As mitocôndrias são organelas semiautonômicas, portadoras do próprio DNA, o mtDNA e responsáveis pela produção de energia celular na forma de ATP, através do processo de fosforilação oxidativa. Atualmente, diferentes tipos de doenças, como distrofias musculares e diversos tipos de câncer, estão associadas à alteração nas moléculas de mtDNA. Na década de 70 um modelo a partir do cultivo celular com brometo de etídio (EtBr) foi desenvolvido com o objetivo de se criar uma linhagem celular depletada de cópias de mtDNA. Desde então os mais variados estudos foram realizados e diversos tipos celulares foram submetidos à depleção do mtDNA. Este projeto teve como objetivos criar um modelo de cultivo celular somático na espécie bovina com depleção de cópias de mtDNA para investigar a resposta da célula a esta condição; avaliar como as células depletadas se comportam na ausência de EtBr, além da utilização destas células no processo de reprogramação celular por indução gênica na tentativa de avaliar o efeito do numero de cópias de mtDNA na indução na espécie bovina. Para tanto foram desenvolvidos três experimentos; Experimento 1- Depleção de mtDNA a partir da utilização do brometo de etídeo; Experimento 2 Repleção do mtDNA; e Experimento 3 Utilização de células bovinas depletadas no sistema de reprogramação nuclear. Todos os experimentos foram avaliados quanto a quantidade de cópias de mtDNA e expressão gênica para os genes Bax, Bcl2 e Tfam. Ademais, os experimentos 1 e 2 foram avaliados quanto a viabilidade celular e apenas o experimento 1 foi avaliado quanto ao crescimento e morfologia celular. O experimento 1 foi avaliado durante o cultivo celular nos períodos D0, D4, D7, D10 e D13, com os grupos experimentais controle (EtBr-C) e tratado com 100 ng/mL de brometo de etídio (EtBr-T), quanto a núero de cópias do mtDNA, o grupo EtBr-T diferiu do grupo EtBr-C (P=0,0459), apresentando menor número de cópias de mtDNA; menor taxa crescimento celular (P<0,05), porém sem alteração na morfologia celular, e na expressão dos genes descritos acima. No experimento da repleção, não houve diferença no número de cópias de mtDNA, entre os grupos EtBr-T e EtBr-R, indicativo de que as células atingiram o estado rho 0 ou que necessitam de mais tempo para ativar a replicação do mtDNA; quanto a viabilidade celular, houve diferença entres os grupos, quanto a expressão gênica, com aumento do Bax e do Bcl-2 para o grupo EtBr-T; O grupo EtBr-R apresentou queda do Bcl-2; para o Tfam houve aumento para o grupo EtBr-T e uma queda para o grupo EtBr-R. Quanto ao experimento 3, não foi possível observar sinais de pluripotência, porém foi detectada uma queda na quantidade de mtDNA dos dois grupos tratados por EtBr (EtBr com e sem Stemcca) e o grupo controle com Stemcca. Para analise de expressão gênica, não houve diferenças entre os grupos em relação ao Tfam. Quanto ao Bax, os grupos controle com Stemcca, controle sem Stemcca e EtBr sem Stemcca não diferiram, e o ultimo também não apresentou diferença quando comparado ao grupo EtBr com Stemcca. Para o Bcl-2, os grupos controle sem Stemcca e EtBr com Stemcca não apresentaram diferenças entre si; o grupo controle sem Stemcca não apresentou diferença quando comparado aos grupos controle com Stemcca e EtBr sem Stemcca. Concluindo, este trabalho no nosso conhecimento, descreve pela primeira vez a produção de células bovinas Rho 0 e discute sobre a relação da função mitocondrial e o processo de reprogramação celular. / Mitochondria are semi autonomic organelles which present their own DNA (mtDNA); are in charge of cell energy production as ATP through oxidative phosphorylation. Currently, different types of diseases like muscular distrofy; different types of cancer are associated to alterations of mtDNA molecules. In the 70\'s a model based on cell culture with ethidium bromide (EtBr) was developed in order to create a cell line depleted of mtDNA. Since then, a variety of studies were realized; diverse cell types were submited to mtDNA depletion. This project had as objective creating a model of somatic cell culture in bovine species with depletion of mtDNA copies, in order to investigate cell response to this condition; to analyze depleted cell behavior in the absence of EtBr, besides using this depleteded cell in a reprogramming cell process by genic induction in order evaluate the effect of the number of mtDNA copies during induction in bovine species. Therefore three experiments were developed: Experiment-1 Depletion of mtDNA using ethidium bromide. Experiment-2 repletion of mtDNA; Experiment-3 usage of depleted bovine cells in reprogramming nuclear system. Cell experiments were analyzed according to the quantity of mtDNA copies; genic expression for Bax, BCl2; Tfam genes. Also, experiments 1; 2 were analyzed on cell viability; only experiment 1 was analyzed regarding cell morphology; growth. Experiment-1 was analyzed during cell culture on periods D0, D4, D7, D10, D13, with control experimental groups (EtBr-C),; treated with 100 ng/mL ethidium bromide (EtBr-T); relating to mtDNA quantification the EtBr-T group differed from EtBr-C (P=0,0459) presenting a smaller number of mtDNA copies; smaller growth rate (P<0,05); although there was no differences on cell morphology as there was also no difference related to genic expression of the previous stated genes. Repletion experiment showed no differences about the number of mtDNA copies between EtBr-T; EtBr-R groups, indicating this cells reached Rho0 state or that they need more time to activate mtDNA replication; about cell viability, there were no differences among the groups; relating to genic expression there was an increase of Bax; BCl-2 for EtBt-T group; EtBr-R group showed decrease of BCl-2; for Tfam there was an increase for EtBr-T group; a decrease for EtBr-R. Relating to Experiment-3 it was impossible to notice signs of pluripotency, but we could see a decrease in the amount of mtDNA in both groups treated with EtBr (EtBr with; without STEMCCA) as in control group with STEMCCA. Genic expression analysis didn\'t show differences related to Tfam. Regarding to BAX, both control groups (with; without STEMCCA); EtBr without STEMCCA didn\'t differ from each other,; the last one also didn\'t show any difference when compared to EtBt with STEMCCA group. For BCl-2, control group without STEMCCA; EtBr with STEMCCA didn\'t show differences among each other; control group without SEMCCA didn\'t show differences when compared to control group with STEMCCA; EtBr without STEMCCA. Concluding, this work, regarding our knowledge, describes for the first time, production of bovine Rho0 cells; debates about the relationship among mitochondrial function; the process of cell reprogramation.

During normal placental development, a primitive vascular network develops through vasculogenesis and angiogenesis, and is then remodelled through maturation and regression. The mechanism behind this regression is unknown, but data from other systems suggests that it could be due to an endothelial-mesenchymal transition (EndMT). If this is the case, then dysregulated EndMT could lead to increased vascular regression, which could result in placental hypovascularisation. As the placental vasculature is the area of exchange between maternal and fetal circulations, a reduction in its surface area could result in fetal growth restriction (FGR). The hypothesis of this thesis is that EndMT occurs during normal placental development, but is increased during FGR and contributes to placental hypovascularisation. A primary cell model consisting of endothelial and mesenchymal cells was isolated from human first trimester placental villous stroma. These cells were shown to lose CD31 mRNA (n = 1-3) and protein (n = 15) over 4 passages, with no loss of cell viability (n = 8). EndMT-associated transcription factors were also present in these cells at all 4 passages (n = 2-4). When cells were isolated from this mixed cell model based on their CD31-positivity and examined immediately after isolation, a small proportion also expressed αSMA (n = 5). Co-expression of endothelial and mesenchymal markers suggests that an EndMT was occurring. After 24 hours in culture, the proportion of these cells expressing αSMA increased (n = 5), and some cells co-expressed vWF and αSMA, while others lost their CD31-positivity, indicating that these cells had undergone EndMT. Cells isolated based on their CD31-positivity were treated with factors shown to inhibit EndMT in other systems. However, culture with 10µM SB431542 (TGFβ receptor inhibitor; n = 6), 10µM Dorsomorphin (BMP receptor inhibitor; n = 3), or 0.1µM PDGFR-β Tyrosine Kinase Inhibitor IV (n = 3) did not inhibit gain of αSMA by these cells. Culture on Matrigel in endothelial growth medium containing VEGF and FGF also failed to stabilise the endothelial phenotype (n = 3). The possibility that EndMT occurs in placenta in vivo was examined; genes associated with EndMT were shown to be present in placenta (n = 5), and there was limited evidence of CD31 or vWF co-expression with αSMA in tissue. Preliminary evidence was obtained to suggest that expression of EndMT-associated genes was altered in FGR placentas compared to normal. In summary, the data presented in this thesis demonstrate that an EndMT occurs in primary placental microvascular endothelial cells in vitro. Furthermore, these studies provide evidence to suggest that this transition also occurs in vivo and could be altered in placentas from pregnancies complicated by FGR.

Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

Ischaemic stroke occurs as a result of a blockage in one of the brain’s arteries, leading to neuronal injury and death. Although stroke is a major cause of death and disability, there is no widely available treatment. Inflammation occurs in the brain and in the periphery following stroke, and both contribute to the ischaemic damage. Leukocytes such as neutrophils are key mediators of brain damage and inflammation, particularly in the presence of systemic inflammatory challenges such as interleukin-1 (IL-1). Tumor necrosis factor-stimulated gene 6 (TSG-6) is a potent inhibitor of neutrophil migration, and also modulates the immune response by dampening expression of cytokines and stabilising the extra-cellular matrix (ECM). Mesenchymal stem cells (MSCs) have shown immunomodulatory actions in many inflammatory conditions, and their benefit has often been attributed to the production of TSG-6. This work aimed to evaluate the potential of TSG-6 and TSG-6-expressing MSCs as therapies in cerebral ischaemia, and to investigate the expression profile of endogenous TSG-6 in response to stroke. Mice were subjected to middle cerebral artery occlusion (MCAo) followed by reperfusion. We investigated whether IL-1-induced acute brain injury after stroke is reversed by TSG-6, and long-term recovery was evaluated in mice treated with TSG-6 or MSCs. Functional outcomes were assessed, and brains were sectioned and stained for analysis of lesion volume, haemorrhagic transformation, blood-brain barrier (BBB) disruption and neutrophil infiltration. The expression profile of TSG-6 was evaluated in mice allowed to recover for 4h, 24h, 3, 5 or 7 days. TSG-6 expression was determined by quantitative PCR and immunohistochemistry. Treatment with TSG-6 reduced IL-1-induced neutrophil infiltration into the striatum, and led to decreased BBB disruption and haemorrhagic transformation at 24h. Treatment with TSG-6 in the absence of a systemic inflammatory challenge had no significant effect on lesion volume, BBB disruption or haemorrhagic transformation after 7 days reperfusion, however thalamic neutrophil infiltration was significantly reduced. Treatment with human MSCs had no significant effect on behavioural or histological outcomes, however a heightened inflammatory response in MSC-treated mice suggested rejection of the cells by the murine immune system. TSG-6 expression peaked in the ischaemic hemisphere at 5 days post-reperfusion, and was associated with astrocytes in the glial scar surrounding the infarcted tissue. TSG-6 might be a promising therapy for the treatment of stroke in the presence of systemic inflammation. TSG-6-expressing MSCs might provide a broader therapeutic potential, and further work should optimise experimental conditions to prevent rejection of the cells. Expression of TSG-6 within the glial scar suggests a potential role in repair and recovery following ischaemic stroke. Modulating the peripheral immune response remains an attractive and accessible therapeutic target for the treatment of cerebral ischaemia.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-tomesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 knockdown becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT.

Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.

Introduction: Mesenchymal stem cells (MSC) offer a novel cell therapy within tissue engineering and regenerative medicine (TERM)-based strategies, and the prospect of MSC therapies are widening since the discovery of MSCs within multiple locations of the body including bone marrow (BM-MSCs) and adipose tissue, (AD-MSCs). It is highly recognised that an organisms reparative and regenerative potential declines with advancing age, therefore aged patients are one of the primary target populations for TERM applications. Although information is available regarding the effects of patient age on the quality of human BM-MSCs, little and conflicting information currently exists for AD-MSCs. In addition, few studies have compared the quality of freshly isolated and expanded donor-matched BM and AD-MSCs to elucidate the more appropriate cell source. This study investigated the effect of donor age and in vitro ageing on functional behaviour (i.e. senescence state, population kinetics and differentiation potential) of donor-matched BM and AD-MSCs. Methods: The influence of donor age and in vitro ageing on mature (28-55 years) and elderly (75-86 years) donor-matched BM and AD-MSCs was assessed upon isolation (early life-span) and during extended (mid and late lifespan) timepoints through culture. During culture MSCs were characterised for cumulative population doublings (CPDs) and the expression of senescence associated marker genes, p16INK4A, p21 and p53, and transcription factor NANOG. At each lifespan telomere length was assessed along with differentiation efficiency along the osteogenic, adipogenic and chondrogenic lineages through lineage specific marker genes and histological staining. Results: Elderly BM and AD-MSCs displayed similar characteristics in terms of initial CPD number, p21, p53 and NANOG expression, telomere length and differentiation along osteogenic and adipogenic lineages. With increasing donor age there was a significant decline in p16INK4A expression within BM-MSCs, whilst expression of all chondrogenic markers significantly decreased within AD-MSCs. BM and AD-MSCs were comparable for the majority of outcome measures with the exception of chondrogenic differentiation which was superior with BM-MSCs in terms of COL2A1 expression and histological staining for proteoglycans. Donor age had a negative effect on BM-MSCs with long-term culture leading to a significantly longer PD time and decreased telomere lengths. Similar population kinetics was displayed between BM and AD-MSCs during long-term culture. Increasing culture time had effects on differentiation potential for both MSC sources with complete loss of osteogenic capacity and decreased adipogenic capacity; however chondrogenic capacity was only decreased within AD-MSCs. Differentiation potential after long-term culture between BM and AD-MSCs showed similar osteogenic and adipogenic ability yet superior chondrogenic ability was apparent within mature BM-MSCs compared to AD-MSCs, in terms of ECM deposition. Conclusions: In conclusion the source of MSCs for TERM will need to be considered depending upon the type of tissue regeneration required. The clinical outcome would be greater using MSCs during early stages of culture, as culture expansion has detrimental effects on functional properties of both BM and AD-MSCs.

Mesenchymal stem cells (MSCs) are the most widely used source for bone tissue engineering due to their capability of multipotent differentiation. The use of nanotechnology in biomedical applications and therapy has increased in recent years provides an elegant alternative in comparison to current tissue engineering methods. Magnetic nanowires have a high potential in the medical field, as they are biocompatible, are simple to fabricate, possess low cytotoxic effects and can be operated wirelessly via magnetic fields. A nanowire substrate (NW) can provide a surface with tunable elastic properties. Therefore, magnetic nanowires have many promising applications such as in cell therapy, cell separation, cancer treatment, and as a scaffold for cell culture. This thesis explores the effects of alternating magnetic field (AMF) as a biophysical stimulator of osteogenic differentiation of MSCs by culturing the stem cells on a magnetic iron (Fe) NW. To this end, Fe nanowires were fabricated through electrodeposition and interactions between the NW and cells were analysed by electron microscopy. An AMF was applied to the NW in order to induce a vibration. MSCs were exposed to different magnetic field intensities, 250 mT and 50 mT, for different application times, 12 hours on followed by 12 hours off for two days and 24 hours on followed by 12 hours off. Differentiation was determined through the assessment of osteogenic markers at the mRNA level by RT-PCR and at the protein level by flow cytometry and fluorescence microscopy. Different effects were observed on MSCs grown on Fe NWs following exposure to different magnetic field intensities and duration applications. MSC differentiation towards the osteogenic lineage increased with increased field intensities. The most enhanced osteogenic differentiation of MSCs was observed at 250 mT AMF for 12 hours, as evidenced by elevated osteogenic markers at mRNA level compared to that of an AMF free control. Based on these results, we proposed that culturing MSCs on magnetic nanomaterials has the potential to control and promote osteogenesis under magnetic field and without the addition of external differentiation factors. These findings provide a new tool for stem cell research as an effective technology for bone tissue engineering and regenerative medicine.

Osteoporosis is a systemic skeletal disease that affects millions of people worldwide. There are many possible etiologies for osteoporosis, including inherent variables like genetics and sex, and lifestyle variables like diet and exercise. Characterized by low bone mass and increased fracture risk, the disease places a burden on both the patients and the healthcare industry. Therefore, it is vital that research determine the mechanisms by which the risk factors affect BMD so that better diagnosis and treatment options may be developed. The purpose of this study was to examine the relationship between various osteoporosis risk factors and biochemical markers of osteogenic cell activity derived from bone-marrow MSCs. It was hypothesized postmenopausal white women, having the greatest risk for osteoporosis, would have elevated hydroxyproline and decreased ALP, indicative of greater bone resorption. Acetabular reamings were collected from 26 patients (15 males and 11 females) undergoing total hip arthroplasty at Boston Medical Center. MSCs from the reamings were plated and underwent osteoinduction into osteoblasts. The cells were then harvested and assayed for various indicators of cell growth and bone cell activity, such as DNA, ALP, and hydroxyproline. Our hypothesis was generally supported in that postmenopausal white women did have less ALP, an indicator of bone deposition, than premenopausal women and postmenopausal African American women. Additional findings and directions for future studies are further discussed in this paper.