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Xue K.,Shanghai JiaoTong University | Zhu Y.,Shanghai JiaoTong University | Zhang Y.,Shanghai JiaoTong University | Chiang C.,Shanghai JiaoTong University | And 3 more authors.
International Journal of Molecular Medicine | Year: 2012

The local microenvironment may change the ultimate fate of engineered cartilage differentiated from bone marrow stromal cells (BMSCs) after subcutaneous implantation. Chondrogenically differentiated BMSCs directed by growth factors or low-intensity ultrasound are apt to fibrose or vascularize in the subcutaneous environment, while BMSCs implanted in articular cartilage defects can form stable cartilage. We hypothesized that chondrocytes would provide an ideal chondrogenic environment, and thus promote the maintenance of the chondrocytic phenotype in ectopia. To test this hypothesis, we developed a new method to promote chondrocyte development from BMSCs in a chondrogenic environment produced by xenogeneic chondrocytes and compared the subcutaneous chondrogenesis of BMSCs mediated by xenogeneic chondrocytes with that produced by growth factors. These results indicate that subcutaneous chondrogenesis of BMSCs directed by xenogeneic chondrocytes is more effective than that induced by growth factors. BMSCs induced by xenogeneic chondrocytes formed relatively mature cartilage before or after implantation, following 4 weeks of culture, which reduced the induction time in vitro and led to maintenance of a stable cartilage phenotype after subcutaneous implantation.


Zhang C.-M.,Shanghai JiaoTong University | Zhang C.-M.,CAS Shanghai Institutes for Biological Sciences | Zhang C.-M.,Shanghai Stem Cell Institute | Gao L.,Shanghai JiaoTong University | And 7 more authors.
Circulation Journal | Year: 2012

Background: Berbamine, a natural compound from Barberry, was reported to protect myocardium from ischemia/ reperfusion (I/R) injury, but the underlying mechanisms are largely unknown. Methods and Results: Berbamine pretreatment from 10 to 100 nmol/L concentration-dependently improved postischemic myocardial function. Similar protection was confirmed in isolated cardiomyocytes characterized by the attenuation of I/R-induced intracellular free Ca2+ concentration ([Ca2+]i) overloading and the depression of cell shortening and Ca2+ transients, which were partially mimicked but not augmented by calpain inhibitor calpeptin and abolished by mitochondrial ATP-sensitive potassium (mitoKATP) channel inhibitor 5-hydroxydecanoate (5-HD) and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Consistently, I/R-induced increase of calpain activity and decrease of sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity; and protein expression of SERCA2a, desmin, calpastatin and Akt was significantly attenuated by berbamine. In addition, I/R-decreased Akt protein was reversed by calpeptin. Moreover, berbamine further increased I/R-enhanced phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β). These protections were abolished by wortmannin. Furthermore, berbamine significantly attenuated I/R-induced lactate dehydrogenase release, infarct size and contractile dysfunction, and such cardioprotective actions were abolished by wortmannin and 5-HD or mimicked by glycogen synthase kinase-3β (GSK3β) inhibitor SB216763 but without additive effect. Conclusions: These findings suggest that berbamine confers cardioprotection against I/R injury by attenuating [Ca2+]i overloading and preventing calpain activation through the activation of the PI3K-Akt-GSK3β pathway and, subsequently, opening of the mitoKATP channel.


Cao N.,Shanghai JiaoTong University | Liu Z.,Shanghai JiaoTong University | Chen Z.,Shanghai JiaoTong University | Wang J.,Shanghai JiaoTong University | And 11 more authors.
Cell Research | Year: 2012

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to Β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells. © 2012 IBCB, SIBS, CAS All rights reserved.


Fang H.,CAS Shanghai Institutes for Biological Sciences | Fang H.,Ruijin Hospital affiliated to SJTU SM | Yang Y.,CAS Shanghai Institutes for Biological Sciences | Yang Y.,Shanghai Stem Cell Institute | And 11 more authors.
Developmental Cell | Year: 2010

Genome-wide expression analysis of embryonic development provides information that is useful in a variety of contexts. Here, we report transcriptome profiles of human early embryos covering development during the first third of organogenesis. We identified two major categories of genes, displaying gradually reduced or gradually increased expression patterns across this developmental window. The decreasing group appeared to include stemness-specific and differentiation-specific genes important for the initiation of organogenesis, whereas the increasing group appeared to be largely differentiation related and indicative of diverse organ formation. Based on these findings, we devised a putative molecular network that may provide a framework for the regulation of early human organogenesis. Our results represent a significant step in characterization of early human embryogenesis and provide a resource for understanding human development and for stem cell engineering. © 2010 Elsevier Inc.


Wang J.,Shanghai JiaoTong University | Cao N.,Shanghai JiaoTong University | Yuan M.,Shanghai JiaoTong University | Cui H.,Shanghai JiaoTong University | And 7 more authors.
Stem Cells and Development | Year: 2012

MicroRNAs (miRNAs) are important regulators of cell fate decisions, while the miRNAs and their targets in the regulation of stem cell differentiation are largely unidentified. Here we report novel functions of miR-125b/Lin28 axis in the regulation of mouse embryonic stem cell (mESC) lineage specification and cardiomyocyte differentiation. With a MicroRNA Array screen, we identified a number of miRNAs significantly changed during ESC differentiation, among which miR-125b showed a marked reduction during early differentiation. The abundantly expressed miR-125b in undifferentiated mESCs was dramatically downregulated to a level hardly detected during differentiation day 3 to 5, with a concomitant upregulation of Lin28. Ectopically expressing miR-125b did not alter characteristics of undifferentiated mESCs, whereas it impaired the endoderm and mesoderm development, but not the ectoderm, and inhibited cardiomyocyte formation. We further demonstrate that miR-125b targeted the 3′-untranslated region of Lin28 and reduced the abundance of Lin28 at both mRNA and protein levels. Moreover, phenotypes of miR-125b overexpressing cells were mimicked by downregulation of Lin28 and rescued by reintroduction of Lin28. In addition, the impaired cardiogenesis in miR-125b-introduced cells was greatly recovered when mimicking endoderm environment by cultivation with the condition medium from a visceral endoderm-like cell line, END-2. These results reveal that the miR-125b/Lin28 axis is an important regulator of early lineage specification and cardiomyocyte differentiation of ESCs. © Copyright 2012, Mary Ann Liebert, Inc.


Cao N.,Shanghai JiaoTong University | Liao J.,Chinese Academy of Sciences | Liu Z.,Shanghai JiaoTong University | Zhu W.,Shanghai JiaoTong University | And 9 more authors.
Cell Research | Year: 2011

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy. © 2011 IBCB, SIBS, CAS All rights reserved.


Liang J.,Shanghai JiaoTong University | Wang Y.-J.,Shanghai JiaoTong University | Tang Y.,Shanghai JiaoTong University | Cao N.,Shanghai JiaoTong University | And 4 more authors.
Cell Death and Differentiation | Year: 2010

Ca2+ signals generated by inositol 1,4,5-trisphosphate receptors (IP3Rs) are crucial for cellular processes such as apoptosis and differentiation. However, the exact roles of IP3Rs and their contributions to Ca2+ signals in pluripotent embryonic stem (ES) cell behaviors remain largely unknown. In this study, we showed that the expression of type 3 IP3R (IP3R3) was transiently downregulated with a concomitant increase in apoptosis at the early differentiation stage of murine ES cells. Knockdown of IP3R3 by small interfering RNA increased apoptosis in differentiating cells but not in undifferentiated ES cells. Moreover, IP3R3 overexpression had the opposite effect. Consistently, IP3R3 knockdown altered Ca2+ oscillations in differentiating cells but not in undifferentiated ES cells. The apoptosis in differentiating IP3R3-knockdown cells was decreased by chelating intracellular Ca2+ with BAPTA-AM and increased in control ones. Furthermore, IP3R3 knockdown led to a suppression of the expression of mesodermal and mesoendodermal but not ectodermal markers. The differentiation suppressions were further confirmed by the impaired differentiation of mesodermal and some of the endodermal but not ectodermal derivatives. Such defects were partially because of the increased apoptosis in Flk-1 cells. These findings provide the first demonstration of the important role of IP 3R3 in the regulation of apoptosis in early differentiating ES cells and subsequent lineage commitments through modulation of Ca2+ signals. © 2010 Macmillan Publishers Limited All rights reserved.


Fang H.,Shanghai JiaoTong University | Fang H.,CAS Shanghai Institutes for Biological Sciences | Fang H.,University of Bristol | Jin W.,Shanghai JiaoTong University | And 8 more authors.
BMC Systems Biology | Year: 2011

Background: Integrated networks hold great promise in a variety of contexts. In a recent study, we have combined expression and interaction data to identify a putative network underlying early human organogenesis that contains two modules, the stemness-relevant module (hStemModule) and the differentiation-relevant module (hDiffModule). However, owing to its hypothetical nature, it remains unclear whether this network allows for comparative transcriptome analysis to advance our understanding of early human development, both in vivo and in vitro.Results: Based on this integrated network, we here report comparisons with the context-dependent transcriptome data from a variety of sources. By viewing the network and its two modules as gene sets and conducting gene set enrichment analysis, we demonstrate the network's utility as a quantitative monitor of the stem potential versus the differentiation potential. During early human organogenesis, the hStemModule reflects the generality of a gradual loss of the stem potential. The hDiffModule indicates the stage-specific differentiation potential and is therefore not suitable for depicting an extended developmental window. Processing of cultured cells of different types further revealed that the hStemModule is a general indicator that distinguishes different cell types in terms of their stem potential. In contrast, the hDiffModule cannot distinguish between differentiated cells of different types but is able to predict differences in the differentiation potential of pluripotent cells of different origins. We also observed a significant positive correlation between each of these two modules and early embryoid bodies (EBs), which are used as in vitro differentiation models. Despite this, the network-oriented comparisons showed considerable differences between the developing embryos and the EBs that were cultured in vitro over time to try to mimic in vivo processes.Conclusions: We strongly recommend the use of these two modules either when pluripotent cell types of different origins are involved or when the comparisons made are constrained to the in vivo embryos during early human organogenesis (and an equivalent in vitro differentiation models). Network-based comparative transcriptome analysis will contribute to an increase in knowledge about human embryogenesis, particularly when only transcriptome data are currently available. These advances will add an extra dimension to network applications. © 2011 Fang et al; licensee BioMed Central Ltd.

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