(262c) Laminin-511 Inspired 3D Zwitterionic Culture System for Long-Term Human Pluripotent Stem Cell Culture

Human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), possess far-reaching capabilities for clinical applications as they can undergo multi-lineage differentiation into cell and organoid types critical for disease modeling, patient-specific drug screening, and transplantation. The most widely used biomaterial for culturing hPSCs is Matrigel, a matrix harvested from mouse tumors that suffers from batch-to-batch variability, xenogenic contaminants, and nonspecific protein interactions. These limitations have driven the development of 3D synthetic biomaterial alternatives which can be chemically-defined, xenogenic-free microenvironments optimized to fit a specific cell type and yield reproducible results. Many of these synthetic systems, however, suffer from spontaneous differentiation and reduced long-term proliferative capacity. We propose the use of PCB, a non-fouling zwitterionic material that greatly minimizes nonspecific interactions from background factors, as an ideal base material to culture hPSCs in 3D and to screen laminin peptides fit for hPSC culture. Laminins are the first matrix proteins expressed in the embryo at the two-cell stage, and laminin-511 (LN-511) coatings have been shown to successfully maintain hPSC pluripotency by interacting with the α6β1 integrin on hPSCs. By coupling PCB hydrogels with direct biomimetic signals through LN-511 incorporation, a robust, highly effective, and user-friendly 3D culture system can be developed for long-term use in hPSC culture and differentiation.

Four hPSC lines derived from healthy donors, UCSD001i-5-1, UCSD030i-23-2, H1, and H9, were purchased from WiCell. Cells were maintained on Matrigel with mTeSR Plus medium supplemented with 50µg/mL Normocin. Cells were encapsulated in the hydrogels by dissociating the cells, centrifuging, and resuspending the pellet in the polymer, MMP-degradable crosslinker, and laminin-derived peptides. This was plated as 20µL droplets and gelled in 10 minutes at 37°C. Cells were released from the hydrogel by incubation with 0.1% collagenase for 35 min. By evaluating four peptides derived from human LN-511 individually and in combinations of two, three, and four based on cell growth and spheroid characteristics compared to control peptides of RGD, YIGSR, and IKVAV, six peptide combinations were observed to have cell growth similar to or greater than all three controls, to have more uniform distribution of spheroid diameter and circularity, and to have higher alkaline phosphatase expression after 5 days of culture. These top 6 peptide combinations were further assessed by the pluripotency markers NANOG, OCT4, and SOX2 and by the integrins α6 and β1 by RT-qPCR. Based on significantly higher expression of pluripotency markers and an upregulation of the α6 integrin, one peptide combination was selected as the optimal candidate. To evaluate the robustness of PCB and the best peptide for hPSC culture, 4 hPSC lines and 3 stem cell media, E8, mTeSR Plus, and StemFlex, were used against Matrigel, iMatrix, and PEG culture systems. Overall, the results from all 4 cell lines and 3 media demonstrate that PCB-LN has superior cell expansion than Matrigel and iMatrix given the same surface area for culture and that PCB generates more cells and larger spheroids than PEG-LN with StemFlex and E8 media. Additional pluripotency analysis illustrates that PCB-LN-cultured cells have an upregulation of NANOG and OCT4 expression compared to Matrigel and iMatrix and similar expression compared to PEG-LN. Flow cytometry analysis after 1 week of culture in the PCB-LN hydrogel shows approximately 99% OCT4+, NANOG+, SOX2+, SSEA-3+, SSEA-4+, and TRA-1-60+ cells which are consistent with the Matrigel control at one week of culture. Future work includes passaging the cells in PCB-LN for at least 10 passages followed by further flow cytometry analysis of pluripotency markers, teratoma formation, and in vitro differentiations. This culture system has many advantages including (a) noise-free background, (b) ability to add specific cell-adhesive ligands to recapitulate in vivo niche conditions, (c) cell-mediated degradation, (d) user-desired cell release, (e) ability to passage cells repeatedly for long-term culture, and (f) ability to mimic 3D physiological conditions.