Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non‐collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. Results. DFCs were isolated from tooth germs of SD (Sprague—Dawley) rats and then co‐cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization‐associated proteins and genes were up‐regulated after induction, whereas the expression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum‐like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. Conclusions. dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.     

Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages.

BACKGROUND INFORMATION: Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non-collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. RESULTS: DFCs were isolated from tooth germs of SD (Sprague-Dawley) rats and then co-cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization-associated proteins and genes were up-regulated after induction, whereas the expression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum-like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. CONCLUSIONS: dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.     

Nuclear heterogeneity and proliferative capacity of human adipose derived MSC-like cells

Adipose derived stem cells (ADSCs) are MSC-like cells which could be easily used for regenerative medicine. Here, the morphology and proliferative capacity of human ADSCs is discribed. ADSCs were analyzed after one month of cultivation at a density of 10 cells/cm2. 21 colonies were counted. Few atypical cells (huge nuclei and cytoplasm) were found in 9 out of 17 colonies analyzed. ANOVA demonstrated that colonies also differed (P = 0.0025) in nuclei dimensions and scatter in the dimensions in each colony. Nuclei dimensions and cell density logarithms correlated in reverse proportion (-0.7; P = 0.002). Thus, ADSCs were heterogeneous and represented two types of cells: small highly proliferative and large low proliferative cells. Cell heterogeneity observed in some colonies might be due to cells registered at different cell cycle phases. Stable and typical morphology, colony-formation capability and high proliferative capacity of cells indicate visceral adipose tissue as a rich source of ADSCs.     

Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro

Objectives: Schwann cell (SC) transplantation is a promising therapy for peripheral nerve transaction, however, clinical use of SCs is limited due to their very limited availability. Adipose‐derived stem cells (ADSCs) have been identified as an alternative source of adult stem cells in recent years. The aim of this study was to evaluate the feasibility of using ADSCs as a source of stem cells for differentiation into Schwann‐like cells by an indirect co‐culture approach, in vitro. Materials and methods: Multilineage differentiation potential of the obtained ADSCs was assayed by testing their ability to differentiate into osteoblasts and adipocytes. The ADSCs were co‐cultured with SCs to be induced into Schwann‐like cells through proximity, using a Millicell system. Expression of typical SC markers S‐100, GFAP and P75NTR of the treated ADSCs was determined by immunocytochemical staining, western blotting and RT‐PCR. Myelination capacity of the differentiated ADSCs (dADSCs) was evaluated in dADSC/dorsal root ganglia neuron (DRGN) co‐cultures. Results: The treated ADSCs adopted a spindle shaped‐like morphology after co‐cultured with SCs for 6 days. All results of immunocytochemical staining, western blotting and RT‐PCR showed that the treated cells expressed S‐100, GFAP and P75NTR, indications of differentiation. dADSCs could form Schwann‐like cell myelin in co‐culture with DRGNs. Undifferentiated ADSCs (uADSCs) did not form myelin compared to DRGNs cultured alone, but could produce neurite extension. Conclusions: These results demonstrate that this indirect co‐culture microenvironment could induce ADSCs to differentiate into Schwann‐like cells in vitro, which may be beneficial for treatment of peripheral nerve injuries in the near future.     

Effect of Calcitonin Gene-Related Peptide on the Neurogenesis of Rat Adipose-Derived Stem Cells In Vitro

Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.     

Wnt3a promotes differentiation of human bone marrow-derived mesenchymal stem cells into cementoblast-like cells

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.     

Effect of CGRP-Adenoviral Vector Transduction on the Osteoblastic Differentiation of Rat Adipose-Derived Stem Cells

Calcitonin gene-related peptide (CGRP) promotes osteoblast recruitment and osteogenic activity. However, no evidence suggests that CGRP could affect the differentiation of stem cells toward osteoblasts. In this study, we genetically modified adipose-derived stem cells (ADSCs) by introducing the CGRP gene through adenoviral vector transduction and investigated on cellular proliferation and osteoblast differentiation in vitro and osteogenesis in vivo as well. For the in vitro analyses, rat ADSCs were transducted with adenoviral vectors containing the CGRP gene (Ad-CGRP) and were cultured in complete osteoblastic medium. The morphology, proliferative capacity, and formation of localized regions of mineralization in the cells were evaluated. The expression of alkaline phosphatase (ALP) and special markers of osteoblasts, such as Collagen I, Osteocalcin (BPG) and Osteopontin (OPN), were measured by cytochemistry, MMT, RT-PCR, and Western blot. For the in vivo analyses, the Ad-CGRP-ADSCs/Beta-tricalcium phosphate (β-TCP) constructs were implanted in rat radial bone defects for 12 weeks. Radiography and histomorphology evaluations were carried out on 4 weeks and 12 weeks. Our analyses indicated that heterogeneous spindle-shaped cells and localized regions of mineralization were formed in the CGRP-transduced ADSCs (the transduced group). A higher level of cellular proliferation, a high expression level of ALP on days 7 and 14 (p<0.05), and increased expression levels of Collagen I, BPG and OPN presented in transduced group (p<0.05). The efficiency of new bone formation was dramatically enhanced in vivo in Ad-CGRP-ADSCs/β-TCP group but not in β-TCP group and ADSCs/β-TCP group. Our results reveal that ADSCs transduced with an Ad-CGRP vector have stronger potential to differentiate into osteoblasts in vitro and are able to regenerate a promising new tissue engineering bone in vivo. Our findings suggest that CGRP-transduced ADSCs may serve as seed cells for bone tissue engineering and provide a potential way for treating bone defects.     

Dentin sialoprotein and dentin phosphoprotein overexpression during amelogenesis

The gene for dentin sialophosphoprotein produces a single protein that is post-translationally modified to generate two distinct extracellular proteins: dentin sialoprotein and dentin phosphoprotein. In teeth, dentin sialophosphoprotein is expressed primarily by odontoblast cells, but is also transiently expressed by presecretory ameloblasts. Because of this expression profile it appears that dentin sialophosphoprotein contributes to the early events of amelogenesis, and in particular to those events that result in the formation of the dentino-enamel junction and the adjacent "aprismatic" enamel. Using a transgenic animal approach we have extended dentin sialoprotein or dentin phosphoprotein expression throughout the developmental stages of amelogenesis. Overexpression of dentin sialoprotein results in an increased rate of enamel mineralization, however, the enamel morphology is not significantly altered. In wild-type animals, the inclusion of dentin sialoprotein in the forming aprismatic enamel may account for its increased hardness properties, when compared with bulk enamel. In contrast, the overexpression of dentin phosphoprotein creates "pitted" and "chalky" enamel of non-uniform thickness that is more prone to wear. Disruptions to the prismatic enamel structure are also a characteristic of the dentin phosphoprotein overexpressing animals. These data support the previous suggestion that dentin sialoprotein and dentin phosphoprotein have distinct functions related to tooth formation, and that the dentino-enamel junction should be viewed as a unique transition zone between enamel and the underlying dentin. These results support the notion that the dentin proteins expressed by presecretory ameloblasts contribute to the unique properties of the dentino-enamel junction.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.     

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin

Background

Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice.

原儿茶酸促进人脂肪干细胞体外增殖的研究

为了寻找能够促进干细胞增殖的药物,观察了中药益智仁（Alpinia oxyphylln）中提取的原儿茶酸对人脂肪干细胞体外增殖的影响,并对其作用机制进行了初步的探讨．人脂肪干细胞能在体外分化为神经元样细胞,并对凋亡的PC-12细胞起到保护作用．原儿茶酸能够促进人脂肪干细胞的增殖,且呈现明显的剂量依赖性和时间依赖性．流式细胞术检测细胞DNA含量的结果显示,原儿茶酸处理组细胞S期所占比例明显增加,其中,1．5mmol／L原儿茶酸处理组细胞S期所占比例与对照组相比增加2倍以上．同时,该组细胞G2／M期所占比例明显增加,G0／G1期所占比例明显下降．蛋白质免疫印迹结果显示,1．5mmol／L原儿茶酸处理组细胞周期素D1（cyclinD1）的表达明显升高．cyclin D1-siRNA转染显著抑制了原儿茶酸对人脂肪干细胞体外增殖的促进作用．流式细胞术检测细胞表面标志物,成骨诱导和脂肪诱导的结果显示,原儿茶酸处理后,人脂肪干细胞仍保持间充质干细胞多分化潜能的特性．上述结果提示,原儿茶酸有可能在人脂肪干细胞介导的干细胞移植治疗中发挥作用．     

Regulation of adipose‐tissue‐derived stromal cell orientation and motility in 2D‐ and 3D‐cultures by direct‐current electrical field

Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane‐protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose‐tissue‐derived stromal cells (ADSCs) in 2D‐culture on plastic culture dishes and in 3D‐culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L‐lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological‐strength EFs in a homemade EF‐bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time‐lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D‐culture aligned vertically to EF vector and kept a good cell survival rate. In 3D‐culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D‐culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.     

Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.     

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents

BMSCs (bone‐marrow‐derived mesenchymal stem cells) and ADSCs (adipose tissue‐derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis‐related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose‐dependent reduction in cell viability compared with 5‐FU (5‐fluorouracil) treatment. Different expression patterns of apoptosis‐related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy‐induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.     

Effect of cell senescence on the impedance measurement of adipose tissue-derived stem cells

Label-free and real-time monitoring of stem cells based on electrical impedance measurement is increasingly utilized for the quality control of the isolated stem cells to be used in stem cell-based tissue therapy or regenerative medicine. In spite of that the proliferative capacity and multipotency of stem cells are dependent on the type and age of the source tissue, however, the effect of the cell senescence on the impedance measurement of stem cells has not yet been studied. We investigated whether the senescence of adipose tissue-derived stem cells (ADSCs) can be detected by electrical impedance spectroscopy. For this, ADSCs at passage 9 and 31 were prepared and those genetic characteristics and growth kinetics were evaluated by quantitative polymerase chain reaction and cell counting. While the identified ADSCs were grown on the indium tin oxide electrodes, the impedance spectra were measured and interpreted by fitting analysis with an equivalent circuit model. ADSCs at passage 9 adhered on the electrode were small and spindle-shaped whereas the cells at passage 31 were flattened and larger than younger cells. At the beginning of culture time when the cell adhesion occurred, the resistance at 4.6 kHz of passage 31 cells was higher than passage 9 due to the larger size of older cells. Afterwards, the value of passage 9 cells increased higher than passage 31, since younger cells proliferated more than old cells. Therefore, the impedance measurement could characterize the proliferative capacity of ADSCs during expanded culture.