Effects of ALK-5 inhibitor and transforming growth factor-Beta1 in the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into epithelial-like cells

Stem cells from human exfoliated deciduous teeth (SHED) are capable to divide, differentiate and mature to the specific types of cells as well as to replenish themselves to regenerate other living cells. Previous study has showed that SHEDs could differentiate into epithelial-like cells. Yet, the effects of Transforming Growth Factor-Beta1 (TGF-β1) or activin like kinase 5 (ALK-5) inhibitor on SHEDs remain unexplored. Thus, the present study was aimed to investigate the effects of TGF-β1 and ALK-5 inhibitor on SHEDs cultured in Keratinocyte Growth Medium (KGM) on its potential to differentiate into epithelial-like cells employing MTT [(3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] and alamar blue assays, cell morphology analysis, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique and flow cytometry. A serial dilution of TGF-β1 (0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 ng/ml) and ALK-5 inhibitor (0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 μM) concentration was carried out to determine the cell cytotoxicity for each treatment using MTT assay for 72 hours. Afterwards, the three selected concentrations for TGF-β1 (0.3125, 0.625, and 1.25 ng/ml) and ALK-5 inhibitor (0.156, 0.3125, and 0.625 μM) were analysed using alamar blue assay on day 1, 3, 5, 7, and 10 and was done in alpha Minimum Essential Medium (α-MEM) to determine the population doubling time (PDT). The study wasfurther investigated with observation of the cell morphological changes on SHED cultured in KGM only, and with selected concentration of TGF-β1 or ALK-5 inhibitor on day 1, 3, 7, 14, and 21. RNA isolation of SHED culture in three different conditions were harvested at day 1, 3, 7, 14, and 21. Then, the gene expression analysis of stem cell, epithelial cell, and specific genes involved in TGF-β signalling were identified during the differentiation process using Two-step RT-PCR. Apart from that, protein expression analysis of epithelial markers was also determined using flow cytometer on day 1 and 21. Based on MTT assay, 0.3125, 0.625, and 1.25 ng/ml TGF-β1 and 0.156, 0.3125, and 0.625 μM ALK-5 inhibitor showed less cytotoxicity effects (more than 50%) and were selected for proliferation assay. The shortest PDT was represented by 1.25 ng/ml TGF-β1 (75 hours) and 0.625 μM ALK-5 inhibitor (68 hours) and these concentrations including cells in KGM only, showed there were cell morphological changes compared to control. The gene expression profile showed an absence of epithelial markers E-cadherin, ΔNp63, and Keratin5 for gene, and E-cadherin and pancytokeratin for protein expression indicated that the culture conditions unable to induce the differentiation process into epithelial-like cells. However, the presence of stem cell markers (NANOG, nestin, Rex1, and vimentin) and specific molecules involved in TGF-β signalling (TGFβR1, TGFβ1, Smad3, and Smad4) indicated that the cell culture in three different condition induced epithelial to mesenchymal transition (EMT) since the presence of TGFβ1 and Smad3 in TGF-β signalling that have been associated with EMT. Thus, KGM was unable to fully differentiate SHED into epithelial-like cells and hence, SHED were incapable to undergo mesenchymal to epithelial transition (MET)

Expression analysis of notch signaling pathway molecules in SHED cultured in keratinocyte growth medium

Aim: To detect the expression of molecules associated with Notch signaling pathway in stem cells from human exfoliated deciduous teeth (SHED) cultured in specific differentiation medium, namely, keratinocyte growth medium (KGM). Methods: RNA was extracted from SHED harvested on day 1, 3 and 7. RNA was reverse-transcribed to obtain the cDNA and then proceeded with PCR using specific primers for the Notch signaling pathway molecules (Notch1, Jagged-1, Jagged-2 and, Hes1) as well as stem cell marker (Nanog). PCR products were electrophoresed on a 2% agarose gel and stained with SYBR green. Results: Notch-1 was highly expressed in SHED cultured in KGM and showed increase in density as the days progressed, while Jagged-1 showed a decrease. Jagged-2 on the other hand, showed a slight increase on day 3 followed by a decrease on day 7. However, Hes-1 was not expressed in SHED cultured in KGM. Nanog showed expression only on day 3 and gradually increased in expression on day 7. Conclusions: Notch signaling pathway associated molecules; Notch-1, Jagged-1, Jagged-2, and stem cell marker Nanog are expressed in SHED cultured in KGM which may be involved in the differentiation into epithelial-like cells in human dental pulp tissues. Keywords: receptors, notch; gene expression; stem cells; tooth, deciduous; culture media

Effect of perivitelline fluid from horseshoe crab on the expression of cell cycle regulatory genes in human dental pulp stem cells

Abstract Perivitelline fluid (PVF) of the horseshoe crab embryo has been reported to possess an important role during embryogenesis by promoting cell proliferation. This study aims to evaluate the effect of PVF on the expression of cell cycle regulatory genes from human dental pulp stem cells (DPSCs) between different cell passages viz. 4, 5, 6. The cells were treated with a single dose of PVF (26.89 mg/ml) PVF. Gene expression was quantified for CDKNA2A, PTEN, MDM2 and TP53 genes using reverse transcriptase PCR. CDKN2A and MDM2 expression for treated and untreated DPSCs, expressed a similar pattern of expression. The higher expression of CDKN2A showed that the treatment increased cell proliferation and prevented cell senescence. DPSCs with PVF treatment showed increased expression of MDM2 at passage 4 and drastically declined expression at passage 5 and slightly increased at passage 6. TP53 expression of DPSCs treated group showed a higher expression compared to untreated group. On the other hand, the expression of PTEN in DPSCs treated group started to increase from passage 5 to 6. However, on the whole, the PTEN expression was higher than the untreated group in all the passages studied here. The results showed that PVF could enhance cell cycle regulatory gene expression in DPSCs as indicated by the higher expression of all the genes considered in this study at different cell passages in the treated group compared to the untreated group. Mann Whitney test was utilized to determine the significance of cell cycle regulatory genes expression between treated and untreated group. Significant difference in expression of genes between the treated and untreated groups were found at all passages except for CDKN2A gene whereby, its expression was not significantly different at passage 5 though it did express slightly higher in PVF treated DPSCs. Keywords: cell cycle, dental pulp stem cells, gene expression, horseshoe crab, perivitelline fluid

TGFβ-1 and ALK5 inhibitor treatment affects expressions of TGF β signalling pathway associated molecules in SHED cultured in keratinocyte growth medium

Stem cells from human exfoliated deciduous teeth (SHED) can be induced to differentiate into epithelial-like cells when cultured in a differentiation medium. Transforming growth factor beta (TGF-β) signalling pathway plays an important regulatory function in epithelial proliferation and differentiation.TGF-β1 functions through a complex mechanism consisting of TGFβ type II and type I (ALK5) receptor to initiate cellular processes which could be inhibited by SB431542 molecule (ALK5 inhibitor). This study aims to analyse the expression of TGF-β signalling pathway associated molecules, ALK5 and Smad4, in SHED cultured in keratinocyte growth medium (KGM), treated with exogenous TGF-β1 and ALK5 inhibitor. SHED was cultured in KGM treated with TGF-β1 or ALK-5 inhibitor. RNA was extracted on cells harvested on day 1, 3, 7, 14 and 21 and subjected to reverse transcriptase-PCR using specific primers for ALK5, Smad4 and stem cell marker (Nanog). PCR products were electrophoresed on a 2% agarose gel stained with SYBR green. Nanog expression was maintained from day1 to 14 in all samples but was down regulated at day 21. ALK5 was expressed in most of the samples analysed from day 1 to 14 and down regulated at day 21. Smad4 expression was up regulated at day 1 to 3, down regulated from day 7 to 14 and lost its expression at day 21. TGFβ-1 and ALK5 inhibitor treatment affects the expressions of Nanog, ALK5 and Smad4 in SHED cultured in KGM. The involvement of these molecules during the differentiation process of SHED into epithelial-like cells will contribute further in the fundamental aspects of tissue regeneration research and highlights the importance of dental pulp stem cell technology

A review of open-source image analysis tools for mammalian cell culture: algorithms, features and implementations

Cell culture is undeniably important for multiple scientific applications, including pharmaceuticals, transplants, and cosmetics. However, cell culture involves multiple manual steps, such as regularly analyzing cell images for their health and morphology. Computer scientists have developed algorithms to automate cell imaging analysis, but they are not widely adopted by biologists, especially those lacking an interactive platform. To address the issue, we compile and review existing open-source cell image processing tools that provide interactive interfaces for management and prediction tasks. We highlight the prediction tools that can detect, segment, and track different mammalian cell morphologies across various image modalities and present a comparison of algorithms and unique features of these tools, whether they work locally or in the cloud. This would guide non-experts to determine which is best suited for their purposes and, developers to acknowledge what is worth further expansion. In addition, we provide a general discussion on potential implementations of the tools for a more extensive scope, which guides the reader to not restrict them to prediction tasks only. Finally, we conclude the article by stating new considerations for the development of interactive cell imaging tools and suggesting new directions for future research

Expression analysis of Notch signaling pathway molecules in SHED cultured in keratinocyte growth medium

detect the expression of molecules associated with Notch signaling pathway in stem cells from human exfoliated deciduous teeth (SHED) cultured in specific differentiation medium, namely, keratinocyte growth medium (KGM). Methods: RNA was extracted from SHED harvested on day 1, 3 and 7. RNA was reverse-transcribed to obtain the cDNA and then proceeded with PCR using specific primers for the Notch signaling pathway molecules (Notch1, Jagged-1, Jagged-2 and, Hes1) as well as stem cell marker (Nanog). PCR products were electrophoresed on a 2% agarose gel and stained with SYBR green. Results: Notch-1 was highly expressed in SHED cultured in KGM and showed increase in density as the days progressed, while Jagged-1 showed a decrease. Jagged-2 on the other hand, showed a slight increase on day 3 followed by a decrease on day 7. However, Hes-1 was not expressed in SHED cultured in KGM. Nanog showed expression only on day 3 and gradually increased in expression on day 7. Conclusions: Notch signaling pathway associated molecules; Notch-1, Jagged-1, Jagged-2, and stem cell marker Nanog are expressed in SHED cultured in KGM which may be involved in the differentiation into epithelial-like cells in human dental pulp tissue

Characterisation and selection of freshwater cyanobacteria for phycobiliprotein contents

Some cyanobacteria species have a high capacity for accumulating phycobiliprotein contents in their cells. However, there is a lack of information on screening tropical freshwater cyanobacteria, particularly phycobiliproteins. In addition, it is unclear which characteristics of cyanobacteria (morphological and/or growth) could affect phycobiliprotein contents. This study aimed to screen and characterise Malaysian indigenous freshwater cyanobacteria for the growth, biomass, and pigment contents and determine the major characteristic that contributed to the variation of phycobiliproteins. The surface/volume (S/V) ratio, specific growth rate, biomass productivity, and pigment contents of the isolated cyanobacteria were analysed. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to distinguish the factor responsible for phycobiliprotein variations in cyanobacteria. For the phycobiliprotein contents, Arthrospira sp., Pseudanabaena sp., and Synechococcus elongatus showed significantly higher (p < 0.05) amounts of cyanobacterial phycocyanin (C-PC), phycoerythrin (C-PE), and allophycocyanin (C-APC), respectively, than other studied cyanobacteria. This study showed no apparent trend of similarity and difference between the unicellular and filamentous cyanobacteria. In addition, carotenoid contents demonstrated a positive correlation with total phycobiliproteins, C-PC and C-APC. Based on the current findings, Arthrospira sp., Pseudanabaena sp. and Synechococcus elongatus might be the promising candidate to be the C-PC, C-PE and C-APC sources, respectively, for commercial production purposes. The selection of optimal cyanobacterial strain is crucial for efficient phycobiliprotein production. This study underlined the potential of freshwater cyanobacteria in producing respective and total phycobiliproteins. Future studies such as the optimization process should be adopted to improve the phycobiliprotein production of these cyanobacteria significantly

Fucoxanthin production of microalgae under different culture factors: a systematic review

Fucoxanthin is one of the light-harvesting pigments in brown microalgae, which is increasingly gaining attention due to its numerous health-promoting properties. Currently, the production of microalgal fucoxanthin is not yet feasible from an economic perspective. However, the cultivation of microalgae at favourable conditions holds great potential to increase the viability of this fucoxanthin source. Hence, this study aimed to review the fucoxanthin production of microalgae under different conditions systematically. A literature search was performed using the Web of Science, Scopus and PubMed databases. A total of 188 articles were downloaded and 28 articles were selected for the current review by two independent authors. Microalgae appeared to be a more reliable fucoxanthin source compared to macroalgae. Overall, a consensus fucoxanthin production condition was obtained and proposed: light intensity ranging from 10 to 100 µmol/m2/s could achieve a higher fucoxanthin content. However, the optimal light condition in producing fucoxanthin is species-specific. The current review serves as an antecedent by offering insights into the fucoxanthin-producing microalgae response to different culture factors via a systematic analysis. With the current findings and recommendations, the feasibility of producing fucoxanthin commercially could be enhanced and possibly achieve practical and sustainable fucoxanthin production