Stem cell sources for Regenerative Medicine and Cartilage Tissue Engineering

• Main Author: Koh, Sehwon
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2013
• Subjects: Biomedical engineering; Cellular biology
• ISBN: 9781303547485; 1303547481

Stem cells have been studied widely as a potential resource for regenerative medicine as they can proliferate for long-term while maintaining differentiation potency. These properties are used for regeneration and repair of cells and tissues within body. Adult stem cells can be found from almost all type of tissues/organ and used for autologous cell therapy. But they have lower proliferative and differentiation potential compared to pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells. It has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). From our study, we report the derivation of iPSCs from adult canine fibroblast by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in three different culture media (FGF2, LIF, or FGF2 plus LIF). Cells cultured in both FGF2 and LIF expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28, ESC-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1) and showed strong levels of alkaline phosphatase (AP) expression. In vitro differentiation by formation of embryoid bodies (EBs) and by directed differentiation generated cell derivatives of all three germ layers as confirmed by mRNA and protein expression. In vivo, the canine iPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all three germ layers. Array comparative genomic hybridization (array-CGH) and chromosomal fluorescence in situ hybridization (FISH) analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive canine iPSCs from adult fibroblasts by using four transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF is still unknown. For cartilage tissue engineering, it is critical to be able to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation. Here we report an optimized porcine chondrocyte expansion system using serum free media and low oxygen, which minimizes changes in cell differentiation and maximizes their ability to redifferentiate into cartilage producing cells upon induction. Chondrocytes were isolated from pig coastal cartilage and expanded in combinations of serum-free media containing FGF2 (SFM) or media containing fetal bovine serum (SCM) and high (20%) versus low (5%) oxygen. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (P<0.05). AP staining indicated that chondrocytes grown in FBS had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (P<0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (P<0.05) in cells expanded in Low-SFM indicating reduced de-differentiation. This was also supported by expression levels of CD90 and CD105, known markers of dedifferentiation. In vitro re-differentiation capacity of chondrocytes grown in Low-SFM showed similar expression ratio of COL2/COL1 and ACAN/VCAN to cartilage, as well as higher glycosaminoglycan concentration. In conclusion, Low-SFM culture conditions resulted in improved cell growth rate, reduced levels of de-differentiation during expansion, and greater ability to re-differentiate into cartilage upon induction.