and phosphorylated JNK and suppression of mRNA levels of CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78 kDa, and X-box binding protein 1. However, PBA and TUDCA did not decrease MCD diet-induced hepatic steatosis. MCD diet-induced hepatic inflammation, as evidenced by increased plasma alanine aminotransferase and induction of hepatic TNFα expression, was also not reduced by PBA or TUDCA. PBA and TUDCA did not attenuate MCD diet-induced upregulation of the fibrosis-associated genes tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9. ER chemical chaperones reduce MCD diet-induced ER stress, yet they do not improve MCD diet-induced hepatic steatosis, inflammation, or activation of genes associated with fibrosis. These data suggest that although the ER stress response is activated by the MCD diet, it does not have a primary role in the pathogenesis of MCD diet-induced steatohepatitis.     

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.     

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200^pos cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up‐regulated in CD200^pos cells compared to CD200^neg fraction. At the functional level, CD200^pos cells were prone to mineralize the extra‐cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200^pos cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down‐regulated. As dexamethasone has anti‐inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro‐inflammatory cytokines interleukin‐1β and tumour necrosis factor‐α increased CD200 membrane expression but down‐regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro‐inflammatory or pro‐osteogenic, CD200 expression was down‐regulated when nuclear‐factor (NF)‐κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro‐inflammatory cytokines through the same pathway: NF‐κB.     

Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.     

Inter‐regulation of the unfolded protein response and auxin signaling

The unfolded protein response (UPR) is a signaling network triggered by overload of protein‐folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down‐regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species‐specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER‐localized auxin transporters, including PIN5, we define a long‐neglected biological significance of ER‐based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone‐dependent strategy for coordinating ER function with physiological processes.     

GPX7 Facilitates BMSCs Osteoblastogenesis via ER Stress and mTOR Pathway

Emerging evidence indicates extensive oxidative stress is a consequence of obesity which impairs bone formation. Glutathione peroxidase 7 (GPX7) is a conserved endoplasmic reticulum (ER) retention protein, lacking of which causes accumulation of reactive oxygen species (ROS) and promotes adipogenesis. Since the imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cell (BMSC) leads to severe bone diseases such as osteoporosis, it is critical to investigate the potential protective role of Gpx7 in osteogenesis. Here, we provide evidence that deficiency of Gpx7 reduces osteogenesis, but increases adipogenesis in both human BMSCs (hBMSCs) and mouse mesenchymal stem cell line. Interestingly, further studies indicate this defect can be alleviated by the ER stress antagonist, but not the ROS inhibitor, unveiling an unexpected finding that, unlike adipogenesis, lacking of Gpx7 inhibits osteogenesis mediating by induced ER stress instead of enhanced ROS. Furthermore, the mTOR signalling pathway is found down-regulation during osteogenic differentiation in Gpx7-deficient condition, which can be rescued by relief of ER stress. Taken together, for the first time we identify a novel function of Gpx7 in BMSCs’ osteogenic differentiation and indicate that Gpx7 may protect against osteoporotic deficits in humans through ER stress and mTOR pathway interplay.     

In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response

In vitro expansion of mesenchymal stem cells (MSCs) has been implicated in loss of multipotency, leading to impaired chondrogenic potential and an eventual therapeutic effect, as reported in our previous study. However, the precise regulatory mechanism is still unclear. Here, we demonstrate that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) were involved in transformation of MSCs induced by in vitro culture based on the comparative profiling of in vitro cultured bone marrow MSCs at passage 3 (P3 BMSCs) vs. fresh P0 BMSCs by microarray analysis. Indeed, RT-PCR and Western blot analysis showed significantly lower expression levels of three key UPR-related molecules, ATF4, ATF6 and XBP1, in P3 BMSCs than P0 BMSCs. Further, we found that UPR suppression by 4-phenylbutyrate (4-PBA) reduced the chondrogenic potential of P0 BMSCs and further cartilage regeneration. Conversely, UPR induction by tunicamycin (TM) enhanced the chondrogenic differentiation of P3 BMSCs and the therapeutic effect on cartilage repair. Thus, the decline in the chondrogenic potential of stem cells after in vitro culture and expansion may be due to changes in ER stress and the UPR pathway.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

iPSC‐derived mesenchymal stem cells exert SCF‐dependent recovery of cigarette smoke‐induced apoptosis/proliferation imbalance in airway cells

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.     

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.     

Comparison of alternative mesenchymal stem cell sources for cell banking and musculoskeletal advanced therapies

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs.