A interação entre células tumorais e células estromais tem um papel central na progressão neoplásica. As células estromais mesenquimais multipotentes (MSCs) podem se integrar ao microambiente tumoral onde modulam o crescimento dos tumores por meio de distintos mecanismos. Entretanto, pouco se sabe sobre o papel das MSCs na metástase, a principal causa de morte em pacientes com câncer. Utilizando um modelo de melanoma murino ortotópico, nós demonstramos que MSCs obtidas da medula óssea de camundongos (MO-MSCs) ocupam o nicho perivascular nos tumores primários e aumentam 2,5 vezes a incidência de micrometástases pulmonares quando co-infundidas com células de melanoma B16. Observamos ainda que o meio condicionado das MO-MSCs não altera o potencial de colonização pulmonar das células B16 infundidas sistemicamente. Isto indica que as MO-MSCs modulam as fases iniciais da cascata metastática, durante a qual ocorrem os processos de invasão e intravasão nos vasos sangüíneos. Em correlação com estes efeitos pró-metastáticos, o secretoma das MO-MSCs induziu a transição epitélio-mesenquimal (EMT) nas células de melanoma in vitro. Após cultivo em meio condicionado das MO-MSCs, as células B16 adquiriram uma morfologia evidentemente fibroblástica. Ao mesmo tempo, houve o rearranjo dos filamentos de actina e o aumento da expressão de marcadores mesenquimais como fibronectina, vimentina, FSP1, N-caderina e ZEB2, acompanhado da repressão transcricional de E-caderina. A ativação da EMT pelo secretoma das MO-MSCs resultou na aquisição de propriedades metastáticas nas células de melanoma. Após cultivo em meio condicionado de MO-MSCs, as células B16 tiveram seu potencial de ancoragem à fibronectina reduzido, ao passo que houve o aumento na mobilidade e no potencial de invasão em matrizes tridimensionais. Utilizando inibidores competitivos de ATP contra o receptor tirosina-cinase Met, demonstramos que a aquisição de todas as propriedades metastáticas avaliadas e a ativação da EMT nas células de melanoma é mediada pela ativação da via HGF/Met. Estes dados destacam o papel das MOMSCs no microambiente tumoral como fonte perivascular de moléculas indutoras da EMT, cuja ativação leva a aquisição de traços metastáticos nas células de melanoma. Além disso, a inibição da via HGF/Met pode neutralizar os efeitos das MO-MSCs sobre as células tumorais, contribuindo para a repressão de propriedades fundamentais que sustentam a progressão e a disseminação neoplásica. Estas informações são importantes para o desenvolvimento seguro das MO-MSCs como ferramenta terapêutica e demonstram a importância da sinalização entre MSCs e células tumorais na disseminação metastática. Mais especificamente, estas observações reforçam a inibição da via HGF/Met como uma abordagem promissora para o tratamento da metástase. / The crosstalk between tumor cells and stromal cells can profoundly impact tumor progression. Multipotent mesenchymal stromal cells (MSCs) have been reported to integrate the tumor microenvironment where they are described to modulate tumor growth by distinct mechanisms. However, little is known about the impact of MSCs on metastasis, the main cause of death in patients with cancer. Using an orthotopic mouse melanoma model, we showed that mouse bone marrow-derived MSCs (BMMSCs) occupy the perivascular niche within primary tumors and increased by 2.5-fold the incidence of lung micrometastases after co-infusion with B16 melanoma cells. Also, MO-MSCs conditioned medium did not affect the lung colonization ability of systemically infused B16 cells. This indicates that MO-MSCs induces the initial steps of the metastatic cascade, during which the invasion and intravasion occurs. Correlating with these metastatic effects, the BM-MSCs\' secretome activated the epithelial-to-mesenchymal transition (EMT) in B16 cells in vitro. After culture in BMMSCs\' conditioned medium, B16 cells acquired an evident fibroblastic morphology. Simultaneously, we observed the rearrangement of actin filaments and the upregulation of mesenchymal markers such as fibronectin, vimentin, FSP1, Ncadherin and ZEB2. In agreement with the loss of epithelial phenotype, BM-MSCs\' secretome also suppressed E-cadherin expression in B16 cells. The activation of EMT by BM-MSCs leaded to the acquisition of metastatic traits in melanoma cells. After culture in BM-MSCs\' conditioned medium, B16 cells displayed reduced anchorage to fibronectin and increased motility and invasiveness in threedimensional matrix plugs. Inhibition of Met receptor with competitive ATP inhibitors demonstrated that the induction of EMT and the resultant acquisition of metastatic traits are driven by activation of HGF/Met signaling pathway. Taken together, these evidences highlight the role of BM-MSCs as a perivascular source of EMT-inductive signals, whose activation leads to acquisition of metastatic traits in melanoma cells. Furthermore, inhibition of HGF/Met signaling pathway can neutralize the effects of BM-MSCs on tumor cells, thereby allowing the repression of fundamental properties which support tumor progression and metastasis. This information is useful to safely develop BM-MSCs as therapeutic tool and demonstrate the relevance of the signaling between MSCs and tumor cells during metastasis. More specifically, it reinforces that inhibition of Met signaling can be a promissory approach for the treatment of metastasis.

Tissue engineering strategies represent exciting potential therapies to repair cartilage injuries; however, difficulty regenerating the complex extracellular matrix (ECM) organization of native cartilage remains a significant challenge. Cartilaginous ECM molecules, specifically chondroitin sulfate (CS) glycosaminoglycan, may possess the ability to promote and direct MSC differentiation down a chondrogenic lineage. CS may interact with the stem cell microenvironment through its highly negative charge, generation of osmotic pressure, and sequestration of growth factors; however, the role of CS in directing differentiation down a chondrogenic lineage remains unclear. The overall goal of this dissertation was to develop versatile biomaterial platforms to control CS presentation to mesenchymal stem cells (MSCs) in order to improve understanding of the interactions with CS that promote chondrogenic differentiation. To investigate chondrogenic response to a diverse set of CS materials, progenitor cells were cultured in the presence of CS proteoglycans and CS chains in a variety of 2D and 3D material systems. Surfaces were coated with aggrecan proteoglycan to alter cell morphology, CS-based nano- and microspheres were developed as small particle carriers for growth factor delivery, and desulfated chondroitin hydrogels were synthesized to examine electrostatic interactions with growth factors and the role of sulfation in the chondrogenic differentiation of MSCs. Together these studies provided valuable insight into the unique ability of CS-based materials to control cellular microenvironments via morphological and material cues to promote chondrogenic differentiation in the development of tissue engineering strategies for cartilage regeneration and repair.

Human mesenchymal stem cells (MSC) have attracted a great deal of interest for their potential use in regenerative medicine and suppression of the inflammation. Nevertheless, all known therapy protocols require large amounts of MSCs, which can be obtained only by in vitro expansion. One of the most important methodological problems is associated with the use of animal-derived components in the cell culture medium. The main aim of the current research was to elucidate the influence of different serum substitutes on the proliferation, differentiation, expression of cell surface markers, and total protein expression of mesenchymal stem cells derived from human adipose tissue. In addition we were aiming to determine the features of mesenchymal stem cell populations from an exfoliated deciduous tooth (SHED) and their response to the multifunctional proinflammatory protein alpha1-antitrypsin. Our results indicate that adipose tissue derived MSCs cultivated in the presence of fetal calf serum and allogeneic human serum display similar properties, while synthetic serum substitute induces increase in growth and differentiation potential of MSCs. Moreover, our results indicate, that synthetic serum substitute also activates transcription of genes related to adipogenic and osteogenic differentiation and diminishes expression of cell surface marker CD146. In the present study, we used a proteomic approach that allowed us to compare protein expression signatures between primary cell... [to full text] / Žmogaus suaugusio organizmo mezenchiminės kamieninės ląstelės (MKL) pradėtos sėkmingai naudoti pažeistų audinių regeneracijai ir uždegiminio proceso slopinimui. Šiuolaikiniams terapijos protokolams yra reikalingi dideli ląstelių kiekiai, todėl prieš naudojimą yra būtina jas padauginti in vitro. Tačiau naudojant skirtingus MKL išskyrimo, in vitro kultivavimo ir padauginimo protokolus, yra sunku lyginti mokslinių tyrimų duomenis ir klinikinių tyrimų rezultatus. Nevienodos kultivavimo sąlygos gali įtakoti MKL funkcines savybes (augimo greitį, gebėjimą diferencijuotis, migracinį aktyvumą ir kt.). Šiame disertaciniame darbe tyrėme auginimo terpių, praturtintų skirtingais serumais (gyvulinės kilmės serumai, žmogaus alogeninis serumas, sintetinis serumas), poveikį žmogaus riebalinio audinio MKL. Taip pat charakterizavome MKL išskirtas iš žmogaus pieninių dantų pulpos ir palyginome šių ląstelių kultūros bei jų dukterinių klonų savybės. Kompleksiškai tyrėme uždegimo antiproteinazės alfa1-antitripsino (AAT) poveikį šioms ląstelėms. Nustatėme, kad, lyginant su kitais tyrime naudotais serumais, sintetinis serumo pakaitalas geriausiai skatino MKL augimą bei gebėjimą diferencijuotis adipogenine ir osteogenine kryptimis. Taip pat, skirtingai nuo kitų naudotų gyvulinės kilmės ir žmogaus serumų, sintetinis serumo pakaitalas veikė kaip adipogeninės ir osteogeninės diferenciacijos indukcijai svarbių genų PPARγ ir Msx2 transkripcijos aktyvatorius. Be to, sintetinis serumo pakaitalas slopino... [toliau žr. visą tekstą]

A interação entre células tumorais e células estromais tem um papel central na progressão neoplásica. As células estromais mesenquimais multipotentes (MSCs) podem se integrar ao microambiente tumoral onde modulam o crescimento dos tumores por meio de distintos mecanismos. Entretanto, pouco se sabe sobre o papel das MSCs na metástase, a principal causa de morte em pacientes com câncer. Utilizando um modelo de melanoma murino ortotópico, nós demonstramos que MSCs obtidas da medula óssea de camundongos (MO-MSCs) ocupam o nicho perivascular nos tumores primários e aumentam 2,5 vezes a incidência de micrometástases pulmonares quando co-infundidas com células de melanoma B16. Observamos ainda que o meio condicionado das MO-MSCs não altera o potencial de colonização pulmonar das células B16 infundidas sistemicamente. Isto indica que as MO-MSCs modulam as fases iniciais da cascata metastática, durante a qual ocorrem os processos de invasão e intravasão nos vasos sangüíneos. Em correlação com estes efeitos pró-metastáticos, o secretoma das MO-MSCs induziu a transição epitélio-mesenquimal (EMT) nas células de melanoma in vitro. Após cultivo em meio condicionado das MO-MSCs, as células B16 adquiriram uma morfologia evidentemente fibroblástica. Ao mesmo tempo, houve o rearranjo dos filamentos de actina e o aumento da expressão de marcadores mesenquimais como fibronectina, vimentina, FSP1, N-caderina e ZEB2, acompanhado da repressão transcricional de E-caderina. A ativação da EMT pelo secretoma das MO-MSCs resultou na aquisição de propriedades metastáticas nas células de melanoma. Após cultivo em meio condicionado de MO-MSCs, as células B16 tiveram seu potencial de ancoragem à fibronectina reduzido, ao passo que houve o aumento na mobilidade e no potencial de invasão em matrizes tridimensionais. Utilizando inibidores competitivos de ATP contra o receptor tirosina-cinase Met, demonstramos que a aquisição de todas as propriedades metastáticas avaliadas e a ativação da EMT nas células de melanoma é mediada pela ativação da via HGF/Met. Estes dados destacam o papel das MOMSCs no microambiente tumoral como fonte perivascular de moléculas indutoras da EMT, cuja ativação leva a aquisição de traços metastáticos nas células de melanoma. Além disso, a inibição da via HGF/Met pode neutralizar os efeitos das MO-MSCs sobre as células tumorais, contribuindo para a repressão de propriedades fundamentais que sustentam a progressão e a disseminação neoplásica. Estas informações são importantes para o desenvolvimento seguro das MO-MSCs como ferramenta terapêutica e demonstram a importância da sinalização entre MSCs e células tumorais na disseminação metastática. Mais especificamente, estas observações reforçam a inibição da via HGF/Met como uma abordagem promissora para o tratamento da metástase. / The crosstalk between tumor cells and stromal cells can profoundly impact tumor progression. Multipotent mesenchymal stromal cells (MSCs) have been reported to integrate the tumor microenvironment where they are described to modulate tumor growth by distinct mechanisms. However, little is known about the impact of MSCs on metastasis, the main cause of death in patients with cancer. Using an orthotopic mouse melanoma model, we showed that mouse bone marrow-derived MSCs (BMMSCs) occupy the perivascular niche within primary tumors and increased by 2.5-fold the incidence of lung micrometastases after co-infusion with B16 melanoma cells. Also, MO-MSCs conditioned medium did not affect the lung colonization ability of systemically infused B16 cells. This indicates that MO-MSCs induces the initial steps of the metastatic cascade, during which the invasion and intravasion occurs. Correlating with these metastatic effects, the BM-MSCs\' secretome activated the epithelial-to-mesenchymal transition (EMT) in B16 cells in vitro. After culture in BMMSCs\' conditioned medium, B16 cells acquired an evident fibroblastic morphology. Simultaneously, we observed the rearrangement of actin filaments and the upregulation of mesenchymal markers such as fibronectin, vimentin, FSP1, Ncadherin and ZEB2. In agreement with the loss of epithelial phenotype, BM-MSCs\' secretome also suppressed E-cadherin expression in B16 cells. The activation of EMT by BM-MSCs leaded to the acquisition of metastatic traits in melanoma cells. After culture in BM-MSCs\' conditioned medium, B16 cells displayed reduced anchorage to fibronectin and increased motility and invasiveness in threedimensional matrix plugs. Inhibition of Met receptor with competitive ATP inhibitors demonstrated that the induction of EMT and the resultant acquisition of metastatic traits are driven by activation of HGF/Met signaling pathway. Taken together, these evidences highlight the role of BM-MSCs as a perivascular source of EMT-inductive signals, whose activation leads to acquisition of metastatic traits in melanoma cells. Furthermore, inhibition of HGF/Met signaling pathway can neutralize the effects of BM-MSCs on tumor cells, thereby allowing the repression of fundamental properties which support tumor progression and metastasis. This information is useful to safely develop BM-MSCs as therapeutic tool and demonstrate the relevance of the signaling between MSCs and tumor cells during metastasis. More specifically, it reinforces that inhibition of Met signaling can be a promissory approach for the treatment of metastasis.

The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...

Background: Since its emergence in December 2019, SARS-CoV-2, the coronavirus responsible for COVID-19, has spread across the globe, infected millions of people and caused several million deaths. One promising intervention to combat the ongoing COVID-19 pandemic is mesenchymal stem/stromal cells (MSCs). Many trials were registered at the onset of the pandemic to determine the safety and efficacy of MSCs in COVID-19 patients. However, currently published studies are underpowered to provide an estimate of safety and efficacy on their own. Thus, a living systematic review (SR) is needed to establish the benefits and drawbacks of MSCs for COVID-19 on a relevant timescale. Methods: Systematic literature searches were conducted on Feb 3rd, 2021 and November 15th, 2021 to identify all English-language, full-text, clinical studies examining MSCs to treat COVID-19. (PROSPERO:CRD42021225431). Findings/Conclusions: Our first search identified nine studies (4 controlled) examining the use of MSC derived products to treat COVID-19 patients. This first iteration of our SR revealed that MSCs were safe and reduced mortality in patients suffering from COVID-19. However, risk of bias (RoB) and poor adherence to ISCT cell product characterization guidelines limited the strength of our conclusions. In the second iteration of our living SR, we only included controlled studies to strengthen our conclusions. We identified eleven controlled studies (5 RCTs). MSCs continued to demonstrate safety and efficacy at reducing mortality at study endpoint (RR: 0.50 [0.34 to 0.75, 95% CI, p=0.0006, I2=0%]). However, we continued to encounter barriers which prevented us from drawing more definitive conclusions. A master protocol appears necessary to facilitate the accelerated accumulation of high-quality evidence where standardized outcome reporting and consistent product characterization allow for a more definitive and timely estimate regarding the safety and efficacy of this cell-based therapy for COVID-19.

Dans cette thèse, nous avons combiné les approches des cultures single-cell, de cytométrie en flux,des analyses de métabolisme énergétique et de génétique moléculaire afin d’explorer les effets del’Acétate d’α-Tocopherol (α-TOA) sur les cellules stromales mésenchymateuses (MStroC) et leurs souspopulations fonctionnelles (cellules souches et progénitrices mésenchymateuses). L’autre but était de tester une molécule de miR-210 par rapport à son utilisation potentielle comme « hypoxia mimicking molecule ». Après avoir démontré l’hétérogénéité de la population MStroC et conclu que la population de premier passage est appropriée pour des expérimentations ultérieures, nous avons trouvé que l’α-TOA présentait un effet positif sur le maintien de la capacité proliférative élevée des cellules souches mésenchymateuses. Cet effet est accompagné d’une atténuation de l’activité de la chaîne de transport d’électrons (ETC) qui pourrait d’autre part expliquer l’accroissement modéré du niveau des Reactive Oxygen Species mitochondriales (mtROS) que nous avons détectées. L’augmentation du niveau de mtROS pourrait être associée à une dégradation de protéine HIF-1 dans la population MStroC exposée à l’α-TOA. Bien que nous n’ayons pas détecté d’augmentation compensatoire de la glycolyse, les phénomènes observés représentent en partie la réponse cellulaire complexe au faible niveau d’O2. Il a été établi que ce phénomène était relié au maintien de primitivité des cellules souches. Le mécanisme exact reste à clarifier ainsi que son potentiel translationnel. En outre, nous avons apporté la preuve que miR-210 fait partie intégrante de la réponse des MStroC à la faible concentration en O2. Dans cette étude, nous avons montré que l’augmentation de l’expression de miR-210 sur une période courte (jusqu’à 24 heures) et après une période étendue (jusqu’à 72 heures) d’exposition des MStroC à une faible concentration en O2. De plus, nous avons prouvé que ce micro ARN pouvait être régulé par les deux facteurs transcriptionnels HIF-1 et HIF-2, nous laissant penser que ceci faisait partie intégrante de la réponse des MStroC à une faible concentration en O2. Jusqu’à présent, nos données suggèrent que miR-210 est digne d’intérêt en tant que bonne molécule « hypoxia mimicking ». / In this thesis, we combined approaches of single-cell cultures, flow-cytometry, energetic metabolismanalysis and molecular genetics in order to get insight in the effects of α-Tocopherol-Acetate (α-TOA)on Mesenchymal Stromal Cells (MStroC) and their functional subpopulations (mesenchymal stem and progenitor cells). The other aim was to test a miR-210 molecule with respect to its potential use as hypoxia mimicking molecule. After defining MStroC population heterogeneity and concluding that the first passage population is convenient for further experiments, we demonstrated that α-TOA exhibits a positive effect on the maintenance of high proliferative capacity of mesenchymal stem cells. This effect could be associated with an attenuation of electron transport chain (ETC) activity, which, on the other hand could explain moderate increase in the level of mitochondrial Reactive Oxygen Species (mtROS) we detected. The increase in mtROS level could be associated with a decreased HIF-1 alpha protein degradation in MStroC exposed to α-TOA. Although we did not detect a compensatory increase in glycolysis, the observed phenomena depict part of a complex cellular response to the low O2 that is demonstrated to be related with maintenance of stem cell primitiveness. The exact mechanism remains to be elucidated as well as its translational potential. In addition, we provided new evidences that miR-210 is integral part in MStroC response to low O2. In the study, we showed increased in miR-210 expression in a short-term (up to 24 hours) and after extended (up to 72 hours) MStroC exposed to low O2. Moreover, we demonstrated that this micro- RNA could be regulated by both HIF-1 and HIF-2 transcriptional factors, suggesting it as integral part of MStroC response to low O2. So far, our data suggest that miR-210 is worthy to be considered as good hypoxia mimicking molecule.

L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires. / The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The common choice of medium for culturing pig umbilical cord matrix stem cells (PUCs) is high glucose Dulbecco’s Minimum Essential Medium (HG-DMEM) supplemented with fetal bovine serum (FBS). FBS is a chemically undefined supplement that encourages attachment of explants and cells and is useful for long-term proliferation in an undifferentiated state. Removing FBS from the culture medium would decrease the possibility of microbial contamination and might produce more consistent results. A defined medium would facilitate experiments to determine requirements for specific growth factors and nutrients. Starting PUCs in a FBS-free environment proved to be a challenge. The results of 15 experiments testing various media, supplements, and culture conditions indicate that PUCs initially plated in an FBS-free environment do not attach as readily as those in HG-DMEM supplemented with FBS. PUCs were collected using enzyme digestion of the whole cord or by plating explants from the cord in culture medium. In the final experiment PUCs were seeded in 24-well plates (5.0 * 10[superscript]4 viable cells per well) with a collagen coating and cultured in Knock-out DMEM (KO-DMEM) with basic fibroblast growth factor (5ng/mL) and platelet derived growth factor (5ng/mL) in a low oxygen atmosphere (5% O[subscript]2/ 5% CO[subscript]2/ 90% N[subscript]2). The total non-adherent cell count at passage 1 was 1.78 * 10[superscript]5 +or- 3.68 * 10[superscript]4 and the total adherent cells were 2.58 * 10[superscript]5 +or- 9.29 * 10[superscript]4. The well confluence during initial cell proliferation appeared similar to cells cultured in the control media with 20% FBS (total adherent cells = 6.40 * 10[superscript]5 +or- S.E. 1.61 * 10[superscript]5 and total non-adherent cells = 2.88 * 10[superscript]5 + 7.60 * 10[superscript]4). However the number of adherent cells recovered for passage 2 was considerably less for cultures in FBS-free media than for the control group. Serum may affect attachment by providing attachment factors or it could change expression of integrins or other attachment molecules on the PUCs that enhance attachment to plastic or other substrates. In future studies the requirements for attachment of PUCs should be further evaluated.

BACKGROUND:The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion.RESULTS:We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting.CONCLUSION:We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.