Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes if an appropriate cellular environment is provided. Notch signals exchanged between neighboring cells through the Notch receptor can eventually dictate cell differentiation. In our study, we show that MSC differentiation into cardiomyocytes is dependent on the Notch signal. METHODS: We created a myocardial infarction model in rat by coronary ligation, administered direct intramyocardial injection of DAPI-labeled MSC immediately, and observed the differentiation of MSCs after 14 days by immunofluorescence staining against troponin T. We cultured MSCs and cardiomyocytes in four ways, respectively, in vitro. (1) MSCs cocultured with cardiomyocytes obtained from neonatal rat ventricles in a ratio of 1:10. (2) The two types of cells were cultured in two chambers separated by a semipermeable membrane as indirect coculture group. (3) Notch receptor-soluble jagged1 protein was added to indirect coculture group. (4) Both jagged1 protein and gamma-secretase inhibitor-DAPT were added to indirect coculture group. Two weeks later, we observed the differentiation percentage, respectively, by immunofluorescence staining. RESULTS: We found the differentiation of MSCs which were close to cardiomyocytes in vivo. The differentiation percentage of the four cell culture group was 30.13+/-2.16%, 12.52+/-1.18%, 26.33+/-2.20%, and 13.08+/-1.15%. CONCLUSIONS: MSCs can differentiate into cardiomyocytes in vitro and in vivo if a cardiomyocyte microenvironment is provided. 2. Cell-to-cell interaction is very important for the differentiation of MSCs into cardiomyocytes. 3. Jagged1 protein can activate Notch signal and enhance the differentiation of MSC into cardiomyocyte, while the effect can be inhibited by DAPT.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.     

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.     

人间充质干细胞对乳腺癌细胞生长的影响研究

目的通过构建间充质干细胞(MSC)与乳腺癌细胞间相互作用的共培养模型,探讨MSC对乳腺癌细胞生长的影响。方法用含荧光基因第三代自身失活慢病毒载体感染人类脐带分离提取的MSC和乳腺癌细胞MDA-MB-231、MCF-7,以单独培养的乳腺癌细胞MDA-MB-231和MCF-7分别设立对照,2种乳腺癌细胞分别与MSC共培养,检测乳腺癌细胞在MSC作用下增生能力的改变,流式细胞术检测共培养后细胞表面标记物表达。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,多重比较采用Dunnet-t检验。结果MSC在与乳腺癌细胞共培养过程中促进肿瘤细胞生长,第3天共培养组乳腺癌MDA-MB-231细胞数高于单独MDA-MB-231培养组[(5.50±0.71)×10^3个比(1.63±0.41)×10^3个],培养至第7天,两组间MDA-MB-231细胞数差异进一步增大[(81.25±7.40)×10^3个比(26.25±4.15)×10^3个],差异具有统计学意义(P均<0.001);共培养后MSC促进乳腺癌细胞表达干细胞特有标记物CD90,MCF-7从共培养第2天CD90表达率(1.38±0.30)﹪升高至第9天(92.45±2.04)﹪。在共培养中MSC围绕肿瘤细胞集落方式生长,在形态上变长,并发现一种新型混合细胞(hybrid融合细胞)同时表达绿色和红色荧光,且对化疗药物更敏感。结论MSC促进乳腺癌细胞的生长,伴随MSC形态学改变和hybrid融合细胞出现,乳腺癌细胞获得MSC特有CD90表达。     

Mesenchymal stem cells induce dermal fibroblast responses to injury

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.     

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament.     

The interaction between co-cultured human nucleus pulposus cells and mesenchymal stem cells in a bioactive scaffold

Mesenchymal stem cells (MSCs) can differentiate into nucleus pulposus (NP) cells upon being co-cultured with NP cells. Important growth factors and morphogens secreted by MSCs during the differentiation process also enhance the biological properties of NP cells. In this study, the interactions between human NP cells and MSCs co-cultured in different cell-ratio (100% NP, 75% NP with 25% MSCs, 50% NP with 50% MSCs, 25% NP with 75% MSCs, and 100% MSCs) in a three-dimensional gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold were examined. Results showed that the cell proliferation was increased when NP and MSCs were co-cultured. Real-time PCR and immunohistochemical staining revealed that all co-culture groups produced type II collagen which represent normal NP cells but not type I collagen secreted by degenerated NP cells. FADD expression, which modulates cell survival and extracellular matrix homeostasis, was maintained in a stable status for co-cultured groups. The cultures containing 75% NP cells with 25% MSCs showed high level of collagen production and glycosaminoglycan content. Moreover, 75% NP cells with 25% MSCs had upregulated SOX9 that contributes to the improvement in type II collagen mRNA expression and protein production. These findings showed the NP/MSC cell-ratio influenced the cell functions dramatically. The co-culture of NP/MSC cells in a bioactive scaffold is a promising treatment for intervertebral disc diseases.     

miR-124 promotes proliferation and differentiation of neuronal stem cells through inactivating Notch pathway

Background

Neural stem cells (NSCs) are able to differentiate into neurons and astroglia. miRNAs have been demonstrated to be involved in NSC self-renewal, proliferation and differentiation. However, the exact role of miR-124 in the development of NSCs and its underlying mechanism remain to be explored.

Methods

Primary NSCs were isolated from embryos of Wistar rats. Immunocytochemistry was used to stain purified NSCs. miR-124, Delta-like 4 (DLL4), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), HES1, HEY2, and cyclin D1 (CCND1) expressions were detected by qRT-PCR and western blot. The interaction between miR-124 and DLL4 was confirmed by luciferase reporter assay. Cell proliferation was assessed by MTT assay.

Results

NSCs could self-proliferate and differentiate into neurons and astrocyte. miR-124 was up-regulated and DLL4 was down-regulated during NSC differentiation. DLL4 was identified as a target of miR-124 in NSCs. Ectopic expression of miR-124 or knockdown of DLL4 promoted the proliferation and the formation of NSCs to neurospheres. Moreover, miR-124 overexpression or DLL4 down-regulation improved β-tubulin III expression but decreased GFAP expression in NSCs. Furthermore, enforced expression of DLL4 partially reversed the effects of miR-124 on NSCs proliferation and differentiation. Elevated expression of miR-124 suppressed the expressions of HES1, HEY2, and CCND1 in NSCs, while these effects were attenuated following the enhancement of DLL4 expression.

Conclusion

miR-124 promoted proliferation and differentiation of NSCs through inactivating Notch pathway.

     

Roles of bHLH genes in neural stem cell differentiation

Neural stem cells change their characteristics over time during development: they initially proliferate only and then give rise to neurons first and glial cells later. In the absence of the repressor-type basic helix-loop-helix (bHLH) genes Hes1, Hes3 and Hes5, neural stem cells do not proliferate sufficiently but prematurely differentiate into neurons and become depleted without making the later born cell types such as astrocytes and ependymal cells. Thus, Hes genes are essential for maintenance of neural stem cells to make cells not only in correct numbers but also in full diversity. Hes genes antagonize the activator-type bHLH genes, which include Mash1, Math and Neurogenin. The activator-type bHLH genes promote the neuronal fate determination and induce expression of Notch ligands such as Delta. These ligands activate Notch signaling and upregulate Hes1 and Hes5 expression in neighboring cells, thereby maintaining these cells undifferentiated. Thus, the activator-type and repressor-type bHLH genes regulate each other, allowing only subsets of cells to undergo differentiation while keeping others to stay neural stem cells. This regulation is essential for generation of complex brain structures of appropriate size, shape and cell arrangement.     

Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs)

Notch signaling is an evolutionarily conserved cell–cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.     

Bone mesenchymal stem cell‐conditioned medium attenuates the effect of oxidative stress injury on NSCs by inhibiting the Notch1 signaling pathway

Numerous studies have demonstrated the therapeutic effect of bone mesenchymal stem cells on spinal cord injury (SCI), especially on neural stem cells (NSCs). However, the predominant mechanisms of bone mesenchymal stem cells (BMSCs) are unclear. Recently, some researchers have found that paracrine signaling plays a key role in the therapeutic capacity of BMSCs and emphasized that the protective effect of BMSCs may be due to paracrine factors. In this study, we aimed to investigate the potential mechanisms of BMSCs to protect NSCs. NSCs were identified by immunocytochemistry. The oxidative stress environment was simulated by H₂O₂ (50, 100, 200 μM) for 2 h. The apoptotic rate of the NSCs was detected via flow cytometry. Lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) activity were evaluated via corresponding assay kits. Western blot was used to detect the expressions of Notch1, HES1, caspase‐3, cleave caspase‐3, Bax, and Bcl‐2. We found that H₂O₂ could significantly induce the apoptosis of NSCs, increase LDH, MDA levels, and decrease SOD activity by activating the Notch1 signaling pathway. DAPT (the specific blocker of Notch1) and BMSC‐conditioned medium (BMSC‐CM) could significantly prevent the apoptotic effect and oxidative stress injury on NSCs that were treated with H₂O₂. We also revealed that BMSC‐CM could decrease the expression of Notch1, Hes1, cleave caspase‐3, Bax, and increases the expression of Bcl‐2 in NSCs, which was induced by H₂O₂. These results have revealed that BMSC‐CM can neutralize the effect against oxidative stress injury on the apoptosis of NSCs by inhibiting the Notch1 signaling pathway.     

KRT1 gene silencing ameliorates myocardial ischemia–reperfusion injury via the activation of the Notch signaling pathway in mouse models

Myocardial ischemia and reperfusion injury (MIRI) includes major drawbacks, such as excessive formation of free radicals and also overload of calcium, which lead to cell death, tissue scarring, and remodeling. The current study aims to explore whether KRT1 silencing may ameliorate MIRI via the Notch signaling pathway in mouse models. Myocardial tissues were used for the determination of the positive rate of KRT1 protein expression, apoptosis of myocardial cells, creatine kinase (CK) and lactate dehydrogenase (LDH) expression, expression of related biomarkers as well as myocardial infarction area. The transfected myocardial cells were treated with KRT1-siRNA, Jagged1, and DAPT (inhibitor of Notch-1 signaling pathway). The expression of KRT1, NICD, Hes1, Bcl-2, and Bax protein was detected. The MTT assay was applied for cell proliferation and flow cytometry was used for cell apoptosis. Mice with MIRI had a higher positive rate of KRT1 protein expression, apoptosis of myocardial cells, CK and LDH expression, myocardial infarction area, increased expression of MDA, NO, SDH, IL-1, IL-6, TNF-α, CRP, KRT1, Bax protein, CK, and LDH, and decreased expression of SOD, NICD, Hes1, and Bcl-2. The downregulation of KRT1 led to decreased expression of KRT1 and Bax protein, increased expression of NICD, Hes1, and Bcl-2, decreased cell apoptosis, and improved cell proliferation. The inhibition of the Notch signaling pathway leads to reduced expression of Bax, increased expression of NICD, Hes1, and Bcl 2, and also decreased cell apoptosis and increased cell proliferation. Our data conclude that KRT1 silencing is able to make MIRI better by activating the Notch signaling pathway in mice.     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

Notch信号在盐酸法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化过程中的作用

目的:探讨Notch信号通路在盐酸法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经元分化中的作用。方法:实验分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs转染后荧光表达情况;RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化;免疫细胞化学法检测Notch1、神经元烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化;MTT方法检测细胞存活率。结果:①siRNA转染72h,MSCs荧光表达最强,转染率可达91.3%±4.2%;同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P0.05);MTT提示转染组细胞存活率也显著减少(P0.05)。②盐酸法舒地尔可以诱导MSCs向神经元分化,其中以转染组诱导效果最佳,NSE、NF-M的表达率显著的高于其它各组(P0.05)。结论:盐酸法舒地尔在诱导大鼠MSCs向神经元分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经元分化。