Identifying the sites and mechanisms involved in haematopoietic stem cells (HSCs) during development would improve our understanding of how to induce HSCs from alternative sources like embryonic stem cells, while offering insight into pathways involved in HSC-related diseases such as leukaemia. Adult-type HSC, or long-term reconstituting HSCs (LTR-HSCs), are widely defined as cells capable of reconstituting the entire haematopoietic system of a lethally irradiated adult recipient. The first LTR-HSCs emerge and expand in the aorta-gonad-mesonephros (AGM) region of the mid-gestation mouse embryo. Recently, the development of a novel reaggregate culture system has provided a valuable tool to identify key cell populations involved in LTR-HSC development. This system allows the mechanical dissociation of the E11.5 AGM region prior to culture whilst maintaining its ability to autonomously expand LTR-HSCs. Here, I show that reaggregate LTR-HSCs are CD45+Sca1+c-kit+CD31med and that IL-3, SCF, and Flt3l are required in order to achieve an optimal 150 fold LTR-HSC expansion. I also characterise the pattern of Runx1 expression in the adult and E11.5 AGM region of our novel Runx1EGFP reporter mouse and identify a population of EGFP+CD45-VE-cadherin- cells in the E11.5 AGM region that disappears during reaggregate culture. Finally, using the E11.5 AGM reaggregate culture, I show that while uro-genital ridges are potentially required for optimal LTR-HSC expansion, most LTR-HSCs are derived from the dorsal aorta (Ao) region, and that the dorsal aspect of the dorsal aorta (AoD) can contribute to the reaggregate LTR-HSCs compartment.

Sexual dimorphism has been long noted in human neurobiology, apparent most notably in sex-biased distribution of multiple neurological disorders or diseases, from autism spectrum disorder to Parkinson's disease. With the advances in molecular biology, genetics and epigenetics have come into focus as key players in sexually dimorphic neural development; and yet, many studies in the field of neuroscience overlook the importance of sex for the human brain. For this project, human embryonic and neural stem cells were chosen for three main reasons. Firstly, they provide an easily obtainable, scalable and physiologically native model for the early stages of development. Secondly, neural stem cells populations are retained within the adult human brain, and are implicated to play a role in cognition and mental illness, and as such are of interest in themselves. Thirdly, stem cell lines are widely used in research, including clinical trials of transplantation treatments, and for this reason should be meticulously examined and characterized. Here, the morphology, behaviour, and expression of selected genes in four stem cell lines, two of female and two of male origin, was examined in side-by-side comparisons prior to and during neuronal differentiation using a variety of methods including light microscopy, time-lapse two-photon microscopy, quantitative real-time PCR and immunocytochemistry. The obtained results have shown previously uncharacterised differences between those cell lines, such as a higher rate of proliferation but a slower rate of neuronal differentiation in male cell cultures compared to female cells cultivated in the same conditions, and a sex-biased expression of several markers of neuronal maturation at late stages of differentiation, as well as diverse patterns of expression of X- and Y-linked genes involved in stem cell proliferation and neural development.

The complexity of life exhibited in humans and other living creatures has drawn many to investigate the principles associated with organismal growth and development. A few broad questions: How do tissues develop into specified organs? How are these tissues maintained? Do they become different tissues? Scientific research has incessantly been seeking answers to these as well as a plethora of other questions. While on a quest to better understand developmental biology, investigators discovered unique populations of stem cells within a variety of tissues, which retain both varying degrees of developmental plasticity and their potential for self-regeneration. This thesis provides a brief review discussing the development and history of stem cells in medicine and associated research on these cells and their potential clinical applications. Substantial attention has been paid to pluripotent embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) which are able to be recapitulate ESC properties through the in vitro reprogramming of somatic cells. While, the ethical and legal issues have greatly hindered the use of ESCs this has made the benefit of iPSCs so great. An interconnected network of pluripotency-associated genes, integrates external signals and exerts control to maintain the state of pluripotency. Recent research has proven the pluripotency regulatory network to be flexible such that the underlying principles promise unprecedented opportunities for scientific study and regenerative medicine. Additional topics reviewed here include vast clinical applications of stem cells as well as their notable limitations.

Lin, Ka Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-122). / Abstracts in English and Chinese. / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iii / Acknowledgment --- p.V / Abstracts of Publications --- p.vi / Abbreviations --- p.viii / List of Figures --- p.ix / List of Table --- p.X / Contents --- p.xi / Chapter Chapter I --- Introduction --- p.Page / Chapter 1.1 --- Pluripotent Stem Cell --- p.1 / Chapter 1.1.1 --- Characteristic of pluripotent stem cells --- p.1 / Chapter 1.1.2 --- Origin of pluripotent stem cells --- p.1 / Chapter 1.1.2.1 --- Embryonic carcinoma cells --- p.2 / Chapter 1.1.2.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.2.3 --- Epiblast stem cells --- p.2 / Chapter 1.1.2.4 --- Embryonic germ cells and adult germline stem cells --- p.3 / Chapter 1.1.2.5 --- Induced pluripotent stem cells --- p.3 / Chapter 1.1.3 --- Pluripotency in Embryonic Stem Cells --- p.4 / Chapter 1.1.3.1 --- Extrinsic signal governing pluripotency --- p.5 / Chapter 1.1.3.1.1 --- LIF signaling --- p.5 / Chapter 1.1.3.1.2 --- BMP signaling --- p.6 / Chapter 1.1.3.1.3 --- Other signaling pathways --- p.6 / Chapter 1.1.3.2 --- Intrinsic sternness factors --- p.7 / Chapter 1.1.3.2.1 --- Oct4 Expression in Embryonic Stem cells --- p.7 / Chapter 1.1.3.2.2 --- Sox-2 Expression in Embryonic Stem Cells --- p.8 / Chapter 1.1.3.2.3 --- Nanog Expression in Embryonic Stem Cells --- p.9 / Chapter 1.1.3.2.4 --- "Transcriptional Regulation of Oct-4, Nanog and Sox-2 in Embryonic Stem Cells" --- p.10 / Chapter 1.1.3.2.5 --- Others pluripotent genes --- p.11 / Chapter 1.1.3.2.5.1 --- Utfl --- p.11 / Chapter 1.1.3.2.5.2 --- Rexl --- p.11 / Chapter 1.1.3.2.5.3 --- Esrrb --- p.12 / Chapter 1.1.3.2.5.4 --- Eras --- p.12 / Chapter 1.1.3.2.5.5 --- Tell --- p.12 / Chapter 1.1.3.2.5.6 --- Dnm3tl --- p.13 / Chapter 1.1.3.2.5.7 --- Dppa3 --- p.13 / Chapter 1.1.3.2.5.8 --- Dppa4 --- p.14 / Chapter 1.1.3.2.5.9 --- Dppa5 --- p.14 / Chapter 1.1.3.2.5.10 --- Klf2 --- p.15 / Chapter 1.2 --- Somatic cell reprogramming --- p.16 / Chapter 1.2.1 --- Definition of reprogramming --- p.16 / Chapter 1.2.2 --- The history of reprogramming --- p.16 / Chapter 1.2.2.1 --- Reprogramming by nuclear transfer --- p.17 / Chapter 1.2.2.2 --- Reprogramming by fusion with ES or EC cells --- p.18 / Chapter 1.2.2.3 --- Reprogramming with defined factor --- p.19 / Chapter 1.3 --- Induced pluripotent stem cells --- p.20 / Chapter 1.3.1 --- Transcription factor used for reprogramming to iPS cells --- p.20 / Chapter 1.3.1.1 --- Klf4 --- p.20 / Chapter 1.3.1.2 --- c-Myc --- p.21 / Chapter 1.3.2 --- Cornerstone of iPSC generation --- p.22 / Chapter 1.3.3 --- Major events in the reprogramming process --- p.23 / Chapter 1.3.4 --- Gene delivery systems for ips cell generation --- p.26 / Chapter 1.3.5 --- Culture system for embryonic stem cells and iPSC --- p.28 / Chapter 1.3.4.1 --- Feeder and serum used cell culture system --- p.28 / Chapter 1.3.4.2 --- Serum-free culture condition --- p.29 / Chapter 1.3.5 --- Differentiation potential of iPSC --- p.30 / Chapter 1.3.5.1 --- In vitro stringency tests --- p.30 / Chapter 1.3.5.2 --- In vivo stringency test --- p.30 / Chapter 1.3.5.3 --- In utero stringency test --- p.31 / Chapter 1.4 --- Mouse Lewis lung carcinoma-D 122 --- p.32 / Chapter 1.5 --- Dendritic cell vaccine in cancer immunotherapy --- p.33 / Chapter 1.5 --- Green Fluorescence protein Reporters --- p.35 / Chapter 1.5.1 --- GFP reporters in embryos and stem cell --- p.35 / Chapter 1.5.2 --- copGFP --- p.35 / Chapter 1.6 --- Aim of study --- p.36 / Chapter Chapter II --- Methods and Materials / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Synthetic oligos used in polymerase chain reaction (PCR) --- p.37 / Chapter 2.1.2 --- DNA clones used in the study --- p.39 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.39 / Chapter 2.1.4 --- Materials for RNA manipulation --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.6 --- Kits --- p.41 / Chapter 2.1.7 --- Bacteria strain and culture reagents 41 / Chapter 2.1.8 --- Culture media and reagents --- p.42 / Chapter 2.1.8.1 --- General culture media and reagents --- p.42 / Chapter 2.1.8.2 --- Traditional ES medium --- p.42 / Chapter 2.1.8.3 --- Feeder-free Serum-free ESGRO medium --- p.42 / Chapter 2.1.9 --- Cell lines used --- p.43 / Chapter 2.1.10 --- Instrumentation --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Cell culture --- p.44 / Chapter 2.2.1.1 --- Routine cell culture --- p.44 / Chapter 2.2.1.2 --- Resuscitation and culture from frozen stock --- p.44 / Chapter 2.2.1.3 --- Passage of cells --- p.44 / Chapter 2.2.1.4 --- Cryopreservation of cells --- p.45 / Chapter 2.2.1.5 --- Mouse ES cells culture --- p.45 / Chapter 2.2.1.5.1 --- Passage and maintenance of SNL --- p.45 / Chapter 2.2.1.5.2 --- Inactivation and plating of SNLs (Feeder preparation) --- p.45 / Chapter 2.2.1.5.3 --- Cryopreservation (freezing) of SNLs --- p.46 / Chapter 2.2.1.6 --- Mouse ES cells culture in feeder-free culture conditions --- p.46 / Chapter 2.2.1.6.1 --- Preparation of gelatin coated plates --- p.46 / Chapter 2.2.1.6.2 --- Thawing mouse ES cells --- p.46 / Chapter 2.2.1.6.3 --- Passage of mouse ES cells --- p.47 / Chapter 2.2.1.6.4 --- Freezing mouse ES cells --- p.47 / Chapter 2.2.1.7 --- ES cells differentiation-Formation of embryoid bodies (EBs) --- p.47 / Chapter 2.2.1.8 --- Direct differentiation by retinoic acid --- p.48 / Chapter 2.2.1.9 --- Generation of iPS --- p.48 / Chapter 2.2.2 --- Cell transfections --- p.48 / Chapter 2.2.2.1 --- Lipofectamine 2000 transfection --- p.48 / Chapter 2.2.2.2 --- Nucleofection --- p.49 / Chapter 2.2.2.2.1 --- Optimization of nucleofection --- p.49 / Chapter 2.2.2.2.2 --- Nucleofection condition --- p.49 / Chapter 2.2.3 --- Nucleic acid --- p.49 / Chapter 2.2.3.1 --- Genomic DNA isolation --- p.49 / Chapter 2.2.3.2 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.2.3.3 --- RNA and genomic DNA quantification --- p.50 / Chapter 2.2.3.4 --- Reversed transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.3.4.1 --- RNA isolation and Reverse transcription (RT) --- p.50 / Chapter 2.2.3.4.2 --- Polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.3.4.3 --- Real-time polymerase chain reaction (qRT- PCR) --- p.52 / Chapter 2.2.3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 2.2.3.6 --- Genomic PCR for bisulfite sequencing --- p.53 / Chapter 2.2.4 --- Bacteria and Plasmid preparation --- p.54 / Chapter 2.2.4.1 --- Preparation of competent cells --- p.54 / Chapter 2.2.4.2 --- Heat-shock transformation --- p.54 / Chapter 2.2.4.3 --- Midi prep of plasmid --- p.54 / Chapter 2.2.5 --- Cell Staining --- p.55 / Chapter 2.2.5.1 --- Alkaline phosphatase staining --- p.55 / Chapter 2.2.5.2 --- Immunofluorescence --- p.55 / Chapter 2.2.6 --- Flow cytometry --- p.56 / Chapter 2.2.7 --- Animal Handling --- p.56 / Chapter Chapter III --- Results / Chapter 3.1 --- Generation of Nanog-reporter-GFP-D 122 --- p.57 / Chapter 3.2 --- Nucleofection optimization for D122 --- p.60 / Chapter 3.3 --- Generation ofD122-iPS --- p.65 / Chapter 3.3.1 --- Plasmid construct used in the study --- p.65 / Chapter 3.3.2 --- Protocol of D122-iPS generation --- p.67 / Chapter 3.3.3 --- Reprogramming Efficiency of D12´2ؤreNanog cells --- p.69 / Chapter 3.4 --- Expression of pluripotency markers upon reprogramming --- p.70 / Chapter 3.4.1 --- Alkaline Phosphatase staining --- p.70 / Chapter 3.4.2 --- Nanog-GFP expression --- p.72 / Chapter 3.4.3 --- Pluripotency gene expression upon reprogramming --- p.74 / Chapter 3.4.4 --- GFP positive D122 reNanog Colonies --- p.79 / Chapter 3.5 --- Characterization of the D122-iPS-lC --- p.80 / Chapter 3.5.1 --- Morphology of D122-iPS-lC --- p.80 / Chapter 3.5.2 --- Pluripotency gene expression --- p.82 / Chapter 3.5.3 --- Pluripotency markers SSEA-1 and Oct4 --- p.85 / Chapter 3.5.4 --- Bisulfite genomic sequencing --- p.88 / Chapter 3.5.5 --- Differentiation of the D122-iPS-lC --- p.90 / Chapter 3.5.5.1 --- Embryoid body formation by hanging drop --- p.90 / Chapter 3.5.5.2 --- Retinoic acid induced differentiation --- p.92 / Chapter Chapter IV --- Discussion / Chapter 4.1 --- General Discussion --- p.96 / Chapter 4.1.1 --- Cancer immunotherapy and dendritic cells --- p.96 / Chapter 4.1.2 --- Dendritic vaccine and tumor antigen --- p.97 / Chapter 4.1.3 --- Induced pluripotent stem cell technology and dendritic cells --- p.98 / Chapter 4.1.4 --- Tumor antigen presentation and dendritic cells --- p.98 / Chapter 4.1.5 --- D122 and cancer immunotherapy --- p.99 / Chapter 4.1.6 --- Method to introduce transcription factors for reprogramming --- p.100 / Chapter 4.1.7 --- Kinetics of reprogramming --- p.101 / Chapter 4.1.8 --- Culture condition for reprogramming D122_reNanog --- p.102 / Chapter 4.1.9 --- Reprogramming efficiency --- p.103 / Chapter 4.1.10 --- Establishment of D122-iPS-lC --- p.103 / Chapter 4.1.11 --- Differentiation of D122-iPS-1C --- p.104 / Chapter 4.2 --- Future Work --- p.106 / Chapter 4.3 --- Conclusion --- p.107 / Chapter Chapter V --- Bibliography --- p.108 / Appendix --- p.124

It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.

A Comparison of Bone Marrow Derived and Adipose Derived Stem Cells in Point of Care Goat Non-Instrumented Posterolateral Intertransverse Spinal Fusion Daria Brigitte Neidre, Ph.D. The University of Texas at Austin, May, 2010 Supervisor: Roger P. Farrar Concentrated bone marrow containing mesenchymal stem cells (BMSCs) in combination with osteoconductive scaffolds has been used in orthopaedics to replace the need for iliac crest bone grafts. Autologous BMSC volume is limited, but adipose tissue represents a large reservoir of stem cells; adipose derived stem cells (ADSCs). To test these cells, a large animal model using goats was selected due to their similarities to humans in loading conditions of the spine, trabecular bone structure of the vertebrae, and their common use in testing orthopaedic therapies as a clinically relevant model. The aim of this study is to characterize cell surface markers of the isolated cells through flow cytometry, compare goat BMSCs and ADSCs using multilineage differentiation into the osteogenic and adipogenic lineages, and utilize them in a “Point-of-Care” non-instrumented posterolateral lumbar spinal fusion. Both BMSCs and ADSCs were confirmed as stem cells through lack of expression of markers CD34, CD45, CD90, and CD105, which is supported by literature. Both cell types also differentiated into both the adipogenic and osteogenic lineages. Although we had positive in vitro results, we had limited in vivo results. There were no differences between BMSCs, ADSCs and control implantation in identifiable spinal fusion at 3 or 6 months through radiographs or CT scans. Additionally, there were no differences between groups at 6 months in biomechanical testing, histology and microradiographs. Although our in vivo results were lacking in demonstrating fusion at 6 months, this study is the first of it’s kind to investigate a large animal model comparison of BMSCs and ADSCs in spinal fusion and demonstrated that “Point-of-Care” stem cells derived from either bone marrow or adipose tissue demonstrated the potential for bone formation. The in vivo results suggests that this model can be used for stem cell research in orthopaedics, but further research needs to be performed to determine their use, proper scaffold and potential osteoinductive materials needed for solid fusion results in the in vivo model. / text

Thesis (MTech (Nursing))--Cape Peninsula University of Technology, 2017. / Stem cell transplantation has become one of the standard methods of treatment for patients with malignant and benign blood disorders. The multidisciplinary team interacting with these patients and their families, must be knowledgeable concerning the appropriate quality health care. The objectives of the study were to explore the knowledge of the members of the health care team in terms of the processes that need to be adhered to with stem cells transplantation, as well as exploring the perceptions amongst the health care team members and their reactions towards patients undergoing stem cell transplantation. An exploratory research design with a qualitative approach was employed. Data collection took place at two stem cell transplant units in the Western Cape, using non-probability purposive sampling technique. The health care team members included a medical doctor, dietician, physiotherapist, social worker, radiographer and nursing staff. Data was collected by face-to-face personal interviews which were transcribed and analysed by using coding and thematic analysis. The majority of the professional participants could identify the processes for stem cell transplantation, which affirmed their knowledge. The non-professional health care team member, could also identify the types of methods and processes of stem cell transplantation. Participants stated that the health care team members had passion for this treatment option. Some participants felt it to be emotionally challenging to work in the environment, especially with paediatric patients and the dying. However, some health care team members could detach themselves emotionally from the patients. The team stated that the stem cell transplanted patients need special care to overcome all challenges experienced, but were positive about treatment. It is evident that management of stem cell transplanted patients is complicated and the health care team members must have knowledge, skills and the appropriate attitude to practice in these units. This study emphasised how vital it is that stem cell transplantation be included in the training programs of the multidisciplinary team. Health care practitioners in the field must stay abreast with stem cell research in order to effectively conduct health promotions for patients and staff. In addition, hematology and transplant awareness campaigns should also be conducted in order to educate society and suggest referrals if necessary.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic cell transplantation (HCT) is an important modality used to treat patients with hematologic diseases and malignancies. A better understanding of the biological processes controlling hematopoietic cell functions such as migration/homing, proliferation and self-renewal is required for improving HCT therapies. This study focused on the role of two biologically relevant proteins, dipeptidylpeptidase IV (DPPIV/CD26) and Ras homologue enriched in brain 2 (Rheb2), in modulating hematopoietic cell engraftment. The first goal of this study was to determine the role of the protein DPPIV/CD26 in modulating the engraftment of human umbilical cord blood (hUCB) CD34+ stem/progenitor cells using a NOD/SCID mouse xenograft model, and based upon previous work demonstrating a role for this enzyme in Stromal-Derived Factor-1/CXCL12 mediated migration and homing. Related to this first goal, pretreatment with an inhibitor of DPPIV/CD26 peptidase activity increased engraftment of hUCB CD34+ cells in vivo in recipient Non Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice while not disturbing their differentiation potential following transplantation. These results support using DPPIV/CD26 inhibition as a strategy for enhancing the efficacy of cord blood transplantation. The second goal was to determine, by overexpression, the role of the Rheb2 in affecting the balance between proliferation and in vivo repopulating activity of mouse hematopoietic cells. Rheb2 is known to activate the mammalian target of rapamycin (mTOR) pathway, a pathway important in hematopoiesis. Rheb2 overexpression increased the proliferation and mTOR signaling of two hematopoietic cell lines, 32D and BaF3, in response to delayed IL-3 addition. In primary mouse hematopoietic cells, Rheb2 overexpression enhanced the proliferation and expansion of hematopoietic progenitor cells (HPCs) and phenotypic hematopoietic stem cells (HSCs) in vitro. In addition, HPC survival was enhanced by Rheb2 overexpression. Using in vivo competitive repopulation assays, Rheb2 overexpression transiently expanded immature HPC/HSC populations shortly after transplantation, but reduced the engraftment of total transduced cells. These findings support previous work showing that signaling proteins able to enhance the proliferative status of hematopoietic stem cells often cause exhaustion of self-renewal and repopulating ability. These studies of hematopoietic engraftment modulated by both of these molecules provide information which may be important to future work on HCT.

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.