Chondroitinase ABC and Chondroitin 4-Sulfate as Potent Inducers for Neural Differentiation of Human-Induced PluripotentStem Cells

Background and Aim: A lot of studies focused on the human inducedpluripotent stem cells (hiPSCs) for treating patients who suffered fromspinal cord damage. Chondroitinase is a bacterial enzyme, whichallows new nerve fibers to grow across the damaged nerve. In this study,neural differentiation of human induced pluripotent cells (hiPSCs) wasinvestigated qualitatively in the presence of chondroitinase ABC andchondroitin 4-sulfate as its substrate.Methods: The cytotoxicity of chondroitin 4-sulfate was evaluated byAcridine Orange (AO) staining and MTT assay at 10, 50, 100, 200 and500 μg/mL concentrations. hiPSCs derived embryoid bodies were platedon gelatin-coated dishes and were treated with an induction mediumincluding forskolin and IBMX. Neural differentiation of hiPSCs was donein the presence of chondroitinase ABC and chondroitin 4-sulfate on days7 and 14. To confirm the neural differentiation, Von Kossa staining andimmunocytochemistry (ICC) analysis for MAP-2 protein as the neuronalmarker were performed.Results: About 100 μg/mL was selected for neural differentiation ofhiPSCs based on MTT assay and AO staining results. The ICC staining ofhiPSCs showed that the expression of MAP-2 in differentiated hiPSCs insupplemented media containing forskolin, IBMX, chondroitinase ABC,and chondroitin 4-sulfate was significantly higher than induction media without enzyme and substrate. The results of von Kossa staining weremore intense in wells containing enzyme and substrate, compared toother wells. In addition, neural differentiation of hiPSCs on day 14 washigher than day 7 for ICC analysis and von Kossa staining.Conclusion: In conclusion, our results indicated high efficiencyfor differentiation of hiPSCs into neural lineages in the presence ofchondroitinase and chondroitin 4-sulfate on day 14 after induction.

Keywords:

Chondroitinase ABC , Chondroitin 4-sulfate , Neural differentiation , Human-induced Pluripotent stem cells