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Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53‐independent quiescent state in immature hepatoma cells and in pluripotent stem cell‐derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity‐regulated growth arrest followed by cell maturation, mediated by activation of NF‐κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long‐term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely. [ABSTRACT FROM AUTHOR]

Although human pluripotent stem cell (PSC)‐derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC‐derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so‐called concentroids. Such concentroids consisted of a pro‐angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia‐dense outer layer. Incorporating PSC‐derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal‐derived pericyte‐like mural cells co‐localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC‐containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC‐only concentroids. This PSC‐derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood‐brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics. [ABSTRACT FROM AUTHOR]

TDP‐43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP‐43, are one of the common causes of familial ALS. In this study, we investigate TDP‐43 protein behavior in induced pluripotent stem cell (iPSC)‐derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP‐43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP‐43, C‐terminal fragments, and phospho‐TDP‐43. By generating iPSC lines with allele‐specific tagging of TDP‐43, we find that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP‐43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP‐43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP‐43 pathology. SYNOPSIS: Mutant TDP‐43 iPSC‐derived motor neurons recapitulate ALS‐related phenotypes. The use of allele‐specific tagged cell lines allowed us to directly link TDP‐43 pathology with axonal transport defects in patient iPSC‐derived motor neurons. We found that both the pathological hallmarks and axonal transport defects can be rescued by genetic correction of the mutation using CRISPR/Cas9 or by treatment with a selective HDAC6 inhibitor. Mutant TARDPB iPSC‐derived spinal motor neurons recapitulate TDP‐43‐related ALS phenotypes.Allele‐specific endogenous tagging of TDP‐43 shows that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome.Observed TDP‐43 proteinopathy results in a defect in mitochondrial transport.Pharmacological inhibition of HDAC6 restores the observed TDP‐43 pathologies and the axonal mitochondrial motility. [ABSTRACT FROM AUTHOR]

The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR/Cas9), transcription activator‐like effector nucleases (TALENs), and zinc‐finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above‐mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies. [ABSTRACT FROM AUTHOR]

Background and Purpose: The transient receptor potential (TRP) ion channel TRPM3 functions as a noxious heat sensor, plays a key role in acute pain sensation and inflammatory hyperalgesia in rodents. Despite its potential as a novel analgesic drug target, little is known about the expression, function and modulation in the humans.Experimental Approach: We studied TRPM3 in freshly isolated human dorsal root ganglion (hDRG) neurons and human stem cell-derived sensory (hSCDS) neurons. Expression was analysed at the mRNA level using RT-qPCR. Channel function was assessed using Fura-2-based calcium imaging and whole-cell patch-clamp recordings.Key Results: TRPM3 was detected at the mRNA level in both hDRG and hSCDS neurons. The TRPM3 agonists pregnenolone sulphate (PS) and CIM0216 evoked robust intracellular Ca2+ responses in 52% of hDRG and 58% of hSCDS neurons. Whole-cell patch-clamp recordings in hSCDS neurons revealed pregnenolone sulphate (PS)- and CIM0216-evoked currents exhibiting the characteristic current-voltage relation of TRPM3. PS-induced calcium responses in hSCDS neurons were reversed in a dose-dependent manner by the flavonoid isosakuranetin and by antiseizure drug primidone. Finally, the μ-opioid receptor agonist DAMGO and the GABAB receptor agonist baclofen inhibited PS-evoked TRPM3 responses in a subset of hSCDS neurons.Conclusion and Implications: These results provide the first direct evidence of functional expression of the pain receptor TRPM3 in human sensory neurons, largely mirroring the channel's properties observed in mouse sensory neurons. hSCDS neurons represent a valuable and readily accessible in vitro model to study TRPM3 regulation and pharmacology in a relevant human cellular context. [ABSTRACT FROM AUTHOR]

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low‐density lipoprotein (ox‐LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox‐LDL‐induced reduction of MG53. MAPCs were cultured with and without ox‐LDL (0‐20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N‐acetylcysteine (NAC). Serum MG53 level was measured in ox‐LDL‐treated mice with or without NAC treatment. Ox‐LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox‐LDL‐induced reduction of Akt phosphorylation without protecting MAPCs against ox‐LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox‐LDL‐induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox‐LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox‐LDL‐induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox‐LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox‐LDL through preventing ox‐LDL‐induced reduction of MG53. [ABSTRACT FROM AUTHOR]

Introduction: Murine microglia expressing the Alzheimer's disease–linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia‐like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. Methods: We generated isogenic TREM2+/R47H, TREM2+/−, and TREM2−/− hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/− mouse brains. Results: We demonstrated that tMGs resemble cultured human microglia. TREM2+/− and TREM2−/− hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild‐type hPSC progeny, with no difference for TREM2+/R47H progeny. Discussion: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/− and TREM2−/− progeny. [ABSTRACT FROM AUTHOR]

Background: Neuroinflammation is a pathological hallmark of Alzheimer's disease (AD). Murine studies have revealed that mouse microglia can adopt several cell states in response to disease insults. However, single‐nuclei RNA sequencing studies have found divergent results when recalling those cell states in human post‐mortem tissue. Method: To overcome this limitation, we have sequenced 127,000 single cell human microglia isolated from an AD xenotransplantation model. Result: In response to plaques, human microglia can adopt several cell states including a disease‐associated microglia (DAM) and an antigen‐presenting response (HLA). When reacting to Aβ soluble aggregates, human microglia produces a pro‐inflammatory response cytokine response (CRM). TREM2 and APOE polymorphisms affect the development of the DAM‐HLA response, suggesting an important role of this response in AD. Conclusion: Overall, this multi‐faceted response to amyloid pathologies highlights a complex scenario where these distinct phenotypes might influence the outcome on the disease. [ABSTRACT FROM AUTHOR]

Introduction: Tauopathies are neurodegenerative diseases characterized by TAU protein–related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. Methods: To create a new in vitro tauopathy model, we generated a footprint‐free triple MAPT‐mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats‐FokI and piggyBac transposase technology. Results: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU‐mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. Discussion: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU‐mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening. [ABSTRACT FROM AUTHOR]

Abstract: The hematopoietic system has a very well‐studied hierarchy with the long‐term (LT) hematopoietic stem cells (HSCs) taking the top position. The pool of quiescent adult LT‐HSCs generated during the fetal and early postnatal life acts as a reservoir to supply all the blood cells. Therefore, the maintenance of this stem cell pool is pivotal to maintaining homeostasis in hematopoietic system. It has long been known that external cues, along with the internal genetic factors influence the status of HSCs in the bone marrow (BM). Hypoxia is one such factor that regulates the vascular as well as hematopoietic ontogeny from a very early time point in development. The metabolic outcomes of a hypoxic microenvironment play important roles in functional regulation of HSCs, especially in case of adult BM HSCs. Anaerobic metabolic pathways therefore perform prominent role in meeting energy demands. Increased oxidative pathways on the other hand result in loss of stemness. Recent studies have attributed the functional differences in HSCs across different life stages to their metabolic phenotypes regulated by respective niches. Indicating thus, that various energy production pathways could play distinct role in regulating HSC function at different developmental/physiological states. Here, we review the current status of our understanding over the role that energy production pathways play in regulating HSC stemness. © 2018 IUBMB Life, 70(7):612–624, 2018 [ABSTRACT FROM AUTHOR]

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes. [ABSTRACT FROM AUTHOR]

Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions. [ABSTRACT FROM AUTHOR]

Numerous neurodegenerative and neuromuscular disorders are associated with cell-specific depletion in the human body. This imbalance in tissue homeostasis is in healthy individuals repaired by the presence of endogenous stem cells that can replace the lost cell type. However, in most disorders, a genetic origin or limited presence or exhaustion of stem cells impairs correct cell replacement. During the last 30 years, methods to readily isolate and expand stem cells have been developed and this resulted in a major change in the regenerative medicine field as it generates sufficient amount of cells for human transplantation applications. Furthermore, stem cells have been shown to release cytokines with beneficial effects for several diseases. At present however, clinical stem cell transplantations studies are struggling to demonstrate clinical efficacy despite promising preclinical results. Therefore, to allow stem cell therapy to achieve its full potential, more insight in their in vivo behavior has to be achieved. Different methods to noninvasively monitor these cells have been developed and are discussed. In some cases, stem cell monitoring even reached the clinical setting. We anticipate that by further exploring these imaging possibilities and unraveling their in vivo behavior further improvement in stem cell transplantations will be achieved. [ABSTRACT FROM AUTHOR]

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents. [ABSTRACT FROM AUTHOR]

Cell therapy with bone marrow stem cells ( BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox- LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells ( MAPCs) were treated with ox- LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox- LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox- LDL also generated reactive oxygen species ( ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox- LDL, it failed to prevent ox- LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rh MG53) ox- LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rh MG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox- LDL contributed to the impaired survival of MAPCs. rh MG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia. [ABSTRACT FROM AUTHOR]

Micro RNAs are a class of small non-coding RNAs involved in the transcriptional and post-transcriptional regulation of gene expression. The function of mi RNAs in liver disease including hepatocellular carcinoma ( HCC), hepatitis, and alcoholic liver disease, have been widely studied and extensively reviewed. Increasing evidence demonstrates that mi RNAs also play a critical role in normal liver development and in the fine-tuning of fundamental biological liver processes. In this review, we highlight the most recent findings on the role of mi RNAs in liver specification and differentiation, liver cell development, as well as in the many metabolic functions of the liver, including glucose, lipid, iron and drug metabolism. These findings demonstrate an important role of mi RNAs in normal liver development and function. Further researches will be needed to fully understand how mi RNAs regulate liver generation and metabolic function, which should then lead to greater insights in liver biology and perhaps open up the possibility to correct errors that cause liver diseases or metabolic disorders. [ABSTRACT FROM AUTHOR]

Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT- PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable. [ABSTRACT FROM AUTHOR]

In this review, we discuss the migration and homing ability of mesenchymal stem cells (MSCs) and MSC-like cells and factors influencing this. We also discuss studies related to the mechanism of migration and homing and the approaches undertaken to enhance it. Finally, we describe the different methods available and frequently used to track and identify the injected cells in vivo. [ABSTRACT FROM AUTHOR]

Somatic, also termed adult, stem cells are highly attractive biomedical cell candidates because of their extensive replication potential and functional multilineage differentiation capacity. They can be used for drug and toxicity screenings in preclinical studies, as in vitro model to study differentiation or for regenerative medicine to aid in the repair of tissues or replace tissues that are lost upon disease, injury or ageing. Multipotent adult progenitor cells (MAPCs) and mesenchymal stem cells (MSCs) are two types of adult stem cells derived from bone marrow that are currently being used clinically for tissue regeneration and for their immunomodulatory and trophic effects. This review will give an overview of the phenotypic and functional differences between human MAPCs and MSCs, with a strong emphasis on their immunological characteristics. Finally, we will discuss the clinical studies in which MSCs and MAPCs are already used. [ABSTRACT FROM AUTHOR]

The transcription factor Oct4 is critical to the pluripotency, self-renewal and differentiation of stem cells. The aim of the present study was to investigate the effects of high glucose (HG) on the cell cycle progression of bone marrow multipotent adult progenitor cells (MAPC) and Oct4 expression, as well as the underlying mechanisms. Rat MAPC were cultured in normal (5.5 mmol/L d-glucose) and HG (25.5 mmol/L d-glucose) media for up to 14 days. l- Glucose served as a high osmolarity control. Culture in HG media substantially increased the number of cells in the G0/ G1 phase and decreased the number in the S phase without changing the cell population in the G2 phase. Expression of the cell cycle regulatory protein p21 CIP/ WAF-1 (p21), but not that of p27 KIP-1 (p27), was significantly upregulated in cells cultured in HG media. Significant increases were seen in transforming growth factor (TGF)-β1 levels in cells and MAPC-conditioned medium in the presence of HG, and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was enhanced in cells cultured in the presence of HG medium without any changes in Akt phosphorylation. Neutralizing TGF-β1 antibody effectively prevented HG-induced increases in ERK1/2 phosphorylation, p21 expression and suppression of cell cycle progression of MAPC. Inhibiting ERK1/2 phosphorylation with PD98059 completely blocked HG-induced p21 expression and markedly reversed HG-induced inhibition of cell cycle progression in MAPC. The HG-induced suppression of cell cycle progression was not accompanied by inhibition of cell proliferation or Oct4 expression in these cells. The data indicate that HG facilitates cell cycle arrest of rat MAPC through TGF-β1-induced activation of ERK1/2 signalling and p21 expression, and that Oct4 expression in MAPC is independent of the cell cycle and/or TGF-β1 or ERK1/2 signalling in HG medium. [ABSTRACT FROM AUTHOR]

This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox-LDL for up to 2 weeks. Ox-LDL treatment resulted in a time- and dose-dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct-4 was significantly suppressed in MAPCs by ox-LDL in a dose- and time-dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox-LDL with markedly decreased expression of endothelial markers vWF, Flk-1 and CD31, as well as impaired in vitro vascular structure formation. Ox-LDL-induced apoptosis and inhibition of Oct-4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox-LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct-4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox-LDL promotes apoptosis and inhibits Oct-4 expression and self-renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling. [ABSTRACT FROM AUTHOR]

Proteinases have been implicated in the mobilization of haematopoietic progenitor cells (HPCs) from the bone marrow (BM). Here, we report the involvement of the plasminogen (Plg) system in the haematopoietic recovery following chemotherapy. By using gene-deficient mice, we found that plasmin and its activators tPA and uPA play a role in the haematopoietic recovery upon delivery of the cytotoxic agent 5-fluoro-uracil (5-FU). The impaired haematopoietic recovery of Plg-deficient (Plg−/−) mice after 5-FU was not rescued by depletion of fibrinogen, indicating that it was not due to defective fibrinolysis. Instead, loss of Plg impaired breakdown of fibronectin, VCAM-1 and laminin-BM matrix proteins involved in adhesion of HPCs to their BM microenvironment and in transendothelial migration of HPCs. These findings provide novel insights in how plasmin regulates haematopoietic recovery upon cytotoxic myeloablation. [ABSTRACT FROM AUTHOR]

This study was to investigate the effect of high glucose (HG) on vascular endothelial growth factor (VEGF) expression in bone marrow stem cells and JAK2/STAT-3 signalling. Adult rat bone marrow multipotent progenitor cells (rMAPCs) were cultured to evaluate VEGF expression (both mRNA and protein) with or without exposure to HG for up to 48 hrs using RT-PCR and ELISA. JAK2 and STAT3 phosphorylation in rMAPCs was analysed by Western blotting. With cells in normal media, VEGF mRNA level after 24 hrs of culture was significantly increased by 15 times over baseline (day 0) with detectable level of VEGF protein intracellularly using immunofluorescence staining. Although there was no measurable VEGF in the media after 24 hrs of culture, a significant amount of VEGF was detected in the media after 48 hrs of incubation. VEGF expression was associated with constitutive activation of JAK2 and STAT3 in rMAPCs. However, VEGF mRNA level was significantly reduced without detectable VEGF in the media when rMAPCs exposed to HG for 48 hrs. Tyrosine-phosphorylation of JAK2 and STAT3 and nuclear translocation of phosphorylated STAT3 were significantly decreased in the cells exposed to HG for 48 hrs. When JAK2 and STAT3 phosphorylation was blocked by the selective inhibitor AG490, VEGF mRNA level was significantly decreased in rMAPCs in normal media by 80% with no detectable VEGF in the media. VEGF expression was significantly suppressed in rMAPCs cultured in HG media that was further reduced by AG490. VEGF expression in rMAPCs is impaired by HG possibly through inhibition of JAK2/STAT3 signalling. [ABSTRACT FROM AUTHOR]

Background: The latest generation of interactive cardiac magnetic resonance (MR) scanners has made cardiac interventions with real-time MRI possible. To date, cardiac MRI has been mostly applied to measure myocardial perfusion, viability, and regional function, but now the application of cardiac MRI can be extended to cardiovascular interventions. The purpose of this article is to illustrate the potential of MRI in stem cell therapy for cardiac restoration.Methods: We have applied MRI to (1) interactively target myocardial injections with a novel stem cell delivery catheter, and to compare gadolinium/blue dye injections to pathology; (2) assess myocardial perfusion with MR first pass imaging in an infarct model treated with stem cell therapy versus control animals; (3) measure regional functional changes using myocardial tissue tagging in the same animals.Results: We were able to demonstrate the feasibility and safety of myocardial injections under MR fluoroscopy. The intramyocardial distribution of the blue dye at necropsy correlated well with the extent of gadolinium, as detected with a three-dimensional inversion recovery MR pulse sequence for late enhancement immediately after contrast injection. Preliminary results show that myocardial perfusion reserve and regional wall motion improved in the stem-cell-treated group, compared to a control group.Conclusions: These preliminary results suggest that (1) injections into the LV myocardium can be performed under real-time MRI guidance using a directed catheter approach, and (2) regional myocardial perfusion and function, measured with MRI, both improve after stem cell therapy. This ongoing study demonstrates the potential of MRI for image-guided interventions, combined with detailed evaluation of anatomy, function, perfusion, and viability. [ABSTRACT FROM AUTHOR]

Background: The latest generation of interactive cardiac magnetic resonance (MR) scanners has made cardiac interventions with real-time MRI possible. To date, cardiac MRI has been mostly applied to measure myocardial perfusion, viability, and regional function, but now the application of cardiac MRI can be extended to cardiovascular interventions. The purpose of this article is to illustrate the potential of MRI in stem cell therapy for cardiac restoration. Methods: We have applied MRI to (1) interactively target myocardial injections with a novel stem cell delivery catheter, and to compare gadolinium/blue dye injections to pathology; (2) assess myocardial perfusion with MR first pass imaging in an infarct model treated with stem cell therapy versus control animals; (3) measure regional functional changes using myocardial tissue tagging in the same animals. Results: We were able to demonstrate the feasibility and safety of myocardial injections under MR fluoroscopy. The intramyocardial distribution of the blue dye at necropsy correlated well with the extent of gadolinium, as detected with a three-dimensional inversion recovery MR pulse sequence for late enhancement immediately after contrast injection. Preliminary results show that myocardial perfusion reserve and regional wall motion improved in the stem-cell-treated group, compared to a control group. Conclusions: These preliminary results suggest that (1) injections into the LV myocardium can be performed under real-time MRI guidance using a directed catheter approach, and (2) regional myocardial perfusion and function, measured with MRI, both improve after stem cell therapy. This ongoing study demonstrates the potential of MRI for image-guided interventions, combined with detailed evaluation of anatomy, function, perfusion, and viability. (J Interven Cardiol 2004;17:37–46) [ABSTRACT FROM AUTHOR]

If stem cells fulfill their therapeutic promise, moving them from the laboratory into the clinic will raise several concerns about justice. One concern is that, for biological reasons alone, stem cell‐based therapies might not be available for every patient who needs one. Worse, depending on how we address the problem of biological access, they might benefit primarily white Americans. We can avoid this outcome—although at a cost—by carefully selecting the stem cells we make available. [ABSTRACT FROM AUTHOR]

This study was designed to explore the role of exogenous nitric oxide (NO) in the biological behavior of bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells were used as the source of stem cells. The biological behavior of the stem cells was evaluated by assessing the cell proliferation and expression of Oct-4 with real-time PCR with or without NO donor DETA-NONOate. The cells were induced to differentiate into endothelial cells with or without DETA-NONOate. The stages of differentiation were monitored with morphology and endothelial cell marker von Willebrand factor (vWF). It was found that Oct-4 expression was significantly increased by 57.5% (n=5, p<0.05) in stem cells exposed to DETA-NONOate in a concentration-dependant manner. However, the ceil proliferation was significantly decreased when treated with DETA-NONOate for 48 hrs (1,680+/-26 cells/cm2 for control, 1,221+/-29 cells/era2 for 50 uM, and 543+/-77 cells/cm2 for 100 uM, n=6, p<0.05). When the stem cells were exposed to DETA-NONOate (100uM) for the first 48 hrs during differentiation, vWF expression was increased by 17.7% at day 14 in the differentiating cells. However, vWF expression was decreased in the cells at day 14 or cell death occurred if the cells exposed to DETA-NONOate over 72 hrs during differentiation. To demonstrate dependence of DETA-NONOate on cyclic GMP, experiments were repeated in the presence of guanylyl cyclase inhibitor ODQ or cGMP analog 8-Br-cGMP. The effect of DETA-NONOate on cell proliferation and Oct-4 expression of the stem cells was not affected by ODQ or 8-Br-cGMP, suggesting a cGMP-independent mechanism. These data indicate that exogenous NO plays an important role in the biological behavior and differentiation of bone marrow stem cells through a mechanism independent of cGMP. [ABSTRACT FROM AUTHOR]

The transcription factor signal transducer and activator of transcription 3 (STAT-3) is an important mediator for cell differentiation. Extracellular signal regulated kinas (ERK)I (p44 MAPK) and ERK2 (p42 MAPK) also play an important role in the process of cell differentiation. In the present study, experiments were designed to investigate the expression of STAT-3 and MAPK during stem cell differentiation into endothelial cells and their relationship to the development of endothelial cells. Adult mouse bone marrow multipotent progenitor cells were used as the source of stem cells. The stem cells were induced to differentiate into endothelial cells in serum-free medium in the presence of vascular endothelial growth factor (VEGF). The course of differentiation was closely monitored with endothelial cell markers Von Willebrand factor (vWF) and CD31. The differentiating cells were collected at rain 0, 5, 10, 15, 30, 60, hr 2, 3, 4, 6, 8, 24, day 3, 5, 7, 10, 14, and 21 after the initiation of differentiation to evaluate the expression of STAT-3, MAPK, vWF, CD31, and endothelial nitric oxide synthase (eNOS) with Western blot analysis, quantitative real-time PCR, and immunofluorescence staining. As expected, the differentiating cells started to express, vWF, CD31 and eNOS from day 5 and reached the maximal levels at day 14 during differentiation. VEGF-induced phosphorylation of STAT-3 began 2 hours after initiation of differentiation. STAT-3 phosphorylation increased over time thereafter, and reached a plateau at day 7 during the course of differentiation. No STAT-3 phosphorylation was identified in the undifferentiated bone marrow stem cells. On the other hand, VEGF-induced phosphorylation of MAPK occurred from day 3, reached a plateau at day 7, and remained stable thereafter during the course of differentiation. The fact that phosphorylation of STAT-3 occurred very early, followed by MAPK phosphorylation in the process of endothelial cell development indicates that STAT-3 and MAPK may play a critical role in bone marrow stem cell differentiation into endothelial cells. [ABSTRACT FROM AUTHOR]

This study was to investigate the effect of high glucose (HG) on vascular endothelial growth factor (VEGF) expression in bone marrow stem cells and JAK2/STAT-3 signalling. Adult rat bone marrow multipotent progenitor cells (rMAPCs) were cultured to evaluate VEGF expression (both mRNA and protein) with or without exposure to HG for up to 48 hrs using RT-PCR and ELISA. JAK2 and STAT3 phosphorylation in rMAPCs was analysed by Western blotting. With cells in normal media, VEGF mRNA level after 24 hrs of culture was significantly increased by 15 times over baseline (day 0) with detectable level of VEGF protein intracellularly using immunofluorescence staining. Although there was no measurable VEGF in the media after 24 hrs of culture, a significant amount of VEGF was detected in the media after 48 hrs of incubation. VEGF expression was associated with constitutive activation of JAK2 and STAT3 in rMAPCs. However, VEGF mRNA level was significantly reduced without detectable VEGF in the media when rMAPCs exposed to HG for 48 hrs. Tyrosine-phosphorylation of JAK2 and STAT3 and nuclear translocation of phosphorylated STAT3 were significantly decreased in the cells exposed to HG for 48 hrs. When JAK2 and STAT3 phosphorylation was blocked by the selective inhibitor AG490, VEGF mRNA level was significantly decreased in rMAPCs in normal media by 80% with no detectable VEGF in the media. VEGF expression was significantly suppressed in rMAPCs cultured in HG media that was further reduced by AG490. VEGF expression in rMAPCs is impaired by HG possibly through inhibition of JAK2/STAT3 signalling.