This study further demonstrated the efficacy and flexibility of the new method to generate NC-like cells from hiPSCs under the influence of porcine NP tissue, and it showed the high potential of the hiPSCderived NC-like cells for the future regeneration of nucleus pulposus tissue. The pulverized porcine NP matrix was added to the culture medium either directly or via an insert which allows the matrix to contact with the hiPSCs or not. After the freeze-dried NP tissue was added into the culture medium, it rewetted readily and formed gel-like clumps suspending in the medium. The 9-Cyclopentyladenine monomethanesulfonate plated cells did not attach to the tissue culture plate surface until supplementation of the serum-containing differentiation medium. The contact or non-contact culture modes did not apparently affect the cell attachment process or cell viability in the first 5�C6 days. At approximately 7 days, many of the NP tissue clumps began to attach to the cell layers in the contact-mode culture and cells seemed to grow robustly up to 10 days. Interesting, cells formed compact colonies associating the attached NP matrix. In comparison, many cells began to die at approximately 7 day, and the cell population did not show apparent expansion after 10 days in the non-contact culture. Quantification of the cell number clearly showed the difference; when compared to the initial seeding number, the cell number approximately doubled in the contact culture whereas increased little in the non-contact culture. Transcripts of three notochordal CH-223191 marker genes were examined by RT-PCR. The cells remarkably expressed T, CK-8 and CK-18 genes comparing to the undifferentiated hiPSCs in both contact and non-contact cultures. Note that all gene expressions were measured from a pool of three biological replicates, so they provide a good representation of the average level of each transcript. Protein level expression of T and CK-18 were examined by immunocytochemical method. Both proteins were clearly detected in both cultures, whilst T exclusively in cell nuclei and CK-18 in cytoplasm. The T and CK-18 positive cells each represented approximately 100% of all the examined population in both the contact and non-contact cultures. The total population was determined based on the DAPI staining. The result showed the generated cells are highly homogenous pertaining to the two typical notochordal markers. The NP-like tissues generated by both cell contact and noncontact were further examined on their ECM biochemistry.