Nicotine activates nuclear factor of activated T cells c2 (NFATc2) and prevents cell cycle entry in T cells

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.     

骨髓间充质干细胞诱导成骨细胞模型中活化转录因子4基因的表达

背景：研究发现活化转录因子4为近调控成骨细胞分化和功能的活化因子,在成骨细胞分化过程中起着至关重要的作用。 目的：探索SD大鼠骨髓间充质干细胞成骨诱导过程中活化转录因子4基因的表达变化及其意义。 方法：以全骨髓贴壁法培养SD大鼠骨髓间充质干细胞,传代至第3代细胞时加入成骨诱导剂,在诱导的第0,1,4,7,10,13,16,19天分别采用PT-PCR,Western blot动态监测活化转录因子4基因及蛋白的表达变化,以未进行成骨诱导细胞做对照。 结果与结论：RT-PCR结果显示,活化转录因子4 mRNA的表达随着细胞成骨分化程度加剧而升高,16 d达到峰值。Western blot分析结果显示,活化转录因子4蛋白表达量随着骨髓间充质干细胞骨化程度加剧,表达水平亦呈上升趋势,于16 d达到高峰,19 d维持在高水平状态。实验组各时间点活化转录因子4 mRNA和活化转录因子4蛋白表达与对照组相比差异有均有显著性意义（P＆lt;0.05）。结果表明诱导成骨细胞中活化转录因子4表达增强,提示活化转录因子4的增强与骨髓间充质干细胞成骨分化能力呈正相关。     

Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor

The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time‐ and money‐saving tools that aid the efficient selection of cellular specimens – in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell‐based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage‐like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor‐β and elevated levels of Sox9. Copyright © 2013 John Wiley & Sons, Ltd.     

Size of the embryoid body influences chondrogenesis of mouse embryonic stem cells

For applications in tissue engineering and regenerative medicine, embryonic stem cells (ESCs) are commonly pre-differentiated in the form of embryoid bodies (EBs). The uncontrolled cell differentiation in EBs results in a highly heterogeneous cell population, an unfavourable condition for therapeutic development. The purpose of this study was to determine an optimal size of EBs for chondrogenic differentiation. EBs were produced in suspension culture with mouse ESCs (ES-D3 GL). The 5-day-old EBs were sorted under a microscope by diameter: small EBs (S-EBs, < 100 microm), medium EBs (M-EBs, 100-150 microm) and large EBs (L-EBs, > 150 microm). The three sizes of EBs were cultured separately for 3 weeks in chondrogenic medium. Type II collagen and aggrecan gene expression was significantly upregulated in the S-EBs, when compared with the M-EBs and L-EBs (p < 0.05 and p < 0.001, respectively). Proteoglycans produced by the cells derived from S-EBs were > 50% of the other two groups. In addition, both Oct4 and Sox2 were expressed more in S-EBs than in M-EBs and L-EBs. Type X collagen expression was relatively increased in L-EBs. Slight shifts toward haematopoietic and endothelial differentiation were seen in the L- and M-EBs. In summary, the size of EBs has implications on ESC differentiation. Cells derived from S-EBs have a greater chondrogenic potential than those from M-EBs and L-EBs. The size of EBs can be a parameter utilized to optimize ESC differentiation for tissue engineering.     

In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme‐susceptible and ‐degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan‐like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real‐time RT–PCR analysis for cartilage‐specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage‐specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type‐II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Notch信号通路阻断剂对人脐带间充质干细胞软骨分化的影响

背景：Notch信号通路作为一条在细胞增殖和分化过程中起重要作用的信号通路,目前它在人脐带间充质干细胞在体外向软骨细胞诱导分化过程中的作用仍然未知。目的：首次探讨Notch信号通路特异性阻断剂2,4-二氨基-5-苯噻唑（DAPT）对人脐带间充质干细胞向软骨细胞诱导分化的影响。方法：从人脐带中分离获得间充质干细胞,体外向软骨细胞诱导分化。实验分4组：①无诱导组换成含体积分数5%胎牛血清和1%双抗的DMEM高糖培养基培养。②单纯诱导组换成终浓度为6.25 mg/L胰岛素、6.25 mg/L转铁蛋白、10μg/L转化生长因子β1、0.1μmol/L地塞米松、50 mg/L维生素C、体积分数5%胎牛血清和1%双抗的软骨诱导培养基培养。③二甲基亚砜组在软骨诱导培养基中加入终浓度为0.1%的二甲基亚砜培养。④2,4-二氨基-5-苯噻唑组在软骨诱导培养基中加入终浓度为5μmol/L的2,4-二氨基-5-苯噻唑（溶于二甲基亚砜）培养,二甲基亚砜终浓度为0.1%。结果与结论：诱导人脐带间充质干细胞向软骨细胞分化后,细胞形态由长梭形变为多边形,且甲苯胺蓝染色和免疫荧光染色均呈现阳性;软骨诱导分化后Jag-1、PS-1、Notch-1、Hes-1的基因表达均明显下降（P〈0.01）;添加2,4-二氨基-5-苯噻唑后,与单纯诱导组相比,人脐带间充质干细胞中Jag-1、PS-1、Hes-1（P〈0.01）和Notch-1（P〈0.05）的基因表达显著降低;蛋白聚糖和Ⅱ型胶原蛋白含量均降低（P〈0.01）;蛋白聚糖的基因表达显著降低（P〈0.01）,Ⅱ型胶原蛋白的基因表达也出现一定程度的下降。提示Notch信号存在于人脐带间充质干细胞中,诱导人脐带间充质干细胞向软骨细胞分化后,这种信号强度迅速减弱;2,4-二氨基-5-苯噻唑可能通过Jag-1-Notch-1-Hes-1途径抑制人脐带间充质干细胞向软骨细胞分化。     

两种细胞因子联合诱导骨髓间充质干细胞向神经样细胞分化简

背景：骨髓间充质干细胞可被定向诱导分化为神经样细胞,理论上骨髓间充质干细胞可作为种子细胞应用于周围神经组织工程。目的：用2种细胞因子联合诱导骨髓间充质干细胞分化为神经样细胞,进一步探讨其在周围神经损伤方面的应用。方法：从Wistar大鼠胫骨和股骨提取骨髓间充质干细胞,采用差速贴壁法进行培养和纯化,联合应用碱性成纤维细胞生长因子和表皮生长因子对细胞进行诱导,观察细胞形态变化,并用免疫组织化学方法检测骨髓间充质干细胞中神经元特异性标志物的表达;并研究终止诱导后骨髓间充质干细胞的形态及免疫组织化学方面的变化。 结果与结论：骨髓间充质干细胞经诱导后呈典型神经细胞样改变,有两个或多个突起,突起之间相互连接成网状,可见细胞核及核仁,免疫组化检测神经元特异性烯醇化酶、神经丝蛋白及突触素蛋白表达阳性。撤除诱导条件后大部分细胞逐渐恢复原来的成纤维细胞样形态,上述3种蛋白的表达量明显下降。表明应用神经营养因子可诱导骨髓间充质干细胞分化为神经样细胞,但其形态及组织学变化仅能维持一段较短的时间。     

Conditioned medium as a strategy for human stem cells chondrogenic differentiation

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow‐derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly‐derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three‐dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage‐related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of double growth factor release on cartilage tissue engineering

The effects of double release of insulin‐like growth factor I (IGF‐I) and growth factor β1 (TGF–β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid‐co‐glycolic acid) (PLGA) and poly(N‐isopropylacrylamide) (PNIPAM) carrying IGF‐I and TGF–β1, respectively. On tissue culture polystyrene (TCPS), TGF‐β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF‐I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200–400 µm) PLGA scaffolds. It was observed that the combination of IGF‐I and TGF‐β1 yielded better results in terms of collagen type II and aggrecan expression than GF‐free and single GF‐containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. Copyright © 2011 John Wiley & Sons, Ltd.     

异种关节软骨与MSCs体外共同培养的实验研究

目的提供一种新的种子细胞获得方法,解决软骨缺损修复过程中软骨数量不足的困难。方法人骨髓间充质干细胞体外扩增诱导分化后与兔的关节软骨细胞按不同比例共同培养利用real-time PCR技术分析细胞表型的表达情况。结果实验组2种细胞共培养4周后,混合培养显示正反馈调节软骨细胞的增殖,软骨细胞外基质、SOX9、Ⅱ型胶原基因表达增加,软骨细胞生成明显增加。结论间充质干细胞以旁分泌或自分泌的方式调节间充质干细胞向软骨细胞的转化。     

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 µg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP‐spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal‐to‐noise ratios on T₂‐SE and T₂*‐GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose‐dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose‐dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant. Copyright © 2009 John Wiley & Sons, Ltd.     

Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFβ1‐induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4 days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFβ1‐induced group. At 21 days, the TGFß1‐treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21 days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link‐p gene expression compared with the non‐induced group, and to similar levels as the TGFβ1‐induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three‐dimensional (3D) micro‐environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFβ1. This might lead to an interesting alternative when considering short‐term triggering in a one‐step surgical procedure for the treatment of cartilaginous defects. Copyright © 2011 John Wiley & Sons, Ltd.     

视网膜神经节细胞培养上清液对骨髓间充质干细胞向神经元样细胞的诱导作用

目的探讨大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs)体外诱导分化成神经元样细胞的能力。方法用视网膜条件分化液诱导传代的rMSCs,待细胞分化后进行形态学观察,并用免疫细胞化学方法方法进行鉴定。结果诱导细胞2 d后即可见有神经元样细胞出现,7 d神经元样细胞占细胞总数的(27±1.0)%,Nestin染色阳性、MAP-2染色阳性。结论体外rMSC能扩增、传代,并被诱导分化成神经元样细胞。     

Chondrocytes and bone marrow-derived mesenchymal stem cells undergoing chondrogenesis in agarose hydrogels of solid and channelled architectures respond differentially to dynamic culture conditions

The objective of this study was to investigate how a combination of different scaffold architectures and rotational culture would influence the functional properties of thick cartilaginous tissues engineered using either chondrocytes or bone marrow-derived mesenchymal stem cells (BM-MSCs). Expanded porcine chondrocytes and BM-MSCs were suspended in 2% agarose and cast in custom-designed moulds to produce either regular solid or channelled construct cylinders. The study consisted of three seperate experimental arms. First, chondrocyte and BM-MSC constructs were cultured in free swelling conditions for 9 weeks. Second, constructs were subjected to rotational culture for a period of 3 weeks. Finally, BM-MSC-seeded constructs were subjected to delayed rotational culture, in which constructs were first cultured for 3 weeks in free swelling conditions, followed by an additional 3 weeks in rotating culture conditions. Constructs were supplemented with TGFβ3 during the first 3 weeks of all experiments. The introduction of channels alone had little effect on the spatial patterns of tissue accumulation in either chondrocyte- or BM-MSC-seeded constructs. The two cell types responded differentially to rotational culture, resulting in the formation of a more homogeneous tissue in chondrocyte-seeded constructs, but significantly inhibiting chondrogenesis of BM-MSCs. This inhibition of chondrogenesis in response to dynamic culture conditions was not observed if BM-MSC-seeded constructs were first maintained in free swelling conditions for 3 weeks prior to rotation. The results of this study demonstrate that bioreactor culture conditions that are beneficial for chondrocyte-based cartilage tissue engineering may be suboptimal for BM-MSCs.     

人脐带间质干细胞的分离纯化及生物学特性鉴定

近年来,间质干细胞(mesenchymal stem cells,MSC)以其易于在体外分离培养、高增殖潜能等生物学特性,已经成为备受关注的治疗载体.本文探讨人脐带间质干细胞(human umbilical cord mesenchymal stem cell,hUCMSC)的分离培养及其生物学特性.     

骨髓间充质干细胞体外诱导分化为神经样细胞的机制

目的探索骨髓间充质干细胞 (MSCs)在体外诱导分化为神经样细胞的机制。方法分离培养大鼠MSCs ,用二甲基亚砜 (DSMO)和丁羟茴醚 (BHA)诱导分化 ,检测诱导分化前、预诱导 2 4h、诱导分化后 6h、2 4h和 48h神经细胞和神经干细胞的特异性标记蛋白的表达。结果诱导分化后 ,大部分MSCs变成双极、多极和锥形 ,并相互交织成网络结构 ,巢蛋白 (Nestin)在诱导分化前不表达 ,在诱导分化后 6h达到最高 ,2 4h和 48h逐渐降低。神经元特异性核蛋白 (NeuN )在诱导分化前不表达 ,在诱导分化后 6h出现表达 ,2 4h和 48h表达增强。结论MSCs经体外诱导先分化为神经干细胞 ,然后分化为神经元样细胞。