A novel serum-free medium for the isolation, expansion and maintenance of stemness and tissue-specific markers of primary human periodontal ligament cells.

PURPOSE: Periodontal ligament (PDL) cell cultures are classically maintained in serum-containing media. However, unwanted side-effects of these conditions on cellular and molecular characteristics demand a serum-free alternative. Even though these limitations are well known and efforts for the development of adequate serum-free alternatives have been made, these approaches for replacement remained unsuccessful so far. This study aimed at developing a well-defined, serum-free formulation supporting both isolation from tissue samples and efficient expansion of PDL cells. Here, of particular focus was the perpetuation of tissue-characteristic markers detectable in primary tissues and of stemness features. BASIC PROCEDURES: Primary PDL cell cultures from generally healthy human donors (n = 3) were maintained in basal media N2B27 and E6 together with different concentrations of growth and attachment factors. Cell proliferation was recorded via microscopy and WST assay. Gene expression of RUNX2, Periostin, ALP, CD73, CD90, CD105, CD45, SOX10 and SOX2 was compared to primary PDL explants via qRT-PCR. Immunocytochemistry was performed for anti-CD105, SSEA-3, CD271, HNK1. Serum-containing sDMEM medium served as control. MAIN FINDINGS: N2B27 medium substituted with 25 ng/mL EGF, 25 ng/mL IGF1, 0.5 mg/mL Fetuin plus gelatine coating (designated N2B27-PDLsf) emerged as potent serum-free formulation ensuring adequate culture isolation and expansion. Here, PDL primary tissue signature markers RUNX2 and Periostin remained stable in N2B27-PDLsf compared to controls (229.0-fold ±101.0 and 83.2-fold ±9.6 increase). Additionally, stemness markers ALP and CD105 were significantly upregulated on transcriptional, and CD105 and SOX2 on protein level. PRINCIPAL CONCLUSIONS: This investigation identified a novel serum-free medium for the isolation, and expansion of primary human PDL cells with constantly high proliferation rates. Here, purity and stemness properties are maintained. Thus, N2B27-PDLsf represents a valid replacement for serum-containing media in PDL cultures.

Modeling psychiatric disorders: from genomic findings to cellular phenotypes.

Major programs in psychiatric genetics have identified >150 risk loci for psychiatric disorders. These loci converge on a small number of functional pathways, which span conventional diagnostic criteria, suggesting a partly common biology underlying schizophrenia, autism and other psychiatric disorders. Nevertheless, the cellular phenotypes that capture the fundamental features of psychiatric disorders have not yet been determined. Recent advances in genetics and stem cell biology offer new prospects for cell-based modeling of psychiatric disorders. The advent of cell reprogramming and induced pluripotent stem cells (iPSC) provides an opportunity to translate genetic findings into patient-specific in vitro models. iPSC technology is less than a decade old but holds great promise for bridging the gaps between patients, genetics and biology. Despite many obvious advantages, iPSC studies still present multiple challenges. In this expert review, we critically review the challenges for modeling of psychiatric disorders, potential solutions and how iPSC technology can be used to develop an analytical framework for the evaluation and therapeutic manipulation of fundamental disease processes.

Disease-specific iPS cell models in neuroscience.

Neurodegenerative diseases are a heterogeneous group of sporadic or familial disorders of the nervous system that mostly lead to a progressive loss of neural cells. A major challenge in studying the molecular pathomechanisms underlying these disorders is the limited experimental access to disease-affected human nervous system tissue. In addition, considering that the molecular disease initiation occurs years or decades before the symptomatic onset of a medical condition, these tissues mostly reflect only the final phase of the disease. To overcome these limitations, various model systems have been established based on gain and loss-of-function studies in transformed cell lines or transgenic animal models. Although these approaches provide valuable insights into disease mechanisms and development they often lack physiological protein expression levels and a humanized context of molecular interaction partners. The generation of human induced pluripotent stem (hiPS) cells from somatic cells provides access to virtually unlimited numbers of patient-specific cells for modeling neurological disorders in vitro. In this review, we focus on the current progress made in hiPS cell-based modeling of neurodegenerative diseases and discuss recent advances in the quality assessment of hiPS cell lines.

Cognitive therapy for social phobia: individual versus group treatment.

Cognitive-behavioural group treatment is the treatment of choice for social phobia. However, as not all patients benefit, an additional empirically validated psychological treatment would be of value. In addition, few studies have examined whether a group treatment format is more effective than an individual treatment format. A randomized controlled trial addressed these issues by comparing individual cognitive therapy, along the lines advocated by Clark and Wells (Clark, D.M. and Wells, A., 1995. A cognitive model of social phobia. In: R. G. Heimberg, M. Liebowitz, D. Hope and F. Schneier (Eds.), Social Phobia: Diagnosis, assessment, and treatment (pp. 69-93). New York: Guilford.), with a group version of the treatment and a wait-list control condition. 71 patients meeting DSM-IV criteria for social phobia participated in the trial, 65 completed the posttreatment assessment and 59 completed a six-month follow-up. Social phobia measures indicated significant pretreatment to posttreatment improvement in both individual and group cognitive therapy. Individual cognitive therapy was superior to group cognitive therapy on several measures at both posttreatment and follow-up. The effects of treatment on general measures of mood and psychopathology were less substantial than the effects on social phobia. The results suggest that individual cognitive therapy is a specific treatment for social phobia and that it's effectiveness may be diminished by delivery in a group format.

Altered directionality in the Cre-LoxP site-specific recombination pathway.

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.