Thèses sur le sujet « Human Skeletal muscle derived stem cells »

The long-term treatment of injured, aging, or pathological skeletal muscle using stem cell therapy requires an abundant source of skeletal muscle progenitors (SMP) that are capable of self-replenishment. While adult SMPs—known as satellite cells and marked by PAX7 expression—can be collected from healthy donors, these satellite cells have limited replication potential once extracted, and may have difficulties providing sufficient numbers for therapy. Therefore, we sought to utilize the near-unlimited replication potential of human embryonic stem cells (hESC) to generate large quantities of SMPs in vitro. We developed a 50-day directed hESC differentiation that produced cultures with up to 90% myogenic identity; roughly 43 ± 4% become PAX7+ SMPs, and 47 ± 3% of cells become skeletal myocytes. We also performed gene expression profiling on our differentiating cultures to better understand in vitro skeletal myogenesis, and to better characterize in vitro hESC-derived SMPs, which remain poorly understood relative to adult satellite cells. 50-day cultures shared gene expression profiles more similar to quiescent rather than activated satellite cells, featuring a number of genes related to FOS/JUN, NOTCH, and TGFB-signaling. Day 50 cultures also expressed surface proteins known to mark adult or embryonic SMPs: CD82, CXCR4, ERBB3, NGFR, and PDGFRA. Transplanting 50-day cultures into cardiotoxin or BaCl2 injured immunodeficient murine muscle showed donor human cells persisted within the host muscle for 1 – 2 months post-injection; however, donor cells were confined to the interstitial space and did not contribute to host myofibers or the satellite cell niche. Together, these studies provide a tool for generating large quantities of embryonic skeletal muscle, and a gene expression resource that can provide insight into signaling factors that might improve or accelerate SMP development, or provide putative new surface receptors that may isolate embryonic SMPs better suited for in vivo transplantation.

Skeletal myogenesis is a tightly coordinated process resulting from the temporal expression of signalling cascades that specify myogenic cell fate. Identification of signalling pathways and small molecules that can modulate this developmental process, continues to be an active area of research. Utilising the pluripotent nature of human embryonic stem cells (hESC) and combined with next generation sequencing, we demonstrate our in vitro skeletal muscle differentiation system accurately recapitulate major skeletal muscle developmental process. We show myotubes formation can be further enhanced using a combination of anabolic factors and myokines. Multi-omics analysis revealed oxidative phosphorylation as the major up-regulated pathway, suggesting energy metabolism is coupled to enhanced skeletal muscle differentiation. Finally, to identify novel drug candidates that could reinforce muscle strength, we performed a high throughput drug screening of over 1000 drugs in hESC derived skeletal muscle cells (hESC-SkMC) and identified several candidate compounds that significantly increased the muscle marker Myosin Heavy Chain (MyHC) expression level. We further demonstrate this enhanced muscle differentiation is also closely associated with an up-regulation of energy metabolism. Together, this work presents a genetic dissection of hESC-SkMC development in vitro, which may assist in the identification and development of more effective drug therapies targeting skeletal muscle development or diseases.

Thousands of people in the United Kingdom suffer from primary muscle disease and secondary dysfunction is an increasingly recognised cause of muscle-related morbidity. Patients suffering from these conditions face significant disability and few treatment options. Much excitement has surrounded the therapeutic implications of stem cell research, including potential treatment for muscle disease.

Stem cell therapy is considered one of the most promising approaches against different neurodegenerative disorders, including Amyotorophic Lateral Sclerosis (ALS). The evidence that the systemic injection of human cord blood mononuclear cells (HuCB-MNC) was able to reduce the clinical outcomes and increase the lifespan in a murine model of fALS1, the SOD1G93A mouse, even if localized far from affected motor neurons, opens the way for new possible candidates and alternative ways of administration. Here the effect of human skeletal muscle-derived stem cell (SkmSCs) was investigated by single administration in lateral ventricles in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating clinical progression, we found that SkmSCs (previously labeled with the super paramagnetic contrast agent Endorem™ and/or with the fluorescent nuclear dye Hoeschst 33258): 1) spread along the whole ventricular system as far as the ependymal canal at the spinal cord level; 2) remained for a longer time in the Wr than in the healthy mice, and; 3) did not significantly migrate to the parenchyma. Similar to the SOD1G93A mice treated with HuCB-MNCs, the transplantation of SkmSCs: 1) significantly improved the disease progression of ALS-related Wr motorneuropathology; 2) this effect was not associated with a migration of SkmSCs close to the degenerating motor neurons. Very interestingly, we also found that cell grafting in the Wr brain ventricles significantly increased the gene expression of anti-inflammatory cytokines or chemokines activated in the inflammatory response. These results further confirm the consistency of the hypothesis of the bystander effect of stem cells in motor neurodegenerative disorders by a mechanism of action aimed at reducing the neuroinflammatory response.

Regenerative medicine along with tissue engineering represent two closely related fields leading promising advances for the treatment of numerous musculoskeletal diseases and injuries. Nevertheless, new efforts are urgently needed to design a successful therapeutic approach for muscular disorders, aiming at identifying a functional stem cell population and biomaterial scaffolds in which cells and growth factors could be embedded. In this context, recent studies have suggested that reprogramming of somatic cells by defined transcription factors into induced pluripotent stem cells (iPS), as source for generating autologous muscle progenitor cells (MPs), overcomes several limitations related to adult myoblast therapy. The prospect of an unlimited cell source combined with properties such as a more proliferative capacity in vitro, suggesting a better regenerative capacity in vivo models, indicates that iPS could be a promising candidate for stem cell therapy to regenerate skeletal muscle. iPS have been shown to retain specific features that are remnants of epigenomes and transcriptomes of the donor tissue termed ‘epigenetic memory’. Given to these findings, during the first part of the present study, we generated iPS derived from skin fibroblasts and pericytes (known to have a remarkable myogenic capacity) from the same donor to determine whether the epigenetic memory could influence iPS properties, preferentially generating cells similar of the donor somatic cell type. Until now, different approaches have been reported to generate MPs from iPS. So far, these methods present limitations such as low efficiency/reproducibility and usually involve cell sorting for enrichment or forced expression of skeletal master genes risking undesired genetic recombination. Recently, substantial interest is mounting regarding extracellular vesicles (EVs) and their involvement in many cellular processes, including myogenesis. We explored the possibility to use EVs as "physiological liposomes" enriched with myogenic factors to trigger skeletal myogenesis. To this end, during the second part of the study we developed a new transgenic-free approach to obtain transplantable MPs by means of defined factors and extracellular vesicles (EVs) secreted from differentiated mouse skeletal myoblasts. We established a novel, robust stepwise protocol by treating iPS with a WNT agonist, CHIR 99021 and myotubes-derived EVs. Thus, this method has two main advantages: (i) studying molecular mechanisms of myogenesis which is overpassed in case of genetic manipulation; (ii) muscle progenitors are not terminally differentiated, and therefore have a better repair potential following transplantation. One of the major hurdles of stem cell therapy for skeletal muscle regeneration is the massive death following transplantation. Biomaterials exhibit immune protection properties and would ensure an artificial microenvironment which permits them to interact with host cells and exert their therapeutic benefits. With the purpose of a better engraftment, we employed Poly (ethylene glycol) (PEG) -fibrinogen hydrogel (PF) as cell carrier for skeletal muscle regeneration. When transplanted in a αsarcoglycan knockout/severe combined immunodeficiency beige (α-SGKO/SCIDbg) mice, PF-embedded myogenic progenitor cells exhibited stable long-term engraftment and participated in muscle regeneration by fusing with existing muscle fibers. Importantly, no teratoma and no abnormal structure were detected in the muscles transplanted with MPs Finally, our finding and differentiation system provide an effective method that facilitates further utilization of iPS .

Le diabète de type 1 (DT1) est caractérisé par des niveaux élevés de glucose en raison de la destruction des cellules ß pancréatiques sécrétrices d'insuline. Cependant, les thérapies actuelles de remplacement des cellules bêta du pancréas impliquant la transplantation d'îlots pancréatiques sont techniquement difficiles et limitées par la disponibilité de don d'organes. Bien que les cellules souches embryonnaires et les cellules souches pluripotentes induites soient intensément étudiées, aucune de ces deux sources de cellules souches ne peut être utilisée directement sans le risque de développement de tumeurs. Les cellules souches dérivées du muscle squelettique (MDSC) sont une source de cellules alternative intéressante car elles sont multi-potentes et peuvent donc se différencier vers plusieurs lignages cellulaires tels que des cellules cardiaques à battement autonome “pacemaker-like” et des cellules neuronales. Par conséquent, nous avons émis l'hypothèse qu'elles pourraient se différencier en lignées de type pancréatique. Les objectifs de cette étude étaient donc d'étudier le potentiel des MDSC (1) à se différencier in vitro en cellules beta pancréatiques exprimant l'insuline et (2) à se différentier in vivo dans le pancréas et ainsi réduire l'hyperglycémie chez la souris modèle d'un diabète de type 1. Dans cette étude, les MDSC de muscle de souris ont été isolées via une série de passages des cellules les moins adhérentes en culture. Les cellules souches ainsi isolées peuvent adhérer sur une couche de cellules de types fibroblastes ou sur une matrice extra-cellulaire de type laminine pour ensuite se différentier in vitro ou bien être utilisées comme cellules souches MDSC non-adhérentes et non différentiées pour les études in vivo. In vitro, les MDSC peuvent se différencier spontanément en agrégats de cellules formant des îlots et exprimant des marqueurs de cellules bêta identifiés par immunofluorescence et analyse “PCR transcription inverse”. Ceci a été confirmé par immuno-analyse montrant l'expression des protéines nécessaires à la fonction des cellules ß, comme Nkx6.1, MafA et Glut2. Les MDSC différenciées en aggrégats cellulaires de type îlots pancréatiques montrent une sécrétion d'insuline en réponse au glucose in vitro. Cependant, dans des modèles murins de DT1 induit par la streptozotocine, l'injection intra-péritonéale des MDSC n'a pas permis de rétablir chez les souris diabétiques une normoglycémie du glucose sanguin en dépit d'un engreffement des MDSC dans les tissus pancréatiques. Ces données montrent que les MDSC peuvent constituer une source de cellules souches alternative intéressante pour le traitement du diabète
Type 1 Diabetes (T1D) is characterized by high and poorly controlled glucose levels due to the destruction of insulin-secreting pancreatic ß-cells. However, current ß-cell replacement therapies, involving pancreas and pancreatic islet transplantation are technically demanding and limited by donor availability. While embryonic stem cells and induced pluripotent stem cells are intensely investigated, neither can be used due to safety issues. Skeletal muscle-derived stem cells (MDSC) are an attractive alternative cell source as they have the potential to undergo multilineage differentiation into beating pacemaker-like cells and neuronal cells. Hence, it is hypothesised that they can differentiate into pancreatic lineages. This led to the goals of this study, which were (1) to investigate the potential of MDSC to differentiate into mature insulin expressing cells in vitro and (2) to reduce hyperglycemia in mouse model type 1 diabetes. In this study, MDSC were isolated from mouse via a serial pre-plating based on the adhesive characteristics of cultured cells, in which the cells of interest adhered to plates at a later time for in vitro differentiation, while the non-adherence undifferentiated MDSC were used for in vivo study. The MDSC were found to spontaneously differentiate into islet-like aggregates and expressed ß-cell markers in vitro, as determined by immunofluorescence and reverse transcription PCR analyses. This was further confirmed by immunoblotting analysis showing expression of proteins required for ß-cell function, such as Nkx6.1, MafA and Glut2. The differentiation of MDSC into islet-like clusters demonstrated glucose responsiveness in vitro. In streptozotocin-induced T1D mouse models, intraperitoneal injection of the undifferentiated MDSC did not restore the blood glucose levels of the diabetic mice to normoglycemia despite successful engraftment of MDSC into the pancreatic tissues. Taken together, these data show that MDSC may serve as an alternative source of stem cells for the treatment of diabetes

Every year several patients have to deal with bone tissue loss due to trauma or diseases. Bone tissue engineering aims to restore or repair musculoskeletal disorders through the development of bio-substitutes that require the use of cells and scaffolds which should possess both adequate mechanical properties and interconnecting pores to allow cellular infiltration, graft integration and vascularization. The ideal cell for tissue engineering should possess a potential plasticity with the ability to functionally repair the damaged tissue, and it should be available in large amount. Mesenchymal stem cells (MSCs) are present in many adult tissues, and adipose tissue represents an attractive source of MSCs for researchers and clinicians of nearly all medical specialties. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow, placenta, and umbilical cord blood in morphology, immunophenotype, and differentiation ability, and they represent a promising approach of bone regeneration. Additional features of ASCs are their immunoregolatory and anti-inflammatory properties both in vivo and in vitro and their low immunogenicity. Since several years our laboratory is studying mesenchymal stem cells isolated from human and animal adipose tissues. Human ASCs (hASCs) have been characterized by their immunophenotype, their self-renewal potential, and they have been induced to differentiate towards adipogenic, osteogenic and chondrogenic lineages. The ability of hASCs to grow in the presence of several scaffolds has also been tested. hASCs adhered to the surface of tested biomaterials, filling the pores and forming a 3D web-like structure, allowing these progenitor cells to osteo-differentiate more efficiently respect to cells maintained on polystyrene. Since our interest was to regenerate muscle-skeletal defects by ASCs in pre-clinical models, we first studied ASCs isolated from adipose tissue of rat (rASCs), rabbit (rbASCs) and pig (pASCs), considered good models in the orthopaedic field. We have shown that animal ASCs behaved similarly to the human ones, and, in collaboration with the Faculty of Veterinary Medicine of University of Milan and the IRCCS Galeazzi Orthopaedic Institute of Milan, we have tested the ability of autologous ASCs to regenerate a full-thickness critical-size bone defect in rabbits. The experimental study was conducted on the tibiae of 12 New Zealand rabbits, and from 6 rabbits out of 12 we have collected adipose tissue from the interscapular region. We have isolated 2.8x105±1.9x105 rbASCs per ml of raw tissue, and after 3-4 days in culture the cells showed the typical fibroblast-like morphology. One week later, all the 6 cellular populations started to steadily proliferate, and they generated fibroblast (CFU-F) and osteoblast (CFU-O) colonies, highlighting the presence of osteogenic progenitors. Indeed, when rbASCs were induced to osteo-differentiate, either after 7 and 14 days, we have observed an up-regulation of specific osteogenic markers, such as alkaline phosphatase (ALP, +28.9%), collagen (+105.9%) and extracellular calcified matrix (+168.1%), compared to undifferentiated cells. In parallel, testing HA, the scaffold selected for the in vivo experiment, we found that rbASCs were osteoinduced; indeed the presence of HA granules increased per se the amount of collagen production (+48.2%). 1.5x106 undifferentiated rbASCs were seeded on custom-made HA disks (8 mm Ø x 4 mm ↕), and the day after, each bioconstruct was implanted into the lesion created in the tibia of each rabbit. We had an additional experimental group of defects where the same number of rbASCs were inserted in the lesion as a semi-liquid suspension; moreover, as controls, we treated 6 lesioned tibia with just the scaffolds, and we left 6 untreated lesioned bone. 8 weeks after surgery animals were sacrificed and the tibia explanted. A macroscopic analysis showed no bone resorption, no abnormal bone callous formation, no fractures, infection or inflammatory reactions, and all the bone defects were completely filled without any significant differences among the four groups. Interestingly, in the presence of scaffold seeded with rbASCs, histology and immunohistochemistry showed a new bone tissue more mature and similar to the native bone. These data have also been confirmed by biomechanical tests: indeed, the mechanical properties of the bone defect treated by rbASCs-HA were improved, suggesting that these constructs bore mechanical loading with an increase in stiffness of 19.8% and in hardness of 31.6% respect to just HA treated group, indicating that the bioconstructs made out of autologous rbASCs and hydroxyapatite might ameliorate the treatment for large bone defects. We would suggest the use of ASCs as a safe cellular therapy in future clinical applications where a large bone defect needs to be treated. These promising results on small size animals allow us to plan a new study on large size animals such as minipigs. However, before moving to the clinic, we know that there are several important aspects that need to be faced regarding safeness and the features of the candidate patients: 1. may the “quality” of hASCs be affected by the donor’s physiological or pathological conditions? 2. may the use of pharmacological treatment enhance cellular plasticity of multipotent cells? 3. may the use of immunoselected hASCs ameliorate tissue regeneration in the field of muscle-skeletal? We have addressed some of these aspects, comparing different populations of hASCs from subcutaneous adipose tissue of healthy-young-female donors (hASCs<35 y/o, n=12, mean age 31±4 years, BMI=23.5±1.6), and from middle-age ones (hASCs>45 y/o n=14, mean age 56±7 years, mean BMI=28.4±1.8). The cellular yield of hASCs derived from older donors was 2.5 fold greater than the one of hASCs<35 y/o, whereas hASCs from younger donors were more clonogenic than hASCs isolated from older ones, with an increase of 129%. No significant differences were observed looking at their immunophenotype. When hASCs were induced to differentiate into cells of the adipogenic and osteogenic lineages, the donor’s age did not affect their adipogenic differentiation, whereas the osteogenic one was significantly affected by age both in the absence and in the presence of three-dimensional scaffolds, showing a decreased ALP basal levels of about 10-fold in hASCs>45 y/o respect to hASCs<35 y/o. These results seems to indicate that ASCs from different donors could behave differently. Trying to overcome this aspect we have used different approaches, and we have studied if Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells, might improve the differentiation ability of hASCs. 72 hours treatment with 50 nM Reversine induced hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation) compared to control hASCs. Moreover, since it is known that CD34 and L-NGFR positive cells define a subset of high proliferative and multipotent MSCs, we have immunoselected, these progenitor cells from hASC populations. In contrast to the whole population, the immunoseparated fractions maintained their undifferentiate state and their ability to differentiate much longer during culture. We have shown that both CD34+ and L-NGFR+ hASCs can be used as alternative candidates for tissue engineering and regenerative medicine applications. In particular, due to the improved ability of L-NGFR positive cells to adipo- and chondro-differentiate, they appear an ideal tool in reconstructive plastic surgery and cartilage regeneration. From our data, and the ones from researchers in other fields, we believe that in the near future adipose-derived stem cells might be considered a safe tool in regenerative medicine. Furthermore, to improve this “cellular therapy”, we could either pre-treat ASCs with molecules, such as drugs and/or siRNAs known to affect specific differentiation pathways, or by selecting subpopulations of progenitor cells which may be used as allogenic implants. Next step will be to confirm our in vivo data in a large size animal model such as minipig, and then to test if pre-treated cells or selected population might be used in an autologous and allogenic small size animal model.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder associated with premature aging in various tissues and organs of the afflicted individuals, including accelerated skeletal muscle atrophy. Classical HGPS manifests due to single-base substitution in the LAMNA gene which encodes Lamin A/C proteins. As a result of the mutation, a truncated form of Lamin (known as Progerin) is produced which undergoes persistent farnesylation during post-translational modification. Accumulation of Progerin in the nucleus has been linked to various cellular abnormalities including abnormal nuclear morphologies and altered chromatin organization, among others. However, the exact molecular mechanisms leading to skeletal muscle atrophy have not yet been elucidated. In this study, the iPSC approach was implemented in order to study the skeletal muscle phenotype of HGPS by generating and characterizing a population of Pax7 positive skeletal muscle precursor cells (SMPs). During the course of this project, we have demonstrated the need for excessive optimization of the previously developed directed differentiation protocol for successful application on induced Pluripotent Stem Cells. Furthermore, we have successfully modified the protocol to allow for a more rapid expansion of the SMPs through regular passaging of the myogenic cells starting on day 20 of differentiation. Additionally, this new method produced more uniform distribution of the myogenic cells and allowed for successful freezing/thawing of the myogenic cells. When compared to the controls, the HGPS-derived SMPs did not appear to be defective in formation, proliferation or differentiation. Abnormal nuclear morphology and DNA damage, documented in HGPS fibroblasts and vascular smooth muscle cells, were not detected the in myogenic cells. Furthermore, we were not able to detect Progerin protein accumulation in the generated myogenic cultures, offering an explanation for the absence of these phenotypes in the skeletal muscle system.

Skeletal muscle is a tissue with remarkable regenerative abilities but a wide range of disorders can impair its functions. At present, there is no effective therapy for most of these pathologies and this represents a significant unmet medical need. The pursuit of therapeutic strategies has led to the development of different encouraging approaches. Importantly, tissue engineering is also emerging as a promising discipline for the establishment of new platforms with therapeutic relevance for muscle disorders. Generating human artificial skeletal muscles in vitro would indeed provide an invaluable tool for disease modelling, drug screening and tissue replacement. Nevertheless, skeletal muscle tissue engineering is extremely challenging and at present no commonly endorsed model is available. Specifically, one of the bottlenecks is the use of primary cells, which hold the drawback of scarce availability and reduced proliferation and differentiation potential in vitro thus limiting their use. Here I describe the generation of 3D artificial mini-muscles from human pluripotent stem cells derived from healthy donors and patients with muscular dystrophy. This has never been reported before and the use of pluripotent stem cells offers a virtually unlimited source of myogenic cells. These patient- and disease-specific human artificial mini-muscles recapitulate characteristics of the adult tissue and, importantly, are able to engraft into immunodeficient mice. Finally, I show that other isogenic cell types present in normal muscle tissue (such as endothelial cells and pericytes) can be derived and combined together with the same patient-specific myogenic cells, thus generating a multi-lineage artificial mini-muscle. This complex platform could provide a valuable tool for skeletal muscle disease modelling, drug screening and tissue replacement ultimately leading to the development of new therapies for muscle diseases.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011.
Includes bibliography.
ENGLISH ABSTRACT: Background: IL-6 belongs to a cytokine super-family known to affect cell proliferation, although other family members are better characterized. Proliferation promoting factors (IL-6) compete with differentiation promoting factors (myogenic regulatory factors: MyoD and myogenin) to affect cell cycle. Cell cycle progression is assessed by determining the proportion of cells shifting from arrest to chromatin synthesis and mitosis phases (G0/G1 and S and G2/M respectively). Methods: This study assessed the effects of IL-6 on cell cycle progression and proliferation vs. differentiation of C2C12 skeletal myoblasts. Physiological doses (10 or 100 pg/ml) were compared to a high dose (10 ng/ml), with exposure lasting 48 hours (addition of IL-6 dose to proliferation medium at 0 and 24 hours). Acute signaling downstream of the IL-6 gp130 receptor was assessed after the first exposure. Results: Propidium iodide analysis of nuclear material using flow cytometry indicated shifts in forward scatter. Both Low and Medium doses shifted a greater proportion (p<0.05) of cells from G0/G1 to S and G2M phases at 24 hours and all doses resulted in the same shift (p<0.05) at the 48 hour time point. However, the High dose significantly (p<0.05) increased myogenin expression at the 48 hour time point. Microscopy indicated that confluence was prevented by low seeding density and did not influence the result. Cells harvested at 5 minutes post stimulation indicated that all doses significantly increased STAT3 phosphorylation. 10 minutes post stimulation the High dose group sustained elevated levels of STAT3 phosphorylation. Conclusions: Low and medium doses of IL-6 increase proliferation in a muscle satellite cell line by activating cell division and allowing myoblasts to remain in the active cell cycle. High doses of IL-6 increase differentiation by mediating upregulation of myogenic regulatory factors and this is thought to be due to prolonged STAT3 activation. Physiological control of myoblast behaviour by cytokines is evident and such control would be influenced by the severity of the endogenous cytokine response to various stimuli.
AFRIKAANSE OPSOMMING: Agtergrond: IL-6 behoort aan n sitokien super-familie bekend vir die affektering van sel verspreiding, alhoewel ander familie lede beter gekenmerk is. Bevordering van verspreiding faktore (IL-6) kompeteer met bevordering van differensiasie fatore (myogenic regulatory factors: MyoD en myogenin) om die sel siklus te affekteer. Sel siklus progressie word geassesseer deur die bepaling van die proporsie selle wat verskuif van arrestasie na chromatien sintese en mitose fases (G0/G1 en S en G2/M onderskeidelik). Metodes: Hierdie studie het die effekte van IL-6 op die progressie van die sel siklus geassesseer asook die proliferasie vs. differensiasie van C2C12 skelet spier satelliet selle. Fisiologiese dosisse (10 en 100 pg/ml) was vergelyk tot n hoog dose (10 ng/ml), met blywende blootstelling van 48 uur (byvoeging van IL-6 dose tot verspreidings medium op 0 and 24 uur). Akute sein stroomaf van die IL-6 gp130 reseptor was ook geassesseer na die eerste blootstelling. Resultate: Propidium iodide analise van kern materiaal deur vloei sitometrie het voorwaarts verskuiwing aangedui. Beide Laag and Medium doses het n groter proporsie (p<0.05) selle verskuif van die G0/G1 tot die S en G2M fases na 24 uur en alle dosisse het gelei in die selfde verskuiwing (p<0.05) by die 48 huur tyd punt. Alhoewel die Hoog dose myogenin uitdrukking aansienlik (p<0.05) verhoog het na 48 uur. Mikroskopie het aangedui dat samevloeiing voorkom was deur n lae loting digtheid en dit het nie resultate geaffekteer nie. Selle wat geoes was 5 minute na stimulasie het aangedui dat alle dosisse STAT3 fosforilasie laat toeneem het. 10 minute na stimulasie het die Hoog dose groep volgehoue vlakke van STAT3 fosforilasie besit. Gevolgtrekkings: Laag en Medium dosisse van IL-6 verhoog verspreiding in n spier satelliet sel lyn deur die aktivering van sel deling en deur selle toe te laat om in die aktiewe sel siklus te bly. Hoog dosisse van IL-6 verhoog differensiase deur bemiddelende opstoot van myogenic regulatory factors en die gedagte is dat dit bewerkstellig word deur aanhoudende aktivering van STAT3. Fisiologies beheer van satelliet selle deur sitokiene is duidelik en die beheer sal beinvloed word deur die erns van die endogene sitokien reaksie op verskillende stimuli.

Skeletal muscles are composed of thousands of muscle fibres (muscle cells), densely packed together in parallel and surrounded by connective tissue sheaths. These fibres are multinuclear in nature, which allows for the control and regulation of the highly organised, protein rich cellular interior. The primary function of skeletal muscle is to produce force, which allows for movement to occur or posture to be maintained, and the regulation of this function is in turn reliant on the expression of specific genes and proteins. Skeletal muscle exhibits a high degree of plasticity, and can adapt in response to stimuli such as increased/decreased use, metabolic perturbations or changes in the systemic environment which often occur as a result of exercise, ageing, disuse or disease. Examining responses and adaptations in skeletal muscle in vivo are challenging due to experimental restrictions, and studies are limited by ethical issues surrounding experimentation on human beings and indeed on animals following the principals of refinement, reduction and replacement. Thus in vitro studies are often conducted in order to further understand mechanisms involved in adaptation. However, the environment to which skeletal muscle cells are exposed to in vitro is far removed from that in the body, and the resulting cellular architecture is often abnormal in morphology. Tissue engineered skeletal muscle has shown much promise in rectifying these issues, as cells can be grown on/within a matrix which is biologically relevant and engineered to grow in a uniaxial manner in parallel to one another. However, this field is in its relative infancy, and to date little data exists with regards the behaviour and characteristics of human muscle derived cells (MDCs) in tissue engineered constructs. In this thesis, human skeletal MDCs were obtained, characterised and subsequently cultured in a suitable model for tissue engineering purposes. MDCs were seeded on to a fibrin based hydrogel, which self-assembled over time to form a cylindrically shaped construct held in place between two anchor points. In ii this model, the cells were shown to align uniaxially and in parallel to one another in a fascicular like structure. The model was improved in terms of biomimicity and maturation by both increasing the seeding density of the MDCs, and by increasing the ratio of myogenic to non-myogenic cells. These models appear to promote the development of a slow muscle, as evidenced by the favourably high levels of MYH7 transcription in comparison to other isoforms, and showed suggestions of sarcomeric organisation as indicated by the classically striated pattern of protein organisation when myosin heavy chain immunostaining was conducted. The work conducted in the final chapter of this thesis focussed on developing a system capable of assessing and quantifying the force produced by these tissue engineered human skeletal muscle constructs when electrically stimulated. Further work in this area should aim to determine these functional characteristics and thereafter use the model for physiological, cellular and molecular studies in exercise science.

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007.
Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

Tissue engineered vascular grafts with long term patency are in great need in the clinics. An accessible source of human smooth muscle cell (SMC) is important for constructing functional vascular grafts. Human mesenchymal stem cells from the umbilical cord (UCMSCs) exhibit multi-lineage differentiation abilities, including the potential to differentiate towards vascular lineages such as SMCs. MicroRNAs (miRNAs) are short non-coding regulatory RNAs. They widely participate in regulation of stem cell differentiation and may play an important role in SMC differentiation. Understanding how to generate SMCs from UCMSCs as well as its underlying mechanism might greatly contribute to our knowledge of manufacturing functional vascular grafts. We hypothesise that vascular grafts could be generated with SMCs differentiated from human UCMSCs, and further explore the role of miRNAs in the differentiation process. We utilised transforming growth factor β 1 (TGFβ1) to stimulate the UCMSCs differentiation towards SMCs. A panel of SMC markers including αSMA, SM22, Calponin and SMMHC were highly upregulated both at the gene expression and the protein level at day 5 of TGFβ1 treatment. Micro-RNA (miR) array analysis showed that miR-503 was increased at early time points (6 h and 24 h) after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. We further demonstrated that miR-503 mimics promoted SMC differentiation both at the gene expression and the protein level and miR-503 inhibitors downregulated SMC markers at the protein level. Smad7, which is a negative regulator of TGFβ1-related signalling pathways, was identified to be a direct target of miR-503 by luciferase reporter experiments. The expression level of miR-503 was Smad4-dependent as shown by the Smad4 knockdown experiments. Also, Smad4 was demonstrated to be enriched at the promoter region of miR-503 as shown by Chromatin immunoprecipitation experiments. In addition to miR-503, miR-222-5p was also downregulated in the differentiation process. The gain-of-function study with the treatment of miR-222-5p mimics significantly inhibited the induction of SMC markers Calponin and αSMA both at the gene expression and protein level during differentiation. αSMA was confirmed to be a direct target of miR-222-5p. Moreover, ROCK2, which could mediate SMC differentiation through RhoA/ROCK pathway, was downregulated by miR-222-5p mimics both at the gene expression and protein level. The 3’-UTR segment of ROCK2 was identified to be a direct target of miR-222-5p. Finally, SMCs differentiated from UCMSCs exhibited the ability to migrate into decellularised mouse aorta grafts. Seeding of the cells onto the decellularised scaffold gave rise to vascular graft with smooth muscle layer that is comparable to its analog of the native vessel. In conclusion, we demonstrated the potential of using hUCMSCs-derived SMCs to generate vascular grafts which are in critical need in the clinics.

La myopathie myofibrillaire est une maladie neuromusculaire à évolution lente caractérisée par de graves troubles musculaires causés par des mutations dans le gène codant pour des protéines du cytosquelette. L'un des gènes affectés en relation avec le développement de la MFM est DES. Des mutations dans le gène de la desmine entraînent des myopathies des muscles squelettiques et cardiaques. Cependant, les évènements qu'elles entraînent et qui sont à l’origine des phénotypes pathologiques cardiaques restent mal connus. Mon objectif est de créer un modèle in vitro de MFM basé sur des cellules souches pluripotentes humaines afin d'étudier le rôle des mutations spécifiques dans la desmine sur le développement et la fonction des cellules cardiaques. Pour atteindre cet objectif, en collaboration avec les docteurs A. Behin, K. Wahbi et la société Phenocell, nous avons généré des iPSC à partir des cellules sanguines périphériques de patients souffrant d'une forme de cardiomyopathie induite par une mutation de la desmine. Les lignées iPSC générées contenant les mutations du gène codant la desmine ont permis d’étudier le rôle d’une mutation dans la spécification et la fonction des cardiomyocytes. La bioénergétique mitochondriale, la structure cellulaire et la fonction contractiles ont été évaluées au niveau cellulaire. En conclusion, il convient de noter que les mutations de la desmine conduisent à une désorganisation des structures des sarcomères dans les cardiomyocytes et à une perturbation de l'expression des protéines mitochondriales. Ce qui conduit à une altération des fonctions de la mitochondrie. Ces données permettent d’améliorer notre compréhension des mécanismes moléculaire qui sous-tendent le développement de la MFM
Myofibrillar Myopathy is a slowly progressive neuromuscular disease characterized by severe muscular disorders caused by mutations in the gene encoded cytoskeletal proteins. One of the genes described in connection with the development of MFM is DES. Mutations in the desmin gene lead to skeletal and cardiac muscles myopathies. However, the cardiac pathological consequences caused by them remain poorly understood. My objective is to create an in vitro human stem cell model of MFM to specifically investigate the role of patient-specific mutations in desmin on cardiac lineage development and function. To achieve that objective, in collaboration with Drs. Behin and K. Wahbi and Phenocell, we generate patient-specific iPSC from peripheral blood cells of the patient suffering severel form of desmin-deficient cardiomyopathy. The generated iPSC lines carrying DES gene mutations enable a powerful examination of the role of desmin mutation on cardiomyocyte specification and function. Bioenergetic, structural, and contractile function will be assessed in a single cell. In conclusion, it should be noted that desmin mutations lead to a disorganization of sarcomere structures in cardiomyocytes and to a perturbation of mitochondrial protein expression. This leads to a distortion of functions in the mitochondria. These data facilitate the understanding of the molecular pathway underlying the development of desmin-related myopathy. And the system we have created could also allow us to better evaluate the correlation between the desmin genotype and phenotype in terms of effect on the heart

M.Tech.
Stem cells possess self-renewal capacity, long-term viability, and multilineage potential. Stem cells play important roles in normal physiological and disease processes, they also have great therapeutic potential. However, there have been controversies surrounding stem cells in political, religious and ethical arenas. Although the use of certain stem cells (i.e. embryonic stem cells) and the means by which they are obtained contravene certain basic ethical laws, researchers have developed methods with which to ethically obtain and create stem cell lines. Stem cells can be classified as either: totipotent, pluripotent, multipotent, oligopotent and unipotent (Moore, 2007). Totipotent cells have the ability to differentiate into all cell types of an embryo, including the extra-embryonic and post embryonic tissues and organs. Pluripotent cells have the potential to differentiate into almost all tissues found in an embryo (including germ cells), but are not capable of giving rise to supporting cells and tissues. Multipotent stem cells have progeny of several differentiated cell types - but all within a particular tissue, organ, or physiological system. A good example of multipotent cells, are the haematopoietic stem cells that produce blood cell-restricted progenitors, as well as all cell types and elements, such as platelets, that are normal components of blood. Oligopotent stem cells produce two or more lineages within a specific tissue, such as neural stem cells that are able to produce subsets of neurons in the brain. Unipotent cells self-renew, as well as give rise to a single mature cell type, a prime example being the spermatogonial stem cells, that give rise to spermatozoa (Moore, 2007). Adult human subcutaneous adipose tissue contains cells with multilineage developmental plasticity like marrow-derived mesenchymal stem cells (Strem et al., 2005, Tong et al., 2000). Adipose derived stem cells can be obtained in abundance and can differentiate into osteogenic, adipogenic, myogenic and chondrogenic lineages when treated with appropriate growth factors.

博士
國防醫學院
生命科學研究所
102
Skeletal muscle (SkM) comprise approximately 40% of human body weight. This important tissue declines progressively with age, which its endogenous stem cell—the satellite cell—cannot reverse. Mesenchymal stem cells (MSC) are post-natal progenitor/stem cells that possess multilineage mesodermal differentiation capacity, including towards SkM. Adult bone marrow (BM) is the best-studied source of MSCs; however, aging also decreases BMMSC numbers and can adversely affect differentiation capacity. Therefore, we asked whether human sources of developmentally early-stage mesenchymal stem cells (hDE-MSCs) isolated from embryonic stem cells, fetal bone, and term placenta could be cellular sources for SkM repair. Under standard muscle-inducing conditions, hDE-MPCs differentiate towards a SkM lineage rather than cardiomyocytic or smooth muscle lineages, as evidenced by increased expression of SkM-associated markers and in vitro myotube formation. In vivo transplantation revealed that SkM-differentiated hDE-MSCs can incorporate into host SkM tissue efficiently in a mouse model of SkM injury. In contrast, adult BMMSCs do not express SkM-associated genes after in vitro SkM differentiation nor engraft in vivo. Further investigation of possible factors responsible for this difference in SkM differentiation potential revealed that, compared to adult BMMSCs, hDE-MSCs expressed higher levels of serum response factor (SRF), a transcription factor critical for SkM lineage commitment. Moreover, knockdown of SRF in hDE-MSCs resulted in decreased expression of SkM-related genes after in vitro differentiation and decreased in vivo engraftment. Our results implicate SRF as a key factor in age-related SkM differentiation capacity of MSCs, and demonstrate that hDE-MSCs are possible candidates for SkM repair.