Abstracts

Human induced pluripotent stem cells (hiPSCs) aregenerated from human somatic cells using defined transcriptionfactors. These cells possess characteristics very similar to thatof human embryonic stem cells including the ability todifferentiate into cell types of all three germ layers. HiPSCs showgreat potential in clinical researches like drug screening andregenerative medicine, that all require large amount of cellscultured under well-defined conditions. The most common culturemethods used for hiPSCs are 2D culture methods using Matrigel orvitronectin coated culture plates or flasks. 2D culture methodsrequire large surface area to produce the same amount of cellscompared to 3D methods. In addition, cells cultured in 2D cultureenvironment are far from that in vivo. In this study, we developeda robust 3D culture condition based on hiPSC-qualified PGmatrix(PGmatrix-hiPSC) hydrogel. This 3D culture system provide hiPSCswith well-defined, more in vivo-like environment that encapsulatecells in liquid rich hydrogel with appropriate oxygen supply thatresembles the hypoxia condition in vivo. Two hiPSC lines growncontinuously in PGmatrix-hiPSC showed higher total populationexpansion and higher viability, with more consistency compared tothe same cell lines grown in 2D on Matrigel or Vitronectin-XF.After grown in 3D PGmatrix-hiPSC for over 25 passages, majorpluripotency markers, such as Oct4, Sox2, Nanog, and SSEA4 areexpressed in most hiPSCs examined by flow cytometry. RT-qPCR alsoconfirmed adequate expression levels of major pluripotency relatedgenes. In addition, karyotype analysis of hiPSC after 37 passagesin 3D PGmatrix-hiPSC was found normal. The same hiPSC linescultured continuously in parallel in 2D and 3D showed differencesin gene expression and surface marker TRA-1-81 expression. Theseresults indicated the 3D PGmatrix-hiPSC system is likely superiorin maintaining hiPSC growth as well as pluripotency. The findingsalso suggest that it is very important to study cells in 3D cultureenvironment to better understand the mechanism of pluripotencymaintenance.Advisors/Committee Members: X. Susan Sun.