Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

Abstract

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

“…P-IA hydrogels grafted with vitronectin-derived oligopeptides, which has a storage modulus of 25.3 kPa (24 h crosslinking time), supported the long-term culture of human iPS and ES cells for over 10-20 passages. Particularly, P-IA hydrogels grafted with a joint segment (P-IA-24h-VN1G) or a dual chain (P-IA-24h-VN2G) supported the pluripotency of human iPS and ES cells, which were prepared with a relatively lower concentration of oligoECM (200-500 μg/mL) than P-IA-VN1 hydrogels, which necessitated using a high concentration of oligoECM (>1000 μg/mL) to maintain the pluripotency of human iPS and ES cells 28,32 .…”

“…Therefore, it is important to develop another type of hydrogel, of which the stiffness can be controlled by crosslinking of the hydrogels. For this purpose, bioinert hydrogels were developed, which were prepared from polyvinyl alcohol-co-itaconic acid (P-IA) with a different stiffness, which was controlled by the crosslinking degree with a changing crosslinking time 25,26,27,28,29,30,31,32 . The stem cells can be cultivated on nonmodified P-IA hydrogels, as well as P-IA hydrogels grafted with extracellular matrices (ECMs) and oligopeptides.…”

Chromatin Interaction Changes during the iPSC-NPC Model to Facilitate the Study of Biologically Significant Genes Involved in Differentiation

“…The use of human iPSCs was approved by the Institutional Review Board (IRB) of Yonsei University (Permit Number: 7001988-201802-BR-119-01E). We cultured iPSC on Matrigel (354277, BD Biosciences (San Jose, CA, United States)) with Essential 8 medium (A1517001, Invitrogen (Carlsbad, CA, United States)) coating [ 20 , 21 , 22 ]. For the differentiation of iPSC into NPC, an embryoid body (EB) was generated through the culturing of human iPSCs for 5–6 days on non-adherent petri dishes in Essential 8 (Invitrogen) with additional supplement 5 μM dorsomorphin (DM) (P5499, Sigma-Aldrich (St. Louis, MO, United States)) and 5 μM SB431542 (Sigma-Aldrich)).…”

Clinical-Grade Human Pluripotent Stem Cells for Cell Therapy: Characterization Strategy

“…Colonies are typically flat, contain small round cells with large nuclei with prominent nucleoli [70,71]. This morphology is typical for feeder dependent culture and may differ on various surfaces [72,73].…”