Semester

Spring

Date of Graduation

2014

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Exercise Physiology

Committee Chair

Ming Pei

Committee Co-Chair

Stephen E. Alway

Committee Member

John M. Hollander

Committee Member

Emidio E. Pistilli

Committee Member

Yon Rojanasakul

Abstract

Cartilage defects caused by injuries of the knee affect about 900,000 Americans annually, resulting in more than 200,000 surgical procedures. Cartilage repair remains a major challenge due to its limited healing capacity. Current cell-based therapy using autologous chondrocyte implantation has been developed for decades and promising results have been observed in clinic. However, the shortage of autologous chondrocytes and their uncertain long-term effectiveness have led researchers to find alternative solutions. Stem cells from various tissues have been shown to be potential sources of chondrocytes. Among them, synovium-derived stem cells (SDSCs) have been suggested as tissue-specific stem cells for chondrogenesis. However, a major obstacle challenging the cartilage tissue engineering is cell senescence, which is due mainly to extensive ex vivo passaging and elderly donors. A reconstructed ex vivo microenvironment that can maintain or enhance stemness is urgently needed for facilitating large-scale tissue engineering. To facilitate ex vivo expansion, the conventional methods for stem cell expansion were extensively investigated. We first compared the influence of low- and high-seeding density on human SDSC stemness during ex vivo expansion. Low-density seeding expansion yielded SDSCs with enhanced proliferative and multi-differentiation capacities compared to high-density seeding though it is not highly efficient. Downregulation of ERK1/2 and JNK and upregulation of p38 might be attributed to the retained "stemness" under low-density expansion. We next compared the impact of hypoxia, fibroblast growth factor-2 (FGF-2) supplementation and a novel approach of SDSC-deposited decellularized extracellular matrix (DECM) on SDSC stemness. DECM expansion enhanced greater SDSC proliferation while retaining stem cell characteristics, compared to FGF-2 supplementation alone. The combination of hypoxia, FGF-2 and DECM contributed to the highest cell number in SDSC expansion, indicating their synergistic effects. Although the chondrogenic index was comparable between DECM expansion and FGF-2 supplementation, which were much higher than expansion on plastic flask alone, the observations that FGF-2 induced hypertrophic marker genes suggested the superiority of DECM in enhancing SDSC self-renewal while retaining stemness. Other potential cell sources for depositing DECM were also evaluated. We found that, besides SDSCs, adipose- or urine-derived stem cells and dermal fibroblasts could also deposit the DECM, which could enhance SDSC self-renewal and chondrogenic potential without concomitantly enhancing adipogenic and osteogenic potentials. These findings suggest that, given an optimal DECM substrate, the chondrogenic potential within the tissue-specific SDSC could be substantially enhanced. We further characterized human fetal synovial fibroblasts as fetal SDSCs as they possessed the multi-lineage differentiation capacities and mesenchymal stem cell surface marker expression. Fetal SDSC-derived DECM expansion not only increased cell number and enhanced chondrogenic potential, it also lowered SDSC senescence marker expression while enhancing MSC marker expression compared to expansion on plastic flasks alone. As cell senescence is a limiting factor for tissue regeneration, we then investigated whether the DECM derived from fetal SDSCs referred to as a young stem cell microenvironment could be used for rejuvenating adult SDSC. We found that fetal SDSC-derived DECM (FE) was superior to adult SDSC-deposited DECM (AE) in promoting SDSC proliferation and chondrogenic potential. Further investigation revealed that unique proteins in FE might be responsible for the rejuvenation effect and advantageous proteins in AE might contribute to differentiation more than proliferation. Compared to AE, the lower elasticity of FE yielded expanded SDSCs with lower elasticity, which could be responsible for the enhancement of chondrogenic differentiation. MAPK and noncanonical Wnt signals were also actively involved in DECM-mediated SDSC rejuvenation. The young and healthy microenvironment provided by fetal SDSCs could serve as a "fountain of youth" for adult SDSC rejuvenation. Finally, we tested whether the DECM expansion system would also be beneficial to the chondrocyte-like nucleus pulposus cell (NPC) rejuvenation from human herniated discs and whether fetal DECM is superior to adult DECM. Although both SDSC and NPC deposited DECMs (SECM and NECM) significantly enhanced NPC proliferation, only NECM expanded NPCs manifested the increased redifferentiation capacities after chondrogenic induction. NECM is better than SECM in functioning as an expansion system in vitro by promoting NPC proliferation and redifferentiation. In conclusion, we have demonstrated that the DECM deposited by human primary cells or stem cells serves as an ex vivo expansion system for maintaining self-renewal and differentiation potential, which could greatly benefit the future generation of cell-based therapy for cartilage and intervertebral disc regeneration.

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