Treatment for symptomatic atherosclerosis is being carried out by balloon mediated angioplasty, with or without stent implantation, more and more frequently. Although advances with the development of drug eluting stents have improved prognosis, restenosis is still the most limiting factor for this treatment modality. Urotensin-II (UII), a small pleiotropic vasoactive peptide is increasingly being recognized as a contributory factor in cardiovascular diseases. We qualitatively evaluated UII immunoreactivity (IR) in three models of balloon angioplasty mediated restenosis. Specifically, we performed balloon angioplasty in the ilio-femoral arteries of New Zealand White Rabbits (NZWR) fed either a normal chow or high fat diet. In addition, UIIIR was also assessed in stent implanted abdominal aortae of NZWR fed a high fat diet. UII was constitutively expressed in the endothelium of all arterial segments evaluated. Abundant expression of UII was associated with lesion progression, particularly in myointimal cells, and less so in medial smooth muscle cells (SMC). The strongest UII-IR was observed in foam cells of animals fed a high fat diet. We demonstrate abundant expression of UII in regenerating endothelial cells and myointimal cells in vascular lesions following balloon mediated angioplasty and stent implantation in both animals fed a normal chow and high fat diet. Percutaneous coronary angioplasty, a modality for the treatment of atherosclerosis, is being employed more frequently and in more difficult cases every day. Although this procedure has been dramatically improved with the use of stents, especially drug eluting stents, restenosis is still the most limiting factor to its ultimate success. Indeed, although stent implantation reduces the risk of restenosis by 10 folds compared with balloon angioplasty alone . RestenoHuman UII, a potent vasoactive cyclic undecapeptide, has recently been the focus of a number of cardiovascular studies. UII is a vasoactive factor inducing vasoconstriction of some arteries such as the rat aorta, while inducing endothelial mediated vasodilation of other vessels -6. UII iet al. (2004) whom also demonstrated UII immunoreactivity in coronary atherosclerosis an in failed saphenous veins  (age 3 months) weighing 2-4 kg were studied . Animals were divided into the following groups: normal chow diet fed rabbits following balloon angioplasty of iliofemoral arteries, high fat diet fed rabbits following balloon angioplasty of iliofemoral arteries, and rabbits on high fat diet with stented abdominal aorta.All animal work was performed in accordance with institutional guidelines, and in compliance with the guide for the care and use of laboratory animals, published by the national institutes of Health (NIH publication 85-23 revised 85). Male NZWR were housed individually and fed a normal chow or high fat /cholesterol diet containing 2.5% peanut oil and 0.5% cholesterol  ~125 g/day. The animals were adjusted to the high fat diet over 7 days. After 2 weeks on high fat diet, balloon angioplasty (inflation and drag 3X) was performed in the iliac portion of the left femoral artery (Day T0).Following induction of general anesthesia, the animal was placed in dorsal recumbency and its abdominal and ventral femoral areas were shaved. The animal was scrubbed and draped for aseptic surgery, and the drape was cut to expose a muscular branch of the left femoral artery. A 2 cm incision was created next to the muscular branch, and the muscular artery was isolated by blunt dissection. The distal end of the vessel was ligated using 6-0 prolene and a loosely knotted 6-0-prolene suture was placed proximal to the introduction site of the catheter.For balloon angioplasty of the ilio-femoral arteries, a moistened 3.0 french Fogarty balloon catheter (Baxter) was evacuated of air and filled with 0.2 ml of saline. The balloon was checked for leaks and then deflated. Using a sufficient amount of saline to keep the catheter and introduction site moist, the vessel was cut and the catheter introduced into the artery. The balloon catheter was inserted 8-9 cm and pulled back, with a twisting motion, approximately 7-8 cm. To create adequate tension within the vessel, the balloon was inflated 0.09-0.11 ml while being pulled through the artery. This denuding procedure was repeated three times. The muscular artery was ligated as the catheter was withdrawn. The incision site was rinsed with saline and the sub-dermal layer closed using a continuous pattern with 6-0 prolene. Interrupted sutures of 4-0 prolene were used for dermal closure. The contralateral artery from each animal was used as naïve arteries.For abdominal aortic, stenting a sterile stent  was inserted into the end of a section of PE50 tubing. A metal stylet was inserted into the opposite end of the tubing and advanced to the stent. The stent and catheter were moistened with saline and the upper end of the catheter and stylet clamped with mosquito forceps. The femoral artery was cut and the stent-catheter introduced 10 cm into the abdominal aorta. The proximal tie was tightened, the mosquito forceps were released, and the stent was deployed by gently pushing the stent out of the catheter with the stylet. Once the stent is deployed, the catheter was removed and the femoral artery branch was ligated. The incision site was rinsed with saline and the sub-dermal layer closed using a continuous pattern with 6-0 prolene. Interrupted sutures of 4-0 prolene were used for dermal closure. Vet-bond was applied to the closed incision if necessary immediately following surgery; ultrasound was done on each rabbit to confirm proper deployment of the stent in the distal abdominal aorta.Immunohistochemical staining for UII and UT was performed using the avidin–biotin peroxidase method as previously described . MonocloParaffin sections (5μm) were immunostained with a monoclonal antiserum to human U-II. Sections were counterstained with hematoxylin. Negative control sections included immunoabsorption of the antisera with their own antigens, and the use of non-immune serum in place of the primary antibody.The naïve/control arteries showed moderate expression of UII in the vascular endothelial cells, while expression in the media was weak to moderate Fig . One dayIn naïve arteries of animals 7 days post-angioplasty, medial SMCs showed strong UII IR in addition to the prominent EC staining Fig . At 7 daAt 14 days post-angioplasty, naïve arteries demonstrated relatively strong endothelial cell IR while medial SMC staining was moderate Fig . InjuredOne day post-angioplasty the naïve artery demonstrated moderate UII-IR in both the endothelial and medial SMC cells Fig . UII-IR In concordance with observations in arterial segments of animals on normal chow diet, animals on high fat diet exhibited elevated UII expression in both naïve and injured arteries seven days post-angioplasty. Naïve arteries demonstrated moderate to strong UII-IR in the tunica media, while the endothelium demonstrated relatively strong UII-IR Fig . The injAt 14 days post angioplasty, naïve arteries exhibited moderate to strong UII-IR in both the endothelium and in the media Fig . ContralAt 28 days post-angioplasty, Naïve arteries exhibited moderate endothelial and weak medial IR Fig . A relatSections of NZWR aorta on high-fat diet one week following stenting exhibited moderate expression of U-II in endothelial cells while moderate and diffuse UII expression was observed in the thrombus/lesion surrounding the stent Fig . These eAt two weeks, U-II expression remained strong in endothelial cells while it increased within lesions. In fact, both myointimal and foam cells showed strong UII-IR in these animals Fig  & D. IntAt 4 weeks post-stenting there was an evident increase in lesion size/number Fig . The endIn the present study, we qualitatively characterized UII protein immunoreactivity in NZWR on normal chow and high fat diets following angioplasty in the ilio-femoral artery. Furthermore, we also qualitatively assessed UII immunoreactivity in NZWR on a high fat diet following abdominal aortic stenting. Therefore, the present study aimed at evaluating UII expression in a qualitative fashion, across a broad spectrum of vasculopathies, at both early and late time points after percutaneous interventions. Although the study included a large number of animals, the numerous study groups allowed for only an n=4 animals per group which was not appropriate for accurate quantifications. The results showed that there was nominal U-II expression in the endothelium of naïve arteries, while the expression of the peptide in the vascular endothelium and underlying cells of the thickened intima intensified with the progression of the lesion. In addition, UII expression was more prominent in myointimal cells of vascular lesions than in medial SMCs, but was strongest in foam cells of animals fed a high fat diet. Indeed, UII immunoreactivity was more pronounced in arteries from animals fed a high fat diet in large part due to the strong foam cell staining which was absent in animals fed a normal chow diet.There are several key points that can be appreciated from the qualitative demonstration of UII-IR following angioplasty. Firstly, UII was consistently expressed in the endothelium of both ilio-femoral arteries and abdominal aorta of animals on either normal chow or high fat diet, indicating that the endothelium constitutively expresses UII. This is in agreement with our previous report which showed UII immunoreactivity in normal and diseased human aorta .In the acute setting, that is 1 day following angioplasty, UII immunoreactivity was strong just below the internal elastic lamina in angioplasty injured arteries , suggesting a direct effect of the injury response on UII expression. Interestingly, this sub-laminal UII expression had dissipated after three days. However, at 7 days post angioplasty, UII-IR was very strong in injured arteries demonstrating that the stimulus for UII expression is biphasic with an acute up-regulation (noted on day 1) and a chronic phase (noted from day 7-28). In addition, it was interesting to note that that UII-IR was also elevated in naïve arteries (7-days post-angioplasty), which suggests that a circulating humoral factor, induced by arterial injury, has a systemic effect on UII expression in the vasculature.Whether the SMCs involved in lesion formation are dedifferentiated vascular SMCs or are derived from phenotypically abberant SMCs is still a matter of serious debate. However, there is strong evidence that there is in fact a heterogeneous pool of SMCs resident in the media . This heThe prominent UII immunoreactivity associated with myointimal cells and medial SMCs is interesting in light of several studies demonstrating UII as a SMC mitogen , 18. Theet al. which demonstrated that UII induced foam cell formation by increasing ACAT activity and expression (In animals fed a high fat diet it was apparent that UII expression was strongest in foam cells. This was evident in both ilio-femoral arteries and abdominal aorta of animals on the high fat diet but was expectedly absent in those animals on a normal chow diet. This is especially interesting in light of a recent study by Watanabe pression . FurtherIn summary, we show that UII expression is associated with endothelial, myointimal, and foam cells of restenotic lesions in both normo- and hyperlipidemic rabbits following either balloon angioplasty or stent-mediated restenosis. Future studies using a selective UT receptor antagonist like the one we have previously used, or deletion and/or overexpression the UII and UT genes would determine the exact role the urotensin system plays in these disease modalities.


C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35fl allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35∆/∆ organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35∆/∆ organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.In  The mammalian intestinal epithelium is a rapidly self-renewing tissue. Stem cells endow the intestine with its proliferative capacity. Intestinal stem cells reside at the bottom of invaginations of the intestinal epithelium; the crypts of Lieberkühn. The intestinal stem cells are characterized by expression of Lgr5 , they arWnt signaling in intestinal stem cells is activated by Wnt ligands that are expressed in the Paneth cells and cells in the intestinal mesenchyme . Wnt sigC. elegans, Drosophila and mammalian tissue-culture cells have shown that Wls needs to be retrieved back to the trans-Golgi network (TGN) to maintain Wnt secretion. This retrieval route involves AP-2 and clathrin mediated endocytosis of Wls from the plasma membrane [Detailed knowledge has accumulated about the mechanism of Wnt signal transduction in Wnt receiving cells, but the mechanism of Wnt secretion has only recently been uncovered , Sortilin, the polarity protein Crumbs and Wls . Mice were sacrificed 3 days, 1 week, 4 weeks and 8 weeks after Cre induction. Histology and immunohistochemistry was performed as described in [Conditional Vps35 mice were generated by homologous recombination in embryonic stem cells using a targeting construct that is schematically depicted in 2 strain . Recombiribed in . The aniVps35fl/fl;Villin-CreERT2 mouse as described in [Vps35 deletion, 4-OHT  was added to the culture medium for 12 hours. Organoid growth was quantified by scoring the number of buds that had developed 5 days after passaging. Mouse organoids were derived from isolated crypts of the proximal small intestine of a ribed in . The orgribed in . The ENROrganoids were dissolved in TRIzol (Life technologies), RNA was isolated from organoids using an RNAeasy kit (Qiagen) and cDNA was generated using the RT-II kit (Invitrogen) using oligo dT primers. Primers sequences used for RT-PCR: CTGTTGGCTCTCCTTCATCAG; AACTGCACTACTTGGAGGTC, Vps35: GTACACAGACCATCAGCGGC; CCACCCCATCCAGTCTCACT, Cdx2: TGCCATCTGCTTACCAGTGTTGT; ATTCCGTCTTCCCACCACGC, Lgr5: GAGACCGAAGCACCGACTATG; CGGTTTTGACATTGTGTTCGC, Lysozyme: GAACGGGGCCATGGTCAGCA; CATAATTGGTCTGCATGCC.Mucin2: qPCR was performed using the iQ SYBR green reagent in a MiIQ real-time PCR system (Biorad). Relative expression was calculated using the ΔΔCt method relative to Ywhaz expression. Primer sequences used for qPCR: TGACTCTCCTTCCAGATCCCA; TGCCCACACTAGGCTGACA, Ywhaz: TGCAACGATCTACTGTCTCTTTTG; CGGTAGTAGTCACCCTTCATTTTCA.Axin2: Organoids were washed twice in ice cold PBS and taken up in Laemli sample buffer. Samples were boiled for 5 minutes prior to SDS-PAGE and Western blotting following standard procedures. The following antibodies were used for detection: anti-Wls , anti-Vps35 , anti-alpha-Tubulin , anti-mouse-HRP , anti-chicken-HRP (Abcam), anti-rabbit-HRP .Vps35fl allele to inducibly delete Vps35 in the intestinal epithelium of 4 week old mice. We introduced loxP sites in the third and fourth intron of Vps35 by homologous recombination in mouse embryonic stem cells. We crossed the Vps35fl mice to animals carrying a CreERT2 transgene driven by the Villin promotor  injections. We confirmed that recombination occurred in the intestinal epithelium by PCR on genomic DNA obtained from intestinal epithelial cells  stem cell and consist solely of epithelial cells. Organoids form crypt-like buds that contain stem cells and Paneth cells as well as villus-like domains that contain differentiated cells [in vitro by addition of 4-OHT to the culture medium. Using PCR analysis, we found that the recombination of the Vps35fl/fl allele was complete  in standard intestinal organoid culture medium that contains EGF, Noggin and R-spondin (ENR medium). In contrast, deletion of Wnt3 from intestinal organoids causes a characteristic ‘pointy crypt’ phenotype and a loss of Paneth cells in the buds [Wnt3∆/∆ organoids cannot be maintained in regular ENR medium, but need Wnt supplemented in the medium for continuous culturing [We did not observe any major morphological changes in the phenotype of the the buds  and rever Mucin2 . We werethe buds . Furtherulturing . We concVps35∆/∆ organoids and compared it to control organoids. We categorized the organoids based on the number of crypt-like buds that had developed five days after passaging and used this as a proxy for the growth rate , so we could not confirm if Lgr5 is a retromer cargo in intestinal cells.The subtle growth defect that we observed could not be fully rescued by supplementation of Wnt3a in the culture medium. Missorting of other retromer cargos such as the CI-MPR, Sortilin or the polarity protein Crumbs, may negatively influence organoid growth . It was Vps35∆/∆ mice have defects in recovery from injury, or if deletion of Vps35 during development of the intestine causes defects.In our experimental approach, we investigated the role of Vps35 in Wnt signaling and stem cell maintenance in intestinal homeostasis of juvenile and adult mice. In these conditions, retromer mediated recycling of Wls is dispensable. There may be situations, for example during embryonic development or during regeneration after injury, which require enhanced levels of Wnt secretion, and may therefore be more dependent on retromer mediated retrograde transport of Wls. For example, Wnt5a signaling is essential for intestinal tube elongation during development and regeneration of the colon after tissue injury ,31. It wVps35. The mouse strain that carries the floxed Vps35 allele will be a valuable tool to study retromer function during development and regeneration of the intestine as well as in other tissues and in different biological contexts.In summary, we show that the retromer complex is required to maintain high Wls protein levels in intestinal epithelial cells, which is in agreement with the current model of Wls trafficking. Proliferation and the maintenance of stem cells in organoids are however minimally affected by loss of


The symposium, which directly continues the successful Magdeburg meeting series, attracted over 330 colleagues from 29 countries to discuss recent findings and advances in the field. The focus of the 2012 symposium was widened from stroke and traumatic brain injury to neurodegenerative diseases, notably dementia, and more generally the ageing brain. Thereby, emphasis was given on neurovascular aspects of neurodegeneration and stroke including the blood–brain barrier, recent findings regarding the pathomechanism of Alzheimer’s disease, and brain imaging approaches. In addition, neurobiochemical aspects of neuroprotection, the role of astrogliosis, the clinical progress of cell-based approaches as well as translational hurdles and opportunities were discussed in-depth. This review summarizes some of the most stimulating discussions and reports from the meeting.The 7 Vascular disorders of the human brain are a common precondition and a relevant factor of influence for some of the most devastating neurological conditions like stroke and dementia – globally burdening patients, families, and the health care systems. Significant advances have been made regarding our understanding of central pathophysiological aspects of these diseases, as well as possible strategies to counter them. However, we yet have been unable to translate hopeful experimental findings into the clinics to augment the existing, but limited therapeutic options. In the context of ageing industrialized societies with an increasing prevalence of important risk factors like diabetes and arterial hypertension as well as the limited resources for health care worldwide, diseases affecting the cerebral vasculature are one of the most relevant global health problems. Thus, there is an increasing and still unmet need for an even better understanding of stroke and dementia in order to derive novel therapeutic approaches that clearly have the potential to be of benefit for patients.th International Symposium on Neuroprotection and Neurorepair (7th ISN&N) was held from May 2nd to May 5th at the Templin Lake in Potsdam, Germany. In continuation of the Magdeburg meeting series and the 6th ISN&N [th ISN&N was to widen the scientific scope of the meeting series from stroke to dementia and more generally the ageing brain, primarily focusing on Alzheimer’s disease (AD) and vascular dementia. This paper reviews some of the most interesting findings being presented at the 7th ISN&N and gives a compact summary of selected scientific debates that emerged during the meeting.The 7th ISN&N , the meeHolger Braun  demonstrated the sequence of events during small vessel alterations in brains of spontaneously hypertensive stroke prone rats. Accumulations of erythrocytes were shown to be the initial step in the pathological cascade, which is followed by blood–brain barrier (BBB) impairments finally leading to microbleeds that cause necrotic tissue damage in terms of microinfarcts [Nozomi Nishimura  hypothesized that inflammation could play a role in such reductions in blood flow in AD by leukocyte plugging of capillaries. In vivo two-photon excited fluorescence microscopy was used to examine cortical blood flow in mouse models of AD. To determine whether or not individual brain microvessels are stalled, the movement of non-fluorescent blood cells within the dye-labeled blood plasma was monitored [Didier Leys  provided a review of stroke and dementia as consequences of an altered vasculature, which was followed by a lively round table discussion.Cerebral small vessel diseases are a major cause for chronic cerebral ischemia leading to cognitive decline and behavioral changes, but may also induce stroke-like symptoms if vessels in important brain regions are affected. infarcts . Interesonitored . TherebyGregory J. del Zoppo  presented an overview on the barrier structure within the NVU: Since previous research predominantly addressed inter-endothelial cell tight junction complexes, growing evidence indicated that also the adhesion between endothelial cells and the extracellular matrix (arranged amongst others by β1-integrin) contribute to BBB permeability [Berislav V. Zlokovic  stressed the NVU with emphasis on neurodegenerative processes, in particular AD. Thereby, a reduced clearance of β-amyloid peptides is assumed to emerge from BBB alterations, contributing to neuronal dysfunction and finally dementia [Dominik Michalski  focused on side effects of recombinant tissue plasminogen activator (rtPA), currently representing the best available drug-related treatment in acute stroke. Despite the hypothesis that detrimental actions could be attenuated by co-administered hyperbaric oxygen, recent findings failed to support this theoretically forced approach [Mariel G. Kozberg  introduced new approaches to characterize the NVU by in vivo imaging in rodents. Thereby, optical-based techniques were applied to provide information on blood flow, vessel dilations and rate of oxygenation in different types of vessels and the parenchyma. Two-photon microscopy was found to image neurovascular coupling by using intravenously applied agents for concomitant labeling of cells and vessels. Guido Stoll  compared different contrast agents for detecting BBB dysfunction in magnetic resonance imaging (MRI), whereas gadofluorine M was found to best characterize the size of injury after cortical ischemia in mice [In recent years, the BBB received increasing attraction in stroke and neurodegeneration, since the concept of a primary separating role (for instance leading to edema and hemorrhage when the BBB integrity is lost) was added by a functional perspective including inflammatory aspects ,5. Thereeability . This peeability . Berisladementia , thus redementia . Dominikapproach . However in mice . Further in mice , which w in mice . These dth ISN&N, Hans-Ulrich Demuth  emphasized that toxicity in AD is induced by pyroglutamate (PGLU)-Aβ, and furthermore, depends on tau protein. Demuth demonstrated that the cytotoxicity is propagated by a prion-like templating mechanism of Aβ misfolding initiated by PGLU-Aβ and that these PGLU-Aβ-containing low-molecular weight oligomers remain stable for days - in contrast to oligomers not formed by the PGLU-Aβ initiated mechanism [Jens Pahnke  reported that the BBB transporter ABCC1 has an important role in cerebral Aβ clearance and accumulation. Moreover, a pharmacological activation of ABC transporters can slow down the neurodegenerative cascade [While focusing on AD as a major topic of the 7echanism . Jens Pa cascade .th ISN&N focused on promising molecular targets and on mitochondrial processes. The role of a transcription factor family that is denoted as peroxisome proliferator-activated receptor (PPAR) was presented by several speakers. First, Douglas Feinstein  reported on the potential use of PPARγ-sparing thiazolidinediones for the treatment of neurodegenerative diseases. These substances, also known as glitazones, are a class of drugs already used in the treatment of diabetes mellitus type 2. Second, Michael Heneka  described PPARγ as a mediator of protection in AD, and third, Anne Murphy  dealt with the value of a preclinical systematic review to analyze mitochondria as targets of thiazolidinediones. In addition, Georg Reiser  concentrated on the role of mitochondria to describe a major principle for neuroprotection. His report covered the permeability transition in mitochondria [Raghu Vemuganti  reported that ubiquitin-like protein modifications are neuroprotective after stroke.Within the numerous efforts to develop novel or even to improve existing neurochemical approaches, topical sessions of the 7chondria  and therIn vivo imaging of structures and processes in living animals as disease models is commonly used to directly prove a stated experimental hypothesis. In the context of continuous technical advances in imaging techniques and analyzing tools, this research field qualifies to solve two major tasks: imaging of direct functional adaptation and changes in plasticity after brain injury, and further, to characterize the morphological structures on microarchitectural levels. During the meeting, the latest studies which focused on the development of MRI, positron emission tomography (PET) and live cell imaging for this purpose were discussed. Alan Koretsky’s group  recently used high field MRI to distinguish the arterial and venous hemodynamic response in individual intracortical vessels after whisker pad stimulation [Rick Dijkhuizen  brought new insides in the reorganization and functional recovery after stroke by comparing graph-based analyses of fMRI signals in gray matter, quantification of fractional anisotropy in white matter by diffusion tensor imaging (DTI) and functional recovery in relation to stroke severity [Jan Klohs  showed an age-dependent reduction in the number of functional intra-cortical microvessels in aged (24 months) arcAβ mice. This reduced density of transcortical vessels (with radii less than 20–80 μm) was attributed to an impaired perfusion and vascular occlusion caused by deposition of Aβ and fibrin [Henryk Barthel  representing a translational approach which impressively linked preclinical and clinical research [Marc Fisher  summarized clinical trials which used imaging criteria as a decision instrument for the application of recanalization therapies. It was shown that especially MRI is useful to identify subgroups which clearly benefit from a consecutive thrombolytic therapy, also at comparatively late stages.mulation . By usinseverity . The grod fibrin . The Floresearch . As an eRoland Veltkamp  emphasized the ‘Janus-faced’ nature of T cells in stroke development. While several groups could meanwhile confirm that CD4+ and CD8+ T cells act detrimental during the acute phase of stroke, the contribution of other T cell subsets, in particular regulatory T cells (Treg), remains controversial. In line with the description of a beneficial role of Treg for stroke recovery and repair, recently published by his group, Veltkamp now outlined a promising approach to pharmacologically enhance Treg immune functions in stroked mice. In contrast, the group of Christoph Kleinschnitz  failed to confirm a neuroprotective effect of Treg during the course of cerebral ischemia. By using a mouse model of selective FoxP3+ Treg ablation they could even identify Treg as key mediators of ischemic neurodegeneration – at least during the early phase after transient middle cerebral artery occlusion [Ulrich Dirnagl  took this controversy as a good example to stress the need for randomized, multicenter preclinical stroke trials. Further emphasis was placed on the role of polymorphonuclear leukocytes and microglia in stroke. Emmanuel Pinteaux  pointed out that central and peripheral inflammations represent key drivers of tissue damage in response to acute brain injury such as stroke, and are associated with poorer outcome in stroke patients. He demonstrated that interleukin (IL)-1 is primarily expressed by activated microglia, and acts on astrocytes to release MMP-9, known to contribute on neuronal cell death. IL-1 was also found to activate the brain endothelium leading to neutrophil infiltration into the brain and neuronal death, a mechanism that is associated with neutrophil MMP-9 activity and the degradation of the extracellular matrix [Jens Neumann  revealed an elevated slow rolling of neutrophil granulocytes on the endothelium followed by an infiltration of these cells into the brain. Contradicting data were shown by Britta Engelhardt , who provided evidence that neutrophil granulocytes do not enter the parenchyma but stay in close proximity to the vessels.The interesting role of the immune system in the pathophysiology of cerebral ischemia was highlighted in the session ‘Balancing Immune Responses in Neurodegeneration’. cclusion . As poter matrix . InvestiMichael V. Sofroniew  presented a comprehensive overview on the role of astrocytes in the normal and damaged CNS and particularly emphasized the morphological and functional heterogeneity of reactive astrogliosis. Beyond reactive astrogliosis and its implications, astrocytes might also contribute to CNS disorders either by a loss of normal, or a gain of detrimental functions. Sofroniew concluded that the understanding of these processes might uncover novel therapeutic targets. Magdalena Götz  highlighted the potential beneficial action of reactive astrocytes to develop neural stem cell properties beyond the classical CNS niches. These characteristics can be observed after different types of CNS injury and are principally regulated by Sonic hedgehog signaling. In vivo imaging of reactive astrogliosis using two-photon microscopy supported the observation of astrocytic heterogeneity: while many astrocytes remained unchanged after focal CNS damage, a subpopulation of astrocytes showed proliferation within the vascular niche. Milos Pekny  first introduced a novel three-dimensional culture system for astrocytes that largely preserved the complex cellular morphology and reduced the baseline activation which is a common drawback of conventional astrocyte cultures. Pekny further provided evidence for a differentiated role of reactive astrogliosis with beneficial effects in the context of AD and Batten disease, but a detrimental action during amyotrophic lateral sclerosis. Finally, he proposed that reactive astrogliosis and its consequences are disease-specific. Additionally, Karsten Ruscher  linked a common treatment of Parkinson’s disease with stroke while reporting on the beneficial effects of levodopa on functional recovery after stroke. As a conclusion, he hypothesized that peri-infarct astrocytes equipped with dopamine receptors may contribute to these effects.Reactive astrogliosis has long been identified as a simple scarring process in the central nervous system (CNS) that ultimately impairs neural regeneration. However, recent evidence challenges this paradigm, notably by combining genetic ablation of intermediary filaments with different models of CNS diseases. Hence, the scientific community is now reconsidering the unidirectional view on reactive astrocytes and is developing a more differentiated approach. Brigitte Onteniente  showed that intracerebrally administered human iPSC-derived neural progenitor cells differentiate into astrocytes, endothelial cells and GABAergic neurons after transient cerebral ischemia in the rat, ameliorating motor deficits and secondary neurodegeneration. Robert Mays, cofounder and Senior Director of Athersys Inc.  introduced the company’s multipotent adult progenitor cell (MAPC) program, which currently includes clinical phase II testing. MAPC were shown to improve functional recovery and to act neuroprotectively by promoting immune system homeostasis following experimental stroke. An open-label, single arm clinical phase I study using intravenous administration of autologous bone marrow mononuclear cells (BM MNC), which also have been thoroughly investigated in preclinical experiments, was conducted by Sean Savitz  and colleagues [7 BM MNC per kilogram bodyweight by iliac crest puncture as well as subsequent density gradient centrifugation and reinfusion of the cells within 72 hours following stroke onset were shown both feasible and safe in 10 patients. A clinical phase IIb study is actually being prepared, also assessing efficacy endpoints. Intraparenchymal stem cell administration is considered beneficial even in subacute and chronic stages after cerebral ischemia [Gary Steinberg  reported encouraging safety results from these trials. In summary, cell-based approaches have tremendously advanced during the last years now being under clinical evaluation. However, it is still unsure whether these approaches can overcome the translational roadblock and show efficacy in human stroke.For almost two decades, cell- and stem cell-based approaches are considered among the most promising options to treat degenerative diseases of the brain for which regenerative treatments have not been available so far. Even though cell-based therapies have not been proven beneficial in human patients, first data are available from clinical phase I and II trials. In parallel, more concepts emerged to late preclinical phases. Induced pluripotent stem cells (iPSC) were reported to have capacities comparable to those of embryonic stem cells, but can be generated from autologous, patient-specific somatic cells . Brigittlleagues . Obtainiischemia ,29. The Wolf-Rüdiger Schäbitz  reported on the most recent drug candidate that got stuck on its way into the clinic. Granulocyte colony-stimulating factor (G-CSF), although being carefully evaluated in experimental studies [Ulrich Dirnagl gave some very interesting examples for relevant results that were ‘found in translation’, thereby proving evidence that preclinical stroke research can be predictive. For example, the penumbra concept was first reported in primate studies and was reconstructed in rodent models while the time window for intravenous thrombolysis [Virtually all approaches to transfer experimental therapies for stroke and dementia into the clinics have failed so far, mainly due to a lack of efficacy and/or safety. While the reasons for this dilemma  are still a matter of debate, it appears likely that this roadblock will be hard to overcome . Wolf-Rü studies  and prov studies , recentlmbolysis  as well th ISN&N attracted more than 330 scientific colleagues from 29 countries on 6 continents to discuss recent findings and novel concepts in several types of brain injury. Personal comments of numerous attendees, a growing interest in the meeting’s topics indicated by steadily increasing numbers of participants as well as very stimulating discussions emerging throughout the meeting underlined the unbroken demand for a forum tailored to discuss our research in the addressed fields. Consequently, the 8th International Symposium on Neuroprotection and Neurorepair will take place from April 9th to April 12th, also going back to the roots of the meeting series in Magdeburg, Germany – were the series originally was started in 1998. The 8th ISN&N will strengthen the focus on pathophysiological aspects of the altered vasculature in the ischemia-affected and ageing brain.The 7AD, Alzheimer’s disease; BBB, Blood–brain barrier; BM MNC, Bone marrow mononuclear cells; BOLD, Blood oxygenation level dependent; CD, Cluster of differentiation; CNS, Central nervous system; DTI, Diffusion tensor imaging; DTPA, Diethylenetriamine penta-acetic acid; DZNE, Deutsches Zentrum für Neurodegenerative Erkrankungen; G-CSF, Granulocyte colony-stimulating factor; IL, Interleukin; iPSC, Induced pluripotent stem cells; IgG, Immunoglobulin G; ISN&N, International Symposium on Neuroprptection & Neurorepair; MAPC, Multipotent adult progenitor cell; MMP-9, Matrix metalloproteinase-9; MRI/fMRI,  magnetic resonance imaging; NVU, Neurovascular unit; PET, Positron emission tomography; PGLU, Pyroglutamate; PPAR, Peroxisome proliferator-activated receptor; rtPA, Recombinant tissue plasminogen activator; SPIO, Superparamagnetic iron oxide particle; Treg, Regulatory T cell; UK, United Kingdom; USA, United States of America.th ISN&N; GR, DM and CK were invited speakers to the conference. None of the authors has to disclaim any other, particularly commercial or financial competing interests.JB, KR, GR, DCW and AK were organizers of the 7th ISN&N.All authors contributed actively to the conception, writing and revision of the manuscript, and approved its final version. JB coordinated the writing process and was the conference president of the 7


COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells. Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the  Induced pluripotent stem cells (iPSCs) are stem cells generated from individual somatic cells by exogenous expression of several transcription factors to initiate the reprogramming process . In receCOL7A1 gene encoding type VII collagen, the major component of anchoring fibrils at the basement membrane zone (BMZ) at the epidermal-dermal junction of the skin. Since anchoring fibrils provide functional integrity to the skin, defective anchoring fibrils caused by lack or deficiency of type VII collagen leads to skin fragility. Therefore, patients with RDEB develop severe skin phenotypes, including repeated skin blistering from minor trauma, as well as mutilating scarring, alopecia, corneal erosions, tooth and nail dystrophy, esophageal strictures, joint contractures, and fusion of fingers and toes [Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering disorder caused by mutations in the and toes . RDEB paand toes . in vivo, several studies using mouse models and mouse/human fibroblasts have shown that dermal fibroblasts also synthesize type VII collagen, suggesting that both cell types are potentially useful as a source of cells for treating RDEB [There is currently no cure for RDEB. One promising strategy for RDEB treatment is to increase the amount of type VII collagen at the epidermal-dermal junction in patients with RDEB to restore skin integrity . Variousing RDEB –10. PrevWe previously reported the generation of PS-iPSCs from patients with RDEB and efficient derivation of functional keratinocytes from normal and RDEB PS-iPSCs, thereby demonstrating the utility of PS-iPSCs for establishing cell- and gene-based therapy for RDEB . In cont We first aimed to develop a differentiation protocol to generate dermal fibroblasts from iPSCs in vitro. In previous studies, ESCs have been differentiated into mesodermal lineages using several methods and reagents. Ascorbic acid is well known to induce mesodermal differentiation of ESCs ,13 and iBased on such studies, we generated embryoid bodies from iPSCs to initiate differentiation, and used medium supplemented with ascorbic acid and TGFβ2 to accelerate mesodermal differentiation to generate fibroblasts from iPSCs . As a re We next examined the characteristics of iPSC-derived cells to define their fibroblast-like properties. We confirmed by RT-PCR that stem cell markers, OCT4 and SOX2, had completely disappeared in iPSC-derived cells after differentiation. In addition, the expression of several markers indicative of mesodermal and fibroblast induction were strongly detected (CD73 and type I collagen) or increased ) in iPSC-derived cells . We comp Moreover, we confirmed by immunostaining that vimentin, a classic marker of the mesodermal lineage, and prolyl-4-hydroxylase beta (P4HB), another marker of fibroblasts, were expressed in iPSC-derived fibroblasts . P4H, co To further interrogate the utility of iPSC-derived fibroblasts for the treatment of RDEB, we next asked whether these cells produced and secreted mature type VII collagen, which would be required to reconstitute anchoring fibrils in the skin of patients with RDEB. Western blot analysis detected a 290kD band of type VII collagen in secreted media from iPSC-derived fibroblasts, as well as from normal human fibroblasts . The lowin vivo functional capacity of iPSC-derived fibroblasts. We found that iPSC-derived fibroblasts combined with normal human keratinocytes grafted in the chamber contributed to reconstitution of normal skin structures on the back of SCID mice, similar to normal human fibroblasts and keratinocytes , a novel tool for gene modification which generates double-strand breaks at specific sites in the human genome . Moreovein vitro 3D skin formation and terminal differentiation capability in 3D skin models [Although homologous recombination-based gene correction in PS-iPSCs from a skin disease has been not yet reported, there has recently been progress made in differentiation of iPSCs into skin cells. For example, our group previously demonstrated that human iPSCs could be differentiated into epidermal keratinocytes, one of main components of the skin . We confn models . These sin vivo [Here, we report the direct differentiation capability of iPSCs into fibroblasts, the second predominant cellular component of the skin. The cells have the capability to treat individuals with RDEB via their production of type VII collagen, and are also able to reconstitute full-thickness skin for regenerative therapy. In addition, fibroblasts may have another role in patients with RDEB in addition to merely supporting epidermal structure from the dermis. Although the mechanisms leading to the development of aggressive SSCs in RDEB patients is not well understood, loss of type VII collagen at the BMZ has been reported to increase the invasive behavior of RDEB-related SCCs . A recenin vivo , indicatPrevious studies have reported generation of fibroblasts from ESCs/iPSCs –38, howeHere, we focused on developing a method of specifically deriving fibroblasts from human iPSCs. Our protocol advances the generation of fibroblasts for future clinical use, because it can directly generate a relatively pure fibroblast population from iPSCs with high efficiency, based on cell surface marker analysis by FACS . There rType VII collagen matures via a polymerization process and the formation of tripeptide structures to generate collagen fibers. Therefore, using immunostaining and western blot analysis, we detected the tripeptide form of type VII collagen in medium cultured from fibroblasts derived from iPSCs by our protocol, demonstrating that our iPSC-derived fibroblasts had the capability to produce and secrete fully mature type VII collagen for the advancement of cell therapy for RDEB. in vivo function of iPSC-derived fibroblasts. Skin reconstitution chamber assays clearly showed that iPSC-derived fibroblasts can reconstruct human skin structures in vivo with normal human keratinocytes   in iPSM iPSC-derived keratinocytes used in this study were generated by a modification of our protocol as previously described . BrieflyRNA was extracted using the RNeasy Mini kit (Qiagen) and DNA was removed by DNase treatment (Invitrogen) to avoid genomic DNA amplification. Complementary DNA was synthesized using total 2µg RNA by Superscript III reverse transcriptase and Oligo(dT) primer (Invitrogen) according to the manufacturer's instructions. PCR reactions were performed with Platinum® PCR SuperMix (Invitrogen). All primer sequences are supplied in The differentiated spindle-shape cells were enzymatically collected and fixed with 4% PFA/PBS for 5 minutes at RT. After PBS wash, cells were blocked in PBS with 10% goat serum. Primary antibodies conjugated with fluorescent molecules  were inciPSC-derived fibroblasts were fixed with acetone/ethanol. After one hour of blocking using 10% goat or donkey serum in PBS, samples were incubated for one hour at room temperature (RT) or overnight at 4°C with primary antibodies. After three rinses with PBS, incubation with appropriate secondary antibodies was performed for 1 hour at RT. To detect human nuclear staining, frozen sections were fixed with 4% PFA in PBS, and performed blocking process using the M.O.M. immunodetection kit (Vector Laboratories) according to the manufacturer's instructions. Human nuclear antibody and secondary antibody were diluted with M.O.M. diluent and applied. Nuclear staining was performed by Vectashield®, mounting medium containing DAPI (Vector Laboratories). Confocal microscopy (Carl Zeiss LSM 5 Exciter) was used to visualize and capture immunostained cells with good resolution. All antibodies are supplied in  In order to collect secreted type VII collagen from FBs and iPSC-FBs, subconfluent cell cultures were fed for 48 hours with serum-free medium supplemented with 50 ug/ml ascorbic acid. For SDS-PAGE analysis, the culture medium was treated with Amicon Ultra-100,000 Centrifugal Filter Devices (Millipore) for concentration and desalting. The samples were separated on a 5% polyacrylamide gel under reducing conditions. Immunoblotting analysis was performed using a rabbit polyclonal antibody against Collagen Type VII  at 1:5,000 dilution. After overnight incubation at 4 degrees, the blot was washed with PBS with Tween 20 for 4 times, 15 min each at RT and then incubated with the goat anti-rabbit secondary antibody  and detected with the SuperSignal West Dura substrate following the manufacturer's instructions (Thermo Scientific). A sterile silicone chamber  was implanted into the back of SCID mice under anesthesia to prevent inward migration of cells from the adjacent skin. Ten million cells were prepared for each epidermal component  and dermal component , and mixed together followed by centrifugation. The resultant cell pellet, comprised of epidermal and dermal cells, was resuspended in PBS and deposited into the chamber. One week after placing cells, the silicone chamber was removed. Skin was harvested from the chamber sites 21 days later to observe reconstituted skin structures .  3D skin equivalents were generated according to a previously described protocol . BrieflyTable S1List of primers used in this study.(DOCX)Click here for additional data file.Table S2List of antibodies used in this study.(DOCX)Click here for additional data file.


The calpain family consists of twomajor isoforms, calpain I and calpain II which require µM and mM Ca2+ concentrationsto initiate their activity. An increase in intracellular Ca2+ level isthought to trigger a cascade of biochemical processes including calpain activation.Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates,leading to the breakdown of cellular architecture and finally apoptosis.The activation of calpain has been implicated in neuronal apoptosis followingspinal cord injuries and neurodegenerative diseases. This review focuses oncalpain with an emphasis on its key role in the proteolysis of cellular proteinsubstrates following apoptosis.Apoptosis, a form of programmed cell death that occurs under physiologicalas well as pathological conditions, is characterized by morphological and biochemicalfeatures. While the importance of caspases in apoptosis is established,several noncaspase proteases (Ca The Greek term "apoptosis" was first used by Kerret al. in 1972. Originally it referred to the "fallingoff" or "dropping off" of petals from flowersor leaves from trees .Apoptosis is an essential mechanism for eliminatingunwanted neuronal cells during the developmentand homeostasis of multicellular organisms. Duringmetamorphosis, cell death is rapidly apparent in bothinsects and amphibians where the larval tissues mustbe replaced by those of the adult . In mammApoptosis is the main cause of cell death observedin pathological conditions in the central nervoussystem. For instance, apoptosis has been describedin neurons and glial cells during spinal cord injuryas well as in many neurodegenerative diseasessuch as amyotrophic lateral sclerosis, Parkinson’sand Alzheimer’s disease .Classical apoptotic cell death can be defined byboth morphological and biochemical characteristicsthat distinguish it from other forms of cell death.One of the earliest events during apoptosis is celldehydration where the loss of intracellular waterleads to cellular shrinkage. Another change, perhapsthe most characteristic feature of apoptosis, isnuclear and chromatin condensation .These events are followed by nuclear fragmentation.The nuclear fragments, together with the constituentsof the cytoplasm (including organelles)are then packaged and enveloped by fragments ofthe plasma membrane to form apoptotic bodies.When apoptosis occurs in vivo, apoptotic bodiesare phagocytized by neighboring cells in the absenceof an inflammatory reaction in the tissue .AnotherThe formation of distinct DNA fragments of nucleosomalsize (approximately 180 bp) is a biochemicalhallmark of apoptosis in most cell lines andtissues . These f2+-independentproteases) in apoptosis have been clearly established,studies have indicated that several othertypes of noncaspase proteases may also play a rolein the execution of apoptosis. Noncaspase proteasesmost closely linked to apoptosis are calpains,cathepsins, granzymes and the proteasome  catalytic subunit anda small (30 kD) regulatory subunit . The80 2+ binding sites, which are E-helix-loop-F-helixmotifs (EF hands). Another EF hand is present atthe beginning of domain III. Domain V, the N-terminal region of the regulatory subunit, is hydrophobicbecause of glycine clustering and may functionas a membrane anchor. Domain VI, the C-terminalend of the small subunit, is a Ca2+ binding regionsimilar to that of the large subunit (Domain II is composed of two subdomains (IIa andIIb). The active site Cys on IIa interacts with boththe substrate and the inhibitory region of calpastatin.The exact function of domain III is unknown.Domain IV is the C-terminal end of the large subunit.It is structurally similar to calmodulin with fiveCa subunit .The activation of calpain has been implicated inneuronal death following spinal cord injury ,multipl2+ concentration is 50-100 nM in resting cells,is another specific calpain substrate that directlyassesses calpain activity in living cells ,37and tCalpain activation in apoptosis was first demonstratedin thymocytes, as measured by calpain autolysis. Calpain3-morpholine] have beenshown to protect both retinal  N, N, N', N'-tetraacetic acid (EGTA) is nota calpain inhibitor, it mimics the effects of calpaininhibitors by chelating extracellular Cad slices and the d slices .The moscalpain . The inhcalpain , limitin2+concentration and calpain activation are thought tobe responsible for the induction of neuronal deathin these diseases  are the primary cause of neuronalpathology in Alzheimer's . HigherThe pathophysiology underlying the degenerationof substantia nigra dopaminergic neurons inParkinson's disease is proposed to be due to mitochondrialdysfunction, leading to increased freeradical production and higher intracellular calciumconcentrations .One hypothesis concerning the mechanism of amutation in the Huntington protein which resultsin neuronal death in Huntington's disease is thatthe mutant Huntington protein induces mitochondrialdefects through the inhibition of mitochondrialcomplex II—succinate dehydrogenase, leading The pathogenesis of amyotrophic lateral sclerosis,a progressive neurodegenerative disorder leadingto motor neuron loss, axonal degeneration, muscularatrophy and death  is thougLoss of myelin proteins in multiple sclerosis occursby protease-mediated breakdown. Since allmajor myelin proteins, including myelin basicprotein and axonal neurofilament protein are thesubstrates of calpain , myelin In all neurodegenerative disorders in whichcalcium homeostasis is altered, calcium dysregulation leads to the pathologic activation ofcalpain . Calpain2+/calmodulin-dependent protein kinase type IV (Calpain also cleaves key cytosolic enzymes involvedin calcium homeostasis such as Ca type IV .Calpain is calcium-activated protease which existsas an inactive proenzyme in the cytosol. When intracellularcalcium level is overloaded, it triggersto convert the proenzyme to its active form. Activatedcalpain then cleaves cytoplasmic and nuclearsubstrates, leading to apoptosis.


Although several etiopathogenetic mechanisms have been proposed, the causes of left ventricular apical ballooning syndrome are still controversial.A 51-year-old Japanese woman consulted the emergency room complaining of the sudden onset of anterior chest pain while shopping. We initially suspected her disease as left ventricular apical ballooning syndrome based on her clinical background and laboratory examinations. However, the initial coronary angiogram demonstrated diffuse lesions in her distal left anterior descending coronary artery, and she was finally diagnosed with apical myocardial infarction. The blood flow in her distal left anterior descending coronary artery had markedly improved in the chronic phase. If the reduced blood flow in her distal left anterior descending coronary artery was induced by coronary vasospasm and the vasospasm was relieved before the coronary angiogram was performed, this case must be diagnosed as left ventricular apical ballooning syndrome.We think this case may promote discussion regarding the pathophysiology of left ventricular apical ballooning syndrome. Left ventricular apical ballooning syndrome (LVBS), also known as takotsubo cardiomyopathy, is an emerging clinical entity presenting with acute chest pain or dyspnea, associated with new ST-T segment elevation mimicking ST-elevated myocardial infarction (STEMI). Among patients presenting with clinically suspected acute coronary syndrome, its prevalence is reported to range from 1.2 to 2.0% and it occurs almost exclusively in postmenopausal women, often being associated with emotional or physical stress [We encountered a case of initially suspected LVBS based on the clinical background. However, coronary angiography (CAG) demonstrated bizarre images of the left anterior descending (LAD) coronary artery. Although the final diagnosis was apical myocardial infarction induced by prolonged reduced blood flow in the distal portion of the LAD coronary artery which extended around the apex, this case may promote discussion regarding the pathophysiology of LVBS considering the specific clinical course.A 51-year-old Japanese woman (body mass index = 21.0) with no medical history consulted the emergency room complaining of the sudden onset of anterior chest pain while shopping. She had no coronary risk factors. Her blood pressure was 192/100mmHg, and pulse was 64/minute and regular. A 12-lead electrocardiogram demonstrated an increased ST-segment in V2 to V5 anterior chest leads  before an angiographic study is conducted, is one of the hypotheses for the pathophysiology of LVBS. Spontaneous early reperfusion can explain phenomena such as minimal enzymatic release and left ventricular stunning rather than necrosis . One argWe found that our patient’s distal LAD artery looked like a withered branch in the acute phase and its appearance was markedly improved in the chronic phase. For this pathophysiology, we have to make a differential diagnosis between coronary vasospasm and SCAD ,15. SCADMoreover, because CAG was not evaluated until 5 months later, we have no idea about the process of recovery of LAD blood flow. If the reduced blood flow in the distal LAD artery was induced by coronary vasospasm and the vasospasm was relieved before CAG was performed, this case must be diagnosed as LVBS. Although this mechanism is only one hypothesis about the cause of LVBS, as is also presented in several previous reports, we think this case is very rare and has implications for investigating the etiology of LVBS in the future.This case report describes a case of apical myocardial infarction with bizarre initial images of LAD coronary artery mimicking LVBS. There have been proposed several etiopathogenetic mechanisms, but the causes of LVBS are still controversial. We think that this case may promote discussion on the pathophysiology of LVBS.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CAG: Coronary angiography; ISDN: Isosorbide dinitrate; IVUS: Intravascular ultrasonography; LAD: Left anterior descending; LVBS: Left ventricular apical ballooning syndrome; OCT: Optical coherence tomography; SCAD: Spontaneous coronary artery dissection; STEMI: ST-elevated myocardial infarction.The authors declare that they have no competing interests.TN is the primary author of this paper. TN and ST took care of the patient. NK, ST, HK, YH, SI, KT, TK, YU and TT made substantial editorial revisions to the manuscript. NK made major contributions to the conception and design. All authors read and approved the final manuscript.


Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes  appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966  Ischemic stroke still represents one of the leading causes of death worldwide Originally, the observation that cerebral vessels hold back hydrophilic molecules from entering the brain was first described by Paul Ehrlich in 1885 Under physiological conditions cerebral endothelial cells ensure the outstanding cerebral barrier function, which is maintained by the critical interaction of pericytes, astrocytes, neurons and the extracellular matrix as parts of the neurovascular unit (NVU) in vitro hypoxia as well as of global and focal cerebral ischemia in rodents  in rats, which is currently believed to best mimic the human pathophysiology Experiments involving animals were performed according to the European Communities Council Directive (86/609/EEC) after protocol approval by local authorities . Generally, efforts were made to minimize the total number of animals and suffering of animals, which were housed in a temperature (21–22°C) and humidity (45–60%) controlled room with 12 hours of light/dark cycle and free access to food and water.2O/30% O2; vaporisator: VIP 3000, Matrix, New York, USA). To avoid anesthesia-related cooling during surgery, the body temperature was adjusted to 37.0°C by a thermostatically controlled warming pad with rectal probe . After surgery, animals spent time on a commercial warming pad (37.0°C) until recovery from anesthesia. Sufficient eMCAO induction was ensured by functional assessment using Menzies score , were subjected to right-sided eMCAO as originally described by Zhang and coworkers 2 and transcardially perfused with either 200 ml saline , or 200 ml saline followed by 200 ml of a phosphate-buffered fixative containing 4% paraformaldehyde and 0.5% glutaraldehyde . After extraction of the brains the samples for fluorescence microscopy were immediately snap frozen in isopentane on dry ice.Four or 24 hours after ischemia onset, FITC-albumin  was intravenously administered to allow fluorescence-based localization of BBB breakdown, indicated by FITC-albumin leakage For fluorescence microscopy, the brains were coronally cut in 10 µm sections on a cryotome . Before starting the staining procedure, the samples were post-fixed with Zinc-formalin for 5 minutes at room temperature followed by several rinses with 0.1 M phosphate-buffered saline, pH 7.4 (PBS). Unspecific binding of the antibody was inhibited by a blocking step applying 5% of normal goat serum (NGS) and 0.3% of Triton X-100 in PBS for 20 min. The primary antibodies mouse anti-occludin , rabbit anti-claudin-5 , rabbit anti-laminin  and guinea pig anti-GFAP  were allowed to incubate over night at 4°C in PBS containing 0.5% NGS. After several washing steps, the sections were reacted with highly purified fluorochromated antibodies  specifically recognizing IgGs from the host species of primary antibodies for 2 hours. Subsequently, sections were washed several times in PBS and coverslipped with fluorescence mounting medium . Omitting the primary antibodies in control experiments resulted in the expected absence of staining. The specimens were analyzed with an Olympus BX51 fluorescence microscope  equipped with a digital camera.For quantification of tight junction integrity, the ratio of tight junction-positive vessels with respect to the total number of vessels was captured in the area of FITC-albumin leakage, as indicator of BBB breakdown, and compared to the corresponding region on the contralateral hemisphere. Therefore, we applied double immunofluorescence staining for occludin as marker for endothelial tight junctions and pan-laminin as a general marker for cerebral vessels For electron microscopy the brains were consecutively cut in 60 µm sections on a vibratome  in cooled PBS. The immunohistochemical conversion of FITC into an electron-dense reaction product was performed according to our previous study After several washing steps in PBS the sections were stained with 0.5% osmium tetroxide in PBS for 30 min. After 5 washing steps the sections were dehydrated using 30%, 50% and 70% of ethanol. Subsequently, the tissue was incubated with 1% uranyl acetate in 70% ethanol for 1 hour. Following further dehydration using 80%, 90%, 96%, 100% ethanol and finally propylene oxide the sections were incubated in Durcupan  and embedded between coated microscope slides and cover slips followed by polymerization at 56°C for 48 hours. After microscopical identification of areas exhibiting FITC-albumin leakage demarked by DAB staining the cover slips were removed and the respective areas were transferred on blocks of resin followed by another step of polymerization at 56°C for 48 hours. Afterwards, the blocks of resin were trimmed down to the areas intended for ultrastructural analysis which were relocated on semi-thin sections and then ultra-thin sections of 60 nm thickness were prepared on an ultramicrotome . Finally, sections were transferred on formvar-coated grids and stained with lead citrate for 6 min. Ultrastructural analysis was performed using a Zeiss EM900 transmission electron microscope and a Zeiss SIGMA electron microscope equipped with a STEM detector, respectively .P<0.05 was considered as statistical significant.The obtained data on tight junction integrity were processed with IBM SPSS Statistics 20 . Thereby, the Wilcoxon test was utilized to check for statistical significance between means of ratios. In addition, results were further stressed by adding the Monte Carlo simulation with 10,000 samples and a confidence interval of 99%. Generally, a According to our previous study For ultrastructural analysis and identification of areas showing FITC-albumin leakage, we made use of a peroxidase-coupled anti-fluorescein IgG and DAB, which served as permanent and electron dense chromogen. To prove the specificity of this technique on glutaraldehyde-treated sections for electron microscopy, control stainings were performed on consecutive vibratome sections. The specificity of the staining was proven by restriction of the DAB signal to the right striatum affected by eMCAO and the absence of any labeling on the contralateral hemisphere . To precTo further assess whether belts of tight junctions are altered in areas of FITC-albumin leakage critical tight junction constituents were analyzed by double fluorescence labeling. In contrast to our own expectations, visualization of the essential transmembrane tight junction constituents occludin and claudin-5 revealed presence and regular morphology of both antigens in control areas as well as in areas of BBB breakdown . PresencP = 0.500; Monte Carlo simulation, P = 0.630).To investigate the question of whether or not there is a statistical significant difference in the expression of critical tight junction proteins between areas of FITC-albumin extravasation and respective contralateral regions, double fluorescence labeling was applied by use of an antibody for laminin as a pan-vessel marker and the critical transmembrane protein occludin to demark tight junction complexes. Using low power (10× objective) magnification localization of the tracer beyond the glial basement membrane ensured precise confinement of analysis to areas showing distinct FITC-albumin leakage into the brain parenchyma. Here, we compared the number of occludin- and laminin-positive vessels in areas of BBB breakdown and their corresponding control areas . As showDue to apparent limitations of fluorescence microscopy to elucidate minor changes of tight junction complexes, electron microscopy was applied to reveal ultrastructural alterations responsible for vascular leakage. Using flat-embedded vibratome sections stained with DAB to demark the applied tracer, we were able to precisely identify regions of BBB breakdown and their corresponding control areas by initial light microscopy . On the As evidence for leakage through altered endothelial tight junctions was lacking, a more detailed comparison between the vasculature on the control hemisphere and areas of FITC-albumin leakage on the ipsilateral hemisphere was performed to search for signs of transcellular leakage. In control areas, the endothelium appeared not to be affected by the experimental procedure. The nuclei exhibited the typical distribution pattern of euchromatin and heterochromatin, but hardly any transcellular vesicles. Adjacent cells within the neurovascular unit appeared normal and extravasation of the tracer was not observed . In contIn addition to the finding of an enhanced vesicle transport through the endothelial layer, a disintegration of endothelial cells was often observed allowing direct extravasation of FITC-albumin into perivascular spaces and the adjacent neuropil. Here, the luminal plasma membrane frequently appeared to be discontinuous. Thus, the former barrier for blood-borne molecules towards the brain parenchyma proper is often constituted by endothelial debris and remnants of the vascular basement membrane only .Finally, we applied electron microscopy at 5 hours after ischemia onset to capture the condition in this early stage of ischemia, and to allow comparison to the previously described 25-hour time point. Thereby, an equal situation was noted, clearly demonstrating regularly established tight junctions in the presence of endothelial damage and FITC-albumin leakage, as well as enhanced vesicle trafficking within the morphologically altered endothelium .in vitro experiments, which are useful to study endothelial function, but not pathophysiological events of the complete NVU including pericytes, astrocytes, neurons and components of the extracellular matrix, which act in concert to maintain the barrier function of the cerebral vasculature Despite their enormous clinical relevance, the underlying mechanisms for ischemia-induced brain edema and hemorrhagic transformation at the endothelial level are still poorly understood. Consequently, the regulation of BBB integrity has achieved high priority rating in today's stroke research In order to minimize artificial alterations, we used an embolic model of ischemic stroke in rats, which is believed to best mimic the human pathophysiology Although alterations of tight junctions have been described in several models of hypoxia and stroke before via changes in the extra-/intracellular calcium level or phosphorylation of proteins, finally leading to variant functional characteristics in vitro model of endothelial cells To rule out that possible alterations of the endothelial tight junctions remain undetected due to limitations of fluorescence microscopy, electron microscopy was applied to analyze ultrastructural changes within the endothelial layer. As previously described However, Reese and Karnovsky who first described endothelial tight junctions as ‘morphological correlate of a BBB’ also considered in the same paper ‘paucity of transcytotic vesicles’ as a mechanism to prevent penetration of hydrophilic molecules into the brain As we were also not able to demonstrate loss of tight junctions, we further analyzed the areas of tracer extravasation for differences in the distribution of transcytotic and pinocytotic vesicles compared to respective control areas. Indeed, we found a remarkable increase of these vesicles in areas of FITC-albumin leakage while control areas exhibited no or scarcely any vesicles at all . Further


Recently, surprising new findings have contradicted central dogmas of commitment of ASCs by showing their plasticity to differentiate across tissue lineage boundaries, irrespective of classical germ layer designations. The present paper supports the plasticity of the bone marrow stem cells (BMSCs), bringing the most striking and the latest evidences of the transdifferentiation properties of the bone marrow hematopoietic and mesenchymal stem cells , the two BM populations of ASCs better characterized. In addition, we report the possible mechanisms that may explain these events, outlining the clinical importance of these phenomena and the relative problems.In contrast to the pluripotent  It has long been believed that the differentiation potential of ASCs is restricted to the production of the cell types normally found in the organ in which ASCs reside. Classical experiments showed that when fragments or cells dissociated from an organ or a tissue are transplanted to a new site or cultured, they tend to maintain their originalcharacter; although they may lose some of their properties, they usually do not acquire characteristics of a different cell lineage . The firThese and similar studies, performed with transplanted BM cells, suggested that BM is a source of different kinds of ASCs which, given the appropriate environmental signals, show pluripotent properties and transdifferentiate into cells of many different organs, including skeletal muscle, heart, liver, and endothelial and even brain cells. Our focus is to critically evaluate the evidence in favor of HSCs and MSCs plasticity. common lymphoid precursors (CLPs) [common myeloid precursors (CMPs) [HSCs are essential for the generation and homeostasis of the blood system. They give rise to all the blood cell types, including lymphocytes, erythrocytes, monocytes, granulocytes, and platelets, and they replenish these cells  Figure . Contrars (CLPs)  and comms (CMPs) . To support the hypothesis that HSCs are able to transdifferentiate into nonhematopoietic cells , severalin vitro studies have challenged the notion of a strictly hierarchical branching model of hematopoiesis. Numerous investigations have shown that both nontransformed and malignant hematopoietic precursors can switch cell types within the hematopoietic lineage [Eu-myc, in which Klinken et al. overexpressed the v-raf oncogene. The v-raf-transfected cells not only expressed myelomonocytic markers (such as the colony-stimulating factor (CSF)-1 receptor and lysozyme), but also retained immunoglobulin rearrangements characteristic of the original cells [v-fms oncogene (encoding a constitutively active form of the CSF-1 receptor) switched into macrophages [c-fms), which is expressed in the pre-B Pax5 knockout cells. Another series of experiments demonstrated that CLPs can be reprogrammed to become myelomonocytic and that lineage plasticity has been observed also within the myeloid/erythroid compartment [Over the past two decades, results from  lineage  , neurofilament heavy chain (NF-HC), and β-III tubulin [Kopen et al. first demonstrated that MSCs injected into the CNS of newborn mice migrated throughout the brain and adopted morphological and phenotypic characteristics of astrocytes and neurons . These f tubulin , 37. Stu tubulin  showed t tubulin  and prot tubulin  further  tubulin . More re tubulin . Thus evin vivo. It was shown that BMMSCs engraftment in lung of mice was enhanced in response to bleomycin exposure and that a small percentage of MSCs, localized to areas of lung injury, resembled epithelial cells and copurified with type II pneumocytes [in vitro [BMMSCs have also been reported to differentiate into various epithelial cell types after systemic administration umocytes . Moreoveumocytes . Recentlin vitro . BMMSCs in vitro , retinalin vitro , cornealin vitro , and tubin vitro . in vitro and in vivo for the treatment of urinary incontinence [β-myosin heavy chain (βMHC), myosin regulatory ligh chain 2 (Myl 2) but also detailed analysis of contractility, excitation-contraction coupling and signalling pathways demonstrated that MSCs can generate functional cardiomyocytes in vitro [MSCs can also differentiate and integrate into muscle cells . A recenntinence . MSCs apntinence . Not onlin vitro .albumin-urokinase promoter (Alb-uPA) transgenic mice or in fumarylacetoacetate-hydrolase-(FAH-)-deficient mice (FAH(−/−)). Transplantation of BMMSCs-derived hepatocyte-like cells into a patient with homozygous familial hypercholesterolemia failed to affect the cholesterol levels [Transdifferentiation of cultured naïve MSCs into hepatocyte-like cells has been claimed to occur by adding specific differentiation media . Recentll levels . Evidencl levels . β3 could be induced to differentiate into osteoblasts and adipocytes [γ2 for adipogenesis. As expected, expression of Cbfa 1 was upregulated during osteogenesis, whereas both Sox 9 and PPARγ2 were downregulated compared with undifferentiated human MSCs. On the other hand, expression levels of all three transcription factors decreased during osteoblast dedifferentiation, which suggested that cells might return to an uncommitted developmental stage from a fully determined cell type (dedifferentiation) [Similar to transdifferentiation observed in the hematopoietic system (lymphoid-to-myeloid and myeloid-to-erythroid switches), transdifferentiation examples have also been reported in mesenchymal system . Song anipocytes . In the tiation) . Thus diThe red lines indicate normal lineage relationships, and the thick green lines represent transdifferentiation within and outside the hematopoietic and mesenchymal lineages. (These switches do not necessary imply direct transitions.) α which can promote certain differentiation phenotypes in MSCs. Other lines of evidence, of microenvironment-dependant reprogramming of MSCs gene expression profile, come from studies on MSCs isolated from adipose tissue (ATMSCs). Thus, chondrogenic differentiation of ATMSC has been observed at enhanced levels under hypoxic conditions where osteogenesis is inhibited. In contrast, enhanced osteogenic differentiation of ATMSC can be induced under normoxia. In order to undergo transdifferentiation and fate changes compared to their own lineage commitment, ASCs need to change or modify their gene expression programs. Therefore, temporary inactivation of cellular memory of transcriptional state is required. It is well known that BM microenvironment, in which HSCs and MSCs reside, provides signals for survival and external control of stem cell activity. In this regard it can be assumed that new microenvironment signals should be able to modulate the cellular memory of transcriptional state and to lead to a switch in stem cell gene expression and in its cellular identity. Transplanted ASCs may recognise heterotopic environments through cell surface receptors, which stimulate signaling transduction pathways connecting the outside of the stem cell with inside responsive transcription factors and regulatory molecules . At the Functional changes of MSCs under hypoxia also include increased secretory activity, that is, of vascular endothelial growth factor and interleukin-6 as well as mobilization and homing by the induction of stromal cell-derived factor-1 expression and the corresponding receptor CXCR4 . In thisCCAAT/enhancer-binding protein beta (C/EBP-β) and Pax. The zinc-finger transcription factor GATA-1 and its cofactor FOG-1 have been found to be essential for erythroid and megakaryocytic differentiation; the physical interaction between GATA-1 and FOG-1 is required for terminal erythroid and megakaryocyte maturation both in vivo and in vitro [β. This downregulation is a prerequisite for commitment to the eosinophil lineage [−/− mice gave rise to several distinct lineages including macrophages, osteoclasts, and dendritic cells [Polycomb (PcG) and Trithorax (trx-G) Genes. These proteins which regulate Hox genes expression pattern and determine segment identity in Drosophila are strongly involved in the regulation of hematopoiesis [Among the transcription factors known having essential roles in hematopoietic lineage decisions, there are GATA-1, Friend of GATA-1 (FOG-1), PU.1, in vitro . PU.1 isin vitro . The cro lineage . Pax5 isic cells ; thus Paopoiesis .γ2 for adipogenesis [bone morphogenic protein 4 (BMP4), Wingless+ Int-1 (Wnt), and fibroblast growth factor 2 (FGF2) signalling, as well as inhibition of Wnt signalling by the factor Dickkopf1 (Dkk1) and the treatment with DNA-demethylating agent 5-azacytidin, (5-aza) [Less is known about genes and molecules involved in MSCs commitment and transdifferentiation. Among the transcription factors upregulated we note Cbfa 1 for induction of osteogenesis, Sox 9 for chondrogenesis, and PPARogenesis . Satija ogenesis . Several (5-aza) . Recentl (5-aza) . What pe (5-aza) .MicroRNAs (miRNA) are a class of noncoding RNAs which bind the 3′UTR of target mRNAs to mediate translational repression in cells. Many miRNAs are specifically expressed during hematopoietic lineage commitments . miR-181in vitro spontaneous cellular fusion between HSCs and totipotent ESCs in coculture [in vitro fusion results in denying the transdifferentiation for in vivo HSCs switching. However, we must not forget that HSCs isolation protocols require manipulation that could expose highly enriched HSCs to concentrated pluripotent precursors types that might mediate cell fusion. In addition, one transplantation study in mice showed the 30%–50% efficiency of HSCs reconstitution of hepatocytes, which is far greater than the frequency (1 ~ 500.000) of HSC-ESC fusion observed [It has been largely assumed that the nuclear reprogramming and transdifferentiation in response to environmental changes are the mechanism by which committed HSCs give rise to multiple cell types. However Terada et al. first reported the surprising results of an oculture . Later, oculture . The queobserved . This meThere is always the possibility that the BM hosts a variety of dedicated tissue-specific stem cells, such as muscle stem cells, neuronal stem cells, and hepatic progenitors, although there is, as yet, no evidence for the presence of these progenitors cells in the BM. It has also been postulated that a universal BMSC exists . In thisα-actin, the Desmosomal Type Junction Proteins Desmoglein 2 (Dsg2), Desmocollin 2 (Dsc2), desmoplakin and plakophilin 2, and the junction protein myozap has not been found [The transdifferentiation potential of HSCs and MSCs and their capacity for tissue renewal and damage repair have attracted much attention among biotechnologists and clinicians . Howeveren found . This isen found . Even ifen found . Monoclonal gammopathy of undetermined significance (MGUS), hematopoietic stem and progenitor cells (HSPCs) differentiate into cells with endothelial features, contributing to the neovessels wall building together with MM endothelial cells (MMECs) [cancer stem cells (CSCs). In gliomas transdifferentiation of CSCs into vascular mural cells contributes to tumor neovascularization [A negative aspect of HSCs transdifferentiation is their contribution to BM neovascularization which represent a problem in those cancers which home and expand in the BM. Ria et al. demonstrated that in patients with multiple myeloma (MM), but not in those with  (MMECs) . Moreove (MMECs) , 73. We  (MMECs) . Finallyrization . AssuminBMSCs are an attractive source of cells for therapy, especially in view of the recent claims that they are remarkably plastic in their differentiation potential when exposed to new environments. spinal cord injury (SCI) [Transplantation of BMSCs is traditionally used for haematological diseases, but there are increasing numbers of clinical trials using BMSCs for the treatment of nonhematological disorders. Xu and Liu resumed the studies carried out in animal models and in humans underlying the therapeutic potential of BMSCs in liver diseases . This pory (SCI)  have beery (SCI) . 4 to 1 in 108 of BM nucleated cells), but the stimulation with mobilizing agents, including cytokines such as Granulocyte Colony-Stimulating Factor (G-CSF) alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or other agents, dramatically increases the release of HSCs from BM to PB [β-thalassemia, Wiskott-Aldrich syndrome, and severe combined immunodeficiency (SCID) as well as in metabolism errors as Hunter's syndrome and Hurler's syndrome [Among BMSCs, HSCs are the only stem cells being routinely used in the clinics . They coBM to PB . HSCs arBM to PB . Allogensyndrome . HSCs trsyndrome . spinal cord injury (SCI), parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, but these current data do not support the possibility that most of the reported effects occur as a result of direct transdifferentiation and cell replacement [MSCs have become a recent focus of interest for cellular therapy in tissue regeneration. Wound healing studies have focused on MSCs as the cell population within the BM that can contribute to cutaneous regeneration . Experimlacement . Some cllacement . The autlacement . A grouplacement .Cord Blood Stem Cells (CBSCs). It was learned that one umbilical cord contains an adequate number of HSCs for a successful engraftment only in low body weight patients (up to 40 kg) to reconstitute their immune system. The total number of cells, comprising hematopoietic progenitors, collected from one umbilical cord is significantly lower (roughly 5 × 106) than from BMSCs or from PB after BMSCs mobilization (roughly 1 × 108) [+ [graft-versus-host disease (GvHD) [in vivo promoting the development of a permissive stroma for the tumor, as was demonstrated in MM [in vitro [BMSCs have a major advantage over stem cells from other organs: they are well defined, easy to isolate, and can be injected systemically reaching other tissues through the bloodstream. Thus they are more suitable than other kinds of stem cells for the therapeutic use. The advantage of BMSCs isolation compared to other types of ASCs  resides in the properties of mobilization and homing. In fact BMSCs migrate from their BM niche to PB and then return to a new site in the BM. Presumably, some of the mechanisms that regulate stem cell trafficking are the same that regulate homing and lodging of BMHSCs during transplantations. The comparison between the clinical use of BMHSCs and BMMSCs shows a greater number of applications for the latter than the former. Although preclinical studies have demonstrated the plasticity of both BMHSCs and BMMSCs, the majority of clinical trials see the use of BMHSCs for the treatment of hematological malignancies in which the capacity of HSCs to reconstitute the hematopoietic system of the patient rather than the transdifferentiation potential is exploited. On the contrary BMMSCs applicability in therapy exploits their potential to differentiate into different cell types and this explains their increased use in the clinical trials of various diseases. However in the treatment of haematological diseases BMHSCs show many advantages compared to other sources of HSCs such as 1 × 108) . Further1 × 108) . BMSCs a1 × 108) . Another 108) [+ . As BMHS 108) [+ . BMMSCs e (GvHD) . In facted in MM . Anotherin vitro . Thus MSin vitro .Although BMHSCs and BMMSCs belong to the most intensely studied stem cell types in cell therapy, comparison of existing preclinical and clinical data is hampered by a poor standardisation and harmonisation concerning protocols for isolation, expansion, and delivery. As with BMHSCs, BMHSCs mobilized into PB contain a mixture of hematopoietic stem cells, progenitor cells, and other kinds of cells. Consequently, the resulting cell preparation that is infused back into patients is not a pure HSCs preparation, but a mixture of HSCs, progenitors, and various contaminants, including T cells and in the case of autologous graft from cancer patients quite possible tumor cells as described previous. BMHSCs normally passed through a device that enriches cells that express CD34+, a marker of both stem and progenitor cells. The use of highly purified HSCs as graft is rare . The maiin vivo situation of the cells, including advantages such as efficient nutrient supply, waste removal, minimal shear stress, and the possibility to control the cultivation via online measreuments of critical values [Efficient expansion of HSCs in culture remains one of the major goals despite their ability to self-renew. Attempts to expand HSCs in tissue culture with known stem-cell stimulators (growth factors and cytokines) have never resulted in a significant expansion of HSCs. Rather, these compounds induce many HSCs into cell division that are always accompanied by cellular differentiation . Comparel values . Another bottleneck of stem cells therapeutics is the way of administration and the cells delivery. In transplants HSCs are generally infused intravenously. For MSCs the researchers have tried to optimize the delivery. In the treatment of cutaneous wounds, most studies have utilized the technically simple method of injecting a cell suspension intradermally into or around the wound defect; however the true therapeutic potential of MSCs appears to be limited due to poor engraftment efficiency and cell retention at the wound site. A fibrin spray system, to topically administer autologous MSCs to nonhealing lower extremity wounds, has been used in human subjects. Stem cells were found to survive within the fibrin layer and migrate into the wound tissue . HydrogePrevious examples demonstrate that questions needs to be answered in applying BMSCs in therapeutics, such as: (i) when to use pure BMSCs preparation in transplants; (ii) whether markers of HSCs, currently known, can distinguish them from their tumor counterparts; (iii) how to improve BMSCs expansion in culture without altering their stemness and differentiation potential; (iv) how to determine the most efficient method of administration of BMMSCs and how to optimize their delivery. Answering these questions will lead to a better standardization of methods and protocols used in the manipulation of BMSCs and to the overcoming of the most common bottlenecks in BM HSCs and MSCs therapeutics.The therapeutic potential of BMSCs as powerful tools in tissue regeneration and engineering has been recognised, and intense efforts are ongoing to harness and direct HSCs and MSCs plasticity. However before HSCs and MSCs are currently used therapeutically in patients with degenerative disorders of the liver, heart, or brain, the properties of such cells must be well characterized, the functionality proved, and the potential risks of their use well defined. Understanding the molecular mechanisms underlying cell fate switching of BMSCs will be an essential contribution to ensuring their safe use in regenerative medicine. Moreover, even if the transdifferentiation events described in most of these studies were rare under physiological conditions, in the future, it will most likely be possible to transplant genetically modified stem cells carrying genes critical for transdifferentiation into desired cell populations. Finally pharmacologic molecules would also be used to directly influence the trans- or redifferentiation potential of ASCs, both prior and after their administration into patients.