Hydrogels as feeder-free scaffolds for long-term self-renewal of mouse induced pluripotent stem cells

Abstract

Expanding undifferentiated induced pluripotent stem (iPS) cells in vitro is a basic requirement for application of iPS cells in both fundamental research and clinical regeneration. In this study, we intended to establish a simple, low cost and efficient method for the long-term self-renewal of mouse induced pluripotent stem (miPS) cells without using feeder-cells and adhesive proteins. Three scaffolds were selected for the long-term subculture of miPS cells over two months starting from passages 14 to 29: 1) a gelatin coated polystyrene (Gelatin-PS) that is a widely used scaffold for self-renewal of mouse embryonic stem (mES) cells; 2) a neutral hydrogel poly(N,N-dimethylacrylamide) (PDMAAm); and 3) a negatively charged hydrogel poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS). Each passaged miPS cells on these scaffolds were cryopreserved successfully and the revived cells showed high viability and proliferation. The passaged miPS cells maintained a high undifferentiated state on all three scaffolds and a high level of pluripotency by expressing differentiation markers in vitro and forming teratomas in SCID mice with derivatives of all three germ layers. Compared to Gelatin-PS, the two hydrogels exhibited much better self-renewal performance in terms of high proliferation rate and level of expression of undifferentiated gene markers as well as efficiency in pluripotent teratoma formation. Furthermore, the PNaAMPS hydrogel demonstrated a slightly higher efficiency and simpler operation of cell expansion than the PDMAAm hydrogel. To conclude, PNaAMPS hydrogel is an excellent feeder-free scaffold because of its simplicity, low cost and high efficiency in expanding a large number of miPS cells in vitro.

Induced Pluripotent Stem Cells for Regenerative Medicine

“…Although much work has been done on ES cells to achieve these goals, only recently have several reports shown efficient expansion of iPS cells and successful long-term expansion of hiPS and hES cells in a defined medium (47–50). Encapsulation into negatively charged hydrogels of poly(2-acrylamido-2-methyl-propane sulfonic acid) (PNaAMPS) was recently shown to maintain mouse iPS cell pluripotency and long-term self-renewal in a feeder-free culture (51). Matrigel-coated polystyrene microcarriers in a stirred tank bioreactor have also been used to successfully expand hiPS cells (52).…”

Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics

“…Among different reasons for such behavior is the absence of the proper environment and cell/cell and cell/ extracellular matrix interactions in vivo. The use of natural or artificial scaffolds and biologically active molecules developed for tissue engineering and organ reconstruction might help to improve cell retention and survival (62)(63)(64). Preclinical studies should address critical issues regarding the ability of the transplanted cells not only to be retained in the target but also to become part of a functional tissue.…”

Bacterial Cellulose

“…Induced pluripotent stem (iPS) cells have been used in recent in vitro research as an alternative to MSCs [46][47][48][49]. Autologous cells can be readily harvested from expendable tissues such as adipose or skin.…”