Biotechnology & Bioinformatics
http://hdl.handle.net/10139/2958
2024-03-29T08:11:12ZArticular Mesenchymal Stem Cell (MSC) and Effects of Growth Differentiation Factor-5 (GDF-5)
http://hdl.handle.net/10139/3016
Articular Mesenchymal Stem Cell (MSC) and Effects of Growth Differentiation Factor-5 (GDF-5)
Wu, YueJia
Objectives. 1. To determine the presence of mesenchymal stem cells (MSC) in rat embryo limb buds and adult rat knee joints. 2. To determine the pharmacological effects of recombinant human GDF-5 (rhGDF-5) on rat MSCs. 3. To determine the effect of rhGDF-5 on load bearing in a rodent model of osteoarthritis. Methods. Fluorescent-Activated Cell Sorting (FACS) was used to identify and purify MSCs from E18 rat embryo and adult rat knee cartilage. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to assess the RNA profile of MSCs. qRT-PCR was specifically used to quantify the expression of chondrogenesis markers including Aggrecan (Agc), Type II collagen (Col2), and Sox9 and osteogenesis markers including Runx2 and Type I collagen. An Alamar Blue (AB) assay was utilized to assess effects of rhGDF-5 on MSC proliferation. An Alkaline Phosphatase (ALP) assay was used to study the effects of rhGDF-5 on MSC differentiation. Neuroprobe migration chambers were used to assess rhGDF-5 effects on chondrocyte chemotaxis. In situ hybridization (ISH) was conducted to identify MSC (expression of chondrogenic RNA biomarkers) in rat embryos and in adult knee joints from normal and osteoarthritic rats. Differential weight bearing analysis was assessed using Bioseb's DWB system.
Results. MSCs were obtained from E18 rat embryo limb buds and adult rat knee joint cartilage. qRT-PCR showed that MSC comprised pre-osteochondrocytes expressing Agc, Col2a1, Sox9, and GDF-5 RNAs. rhGDF-5 treated MSC showed a dose-dependent increase in AB intensity from Day 1 to Day 14, followed by an increase in rhGDF-5 mediated ALP activity on Day 14 and Day 21. The increased ALP activity coincided with increased Agc, Col2a1, Sox9, and GDF-5 RNA expression indicating that rhGDF-5 selectively promoted chondrogenesis. Greater chemotaxis effect of rhGDF-5 was observed with MSC that presumably were more differentiated after 23 days in culture with rhGDF-5. Following 23 days of treatment with rhGDF-5, MSC appeared as condensations and precartilaginous structures. ISH confirmed the presence of GDF-5, Sox9, and Runx2 in rat embryo hind limbs and in the experimentally-induced osteoarthritic joint of adult animals. DWB analysis revealed significant differential joint loading in the Medial Meniscus Tear (MMT) injured knee, but no reverse differential weight bearing was detected in rhGDF-5 treated rats after a 9-week treatment period. Conclusion. Mesenchymal stem cells are present in rat embryonic limb buds and adult rat knee joint. Our studies show that rhGDF-5 may promote an increase in metabolic activity of MSCs prior to commitment to the chondrocyte lineage. The positive chemotaxic effect of GDF-5 suggests a role in MSC cell homing, cell condensation, and formation of pre-cartilaginous structures. Taken together, GDF-5 is a selective chondrocyte differentiation factor. The presence of MSC in the damaged joint suggests that rhGDF-5 may act to differentiate endogenous MSC to chondrocytes.
2010-09-20T00:00:00ZOptimization of Hydroxyapatite β-Tricalcium Phosphate – Fibrin Constructs for Human Mesenchymal Stem Cell Proliferation and Mechanical Strength
http://hdl.handle.net/10139/3015
Optimization of Hydroxyapatite β-Tricalcium Phosphate – Fibrin Constructs for Human Mesenchymal Stem Cell Proliferation and Mechanical Strength
Fard, Roxana
Bone graft substitutes are commonly used as an alloplastic source for complex bone repair. Human mesenchymal stem cells (hMSCs) have become an idealistic source for bone repair and regeneration due to their potential to differentiate into osteogenic precursors. The purpose of engineering synthetic bone grafts it to successfully find a substitute that is biocompatible, bioresorbable, and has osteoconductive characteristics. The purpose of this study is to construct a bone biocomposite with an optimal amount of biphasic hydroxyapatite/β-tricalcium phosphate (HA-TCP) powder to promote hMSC proliferation with sufficient mechanical stiffness. Results have indicated an increase in metabolic proliferation over a 2-week time period. The constructs seeded with hMSCs exhibited a 3 to 9 fold or greater increase in proliferation depending on the formulation of the construct. This work demonstrates that higher volumes of HA-TCP promote hMSC proliferation in the constructs while maintaining sufficient mechanical stiffness. Optimizing the components of the scaffolds will allow for the most innovative biomimetic bone composite for mesenchymal stem cell differentiation into osteoblasts in an in vivo model.
2010-09-25T00:00:00ZIn Vitro and In Vivo Characterization of Cancer Stem Cells in Primary Colorectal Cancer Models
http://hdl.handle.net/10139/2959
In Vitro and In Vivo Characterization of Cancer Stem Cells in Primary Colorectal Cancer Models
Gehani, Rohit
The existence of cancer stem cells (CSC) postulates that tumors are organized as a cellular hierarchy and that tumor initiation, growth and cellular heterogeneity are driven by a subset of cells with stem cell like properties. The CSCs are endowed with the ability to self-renew and thereby to proliferate indefinitely. At a functional level, CSCs are characterized by their ability to regenerate in vivo from a single cell into the full spectrum of histology of the tumor of origin and to form spheroid colonies in vitro in an anchorage independent environment.
The specific aims for this project include setting up assays that will enable the quantification and the characterization of CSCs and evaluating cell surface markers to enrich for CSC. Additionally, in our in vivo assays, we will compare different mice strains as hosts to our in vivo assays.
In order to assess the frequency of CSCs within a solid tumor, we established an in vitro and in vivo limited dilution assay (LDA). A known number of cells were seeded in a low attachment well with stem cell growth media, and the number of spheroids that grew was counted by Optronix GelCount. In an in vivo LDA, immune deficient mice were inoculated with a specific number of cells, and the number of tumor bearing mice was counted.
In order to optimize our methods, immortalized colorectal cancer (CRC) cell lines COLO205 and T84 were used as tools to develop LDAs. We were able to determine that the sphere and tumor-forming efficiency of COLO205 was superior to T84, resulting in the primary use of COLO205 cells for assay development. We also aimed to compare the tumor initiating capacity of the COLO205 cell line in order to investigate the effect of the mouse immune system on the read-out of the in vivo tumor formation assay. The result of this experiment was inconclusive; no tumors grew, which is a defect that we could pinpoint to a loss of viability of sorted tumor cells by FACS.
We worked with four colorectal cancer models derived from patient tumors and continuously passaged in immune deficient mice. Two models were established from primary tumors obtained from the Amgen Tissue Bank. These tumors were dissociated and cultured in serum-free stem cell growth media on low attachment plates. The other two models were obtained from a company that had continuously passaged the tumors in immune deficient mice. We used these two models to test mouse immune background and effects of irradiation on tumor initiation.
Antibodies to extracellular proteins epithelial cell adhesion molecule (EpCAM) and CD133 were used to enrich for a homogenous population of CSCs by sorting protocols. We could not confirm that either of these is a good marker but did find that FACS sorting may affect tumorigenicity and viability of CSCs in our CRC models. The outcome of the CSC enrichment procedure will be important to enable further characterization at the molecular and cellular level of CSC and the identification of potential therapeutic targets that could selectively eliminate CSCs.
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2010-09-25T00:00:00Z