Cell therapies for tendons: old cell choice for modern innovation.

Although tissue engineering and cell therapies are becoming realistic approaches for medical therapeutics, it is likely that musculoskeletal applications will be among the first to benefit on a large scale. Cell sources for tissue engineering and cell therapies for tendon pathologies are reviewed with an emphasis on small defect tendon injuries as seen in the hand which could adapt well to injectable cell administration. Specifically, cell sources including tenocytes, tendon sheath fibroblasts, bone marrow or adipose-derived stem cells, amniotic cells, placenta cells and platelet-derivatives have been proposed to enhance tendon regeneration. The associated advantages and disadvantages for these different strategies will be discussed and evolving regulatory requirements for cellular therapies will also be addressed. Human progenitor tenocytes, along with their clinical cell banking potential, will be presented as an alternative cell source solution. Similar cell banking techniques have already been described with other progenitor cell types in the 1950's for vaccine production, and these "old" cell types incite potentially interesting therapeutic options that could be improved with modern innovation for tendon regeneration and repair.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Étude fonctionnelle et génétique d'une population de lymphocytes T CD4-CD8- impliquée dans la résistance au diabète auto-immun chez la souris

Le diabète auto-immun résulte de la destruction des cellules bêta pancréatiques sécrétrices d’insuline par les lymphocytes T du système immunitaire. Il s’ensuit une déficience hormonale qui peut être comblée par des injections quotidiennes d’insuline d’origine exogène, toutefois il demeure à ce jour impossible de guérir les patients atteints de la maladie. De façon générale, un système immunitaire sain reconnaît une multitude d’antigènes différents et assure ainsi notre défense à l’égard de différents pathogènes ou encore de cellules tumorales. Il arrive cependant que, pour des raisons génétiques et/ou environnementales, les lymphocytes T puissent s’activer de façon aberrante suite à la reconnaissance d’antigènes provenant du soi. C’est ce bris de tolérance qui mène au développement de pathologies auto-immunes telles que le diabète auto-immun. Afin de limiter l’auto-immunité, des mécanismes de sélection stricts permettent d’éliminer la majorité des lymphocytes T présentant une forte affinité envers des antigènes du soi lors de leur développement dans le thymus. Certains de ces lymphocytes réussissent toutefois à échapper à l’apoptose et migrent en périphérie afin d’y circuler en quête d’un antigène spécifiquement reconnu. Il est alors primordial que des mécanismes périphériques assurent le maintien de la tolérance immunitaire en faisant obstacle à l’activation et à la prolifération des lymphocytes T auto-réactifs. L’une des avenues afin d’inhiber le développement de réponses immunitaires aberrantes est la génération de lymphocytes T régulateurs. Ces cellules, d’origine thymique ou périphérique, peuvent arborer différents phénotypes et agissent via de multiples mécanismes afin d’inactiver et/ou éliminer les cellules impliquées dans l’apparition de pathologies auto-immunes. L’utilisation de modèles murins transgéniques a permis la mise en évidence d’une population peu caractérisée de lymphocytes T au potentiel régulateur. En effet, la proportion de ces cellules T n’exprimant pas les corécepteurs CD4 et CD8 (double négatives, DN) a été inversement corrélée à la prédisposition à l’auto-immunité chez ces ii souris. L’objectif principal de cette thèse est de démontrer la fonction immuno-régulatrice des lymphocytes T DN, tout en investiguant les facteurs génétiques responsables du maintien de cette population cellulaire. Nous avons observé que les lymphocytes T DN exercent une activité cytotoxique à l’égard des lymphocytes B de façon spécifique à l’antigène, via la libération de granules cytolytiques contenant du granzyme B et de la perforine. Par ailleurs, nous avons établi qu’un unique transfert adoptif de ces cellules est suffisant afin d’inhiber le développement du diabète auto-immun chez des hôtes transgéniques prédisposés à la maladie. Le recours à des souris déficientes pour l’expression du gène CD47 a permis de constater que la voie de signalisation CD47-Sirp est essentielle dans le maintien de la proportion des lymphocytes T DN. De plus, le locus murin de prédisposition au diabète auto-immun Idd13, qui contient le gène Sirp, a été identifié pour son rôle dans la régulation de la proportion de ces cellules. Finalement, une analyse génétique a révélé que d’autres intervalles génétiques sont impliqués dans le contrôle de la population des lymphocytes T DN. Parmi ceux-ci, un locus situé en région proximale du chromosome 12 a été validé grâce à la création de souris congéniques. Grâce aux résultats présentés dans cette thèse, notre compréhension de la biologie ainsi que de la régulation des lymphocytes T DN est approfondie. Ces connaissances constituent un pas important vers la création de thérapies cellulaires novatrices permettant de prévenir et de guérir diverses pathologies auto-immunes.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Derivação de células tronco de tecido ovariano na espécie canina

Pós-graduação em Medicina Veterinária - FCAV

Immune Regulatory Properties of Allogeneic Adipose-Derived Mesenchymal Stem Cells in the Treatment of Experimental Autoimmune Diabetes

Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell-mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4(+) Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-gamma levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-beta 1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact-dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional beta-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D. Diabetes 61:2534-2545, 2012

[Lineage tracing of epicardial cells during development and regeneration].

Tracing the history of individual cells during embryonic morphogenesis in a structure as complex as the cardiovascular system is one of the major challenges of developmental biology. It involves determining the relationships between the various lineages of cells forming an organ at different stages, describing the topological rearrangements tissues undergo during morphogenesis, and characterizing the interactions between cells in different structures. However, despite the great expectations raised in the field of regenerative medicine, only limited progress has been made in using regenerative therapy to repair the cardiovascular system. Recent research has highlighted the role of the epicardium during cardiac regeneration, but it is still unclear whether it is important for molecular signaling or acts as a source of progenitor cells during this process. Consequently, increasing knowledge about the origin, diversification and potential of epicardial cells during development and homeostasis and under pathological conditions is of fundamental importance both for basic research and for the development of effective cellular therapies. The aims of this article were to provide a general overview of the classical techniques used for tracing cell lineages, including their potential and limitations, and to describe novel techniques for studying the origin and differentiation of the epicardium and its role in cardiac regeneration.

INDUCED-PLURIPOTENT STEM-DERIVED NEURONAL PROGENITOR CELLS AS A NOVEL TREATMENT FOR NEURODEGENERATIVE DISEASES

Cellular therapies, as neuronal progenitor (NP) cells grafting, are promising therapies for patients affected with neurodegenerative diseases like Creutzfeldt-Jakob Disease (CJD). At this time there is no effective treatment or cure for CJD. The disease is inevitably fatal and affected people usually die within months of the appearance of the first clinical symptoms. Compelling evidence indicate that the hallmark event in the disease is the conversion of the normal prion protein (termed PrPC) into the disease-associated, misfolded form (called PrPSc). Thus, a reasonable therapeutic target would be to prevent PrP misfolding and prion replication. This strategy has been applied with poor results since at the time of clinical intervention substantial brain damage has been done. It seems that a more effective treatment aimed at patients with established symptoms of CJD would need to stop further brain degeneration or even recover some of the previously lost brain tissue. The most promising possibility to recover brain tissue is the use of NPs that have the potential to replenish the nerve cells lost during the early stages of the disease. Advanced cellular therapies, beside their potential for cell replacement, might be used as biomaterials for drug delivery in order to stimulate cell survival or the resolution the disease. Also, implanted cells can be genetically manipulated to correct abnormalities causing disease or to make them more resistant to the toxic microenvironments present in damaged tissue. In recent years cell engineering has been within the scope of the scientific and general community after the development of technologies able to “de-differentiate” somatic cells into induced-pluripotent stem (IPS) cells. This new tool permits the use of easy-to-reach cells like skin or blood cells as a primary material to obtain embryonic stem-like cells for cellular therapies, evading all ethical issues regarding the use of human embryos as a source of embryonic stem cells. The complete work proposes to implant IPS-derived NP cells into the brain of prion-infected animals to evaluate their therapeutic potential. Since it is well known that the expression of prion protein in the cell membrane is necessary for PrPSc mediated toxicity, we also want to determine if NPs lacking the prion protein have better survival rates once implanted into sick animals. The main objective of this work is to develop implantable neural precursor from IPS coming from animals lacking the prion protein. Specific aim 1: To develop and characterize cellular cultures of IPS cells from prp-/- mice. Fibroblasts from prp-/- animals will be reprogrammed using the four Yamanaka factors. IPS colonies will be selected and characterized by immunohistochemistry for markers of pluripotency. Their developmental capabilities will be evaluated by teratoma and embryoid body formation assays. Specific aim 2: To differentiate IPS cells to a neuronal lineage. IPS cells will be differentiated to a NP stage by the use of defined media culture conditions. NP cells will be characterized by their immunohistochemical profile as well as by their ability to differentiate into neuronal cells. Specific aim 3: Cellular labeling of neuronal progenitors cells for in vitro traceability. In order to track the cells once implanted in the host brain, they will be tagged with different methods such as lipophilic fluorescent tracers and transduction with GFP protein expression.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

Transgenic Expression of Endothelin Receptor-B Rescues the Aganglionosis Phenotype of Piebald Lethal Mice

Neural crest cells (NCC) are a unique population of cells in vertebrates that arise between the presumptive epidermis and the dorsal most region of the neural tube. During neurulation, NCC migrate to many regions of the body to give rise to a wide variety of cell types. NCC that originate from the neural tube at the levels of somite 1-7 colonize the gut and give rise to the enteric ganglia. The endothelin signaling pathway has been shown to be crucial for proper development of some neural crest derivatives. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene exhibit similar phenotypes characterized by hypopigmentation, hearing loss, and megacolon. Thesephenotypes are due to lack of melanocytes in the skin, inner ear and enteric ganglia in the distal portion of the colon, respectively. It is well established that Ednrb is required early during the embryonic development for normal innervation of the gut. However, it is not clear if Ednrb acts on enteric neuron precursor cells or in pre-committed NC precursors. Additionally, it is controversial whether the action of Ednrb is cell autonomous or non- autonomous. We generated transgenic mice that express Ednrb under the control of the Nestin second intron enhancer (Nes) which drives expression to pre-migrating NCC. These mice were crosses to the spontaneous mouse mutant piebald lethal, which carriers a null mutation in Ednrb and exhibits enteric aganglionosis. The Nes-Ednrb was capable of rescuing the aganglianosis phenotype of piebald lethal mutants demonstrating that expression of Ednrb in pre-committed precursors is sufficient for normal enteric ganglia development. This study provides insight in early embryonic development of NCC and could eventually have potential use in cellular therapies for Hirschsprung's disease.

Development of cellular and gene therapies for b[beta]-Thalassemia and sickle cell disease

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients