Top PDF Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Taken together, our results showed that functional EC could be generated from hESC and iPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as ‘‘off-the-shelf’’ format for treatment of tissue ischemia. This study has limitations. First, the precise mechanism of therapeutic benefit for each of the cell types remains unknown. In this study, we showed that only very small numbers of transplanted cells were detected at Day 28. This finding suggests that paracrine actions of the transplanted ECs are the likely mechanisms that Figure 5. Retention of derived endothelial-like cells at Day 7, 14 and 28 after transplantation. (A) Representative photos show the immunohistochemical staining for human nuclear antigen (HNA) over the ischemic hind-limb at Day 7 and 28 after hESC-EC, hiPSC-1-EC, BM-EC or medium injection (upper panel). Red arrow indicated engrafted hESC-EC, hiPSC-EC, BM-EC in the ischemic limb with HNA positive (brown), indicating those cells were from human origin. At Day 7, clusters of HNA positive cells were observed at the injection sites after hESC-EC, hiPSC-1-EC, and BM-EC transplantation but not after medium injection. At Day 28, only a few HNA positive cells were found around the vasculature at the injection sites after hESC-EC, hiPSC-1-EC, and BM-EC transplantation. As compared with Day 28, the numbers of HNA positive cells detected at the injection sites were significantly higher at Day 7 after hESC-EC, hiPSC-1-EC, BM-EC transplantation (*p,0.05, lower panel). (B) Representative photos showing live cells tracking by quantifying luciferase activity using the Xenogen’s in vivo optical imaging systems at Day 7, 14 and 28 after hESC-EC, hiPSC-1-EC, or medium injection (upper panel). Luciferase activity was plot as total flux measurement in photons/second/cm 2 . Declined in intensity of luciferase
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A comparison of the reparative and angiogenic properties of mesenchymal stem cells derived from the bone marrow of BALB/c and C57/BL6 mice in a model of limb ischemia

A comparison of the reparative and angiogenic properties of mesenchymal stem cells derived from the bone marrow of BALB/c and C57/BL6 mice in a model of limb ischemia

Introduction: BALB/c mice and C57/BL6 mice have different abilities to recover from ischemia. C57/BL6 mice display increased vessel collateralization and vascular endothelial growth factor expression with a consequent rapid recovery from ischemia compared with BALB/c mice. Mesenchymal stem cells (MSCs) are one of the main cell types that contribute to the recovery from ischemia because, among their biological activities, they produce several proangiogenic paracrine factors and differentiate into endothelial cells. The objective of this study was to evaluate whether the MSCs of these two mouse strains have different inductive capacities for recovering ischemic limbs. Methods: MSCs from these two strains were obtained from the bone marrow, purified and characterized before being used for in vivo experiments. Limb ischemia was surgically induced in BALB/c mice, and MSCs were injected on the fifth day. The evolution of limb necrosis was evaluated over the subsequent month. Muscle strength was assessed on the 30th day after the injection, and then the animals were sacrificed to determine the muscle mass and perform histological analyses to detect cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration.
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Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

In the present study, both GFP- and hbFGF-expres- sing MSCs differentiated into endothelial cells in vivo. This differentiation capacity is thought to be the mechanism by which cell therapy restores cardiac function (30), improves blood flow, and reduces necrosis in ischemia (13,31). Specifically, MSCs have been shown to differ- entiate into cardiomyocytes, vascular smooth muscle cells, and endothelial cells in ischemic cardiomyopathy (30). Furthermore, in an in vivo stroke model, transplanta- tion of brain-derived neurotrophic factor-differentiated, MSCs improved motor function to a greater degree than transplantation of undifferentiated MSCs (32). However, Figure 4. Overexpressing human basic fibroblast growth factor (hbFGF) in mesenchymal stem cells (MSCs) stimulates angiogenesis in a rat hind limb ischemia model. A-D, Representative images of sections of ischemic gastrocnemius muscles from the phosphate-buffered saline (PBS) control (A), MSC (B), green fluorescent protein (GFP)-MSC (C), and hbFGF-MSC (D) groups immunostained with anti-von Willebrand factor (vWF) antibodies. After counting the vWF- positive cells (microvessels) and muscle fibers, the ratio of microvessels to muscle fibers (E) and microvessel density (F) were determined. *P,0.05, compared to the PBS group;
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Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro . Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRb, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo . These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.
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Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium.

Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium.

cGMP-gated channels were selectively expressed by cells commit- ted to a rod fate, as shown in Figure 5C for a cell with low EGFP expression. Similar currents in response to 8-Br-cGMP were recorded from cells treated according to the differentiation Figure 1. Differentiation of cells from RNS. (A–B) Changes in cells proliferation (BrdU) (A) or cell death (B) were analyzed at different times before and after treatment with differentiation medium at day 5 (indicated by an arrow). Data are derived from 8 fields from 2 independent experiments and represented as mean +/2 s.e.m. (C–D) Real-time PCR analyses of CE markers Mitf (C), Rpe65 (D) and Rlbp1 (D; also expressed in Mu¨ller glia) show lower or similar levels in retinal neurospheres (RNS) compared to the ciliary epithelium (CE) and down-regulation of these genes during differentiation. (E) Real-time PCR analysis confirms higher levels of Nestin and Nanog mRNAs in RNS (black bars) compared to CE (white bars). In C, D and E data derive from the formula 2 2DCt . S26 was used as reference gene. (F) Real-time PCR analysis of retinal progenitors markers (Pax6,
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Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells.

Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells.

We compared the quantity of cytokines expressed in MVs and parent MSCs and found no matter under hypoxic or normoxic conditions most cytokines were expressed in greater quantity in MSCs than MVs, except Angiogenin, VEGF, MCP-1, and VEGF R2 which were expressed at a higher level in MVs. As we mentioned above, the sorting and encapsulating of functional proteins from MSCs into MVs is a very precise and intelligent process. We postulate that Angiogenin, VEGF, MCP-1, VEGF R2 are selectively transferred to MVs from the mother MSCs in a great quantity while the other angiogenesis cytokines just transferred seldom. Accumulating evidence suggest that the angiogenesis- promoting effects of MSCs are predominantly caused by secretion-based paracrine and those cytokines which are secreted seldom could not play a crucial role, indicating that Angiogenin, VEGF, MCP-1, and VEGF R2 might be of greater importance for MVs to promte angiogenes. VEGF, also known as vascular permeability factor (VPF), is a potent endothelial cells specific mitogen and permeability-enhancing factor that has been shown to play a central role in angiogenesis. Activation of VEGFR-2 by VEGF is a critical requirement to induce the full spectrum of VEGF responses. VEGF promotes endothelial cells survival, proliferation and migration through numerous pathways, including activation of the MAPK, extracellular signal-regulated kinase (ERK), p38 and c-jun N-terminal inase (JNK), and Rho-GTPase family members [20]. MCP-1 belongs to CXC chemokine family members, and is able to affect endothelial cells migration, promote angiogenesis and reduce apoptosis by reducing caspase-3 activity [21]. Angiogenin is a 14 Da soluble protein and a member of the ribonuclease (RNase) superfamily. Angiogenin induces angiogenesis by activating vessel endothelial and smooth muscle cells and triggering a number of biological processes including cell migration, invasion, proliferation, and formation of tubular structures. It has been reported that angiogenin plays its functions through exerting its ribonucleolytic activity RNase activity directing towards 28S and 18S rRNA or binding to membrane actin and then inducing basement membrane degradation [22].
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Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

It is widely assumed that autologous iPSCs and their derivatives should be immunologically tolerated by the recipient. However, this dogma was challenged by a study showing T-cell-dependent immune rejection of syngeneic mouse iPSCs (miPSCs) following transplantation [17], in which miPSCs derived via the episomal approach were less prone to immune-mediated attack than those generated using viral vectors. Another study showed that short-term immuno- suppression by inhibiting leukocyte co-stimulatory molecules promoted engraft- ment of embryonic and induced pluripotent stem cells [18]. Recently, it was reported that low immunogenicity of less immunogenic cells could be retained after cell reprogramming and further differentiation [19]. Nevertheless, another finding has demonstrated limited or no immune response including T cell infiltration in tissues derived from autologous iPSCs or allogenic ES cells [20]. Interestingly, it was shown that hiPSCs-derived CD34 + hematopoietic progenitor
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Xinghui Song1 , Yanwei Li1 , Xiao Chen2

Xinghui Song1 , Yanwei Li1 , Xiao Chen2

In this study, ESCs were cultured on mouse embry- onic fibroblasts. These cells can be cultured, passaged and cryopreserved, and retain their proliferative capacity after continuous passage. Several totipotency molecular markers with an important role in maintaining the capacity for self-renewal (Eiges et al., 2001; Richards et al., 2002) oc- cur in these cells, including stage-specific embryonic anti- gens (SSEA-1, SSEA-3, SSEA-4), transcriptional regulation antigens (TRA-1-60, TRA-1-81), homologous protein transcription factor (Nanog) and transcription fac- tors OCT3\4 and SOX-2. As shown here, three germ layer teratomas were also generated. These results indicate these cells are totipotent and that the method for establishing them is feasible. MSCs were generated from ESCs. With continuous passage, the cells began to form fibroblast colo- nies. MSCs can proliferate indefinitely while retaining their potential for differentiation, as shown by the identification of totipotency factors. Indeed, MSCs were totipotent and could differentiate into osteoblasts, chondrocytes and adipocytes after stimulation with a specific inducer. MSCs can therefore be regarded as seed cells for tissue engineer- ing. The relatively controllable differentiation may provide a new approach for adipose tissue engineering.
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Dental Press J. Orthod.  vol.16 número6 en a17v16n6

Dental Press J. Orthod. vol.16 número6 en a17v16n6

Introduction: Stem cells (SCs) are capable of inducing tissue regeneration and are, there- fore, potentially therapeutic. Similarly to bone marrow and umbilical cords, dental pulp is one of the available sources of SCs. The fact that these cells are easily accessible and that deciduous teeth are not vital organs, and are normally discarded after exfoliation, make them particularly attractive for use in safety and viability tests. Objective: To de- scribe the collection, isolation and culture of SCs obtained from the pulp of deciduous teeth as well as their characterization by flow cytometry, and the induction of differen- tiation into osteogenic and adipogenic lineages. Methods: SCs were obtained in a rela- tively straightforward manner and showed good proliferative capacity, even from a small amount of pulp tissue. Results: Analysis by flow cytometry confirmed the characteristics of mesenchymal SCs with low expression of CD34 and CD45 antigens, which are mark- ers for hematopoietic cells, and high levels of expression of CD105, CD166, CD90 and CD73 antigens, which are markers for mesenchymal SCs. Cell plasticity was confirmed by identifying calcium deposits in cultures that received osteogenic medium, and in- tracellular lipid accumulation in adipogenic cultures that received adipogenic medium. Conclusions: SCs collected from deciduous teeth show promising potential for applica- tion in tissue regeneration. Therefore, it is important that knowledge about the existence and characteristics of this source of stem cells be disseminated among dentists and that the technique, its limitations and possible indications are highlighted and discussed. Abstract
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Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells.

Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells.

Cells were transferred to gelatin-coated slides containing differentiation medium, allowed to attach overnight, and then fixed with 4% paraformaldehyde (Electron Microscopy Sciences, http://www.emsdiasum.com/microscopy) for 15 min at room temperature. Cells were blocked with 1% (w/v) BSA and stained for 1 h with anti-human primary antibodies specific for smooth muscle a-actin (a-SMA, 1A4, Dako, http://www.dako.com/), smooth muscle myosin heavy chain (SM-MHC, SMMS-1, Dako) and calponin (CALP, Calponin1, Santa Cruz Biotec, http://www. scbt.com/). In each immunofluorescence experiment, an isotype- matched IgG control was used. Binding of primary antibodies to specific cells was detected with anti-mouse IgG Cy3 conjugate or anti-mouse IgG FITC (both form Sigma). Cell nuclei were stained with 4 9, 69-diamidino-2-phenylindole (DAPI) (Sigma) and the slides examined with either a Zeiss fluorescence microscope or Zeiss LSM 50 confocal microscope.
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Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulat- ed by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down- regulate the surface expression of NKp46 on primary murine NK cells following antibody in- jection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activ- ity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose strepto- zotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15- treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.
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Isolation and characterization of a novel strain of mesenchymal stem cells from mouse umbilical cord: potential application in cell-based therapy.

Isolation and characterization of a novel strain of mesenchymal stem cells from mouse umbilical cord: potential application in cell-based therapy.

culture dishes at each passage. Single cell colonies originating from mUC-MSCs were also established using single cell per well dilution in a 96-well plate and microscopically monitoring of growth of each single cell-derived colony was performed daily. The cell from a single colony could be further serially transferred, expanded, and grown to confluence on 1β-well, 6- well, and 100-mm culture dishes. The culture medium used for the growth of mUC-MSCs was modified from that used in a previous study [47]. The medium consisted of low serum and 56% DMEM-LG, γ7% MCDB-β01 (Sigma-Aldrich, St. Louis, MO, USA), with 1× insulin-transferrin-selenium (ITS; Gibco, Life Technologies, Carlsbad, CA, USA), 1× linoleic acid-bovine serum albumin (LA-BSA; Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM ascorbic acid β-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 1 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 100 U Penicillin G and 1000 U streptomycin, β% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 15 ng/mL epidermal growth factor (EGF), 15 ng/mL platelet derived growth factor (PDGF)-BB (both from R&D Systems, Minneapolis, MN, USA) and 10 ng/mL leukemia inhibitory factor (LIF; Millipore, Temecula, CA, USA). mUC- MSCs at passage numbers 1β-β0 were used for characterization and therapeutic treatment.
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Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles.

Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles.

these hESC lines indicating that only minor differences exist. These include three DNA binding proteins with potential role in regulation of gene transcription: ZNF248, ZNF558 and SOHLH2 and proteins with potential membrane functions: MAGEE1, LGALS14 and CLC (Charcot-Leyden crystal protein/Galectn- 10). ZNF248 is a Krueppel C2H2-type zinc-finger DNA binding protein with potential role in transcriptional regulation [16]. ZNF558 is zinc finger DNA binding protein that also binds to Rrp46 which is an exosome subunit and mRNA splicing/ Figure 3. Blastomere-derived hESC lines, SAB-113B and SAB-113D, can be differentiated into derivatives of all three germ layers. In vitro differentiation of SAB-113B (A) and SAB-113D (B) into all 3 germ layers was confirmed by immunocytochemical analysis. aFP – a fetoprotein (endoderm), b3T – bIII-tubulin (ectoderm), SMA – smooth muscle actin (mesoderm). The expression of derivatives of mesodermal – T (Brachyury homolog), WT1 (Wilms tumor 1) and ACTA2 (smooth muscle actin, a2); endodermal – GATA4 (GATA binding protein 4), SOX17 (sex determining region Y- box 17) and AFP (a-fetoprotein); and ectodermal – TUBB3 (tubulin, b3), NES (nestin) and PAX6 (paired box 6) genes were also confirmed by RT-PCR analysis (C). In vivo differentiation was confirmed by histopathological analysis of teratoma tissue obtained 3 months post-initiation under the dorsal flank of immunocompromised NOD/ SCID mice (D). Images of representative areas of H & E stained histological sections of the tumors: derivatives of endoderm – epithelial tissue (Ep) (i); mesoderm – bone tissue fragment (B) (ii) and early cartilage tissue (C) (iii); and ectoderm – neuroectodermal tissue (NE) (iv) and cells with melanin granules (red arrowheads) (iv, inset).
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Stromal cells in the hematopoietic-stem-cell niche

Stromal cells in the hematopoietic-stem-cell niche

Human MSC (hMSC) are characterized by a low ex- pression of major histocompatibility complex (MHC) class I and the absence of costimulatory molecules such as CD80, CD86 or CD40 (12). Moreover, hMSC fail to induce proliferation of allogeneic or xenogeneic lymphocytes. These characteristics support the possibility of exploiting universal donor MSC for therapeutic applications. MSC constitutively express low levels of MHC-I molecules while, as a general rule, they do not constitutively express MHC class II molecules (13). However, recent evidence indicates that MSC can function as antigen-presenting cells and activate immune responses under appropriate conditions (14). Although one study reported constitutive MHC class II expression on MSC (15), several groups reported that both MHC class I and class II molecules are up-regulated following interferon-γ (IFN-γ) treatment, thus inducing a T-cell response to recall antigens (13,14,16).
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J. vasc. bras.  vol.4 número4

J. vasc. bras. vol.4 número4

Precursor endothelial cells, originating in the bone marrow, could be identified as CD34+ and VEGFR2+, although other markers such as AC133+ and CD31+ have been described. During the tissue ischemia, with a drop in the oxygen levels, there is an increase in the production of HIF-1, which in its turn will unleash the increase of several growth factors, notably VEGF. Asahara et al. isolated putative endothelial cell progenitors from peripheral blood by magnetic bead selection on the basis of cell surface CD34+ antigen expression that became spindle-shaped endothelial cells and proliferated for 4 weeks. 7 Bone marrow contains pluripotent CD34+ cells, which are known to give rise to hematopoietic cells. In vitro studies show that they can differentiate into mature endothelial cells. 7,8 A study demonstrated in the dog that CD34+ cells seeded into grafts could enhance vascular graft endothelialization and vessel formation. 9
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UNIVERSIDADE FEDERAL DE SÃO CARLOS CENTRO DE CIÊNCIAS BIOLÓGICAS E DA SAÚDE PROGRAMA DE PÓS–GRADUAÇÃO INTERINSTITUICIONAL EM CIÊNCIAS BIOLÓGICAS

UNIVERSIDADE FEDERAL DE SÃO CARLOS CENTRO DE CIÊNCIAS BIOLÓGICAS E DA SAÚDE PROGRAMA DE PÓS–GRADUAÇÃO INTERINSTITUICIONAL EM CIÊNCIAS BIOLÓGICAS

133. Bakkar N, Wang J, Ladner KJ, Wang H, Dahlman JM, Carathers M, Acharyya S, Rudnicki MA, Hollenbach AD, Guttridge DC. IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis. J Cell Biol, 2008. (180):787 –802. [PubMed: 18299349]. 134. Pan HC, Cheng F, Chen CJ. Post-injury regeneration in rat sciatic nerve facilitated

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Mesenchymal stem cells secretome in Parkinson’s disease regenerative medicine

Mesenchymal stem cells secretome in Parkinson’s disease regenerative medicine

abundant one (Benskey et al., 2016). The precise reason why LBs form and its role in pathogenesis of PD remains unclear (Dickson et al., 2009). However, in recent years, it has become clear that the initial sites displaying LBs are the dorsal motor nucleus of the vagus in the brainstem and the olfactory bulb (which is defined as stage I). This staging concept was firstly proposed by Braak and colleagues (Braak et al., 2003a), demonstrating that the disease most likely progresses in an upward direction via the pons (stage II) to the midbrain (stage III), followed by the basal prosencephalon and mesocortex (stage IV), and eventually reaching the temporal cortex and neocortex (stages V and VI) (Braak et al., 2003b). Therefore, it is only in stage III, when DAergic neuronal death exceeds a critical threshold (i.e. 70-80% of striatal nerve terminals and 50-60% of SNpc perikarya) that motor features of PD become evident, which means, that there is a substantive pre-symptomatic period of the disease that is hidden due to the existence of possible compensatory mechanisms (Bezard et al., 1999; Navntoft and Dreyer, 2016). Indeed, Zigmond and colleagues (Zigmond et al., 1990) proposed a model of compensatory changes, showing that the relationship between DAergic neuronal loss and functional impairments results from adaptive neurochemical changes that occur within the striatum. Recently, this dogma has been challenged, and several reports have shown that the classically accepted dopamine-mediated mechanisms are not the primarily involved in the initial compensation of DA depletion in PD, proposing a series of functional compensatory changes within and outside of the basal ganglia (Bezard et al., 2003; Obeso et al., 2004). In fact, it has been shown that the activation of the subthalamic nucleus (STN) increases the activity of SNpc DAergic neurons. Thus, the loss of DAergic projections and consequent decrease in DA concentration leads to an hyperactivity of the STN before the onset of functional changes in the putamen, suggesting that STN is implicated in the compensatory mechanisms in the initial phases of PD (Bezard et al., 1999; Hamani et al., 2004; Vila et al., 2000). Nevertheless, the precise nature of these compensatory mechanisms, and the reason for their ultimate failure has still been elusive.
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Treatment of critical defects produced in calvaria of mice with mesenchymal stem cells

Treatment of critical defects produced in calvaria of mice with mesenchymal stem cells

Mesenchymal stem cells (MSC) are present in specialized niches in perivascular regions of adult tissues and are able to differentiate into various cell types, such as those committed to repairing. Bone marrow derived MSC from eight young mice C57BL/ 6 gfp + were expanded in culture for repairing critical defects in calvarial bone produced in twenty-four young isogenic adult C57BL/6 mice. The animals were subjected to a cranial defect of 6.0mm diameter and divided into two equal experimental groups. Control group did not receive any treatment and the treated group received a MSC pellet containing 1.0 x 10 7 cells/mL into the defects. The group treated with MSC showed increased angiogenesis and amount of new bone deposited on the defect limits than that observed in the control group. The results demonstrated that transplantation of bone marrow-derived MSC of C57BL/6 gfp + mice to bone critical defects produced in mice calvarial contributes positively to the bone repair process. MSC presets ability to influence the correct functioning of osteoblasts, increases the amount of mobilized cells for the repairing process, speeds up growth, and increases deposition of bone matrix.
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Dissertação para obtenção do Grau de Doutor em Bioengenharia Orientador: Paula Marques Alves, PhD, ITQB-UNL Co-orientador: Manuel Carrondo, Professor, FCT-UNL Co-orientador: Daniel Wang, Professor, MIT

Dissertação para obtenção do Grau de Doutor em Bioengenharia Orientador: Paula Marques Alves, PhD, ITQB-UNL Co-orientador: Manuel Carrondo, Professor, FCT-UNL Co-orientador: Daniel Wang, Professor, MIT

levels of fetal the CYP450 (53, 54) the HepaRG cell line holds promise for hepatic drug induction tests. This latter hepatoma cell line was shown to express nuclear receptors and CYP450 transcripts at levels comparable with primary human hepatocytes (55) and its CYP450 activity induction has been proven for the CYP450 isoforms 1A2, 2C9 and 3A4, among others, as well as the presence of phase II and III drug metabolizing enzymes (57, 60); such results make this cell line an alternative to larger throughput drug metabolism studies, since it can proliferate up to several passages. The main disadvantages of using the HepaRG cell line are its clonal origin (it originated from a hepatocellular carcinoma from one single individual (55)) and the co-culture nature of this system, since HepaRG cell culture contain both hepatocytes and cholangiocytes, making it difficult to quantify the metabolic activity per hepatocyte. The former clonality issue limits the assessment of the naturally occurring inter-individual variability in CYP450 metabolism (42, 61) when using the HepaRG model. For a mid to long term perspective, the differentiation of human pluripotent stem cell lines to hepatocytes is a promising strategy which can potentially deliver an unlimited number of terminally differentiated hepatocytes. Hepatic differentiation protocols have already been established (62, 63) and adapted to large scale culture (64), but the obtained hESC derived hepatocyte-like cells have a strong fetal liver phenotype, namely the expression of Alfa-Fetoprotein and CYP7A1. Further maturation of these cells has been a subject of several publications (65-69) showing the expression of mature mRNAs and protein, such as CYP3A4 (70), thus proving the potential of pluripotent stem cell hepatic differentiation for providing hepatocytes for drug development tests.
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Biomimetic synthetic self-assembled hydrogels for cell transplantation

Biomimetic synthetic self-assembled hydrogels for cell transplantation

work of Stupp and co-workers resulted in the proposal of a broad range of PAs comprising a hydrophilic sequence covalently linked to an alkyl chain [94]. These molecules are typically composed by four distinct domains (Fig. 2C): (1) short hydrophobic tail responsible to drive the self-assembly process; (2) β-sheet peptide domain, which, through the formation of hydrogen bonds, promotes the cohesion of the formed nanofibers; (3) charged residues to confer water solubility; and (4) bioactive domain to mediate a specific biological response. Their self-assembly into long and stable nanofibers (Fig. 2C) is driven by the hydrophobic tail and occurs under specific pH, temperature or ionic strength conditions [95]. The resulting structures further undergo a sol-to-gel transition under physiological conditions in the presence of multivalent ions, which allows injection and in situ gel formation [64]. As result of the self-assembly process, the peptide sequence will be orientated towards the aqueous environment. This, in combination with the versatility and modular nature of PAs, allows the easy incorporation of bioactive sequences at the extremity of the peptide domain (Fig. 2C) and the development of bioactive hydrogels. As an example, PAs functionalized with the IKVAV epitope from laminin, revealed higher ability to promote NSC adhesion, migration and differentiation into a neuronal phenotype, as well as neurite outgrowth, when compared to laminin-coated surfaces [67]. These promising outcomes were associated to the high density of IKVAV epitopes present on the nanofibers surface [67]. Therefore, both the epitope density and their presentation on the nanofiber surface were found to contribute to such a pronounced biological response. Also in the context of nerve regeneration, a hydrogel based on a PA engineered with a heparin sulfate mimetic and an IKVAV epitope was developed [96]. The two bioactive moieties have demonstrated a cooperative effect in the promotion of PC12 neuronal-like cells neurite outgrowth, even when in presence of inhibitory components, such as chondrotin sulfate proteoglycans [96]. For application in bone regenerative therapies, a hydrogel was engineered with BMP-receptor binding peptides, termed osteopromotive domains [97]. The developed hydrogels showed ability to promote cell survival and osteoblastic differentiation of human bone marrow stromal cells (BMSCs) [97].
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