Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells

Mesenchymal stem cells (MSCs) have limited ability to differentiate into cardiomyocytes and the factors affect this process are not fully understood. In this study, we investigated the passage (P)-related transdifferentiation potential of MSCs into cardiomyocyte-like cells and its relationship to the proliferation ability. After 5-azacytidine treatment, only P4 but not P1 and P8 rat bone marrow MSCs (rMSCs) showed formation of myotube and expressed cardiomyocyte-associated markers. The growth property analysis showed P4 rMSCs had a growth-arrest appearance, while P1 and P8 rMSCs displayed an exponential growth pattern. When the rapid proliferation of P1 and P8 rMSCs was inhibited by 5-bromo-2-deoxyuridine, a mitosis inhibitor, only P1, not P8 rMSCs, differentiated into cardiomyocyte-like cells after 5-azacytidine treatment. These results demonstrate that the differentiation ability of rMSCs into cardiomyocytes is in proliferation ability-dependent and passage-restricted patterns. These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.     

Induction of Human Bone Marrow Mesenchymal Stem Cells Differentiation into Neural-Like Cells Using Cerebrospinal Fluid

To optimize a technique that induces bone marrow mesenchymal stem cells (BMSCs) to differentiation into neural-like cells, using cerebrospinal fluid (CSF) from the patient. In vitro, CSF (Group A) and the cell growth factors EGF and bFGF (Group B) were used to induce BMSCs to differentiate into neural-like cells. Post-induction, presence of neural-like cells was confirmed through the use of light and immunofluorescence microscopy. BMSCs can be induced to differentiate into neural-like cells. The presence of neural-like cells was confirmed via morphological characteristics, phenotype, and biological properties. Induction using CSF can shorten the production time of neural-like cells and the quantity is significantly higher than that obtained by induction with growth factor (P < 0.01). The two induction methods can induce BMSCs to differentiate into neural-like cells. Using CSF induction, 30 ml bone marrow can produce a sufficient number of neural-like cells that totally meet the requirements for clinical treatment.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Responses of keratinocytes to substrate-bound vitronectin: growth factor complexes

Insulin-like growth factor-1 (IGF-I) can associate with the extracellular matrix protein vitronectin (VN) via select IGF-binding proteins, and the resulting complex stimulates responses in a variety of cell types. As VN can also associate with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we hypothesized that the multimeric nature of VN could be exploited to deliver multiple growth factors to the cell surface. We report here that VN enhances bFGF but not EGF stimulated [(3)H]-leucine incorporation in the HaCAT keratinocyte cell line, with VN synergistically enhancing cell migration in response to both EGF and bFGF when presented as a VN-bound complex. Furthermore, the addition of EGF and/or bFGF to IGF-I:IGFBP-5:VN complexes significantly enhances both [(3)H]-leucine incorporation and migration of HaCAT cells above that induced by IGF:IGFBP-5:VN complexes alone. Indeed, similar responses are observed in primary cultures of human skin keratinocytes, highlighting the potential use of these novel complexes for a wide range of tissue repair applications.     

Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2, NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs.     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells. Supported by National High-Tech Research and Development Program of China (Grant No. 2006AA02A128) and National Natural Science Foundation of China (Grant No. 30670667).     

Epidermal Growth Factor and Basic Fibroblast Growth Factor Have Independent Actions on Mesencephalic Dopamine Neurons in Culture

Abstract: Epidermal growth factor (EGF) and basic fibro-blast growth factor (bFGF) are both trophic for dopamine neurons s in cultures of dissociated embryonic rat mesen-cephaion, but the significance of this apparent overlap in neurotrophic activity is not yet known. In this study, we investigated the mechanisms of action of these two growth factors and the potential relationship between them, Using a nuclease protection assay, we determined that bFGF mRNA was expressed in the cultures. Double-label immunocytochemistry revealed that bFGF immunore-active material could be detected in tyrosine hydroxylase immunoreactive neurons and glial fibrillary acidic protein immunoreactive astrocytes. EGF treatment increased bFGF mRNA content per culture dish. As we have previously demonstrated that EGF exerts its dopaminergic neurotrophic activity via an intermediate cell type, studies were designed to address whether the pathway by which EGF acts on dopaminergic neurons is mediated by the release of bFGF. However, the trophic action of EGF on dopamine neurons, represented by high-affinity neuronal dopamine uptake, could not be blocked by immunoneutralization of bFGF, suggesting that the actions of EGF were not mediated by bFGF release. The time course of the effects of EGF and bFGF on dopamine uptake were similar, with significant increases detectable only after 5 days in culture. Both growth factors were active in the picomolar-to-nannomolar range with maximal trophic activity between 0.4 and 2.5 n M. EGF, however, was the more potent mitogen under these conditions. When cultures were simultaneously incubated with maximal concentrations of EGF and bFGF, the effect on dopamine uptake was significantly greater than with either growth factor alone and, in fact, approximated the sum of the individual effects. On the basis of these results we conclude that these growth factors have independent effects on dopamine neurons of the mesencephalon.     

Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.     

Adult rat bone marrow stromal cells differentiate into Schwann cell-like cells in vitro

Bone marrow stromal cells (MSCs) have the capability of differentiating into mesenchymal and non-mesenchymal lineages. In this study, MSCs isolated from adult Sprague-Dawley rats were cultured to proliferation, followed by in vitro induction under specific conditions. The results demonstrated that MSCs were transdifferentiated into cells with the Schwann cell (SC) phenotypes according to their morphology and immunoreactivities to SC surface markers including S-100, glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Consequently, rat adult MSCs can be induced in vitro to differentiate into SC-like cells, thus developing an abundant and accessible SC reservoir to meet the requirements of constructing tissue engineered nerve grafts for peripheral nerve repair.     

Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.     

Comparison of the Efficiencies of Three Neural Induction Protocols in Human Adipose Stromal Cells

The aim of this study was to compare the neural differentiation potential and the expression of neurotrophic factors (NTFs) in differentiated adipose-derived stem cells (ADSCs) using three established induction protocols, serum free (Protocol 1), chemical reagents (Protocol 2), and spontaneous (Protocol 3) protocols. Protocol 1 produced the highest percentage of mature neural-like cells (MAP2ab⁺). Protocol 2 showed the highest percentage of immature neural-like cells (β-tubulin III⁺), but the neural-like state was transient and reversible. Protocol 3 caused ADSCs to differentiate spontaneously into immature neural-like cells, but not into mature neural cell types. The neural-like cells produced by Protocol 1 lived the longest in culture with little cell death, but Protocol 2 and 3 led to the significant cell death. Therefore, Protocol 1 is the most efficient among these protocols. Additionally, soon after differentiation, the mRNA levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in dADSCs were sharply decreased by Protocol 1 and 2 (acute induction protocol), but not by Protocol 3 (chronic induction protocol). The results indicate that NTFs played an important role in neural differentiation via acute responses to NGF and BDNF, but not chronically during the transdifferentiation process.     

Basic fibroblast growth factor promotes bone marrow stromal cell transplantation-mediated neural regeneration in traumatic brain injury

The current study was designed to evaluate the effects of basic fibroblast growth factor (bFGF) on human BMSC (hBMSC) transplantation-mediated neural regeneration in traumatic brain injury (TBI). Fibrin gel was used as a delivery vehicle to release bFGF locally in the TBI sites in a controlled manner. To test this hypothesis, hBMSCs suspended in fibrin gel containing bFGF were transplanted to rat TBI sites. Transplantation of hBMSCs suspended in fibrin gel without bFGF served as a control. hBMSC transplantation and bFGF treatment showed enhanced neural tissue regeneration than that of the control. The infarction volume and apoptotic activity of the transplanted hBMSCs were significantly decreased, and functional outcomes were significantly improved in the hBMSC transplantation and bFGF treatment group than in the control group. This study demonstrates that bFGF significantly enhances histological and functional recovery when used in hBMSC transplantation therapy in TBI.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.