Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Aug 26, 2015; 7(7): 1064-1077
Published online Aug 26, 2015. doi: 10.4252/wjsc.v7.i7.1064
Simplified three-dimensional culture system for long-term expansion of embryonic stem cells
Christina McKee, Mick Perez-Cruet, Ferman Chavez, G Rasul Chaudhry
Christina McKee, Mick Perez-Cruet, Ferman Chavez, G Rasul Chaudhry, OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI 48309, United States
Christina McKee, G Rasul Chaudhry, Department of Biological Sciences, Oakland University, Rochester, MI 48309, United States
Mick Perez-Cruet, Beaumont Health System, Royal Oak, MI 48073, United States
Ferman Chavez, Department of Chemistry, Oakland University, Rochester, MI 48309, United States
Author contributions: McKee C preformed the majority of experiments, and helped write the manuscript; Perez-Cruet M and Chavez F provided resources; Chavez F synthesized scaffold material; Perez-Cruet M and Chavez F were involved in the editing of the manuscript; Chaudhry GR designed and guided the study, and helped write the manuscript.
Supported by Oakland University and Oakland University-William Beaumont Institute for Stem Cell and Regenerative Medicine (OU-WB ISCRM).
Institutional review board statement: This research is conducted with cell lines purchased from ATCC and has been approved by the Oakland University Institutional Biosafety Committee (IBC protocol number: 1814).
Institutional animal care and use committee: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Oakland University (IACUC protocol number: 14033).
Conflict-of-interest statement: None.
Data sharing statement: None.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Dr. G Rasul Chaudhry, Department of Biological Sciences, Oakland University, 2200 North Squirrel Road, Rochester, MI 48309, United States.
Telephone: +1-248-3703350 Fax: +1-248-3703586
Received: February 21, 2015
Peer-review started: February 22, 2015
First decision: April 27, 2015
Revised: May 27, 2015
Accepted: June 18, 2015
Article in press: June 19, 2015
Published online: August 26, 2015

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.

METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.

RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.

CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.

Keywords: Three-dimensional culture, Pluripotency, Embryonic stem cells, Self-assembling scaffold, Hydrogel

Core tip: The pluripotent nature of embryonic stem cells (ESCs) makes them an ideal source for cell-based therapeutics and regenerative medicine. Efficient and reproducible expansion of ESCs ex vivo is critical for high quality cells for translational applications. However, propagation of ESCs is technically challenging, and often leads to differentiation due to inefficient two-dimensional culture techniques in vitro. To mimic the three-dimensional microenvironment in vivo, self-assembling scaffolds made from thiol-functionalized dextran and polyethylene glycol tetra-acrylate were designed to encapsulate and propagate mouse ESCs. This culture system is simple, robust, efficient and reproducible, permitting long-term maintenance of ESCs without routine passaging and manipulation.