Establishment of parthenogenetic murine embryonic stem cells and their differentiation into osteogenic and chondrogenic cells

Abstract

Damaged tissue repair using stem cells has become a topic of great interest in tissue engineering including orthopedics and dentistry research. Embryonic stem cells (ESCs) are pluripotent and can differentiate into all somatic cell types. ESCs are an alternative solution to hard and soft osseous tissue regeneration and skeletal tissue repair to treat bone diseases and defects using regenerative strategies. The aim of this thesis was to improve the efficiency of stem cell generation and differentiation by modifying traditional methods. In this study, I used a new culture method called the oil-free micro-tube culture method (MTC). This method was shown to be effective for the generation of murine parthenogenetic embryonic stem cell (PESCs) lines. Murine parthenogenetic embryos cultured in MTC showed higher rates of stem cell line generation than those in the traditional micro-drop culture method, and this may be due to the promoted expression of developmentally important genes such as Igf1 and Oct4. PESCs may be a useful alternative stem cell source for tissue repair and regeneration. The defects in full-term development of this cell type enable researchers to avoid the ethical concerns related with ESC research. However, many previous reports have shown that the differentiation potential of PESCs is limited compared to that of ESCs, and this may be related to the abnormal expression of imprinting genes in PESCs. Hence, it is hypothesized that specific-imprinting gene expression promotes the osseous differentiation of PESCs. Insulin like growth factor 2 (IGF2) is a paternally expressed imprinting gene that is therefore not expressed in PESCs naturally. Osteogenic and chondrogenic differentiation of PESCs promoted by IGF2 was demonstrated. The lack of endogenous IGF2 expression can be compensated by exogenous supplementation of this soluble factor in the culture medium resulting in osteogenic and chondrogenic cell differentiation. In osteogenic cell differentiation, gene expression of specific osteoblastic markers was analyzed by real time qPCR. The expression level of osteocalcin, osteopontin, osteonectin, and alkaline phosphatase was 2-fold higher in the group with IGF2 supplementation. An in vivo experiment (critical-sized calvarial defect mouse model) showed the same results on the regeneration of the damaged bone. In chondrogenic cell differentiation, the gene expression of chondrocyte-specific markers was analyzed by real-time qPCR. The expression level of Decorin, Chordin-like 1, Pax1, Aggrecan, Collagen type II and MMP13 was higher in the group with IGF2 supplementation. The group supplemented with IGF2 showed a higher induction of chondrogenic differentiation. Both the osteogenic and chondrogenic induction data show that the supplementation of IGF2 promotes osteogenic and chondrogenic differentiation of PESCs. Collectively, this study presents the whole effective process from establishment to differentiation into osteogenic and chondrogenic cells of PESCs. The findings provide two insights on PESC research. First, as an alternative to the micro-drop culture, the MTC culture method is an effective and affordable embryo culture for generating PESC lines. Second, compensative supplementation of imprinting factor IGF2 may improve the efficiency of differentiation to specific lineages in monogenic stem cells.