Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

Background. Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods. We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results. In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion. hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.

Cell growth kinetics and accumulation of secondary metabolite of Bletilla striata Rchb.f. using cell suspension culture

Bletilla striata (Orchidaceae) is a well-recognized endangered medicinal plant due to inadequate natural reproduction with high market worth. To evaluate the cell growth kinetics and accumulation of secondary metabolites (SMs), the cell suspension culture is proved to be a valuable approach for acquiring the high yield of medicinal parts. An effective cell suspension culture for obtaining B. striata cell growth and its SMs was in vitro induction of callus from B. striata seeds. The cell growth kinetics and accumulation of SMs were analyzed using the mathematical model. Results cell growth kinetic model revealed that the growth curve of B. striata suspension cells was curved as sigmoid shape, indicating the changes of the growth curve of suspension cells. Improved Murashige and Skoog cell growth medium was the utmost favorable medium for B. striata callus formation with the highest cell growth during the stationary phase of cultivation period, the cell growth acceleration was started after 7 days and thereafter gradually decrease at 24 day and then reached to highest at 36 day of cultivation period in both dry weight and fresh weight. The coelonin concentration was peak during exponential growth stage and decreased afterward at the stationary phase in the cell suspension culture. The maximum content of coelonin (about 0.3323 mg/g cell dry weight) was observed on the 18th day of the cultivation cycle while the dactylorhin A and militarine reached highest at 24 day, and p-hydroxybenzyl alcohol at 39 day. This investigation also laid a foundation for multi-mathematical model to better describe the accumulation variation of SMs. The production of SMs had shown great specificity during cells growth and development. This research provided a well-organized way to more accumulation and production of SMs, on scale-up biosynthesis in B. striata cell suspension culture.

DEGRADATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY Pseudomonas fluorescens Strain HH

Pseudomonas fluorescens HH isolated from soil utilized 2,4-Dichlorophenoxyacetic acid (2,4D) as a sole carbon and energy source. The strain completely utilized 1.0 mM of 2,4D within 30 hr. The immobilized Pseudomonas fluorescens HH degraded 2,4D with higher rates compared to the rates of free-suspension cells. The determination of degradation and cell growth kinetics in exponential growth phase of bacteria showed that both fitted with the Edwards model, in which the maximal utilization rates and inhibition coefficient were 0.079 ± 0.008 mM/h and 0.820 ± 0.03 mM, respectively. The addition of glycerol as a cryoprotectant into alginate increased the survival of bacteria in beads during freeze-drying process, which resulted in reducing the adverse effects of bead lyophilization.