Faciliated primary culture and amplification of breast cancer cells and their biological properties / 基础医学与临床

Objective To efficiently builds up and expand breast cancer cells from cancer tissue and to identify their biological properties , provide abundant materials for research and personalized medicine .Methods Feeder cell layer and ROCK inhibitor Y-27632 were employed to faciliate the breast cancer cells;CCK-8 was used to determine the proliferation of the breast cancer cells; Cell cycle distribution was analyzed by flow cytometry; Histochemistry ( FH) assay to show the expression level of CK .The mRNA expression of HER-2, ER, PR and the breast cancer stem cell associated molecules (such as CD44, CD24, etc.) were detected by RT-PCR;STR assay was used for identifying verification of the cells .Results The use of feeder cells and Y-27632 facilitates rapid expand of the original breast cancer cells , and the cells have kept the original features of the tumor .Conclusions To use the method could obtain a large number of cells within a short time , which can promptly be used for the research of per-sonalized medicine .

Engineering Biomaterials for Feeder-Free Maintenance of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) are capable of differentiating into any type of somatic cell, a characteristic that imparts significant therapeutic potential. Human embryonic stem cells and induced pluripotent stem cells are types of hPSCs. Although hPSCs have high therapeutic potential, their clinical relevance is limited by the requirement for animal feeder layers, which maintain their pluripotency and self-renewal. hPSCs grown on animal feeder cells are at high risk for pathogen contamination and can be affected by the immunogenicity of the feeder layer. The presence of animal feeder cells also limits the scalability of hPSCs in culture because of the high cost of culturing and batch-to-batch variations. Therefore, development of feeder-free systems is imperative for robust, lower-cost, xeno-free, scalable culture of hPSCs. Biomaterials engineered with bioactive molecules such as adhesion proteins and extracellular matrix proteins, or synthetic materials such as peptides and polymers, may provide alternative substrates to animal feeder cells. This article reviews biomaterial-based, feeder-free systems for hPSC growth and maintenance, which provide clinically relevant alternatives to feeder cell systems.

Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in vitro

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

Maintenance of undifferentiated state of human embryonic stem cells in chemical defined medium at high clone density without exogenous cell factors / 中南大学学报(医学版)

Objective To establish human embryonic stem cells (hESCs) feeder-independent and cell factor-free culture system. Methods Effect of high and low clone densities of hESCs culture was compared and impact of the clone densities to hESCs culture media was analyzed. Results HESCs could maintain their undifferentiated states at high clone density (34 clones/cm2) without cell factors. At the same time,the bone morphology protein (BMP)-like induction of N2 and B27 supplements (NB) medium could be modulated by the clone density,and high level of BMP-like induction was accompanied by high clone density. Conclusion High clone density of hESCs can change the environments by themselves to maintain the undifferentiated states,which provides a new clue to explore the mechanism of undifferentiated states of hESCs and simplify the culture medium.

Mouse Embryonic Stem Cells Derived Feeder Cells Support the Growth of Themselves / 上海第二医科大学学报

Objective To test whether feeder cells derived from mouse embryonic stem cells(mESCs) could support the growth of mESCs themselves. Methods mESCs were induced to form mouse embryoid bodies(EB),and then fibroblast-like cells were derived from further differentiated mEB(mEB-dF),which served as feeder cells.The undifferentiation of mESCs grown on mEB-dF was confirmed by morphological analysis,colony efficiency and cell differentiation rate of mESCs,immunocytochemistry,alkaline phosphatase staining and RT-PCR.The pluripotency of mESCs grown on mEB-dF was examined by RT-PCR,inducing their differentiation in vivo and in vitro. Results(Forty-eight) fibroblast-like cells lines were derived from the same EB at three periods(d 10,d 15 and d 20),and five of them,mostly derived from d15 EB,were able to maintain mESCs in undifferentiated status and pluripotential ability over 10 passages.mESCs cultured on these feeder cell lines expressed alkaline phosphatase and specific mESCs markers,including SSEA-1,OCT-4,NANOG,and formed EB in vitro and teratomas in vivo.However,the majority of mEB-dF lines(43/48) has no such ability. Conclusion This study not only provides a novel feeder system for mESCs culture,avoiding lot of disadvantages of mouse embryo fibroblasts used as the feeder,but also indicates that fibroblast-like cells derived from mESCs take on different functions.The molecular mechanism of different function of these fibroblast cells is worthy of further investigations.