FAM96A和FAM96B结构与功能的研究进展

96序列相似的家庭成员A和B（family with sequence similarity 96 member A and B,FAM96A和FAM96B）是属于MIP18（MMS19-interacting protein of 18 kD）家族的2个高度保守的同源蛋白,MIP18是与有丝分裂纺锤体相关的MMDX（MMS19-MIP18-XPD）复合体的亚基。研究表明,FAM96A和FAM96B在人胃肠道间质瘤、结肠癌、肝癌、胃癌和乳腺癌等多种肿瘤组织中的表达显著降低,提示其可能是作为潜在的抑癌基因参与肿瘤的发生发展,但目前关于FAM96A和FAM96B在肿瘤发生发展过程中的作用机理并不十分清楚。此外,研究发现FAM96A和FAM96B可通过与其他不同的蛋白质相互作用在体内发挥多种不同的功能。因此,就目前对于FAM96A和FAM96B结构和功能的研究所取得的进展进行了回顾与总结,并对其在肿瘤发生发展中的分子机制和相互作用蛋白鉴定的研究前景进行了展望,以期为临床上将FAM96A和FAM96B作为新的肿瘤诊断标志物和治疗靶点奠定基础,并为揭示二者在体内更多的新功能提供依据。     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

Progress in the use of dental pulp stem cells in regenerative medicine

The field of tissue engineering is emerging as a multidisciplinary area with promising potential for regenerating new tissues and organs. This approach requires the involvement of three essential components: stem cells, scaffolds and growth factors. To date, dental pulp stem cells have received special attention because they represent a readily accessible source of stem cells. Their high plasticity and multipotential capacity to differentiate into a large array of tissues can be explained by its neural crest origin, which supports applications beyond the scope of oral tissues. Many isolation, culture and cryopreservation protocols have been proposed that are known to affect cell phenotype, proliferation rate and differentiation capacity. The clinical applications of therapies based on dental pulp stem cells demand the development of new biomaterials suitable for regenerative purposes that can act as scaffolds to handle, carry and implant stem cells into patients. Currently, the development of xeno-free culture media is emerging as a means of standardization to improve safe and reproducibility. The present review aims to describe the current knowledge of dental pulp stem cells, considering in depth the key aspects related to the characterization, establishment, maintenance and cryopreservation of primary cultures and their involvement in the multilineage differentiation potential. The main clinical applications for these stem cells and their combination with several biomaterials is also covered.     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

Wnt signaling reprograms metabolism in dental pulp stem cells

Human dental pulp stem cells (DPSCs) can differentiate to a wide range of different cell lineages, and share some gene expression and functional similarities with pluripotent stem cells. The stemness of DPSCs can also be pharmacologically enhanced by the activation of canonical Wnt signaling. Here, we examined the metabolic profile of DPSCs during reprogramming linked to Wnt activation, by a short (48 hr) exposure to either the GSK3-β inhibitor BIO (6-bromoindirubin-3´-oxine) or human recombinant protein WNT-3A. Both treatments largely increased glucose consumption, and induced a gene overexpression of pyruvate and mitochondrial acetyl-coA producing enzymes, thus activating mitochondrial tricarboxylic acid cycle (TCA) metabolism in DPSCs. This ultimately led to an accumulation of reducing power and a mitochondrial hyperpolarization in DPSCs. Interestingly, Nile Red staining showed that lipid fuel reserves were being stored in Wnt-activated DPSCs. We associate this metabolic reprogramming with an energy-priming state allowing DPSCs to better respond to subsequent high demands of energy and biosynthesis metabolites for cellular growth. These results show that enhancement of the stemness of DPSCs by Wnt activation comes along with a profound metabolic remodeling, which is distinctly characterized by a crucial participation of mitochondrial metabolism.     

Downregulation of heat shock protein B8 decreases osteogenic differentiation potential of dental pulp stem cells during in vitro proliferation

Objectives

Tissue‐derived stem cells, such as dental pulp stem cells (DPSCs), reduce differentiation capability during in vitro culture. We found that cultured DPSCs reduce expression of heat shock protein B8 (HspB8) and GIPC PDZ domain containing family member 2 (Gipc2). Our objectives were to evaluate the changes in DPSC composition during in vitro proliferation and to determine whether HspB8 and Gipc2 have function in differentiation potential of DPSCs.

Materials and Methods

Different passages of rat DPSCs were evaluated for changes in CD90+ and/or CD271+ stem cells and changes in osteogenic potential. Real‐time RT‐PCR and immunostaining were conducted to determine expression of HspB8 and Gipc2. Expression of the genes in DPSCs was knocked down by siRNA, followed by osteogenic induction to evaluate the function of the genes.

Results

About 90% of cells in the DPSC cultures were CD90+ and/or CD271+ cells without dramatic change during in vitro proliferation. The DPSCs at passages 3 to 5 (P3 to P5) possess strong osteogenic potential, but such potential was greatly reduced at later passages. Expression of HspB8 and Gipc2 was significantly reduced at P11 versus P3. Knock‐down of HspB8 expression abolished osteogenic potential of the DPSCs, but knock‐down of Gipc2 had no effect.

Conclusions

CD90+ and CD271+ cells are the major components of DPSCs in in vitro culture. High‐level expression of HspB8 was critical for maintaining differentiation potential of DPSCs.

     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

The role of lysyl oxidase-like 2 in the odontogenic differentiation of human dental pulp stem cells

Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Identification of FAM96B as a novel prelamin A binding partner

Prelamin A accumulation causes nuclear abnormalities, impairs nuclear functions, and eventually promotes cellular senescence. However, the underlying mechanism of how prelamin A promotes cellular senescence is still poorly understood. Here we carried out a yeast two-hybrid screen using a human skeletal muscle cDNA library to search for prelamin A binding partners, and identified FAM96B as a prelamin A binding partner. The interaction of FAM96B with prelamin A was confirmed by GST pull-down and co-immunoprecipitation experiments. Furthermore, co-localization experiments by fluorescent confocal microscopy revealed that FAM96B colocalized with prelamin A in HEK-293 cells. Taken together, our data demonstrated the physical interaction between FAM96B and prelamin A, which may provide some clues to the mechanisms of prelamin A in premature aging.