Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood. This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could achieve fully reprogrammed state. We demonstrate that loss of microRNA biogenesis pathway abolished the potential of mouse embryonic fibroblasts (MEFs) to be reprogrammed and revealed that several clusters of mES-specific microRNAs were highly induced by four factors during early stage of reprogramming. Among them, miR-93 and 106b were further confirmed to enhance iPSC generation by promoting mesenchymal-to-epithelial transition (MET) and targeting key p53 and TGFβ pathway components: p21 and Tgfbr2, which are important barrier genes to the process. To expand our view of microRNAs function during reprogramming, a systematic approach was used to analyze microRNA expression profile in iPSC-enriched early cell population. From a list of candiate microRNAs, miR-135b was found to be most highly induced and promoted reprogramming. Subsequent analysis revealed that it targeted an extracellular matrix network by directly modulating key regulator Wisp1. By regulating several downstream ECM genes including Tgfbi, Nov, Dkk2 and Igfbp5, Wisp1 coordinated IGF, TGFβ and Wnt signaling pathways, all of which were strongly involved in the reprogramming process. Therefore, we have identified a microRNA-regulated network that modulates somatic cell reprogramming, involving both intracellular and extracellular networks. In addition to microRNAs, in order to identify new regulators and signaling pathways of reprogramming, we utilized small molecule kinase inhibitors. A collection of 244 kinase inhibitors were screened for both enhancers and inhibitors of the process. We identified that inhibition of several novel kinases including p38, IP3K and Aurora kinase could significantly enhance iPSC generation, the effects of which were also confirmed by RNAi of specific target genes. Further characterization revealed that inhibition of Aurora A kinase enhanced phosphorylation and inactivation of GSK3β, a process mediated by Akt kinase. All together, in this dissertation, we have identified novel role of both small non-coding RNAs and kinases in regulating the reprogramming of MEFs to iPSCs.

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS-2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development.