Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-ß.

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-ß(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TßRII, ALK5 as well as TGF-ß1 and the ßA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-ß1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-ß1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-ß signaling by either SB431542 or anti-TGF-ß antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-ß antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-ß/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-ß/Smad3, and to a lesser extent, activin/Smad2 signaling.

Isolation and characterisation of human gingival margin-derived STRO-1/MACS(+) and MACS(-) cell populations.

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS⁺) and STRO-1-negative (MACS⁻) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS⁺ and MACS⁻ cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS⁺ and MACS(-) cell fractions showed plastic adherence. MACS⁺ cells, in contrast to MACS⁻ cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS⁺ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS⁻ cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS⁺ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS⁻ cells demonstrated slight osteogenic potential. Unstimulated MACS⁺ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS⁻ cells demonstrated higher expression of osteonectin (P<0.05; Mann-Whitney). The present study is the first to compare gingival MACS⁺ and MACS⁻ cell populations demonstrating that MACS⁺ cells, in contrast to MACS⁻ cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS⁺ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS⁺ cells are a unique renewable source of multipotent stem/progenitor cells.

Toothbrushing education via a smart software visualization system.

BACKGROUND: The aim of this study is to evaluate the efficiency of a recently developed smart digital toothbrush monitoring and training system (DTS) in terms of correct brushing motion and grip axis orientation in an at-home environment. METHODS: Twenty-one participants (11 test individuals [DTSG] and 10 control individuals [COG]) received instructions on the modified Bass technique (MBT) after their toothbrushing performance was monitored and they received professional tooth cleaning (T0). After 36 hours (T1), without mechanical oral hygiene measures, plaque and gingival indices were recorded, and the brushing technique was reviewed. After randomization, participants individually performed oral hygiene for 6 weeks (T2) with the provided oral hygiene kits. The DTSG group additionally used DTS. During the following 8 weeks (T3), participants used their original brushing devices without any additional interference. Investigators at each visit were masked regarding group identity. Data were statistically evaluated using Mann-Whitney U, Friedman, Wilcoxon, and paired tests and Pearson correlation. RESULTS: At T0, 27.27% of DTSG participants used the MBT correctly (COG, 50%), increasing to 54.55% (COG, 60%) after professional instruction (T1) and further to 90.91% at T2 (COG, 60%) (P <0.001). Plaque scores were reduced in DTSG (P <0.05). At T3, 80% of the DTSG (COG, 40%) totally adopted the MBT (P <0.05). The plaque scores on buccal surfaces of the DTSG showed an additional slight improvement between T2 and T3, in contrast to a decline on oral surfaces (P <0.001). At T2 and T3, the DTSG brushed >120 seconds (COG, 90% and 50%) (P <0.05). CONCLUSION: Apparently, the tested DTS effectively improves the brushing technique and leads to a prolonged learning effect, including improved oral hygiene.