La régénération musculaire implique des interactions fonctionnelles entre différents types de cellules mononucléées. Parmi elles, citons les progéniteurs myogéniques (MPs), qui fusionnent pour générer de nouvelles myofibres en réponse à une blessure, et les cellules immunitaires qui envahissent les muscles endommagés. Des dépôts transitoires fibrotiques et d’adipocytes sont observés dans les muscles en régénération qui cependant persistent dans la dystrophie musculaire de Duchenne (DMD) et au cours du vieillissement. Nous avons démontré que les progéniteurs fibro-adipogéniques (FAPs) exprimant le marqueur de surface PDGFRα, contribueraient au développement des dépôts non myogéniques dans les muscles sains. En effet, ces progéniteurs se différencient en adipocytes blancs fonctionnels, bien qu’étant insensibles à l’insuline, et génèrent des myofibroblastes. Quant à la fibrose des muscles DMD, elle se formerait à partir de la différenciation à la fois des MPs et des FAPs. Dans les muscles sains, les FAPs stimulent la myogenèse des MPs au cours de la régénération, alors que les myotubes et les macrophages pro-inflammatoires inhibent l’adipogenèse et la fibrogenèse des FAPs. Pour les progéniteurs âgés ou dystrophiques, les interactions cellulaires entre les FAPs et les MPs sont perturbées. De manière intéressante, la régulation des FAPs DMD ou âgés peut être restaurée en remplaçant les MPs DMD ou âgés par des MPs jeunes et sains. Nos résultats montrent que les muscles humains contiennent des progéniteurs fibro-adipogéniques qui jouent un rôle central dans la régulation de l’homéostasie musculaire en interagissant avec les progéniteurs myogéniques et les macrophages / Muscle regeneration involves functional interactions between different types of mononuclear cells including myogenic progenitors (MPs) and macrophages. Following injury, damaged muscles are invaded by immune cells and MPs fuse to generate new myofibres. Transient fibrotic and adipocyte deposits are observed in regenerating muscles, which however persist in Duchenne muscular dystrophy (DMD) and during aging. We demonstrated that fibro-adipogenic progenitors (FAPs) expressing the PDGFRα surface marker would contribute to the development of non-myogenic deposits in healthy muscles. Indeed, these progenitors differentiate into functional white adipocytes that have the feature to be insulino-resistant, and give rise to myofibroblastes. Intramuscular fibrosis in DMD patients could be formed from both FAPs and MPs differentiation. In healthy muscles, FAPs stimulate myogenesis of MPs during regeneration, while myotubes and pro-inflammatory macrophages inhibit the adipogenesis and fibrogenesis of FAPs. Cellular interactions between FAPs and MPs are disrupted for DMD or aged progenitors. Interestingly, they are restored if aged or DMD FAPs are replaced by healthy and young MPs. Our results show that the human muscles contain fibro-adipogenic progenitors that play a crucial role in the control of muscle homeostasis by interacting with myogenic progenitors and macrophages

Human induced pluripotent stem cells (iPSCs) represent a scalable cell source for the generation of hematopoietic progenitor cells (iHPCs); however, a lack of efficient iHPC expansion in vitro currently limits translational applications. To address this translational bottleneck, we assessed a panel of stem cell agonist cocktails (SCACs), originally developed to enhance cord-blood derived HSPC (CB-HSPC) expansion, on iHPC expansion. Three SCACs and GAS6 (X2A, X2A+GAS6, SM6, or SMA) were supplemented during iHPC differentiation and subsequent expansion using the STEMdiff™ Hematopoietic Kit. This monolayer differentiation strategy yielded a population of CD34⁺CD43⁺ and CD45⁺CD34⁺ iHPC. SCAC supplementation during iHPC differentiation yielded up to 2.5-fold higher frequency of CD34⁺CD43⁺ hematopoietic progenitors and up to 2.9-fold higher frequency of CD45⁺CD34⁺CD45RA⁻CD90⁺ HSC-like cells compared to non-treated controls. Subsequent SCAC supplementation during 2 weeks of expansion culture also significantly increased iHPC expansion (X2A+GAS6: 3.8-fold, X2A: 3.5-fold, SM6: 2.8-fold, SMA: 2.0-fold). The expanded iHPCs retained high levels of CD34⁺CD43⁺ expression but we observed an increase in the expansion of HSC-like cell fraction. The collective expansion observed with the SCACs was 1.5- to 2.8-fold higher than UM171 treatment alone. Furthermore, all SCAC-supplemented iHPCs retained multilineage potency, producing erythroid and granulocyte-macrophage progenitors in CFU assays. However, prolonged expansion, beyond 7 days, reduced multilineage potential, indicating a limited expansion window. Although optimal timing and composition of SCAC supplementation remains to be refined, these results highlight that exploiting the additive and synergistic effects of multiple small molecules represents a promising approach for enhancing iHPC expansion yields and biomanufacturing.

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.

Introduction: The development of liver fibrosis is characterised by dramatic changes in the biomechanical composition and mechanical properties of the extracellular matrix (ECM). Increases in matrix stiffness associated with inflammation and fibrosis are implicated in promoting cancer development. Clinical studies have demonstrated a close association between increases in liver stiffness and the incidence of hepatocellular carcinoma (HCC). The effect of changes in matrix stiffness on tissue-resident hepatic progenitor cells (HPC) is unknown. Aberrant HPC proliferation has been implicated in the pathogenesis of HCC. It was hypothesised that changes in the stiffness of the cellular microenvironment are important in regulating the behaviour of liver-resident cell populations and may promote the development of HCC. Aims: i) to determine how changes in the stiffness of the cancer cell niche might regulate proliferation, differentiation and chemotherapeutic resistance in HCC; ii) to determine the relationship between changes in liver stiffness and hepatic progenitor cell (HPC) response in rodent models of chronic liver disease; and iii) to determine whether changes in the stiffness of the HPC niche regulate proliferation and differentiation in these cells. A secondary aim of the thesis was to characterise the pattern of histological changes observed in rodent models of chronic hepatic congestion and whether this might provide insight into the effect of oedema and congestion on the development of liver fibrosis. Methods: Cell culture experiments in HCC (Huh7/ HepG2) and HPC cell lines were performed using a system of ligand-coated polyacrylamide (PA) gel supports of variable stiffness. The stiffness of the PA supports (expressed as shear modulus) was altered across a physiological change (1-12kPa) corresponding to values encountered in normal and fibrotic livers. Thiacetamide and carbon tetrachloride (CCl4) models of liver fibrosis were used to investigate the relationship between increasing liver fibrosis, changes in matrix stiffness and HPC response. The pattern of histological changes in the liver in response to hepatic congestion was assessed in two unrelated murine models of dilated cardiomyopathy; the python and CREB S133A mice. Results: Increases in matrix stiffness, as would be encountered in liver fibrosis, promote HCC cell proliferation. Increasing matrix stiffness is associated with enhanced basal and hepatocyte growth factor-mediated signalling though ERK, PKB/ Akt and STAT3. Stiffness-dependent HCC cell proliferation is modulated by β1-integrin and focal adhesion kinase. Increasing matrix stiffness is associated with a reduction in chemotherapy-induced apoptosis in HCC cells. However, following chemotherapy there was an increase in the frequency of clone-initiating cells for cells maintained in a low stiffness environment. Flow cytometry in HepG2 cells demonstrated that culture in a low stiffness environment was associated with an increase in the frequency of the stem cell markers CD44, CD133 and CXCR-4. This effect was further enhanced in the presence of chemotherapy. There is a close association between HPC numbers and liver stiffness measurements in a rat CCl4 model of chronic liver fibrosis. The major expansion in HPC numbers in this model coincides with a similarly large increase in fibrous tissue deposition. In vitro experiments using PA supports demonstrate that increasing matrix stiffness promotes the proliferation of both primary murine HPCs and an immortalised HPC line (BMOL). Changes in matrix stiffness regulate the expression of hepatocyte and biliary markers in BMOL cells. Histological studies in both the Python and CREB S133A models reveal findings consistent with acute on chronic cardiac hepatopathy (ischaemic hepatitis). Features of chronic passive congestion and centrilobular necrosis are present concurrently and develop rapidly. Bridging fibrosis and cirrhosis are not present. Conclusions: Physiologically-relevant changes in matrix stiffness regulate proliferation, differentiation, chemotherapeutic-resistance and stem cell marker expression in HCC cells. Similarly, increases in matrix stiffness are closely correlated to HPC response in vivo and regulate HPC proliferation and differentiation in vitro.

Williams Beuren syndrome Syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development.

Williams Beuren syndrome Syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

<p>Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.</p><p>A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.</p><p>Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.</p><p>The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.</p> / Dissertation

The Nance-Horan syndrome gene (NHS) plays a role in lens, eye and brain development. To date, the function of NHS remains unclear. Recent evidence showed that p53 isoform, Δ113p53, inhibits abnormal cell growth during organogenesis. We show that NHS is expressed in the retinas of Danio rerio and Xenopus tropicalis during key stages of retinogenesis, and that knockdown of the gene resulted in a small eye phenotype in both species. Initially, knockdown of nhsb in zebrafish had no visible defects at 24hpf. But examination of the retina at 48hpf, we see a marked difference in size compared to control embryos. Cell proliferation is a major feature of the developing retina from 24 hpf to 48 hpf. Differentiation of neurons was delayed, while the total number of cells that makes up the volume of the retina was markedly reduced. Here we show that the small retina in nhsb knockdown embryos are due to p53-dependent cell cycle arrest with specific induction of p53 target gene, Δ113p53 and p21. Δ113p53 protects nhsb- knockdown cells from p53-mediated apoptosis. We hypothesize that nhsb overcomes a proliferation restriction in retina progenitor cells during retinogenesis, while knockdown of nhsb increases expression of Δ113p53 and p21, lengthening the cell cycle.