Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Benchmarking and setting intervention targets for key cell count parameters

Herd Companion uses routine milk‐recording records to generate twelve‐month rolling averages that indicate performance trends. This article looks at Herd Somatic Cell Count (SCC) and four other SCC‐related parameters from 252 National Milk Records (NMR) recorded herds to assess how each parameter correlates with the Herd SCC. The analysis provides evidence for the importance of targeting individual cows with high SCC recordings (>200,000 cells/ml and >500,000 cells/ml) and/or individual cows with repeatedly high SCC recordings (chronic high SCC) and/or cows that begin lactation with a high SCC recording (dry period infection) in order to achieve bulk milk Herd SCC below 200,000 cells/ml.

Use of secondary somatic embryos promotes genetic fidelity in cryopreservation of cocoa (Theobroma cacao L.)

The inability to conserve cocoa (Theobroma cacao L.) germplasm via sced storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehydration based cryopreservation system has been developed for cocoa but because the somatic embryos used for freezing arise after a protracted period of callus culture there is concern about maintenance of genetic fidelity during the process. Microsatellite markers for seven of the 10 cocoa linkage groups were used to screen a population of 189 primary somatic embryo-derived emblings and the 43 secondary somatic embryos they gave rise to. Of the primary somatic embryos, 38.1% exhibited polymorphic microsatellite profiles while for secondary somatic embryos the frequency was 23.3%. The same microsatellite markers used to screen another population of 44 secondary somatic embryos cryopreserved through encapsulation-dehydration revealed no polymorphisms. Scanning electron microscopy showed the secondary somatic embryos were derived from cotyledonary epidermal cells rather than callus. The influence of embryo ontogeny on somaclonal variation is discussed.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.