Establishment Of Cell-based Screening And Traditional Chinese Medicines For Growth Enhancers Of Embryonic Stem Cells

The capability of unlimited proliferation as well as differentiation into all types of cellsand tissues make embryonic stem (ES) cells ideal cell sources for tissue engineering and celltherapy. Although several biocompatible matrices and bioreactors have been developed forculturing stem cells, mass production of ES cells is still challenging due to the high costs ofcell growth promoters and pluripotency-maintaining reagents. Nowadays, traditional Chineseherbal medicines (TCHM) are becoming popular because of their therapeutic effects and lowtoxicity. In addition to disease treatments, some herbal medicines can also enhance immunefunctions by activating the proliferation of lymphocytes, increasing the amount of naturalkiller (NK) cells, and scavenging free radicals to resist oxidation. Thus, TCHMs have thepotential to be a new source of growth promoters for stem cells, especially ES cells.In this work, a simple, reliable, high-throughput screening method was developed andused to assess the pharmaceutical effects of extracts of TCHM. This method is based on3-dimensional (3D) cultures in polyethylene terephthalate (PET) fibrous scaffolds of mouseembryonic stem (mES) and human colon cancer and breast cancer cells expressing enhancedgreen fluorescent protein (GFP) after transfection on modified384-well plates with onlinemonitoring of culture fluorescence for dynamic responses of cells to drugs present in culturemedia. Cell responses to deoxycholic acid and the extracts of3TCHM (Ganoderma lucidumspores, Ginkgo biloba, and Epimedium brevicornum) at various concentrations wereinvestigated for their effects on proliferation and cytotoxicity. The screening results wereconsistent with what have been reported in the literature, confirming the reliability of the newscreening approach. Reliability analysis indicated the value of Z factor is in the range of0.5and1.0, verifying the cell-based drug screening (CBDS) platform is feasible forhigh-throughput screening (HTS) of TCHM with low variation.In in vitro mass production test, mESC grew in spinner flask supplemented with0.01%(w/v) Gls for3D dynamic culture reached an18.5folds expansion after5d. Compared to theno drug treated control group (13.6folds), Gls increased the expansion fold of mESC by36.1%, and the fractions of cells expressing both SSEA-1and Oct-4remained at over90%.Two active ingredients, water-soluble polysaccharides (PSS) and lipid-soluble triterpenes(TTS), of Gls were separated and extracted to test the effects on mESC proliferation using3Dfluorescence CBDS platform. Little growth promotion effects were detected when PSS orTTS treated mESC individually. For further screening, the high-throughput cell-based method was applied for screeningTCHM for potential stem cell growth promoters. Mouse ES cells expressing enhanced GFPwere cultured in growth media supplemented with various TCHM extracts. Thedosage-dependent effects of TCHM extracts on cell growth, including proliferation andcytotoxicity, were assessed via GFP fluorescence measurement. Six more TCHM extractswere investigated, and among all the nine tested extracts Panax notoginseng (Pn), RhizomaAtractylodis macrocephalae (Ram), Rhizoma chuanxiong (Rc) and Ganoderma lucidumspores (Gls) showed potential to improve mES cell proliferation. Eleven mixtures of these4TCHMs were then studied, and the results showed that the mixture of Pn and Gls had thestrongest growth promoting effect, increasing the specific growth rate of mES cells by29.5%at a low dosage of0.01%(w/v) Pn/Gls (p <0.01) and34.2%at0.1%(w/v) Pn/Gls (p <0.05)compared to the control. The growth promoting effect of Pn/Gls was further confirmed withES cells cultured in spinner flasks. A29.3-fold increase in the total cell number was achievedin the medium supplemented with0.01%Pn/Gls after5days, while the control culture onlygave a16.8-fold increase. It is noted that mES cells cultured in Pn/Gls supplemented mediacan also preserve pluripotency and without damage on Karyotype.Therefore, the cell-based screening method can provide an efficient and high-throughputway to explore potential stem cell growth promoters from TCHMs, and Pn/Gls screened fromthis method is promising in large-scale production of high-quality ES cells.