Search

Aim: To evaluate in vitro the effect of laser photobiomodulation (PBM) combined or not with 30-nm hydroxyapatite nanoparticles (HANp), on the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) by morphometric analysis using artificial intelligence programs (TensorFlow and ArcGIS). Methods: UC-MSCs were isolated and cultured until 80% confluence was reached. The cells were then plated according to the following experimental groups: G1 –control (DMEM), G2 –BMP-2, G3 –BMP-7, G4 –PBM (660 nm, 10 mW, 2.5 J/cm2, spot size of 0.08 cm2), G5 –HANp, G6 –HANp + PBM, G7 –BMP-2 + PBM, and G8 –BMP-7 + PBM. The MTT assay was used to analyze cell viability at 24, 48 and 72 h. Osteogenic differentiation was assessed by Alizarin Red staining after 7, 14 and 21 days. For morphometric analysis, areas of osteogenic differentiation (pixel2) were delimited by machine learning using the TensorFlow and ArcGIS 10.8 programs. Results: The results of the MTT assay showed high rates of cell viability and proliferation in all groups when compared to control. Morphometric analysis revealed a greater area of osteogenic differentiation in G5 (HANp = 142709,33±36573,39) and G6 (HANp + PBM = 125452,00±24226,95) at all time points evaluated. Conclusion: It is suggested that HANp, whether combined with PBM or not, may be a promising alternative to enhance the cellular viability and osteogenic differentiation of hUC-MSCs. [ABSTRACT FROM AUTHOR]

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) is an organ responsible for crucial processes in living organs, e. g., hematopoiesis. In recent years, Organ-on-a-Chip (OoC) devices have been used to satisfy the need for in vitro systems that better mimic the phenomena occurring in the BM microenvironment. Given the growing interest in these systems and the diversity of developed devices, an integrative systematic literature review is required. We have performed this review, following the PRISMA method aiming to identify the main characteristics and assess the effectiveness of the devices that were developed to represent the BM. A search was performed in the Scopus, PubMed, Web of Science and Science Direct databases using the keywords (("bone marrow" OR "hematopoietic stem cells" OR "haematopoietic stem cells") AND ("organ in a" OR "lab on a chip" OR "microfluidic" OR "microfluidic*" OR ("bioreactor" AND "microfluidic*"))). Original research articles published between 2009 and 2020 were included in the review, giving a total of 21 papers. The analysis of these papers showed that their main purpose was to study BM cells biology, mimic BM niches, model pathological BM, and run drug assays. Regarding the fabrication protocols, we have observed that polydimethylsiloxane (PDMS) material and soft lithography method were the most commonly used. To reproduce the microenvironment of BM, most devices used the type I collagen and alginate. Peristaltic and syringe pumps were mostly used for device perfusion. Regarding the advantages compared to conventional methods, there were identified three groups of OoC devices: perfused 3D BM; co-cultured 3D BM; and perfused co-cultured 3D BM. Cellular behavior and mimicking their processes and responses were the mostly commonly studied parameters. The results have demonstrated the effectiveness of OoC devices for research purposes compared to conventional cell cultures. Furthermore, the devices have a wide range of applicability and the potential to be explored. [ABSTRACT FROM AUTHOR]

Adipose-derived mesenchymal stem cells (AdMSCs) have been reported to ameliorate neurological deficits after acute ischemic stroke. As neuregulin 1 (NRG1, or heregulin 1), a growth factor with versatile functions in the central nervous system, has demonstrated protective effects against ischemic brain injuries, we have generated NRG1-overexpressing AdMSCs in order to investigate whether NRG1-AdMSCs could enhance therapeutic benefits of AdMSCs in ischemic stroke. After AdMSCs were infected with adenoviral NRG1, increased NRG1 secretion in NRG1-AdMSCs was confirmed with ELISA. At 1 d after ischemic stroke that was induced by the occlusion of middle cerebral artery (MCAo) for 60 min in Sprague Dawley (SD) rats, adenoviral NRG1, AdMSCs, NRG1-AdMSCs, or PBS were injected into the striatum and serial neurologic examinations were performed. Administration of NRG1-AdMSCs resulted in significant improvement of functional outcome following stroke compared to AdMSCs- or adenoviral NRG1-treated group, in addition to the reduction in the infarct size evaluated by hematoxylin and eosin staining. When NRG1 expression in the brain was examined by double immunofluorescence to human nuclei (HuNu)/NRG1 and ELISA, NRG1-AdMSCs demonstrated marked increase in NRG1 expression. Moreover, western blot analysis further showed that transplantation of NRG1-AdMSCs significantly increased both endogenous and adenoviral NRG1 expression compared to AdMSCs-treated group. To elucidate molecular mechanisms, NRG1-associated downstream molecules were evaluated by western blot analysis. Expression of ErbB4, a receptor for NRG1, was markedly increased by NRG1-AdMSCs administration, in addition to pMAPK and pAkt, crucial molecules of NRG1-ErbB4 signaling. Taken together, our data suggest that NRG1-AdMSCs can provide excellent therapeutic potential in ischemic stroke by activating NRG1-ErbB4 signaling network. [ABSTRACT FROM AUTHOR]

In all metazoan species, sperm is produced from germline stem cells. These self-renew and produce daughter cells that amplify and differentiate dependent on interactions with somatic support cells. In the male gonad of Drosophila melanogaster, the germline and somatic cyst cells co-differentiate as cysts, an arrangement in which the germline is completely enclosed by cytoplasmic extensions from the cyst cells. Notch is a developmentally relevant receptor in a pathway requiring immediate proximity with the signal sending cell. Here, we show that Notch is expressed in the cyst cells of wild-type testes. Notch becomes activated in the transition zone, an apical area of the testes in which the cyst cells express stage-specific transcription factors and the enclosed germline finalizes transit-amplifying divisions. Reducing the ligand Delta from the germline cells via RNA-Interference or reducing the receptor Notch from the cyst cells via CRISPR resulted in cell death concomitant with loss of germline cells from the transition zone. This shows that Notch signaling is essential for the survival of the germline stem cell lineage. [ABSTRACT FROM AUTHOR]

Freshly isolated, uncultured, autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron, Inc., Houston, TX, USA) is a system for isolating ADRCs from adipose tissue, commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary, enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield, cell viability, live cell yield, biological characteristics, physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then, characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation, enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e., numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences, cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics, physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate. [ABSTRACT FROM AUTHOR]

In an ever-aging society the demand for bone-defect filling grafts continues to gain in importance. While autologous grafting still prevails as the gold standard, allografts and xenografts present viable alternatives with promising results. Physiochemical properties of a graft strongly depend on the processing method such as the decellularization protocol. In addition, the physiochemical characteristics are critical factors for a successful integration of the graft after the implantation and might influence mesenchymal stem cell function in therapeutic approaches combining grafts and autologous mesenchymal stem cells (MSCs). Several decellularization methods have been proposed, however it still remains unclear which method results in favorable physiochemical properties or might be preferred in stem cell applications. In the first part of this study we compared two decellularization approaches resulting in chemically processed allografts (CPAs) or sonication-based processed allografts (SPAs). Each decellularization approach was compared for its decellularization efficacy and its influence on the grafts’ surface texture and composition. In the second part of this study biocompatibility of grafts was assessed by testing the effect of extraction medium on MSC viability and comparing them to commercially available allografts and xenografts. Additionally, grafts’ performance in terms of MSC functionality was assessed by reseeding with MSCs pre-differentiated in osteogenic medium and determining cell adhesion, proliferation, as well as alkaline phosphatase (ALP) activity and the degree of mineralization. In summary, results indicate a more effective decellularization for the SPA approach in comparison to the CPA approach. Even though SPA extracts induced a decrease in MSC viability, MSC performance after reseeding was comparable to commercially available grafts based on DNA quantification, alkaline phosphatase activity and quantification of mineralization. Commercial Tutoplast allografts showed overall the best effects on MSC functionality as indicated by extraction biocompatibility testing as well as by comparing proliferation and osteogenic differentiation. [ABSTRACT FROM AUTHOR]

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances. [ABSTRACT FROM AUTHOR]

High morbidity and mortality are common traits of malignant tumours and identification of the cells responsible is a focus of on-going research. Many studies are now reporting the use of antibodies specific to Clusters of Differentiation (CD) cell surface antigens to identify tumour-initiating cell (TIC) populations in neural tumours. Medulloblastoma is one of the most common malignant brain tumours in children and despite a considerable amount of research investigating this tumour, the identity of the TICs, and the means by which such cells can be targeted remain largely unknown. Current prognostication and stratification of medulloblastoma using clinical factors, histology and genetic profiling have classified this tumour into four main subgroups: WNT, Sonic hedgehog (SHH), Group 3 and Group 4. Of these subgroups, SHH remains one of the most studied tumour groups due to the ability to model medulloblastoma formation through targeted deletion of the Shh pathway inhibitor Patched1 (Ptch1). Here we sought to utilise CD antibody expression to identify and isolate TIC populations in Ptch1 deleted medulloblastoma, and determine if these antibodies can help classify the identity of human medulloblastoma subgroups. Using a fluorescence-activated cell sorted (FACS) CD antibody panel, we identified CD24 as a marker of TICs in Ptch1 deleted medulloblastoma. CD24 expression was not correlated with markers of astrocytes or oligodendrocytes, but co-labelled with markers of neural progenitor cells. In conjunction with CD15, proliferating CD24+/CD15+ granule cell precursors (GCPs) were identified as a TIC population in Ptch1 deleted medulloblastoma. On human medulloblastoma, CD24 was found to be highly expressed on Group 3, Group 4 and SHH subgroups compared with the WNT subgroup, which was predominantly positive for CD15, suggesting CD24 is an important marker of non-WNT medulloblastoma initiating cells and a potential therapeutic target in human medulloblastoma. This study reports the use of CD24 and CD15 to isolate a GCP-like TIC population in Ptch1 deleted medulloblastoma, and suggests CD24 expression as a marker to help stratify human WNT tumours from other medulloblastoma subgroups. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease with no cure. Currently there are only two ALS drugs approved by the FDA, both with a limited therapeutic effect. In the search for drug candidates for ALS, we studied the effect of known stem cell mobilizing agents (treatment) and antimetabolite 5-fluorouracil (5-FU) (anti-treatment) in SOD1G93A model of ALS. Surprisingly, we found that anti-cancer drug 5-FU increases lifespan, delays the disease onset and improves motor performance in ALS mice. Although we were not able to demonstrate the mechanistic basis of the beneficial 5-FU action in ALS mice, our findings suggest that 5-FU or similar drugs are possible drug candidates for the treatment of motor neuron diseases through drug repurposing. [ABSTRACT FROM AUTHOR]

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method–with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AHR) is a ligand activated bHLH transcription factor that belongs to the Per-Arnt-Sim (PAS) superfamily of proteins involved in mediating responses to cellular environment regulating normal physiological and developmental pathways. The AHR binds a broad range of naturally derived and synthetic compounds, and plays a major role in mediating effects of certain environmental chemicals. Although our understanding of the physiological roles of the AHR in the immune system is evolving, there is little known about its role in hematopoiesis and hematopoietic diseases. Prior studies demonstrated that AHR null (AHR-KO) mice have impaired hematopoietic stem cell (HSC) function; they develop myeloproliferative changes in peripheral blood cells, and alterations in hematopoietic stem and progenitor cell populations in the bone marrow. We hypothesized mice lacking AHR expression only within hematopoietic cells (AHRVav1 mice) would develop similar changes. However, we did not observe a complete phenocopy of AHR-KO and AHRVav1 animals at 2 or 18 months of age. To illuminate the signaling mechanisms underlying the alterations in hematopoiesis observed in these mice, we sorted a population of cells highly enriched for HSC function (LSK cells: CD34-CD48-CD150+) and performed microarray analyses. Ingenuity Pathway and Gene Set Enrichment Analyses revealed that that loss of AHR within HSCs alters several gene and signaling networks important for HSC function. Differences in gene expression networks among HSCs from AHR-KO and AHRVav1 mice suggest that AHR in bone marrow stromal cells also contributes to HSC function. In addition, numerous studies have suggested a role for AHR in both regulation of hematopoietic cells, and in the development of blood diseases. More work is needed to define what these signals are, and how they act upon HSCs. [ABSTRACT FROM AUTHOR]

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG—two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos. [ABSTRACT FROM AUTHOR]

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells. [ABSTRACT FROM AUTHOR]

Background: Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called ‘brain sparing’. Therefore, we hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. Results: Here, we studied the function of p53 during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. We showed that p53 loss of function reduced animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, we showed that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. Conclusions: Using genetic approaches, we demonstrate that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals. [ABSTRACT FROM AUTHOR]

CD34+/CD133+- cells are a bone marrow derived stem cell population, which presumably contain vascular progenitor cells and are associated with improved vascular repair. In this study, we investigated whether the adhesion molecules ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule-1), E-selectin und L-selectin, which are involved in homing of vascular stem cells, are upregulated by CD34+/CD133+-stem cells from septic patients and would be associated with improved clinical outcome. Peripheral blood mononuclear cells from intensive care unit (ICU) patients with (n = 30) and without sepsis (n = 10), and healthy volunteers (n = 15) were isolated using Ficoll density gradient centrifugation. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin was detected on CD34+/CD133+-stem cells by flow cytometry. The severity of disease was assessed by the Simplified Acute Physiology Score (SAPS) II. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined by . The expression of VCAM-1, ICAM-1, E-selectin and L-selectin by CD34+/CD133+-stem cells was significantly upregulated in septic patients, and correlated with sepsis severity. Furthermore, high expression of VCAM-1 by CD34+/CD133+-stem cells revealed a positive association with mortalitiy (p<0.05). Furthermore, significantly higher serum concentrations of VEGF and Ang-2 were found in septic patients, however none showed a strong association with survival. Our data suggest, that VCAM-1 upregulation on CD34+/CD133+-stem cells could play a crucial role in their homing in the course of sepsis. An increase in sepsis severity resulted in both and increase in CD34+/CD133+-stem cells and VCAM-1-expression by those cells, which might reflect an increase in need for vascular repair. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is a leading cause of death in the western world, and its incidence increases with patient age. It is also known that with age there occur changes in the levels of certain hormones, including an increase in the secretion of pituitary gonadotropins (PtGs) as a result of the loss of gonadal hormone feedback. We recently reported that functional PtG receptors are expressed in human lung cancer cells, rhabdomyosarcoma cells, and malignant hematopoietic stem cells. Findings: Here we report for the first time that the receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are expressed in primary tumor samples isolated from CRC patients as well as in the established human CRC cell lines HTC116 and HTB37. Moreover, we also report that PtGs stimulate chemotaxis, adhesion, and proliferation of these cell lines. Conclusions: Our results suggest that PtGs play an important and underappreciated role in CRC pathogenesis, and we call for further studies to better define their role in gastrointestinal malignancies and their direct effect on putative CRC cancer stem cells. [ABSTRACT FROM AUTHOR]

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation. [ABSTRACT FROM AUTHOR]

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans are abundant on the cell surface and in the extracellular matrix and have important functions in matrix structure, cell-matrix interaction and signaling. The DS epimerases 1 and 2, encoded by Dse and Dsel, respectively, convert CS to a CS/DS hybrid chain, which is structurally and conformationally richer than CS, favouring interaction with matrix proteins and growth factors. We recently showed that Xenopus Dse is essential for the migration of neural crest cells by allowing cell surface CS/DS proteoglycans to adhere to fibronectin. Here we investigate the expression of Dse and Dsel in Xenopus embryos. We show that both genes are maternally expressed and exhibit partially overlapping activity in the eyes, brain, trigeminal ganglia, neural crest, adenohypophysis, sclerotome, and dorsal endoderm. Dse is specifically expressed in the epidermis, anterior surface ectoderm, spinal nerves, notochord and dermatome, whereas Dsel mRNA alone is transcribed in the spinal cord, epibranchial ganglia, prechordal mesendoderm and myotome. The expression of the two genes coincides with sites of cell differentiation in the epidermis and neural tissue. Several expression domains can be linked to previously reported phenotypes of knockout mice and clinical manifestations, such as the Musculocontractural Ehlers-Danlos syndrome and psychiatric disorders. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA lncR492 as lineage-specific inhibitor of neuroectodermal differentiation. Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells. [ABSTRACT FROM AUTHOR]

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site []. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR “hotspots” were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration. [ABSTRACT FROM AUTHOR]

The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R2 = 0.994). Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells (Skov-3/DDP, A549/DDP, MDA-MB-231, Huh-7) was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy. [ABSTRACT FROM AUTHOR]

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression. [ABSTRACT FROM AUTHOR]

Gastrointestinal motility disorders, severe variants in particular, remain a therapeutic challenge in pediatric surgery. Absence of enteric ganglion cells that originate from neural crest cells is a major cause of dysmotility. However, the limitations of currently available animal models of dysmotility continue to impede the development of new therapeutics. Indeed, the short lifespan and/or poor penetrance of existing genetic models of dysmotility prohibit the functional evaluation of promising approaches, such as stem cell replacement strategy. Here, we induced an aganglionosis model using topical benzalkonium chloride in a P0-Cre/GFP transgenic mouse in which the neural crest lineage is labeled by green fluorescence. Pathological abnormalities and functional changes in the gastrointestinal tract were evaluated 2–8 weeks after chemical injury. Laparotomy combined with fluorescence microscopy allowed direct visualization of the enteric neural network in vivo. Immunohistochemical evaluation further confirmed the irreversible disappearance of ganglion cells, glial cells, and interstitial cell of Cajal. Remaining stool weight and bead expulsion time in particular supported the pathophysiological relevance of this chemically-induced model of aganglionosis. Interestingly, we show that chemical ablation of enteric ganglion cells is associated with a long lifespan. By combining genetic labeling of neural crest derivatives and chemical ablation of enteric ganglion cells, we developed a newly customized model of aganglionosis. Our results indicate that this aganglionosis model exhibits decreased gastrointestinal motility and shows sufficient survival for functional evaluation. This model may prove useful for the development of future therapies against motility disorders. [ABSTRACT FROM AUTHOR]

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells. [ABSTRACT FROM AUTHOR]

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application. [ABSTRACT FROM AUTHOR]

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC) may be harvested from bone marrow (BMSC) and adipose (AMSC) tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma. Methods: Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs) were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic). Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza) and hAMSCs (Invitrogen) for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures. Results: Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines. Conclusions: Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting. [ABSTRACT FROM AUTHOR]

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs. [ABSTRACT FROM AUTHOR]

Background and Methods: In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1α and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions. Conclusions/Significance: Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process. Methods: We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease. Results: Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node-derived T cells in response to nonspecific polyclonal stimuli. Conclusions: The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS. [ABSTRACT FROM AUTHOR]

Cell-based tissue reconstruction is an important field of regenerative medicine. Stem and progenitor cells derived from tooth-associated tissues have strong regeneration potential. However, their in vivo application requires the development of novel scaffolds that will provide a suitable three-dimensional (3D) environment allowing not only the survival of the cells but eliciting their proliferation and differentiation. Our aim was to study the viability and differentiation capacity of periodontal ligament cells (PDLCs) cultured on recently developed biocompatible and biodegradable poly(aspartamide) (PASP)-based hydrogels. Viability and behavior of PDLCs were investigated on PASP-based hydrogels possessing different chemical, physical and mechanical properties. Based on our previous results, the effect of thiol group density in the polymer matrix on cell viability, morphology and differentiation ability is in the focus of our article. The chemical composition and 3D structures of the hydrogels were determined by FT Raman spectroscopy and Scanning Electron Microscopy. Morphology of the cells was examined by phase contrast microscopy. To visualize cell growth and migration patterns through the hydrogels, two-photon microscopy were utilized. Cell viability analysis was performed according to a standardized protocol using WST-1 reagent. PDLCs were able to attach and grow on PASP-based hydrogels. An increase in gel stiffness enhanced adhesion and proliferation of the cells. However, the highest population of viable cells was observed on the PASP gels containing free thiol groups. The presence of thiol groups does not only enhance viability but also facilitates the osteogenic direction of the differentiating cells. These cell-gel structures seem to be highly promising for cell-based tissue reconstruction purposes in the field of regenerative medicine. [ABSTRACT FROM AUTHOR]

Currently, no safe vaccine against leishmaniasis is available. So far, different control strategies against numerous reservoir hosts and biological vectors have not been environment-friendly and feasible. Hence, employing medicinal components and conventional drugs could be a promising approach to developing novel therapeutic alternatives. This study aimed to explore diallyl sulfide (DAS), a dynamic constituent of garlic, alone and in a mixture with meglumine antimoniate (MAT as standard drug) using in vitro and animal model experiments against Leishmania major stages. The binding affinity of DAS and four major defense elements of the immune system (iNOS, IFN-ɣ, IL-12, and TNF-α) was used to predict the predominant binding mode for molecular docking configurations. Herein, we conducted a broad range of experiments to monitor and assess DAS and MAT potential treatment outcomes. DAS, combined with MAT, displayed no cytotoxicity and employed a powerful anti-leishmanial activity, notably against the clinical stage. The function mechanism involved immunomodulation through the induction of Th1 cytokine phenotypes, triggering a high apoptotic profile, reactive oxygen species (ROS) production, and antioxidant enzymes. This combination significantly decreased cutaneous lesion diameter and parasite load in BALB/c mice. The histopathological findings performed the infiltration of inflammatory cells associated with T-lymphocytes, particularly CD4+ phenotypes, as determined by biochemical markers in alleviating the amastigote stage and improving the pathological changes in L. major infected BALB/c mice. Therefore, DAS and MAT deserve further advanced therapeutic development and should be considered as possible candidates for treating volunteer cases with cutaneous leishmaniasis in designing an upcoming clinical trial. [ABSTRACT FROM AUTHOR]

We investigated therapeutic potential of human tonsil-derived mesenchymal stem cells (TMSC) subcutaneously delivered to ovariectomized (OVX) mice for developing more safe and effective therapy for osteoporosis. TMSC were isolated from tonsil tissues of children undergoing tonsillectomy, and TMSC-embedded in situ crosslinkable gelatin-hydroxyphenyl propionic acid hydrogel (TMSC-GHH) or TMSC alone were delivered subcutaneously to the dorsa of OVX mice. After 3 months, three-dimensionally reconstructed micro-computed tomographic images revealed better recovery of the femoral heads in OVX mice treated with TMSC-GHH. Serum osteocalcin and alkaline phosphatase were also recovered, indicating bone formation only in TMSC-GHH-treated mice, and absence in hypercalcemia or other severe macroscopic deformities showed biocompatibility of TMSC-GHH. Additionally, visceral fat reduction effects by TMSC-GHH further supported their therapeutic potential. TMSC provided therapeutic benefits toward osteoporosis only when embedded in GHH, and showed potential as a supplement or alternative to current therapies. [ABSTRACT FROM AUTHOR]