Establishing a Robust In Vitro Embryonic Stem Cell Differentiation Assay to Monitor the Hematopoietic Potential of DELES Clones

Afin d’effectuer des études fonctionnelles sur le génome de la souris, notre laboratoire a généré une bibliothèque de clones de cellules souches embryonnaires (ESC) présentant des suppressions chromosomiques chevauchantes aléatoires – la bibliothèque DELES. Cette bibliothèque contient des délétions couvrant environ 25% du génome murin. Dans le laboratoire, nous comptons identifier de nouveaux déterminants du destin des cellules hématopoïétiques en utilisant cet outil. Un crible primaire utilisant la benzidine pour démontrer la présence d'hémoglobine dans des corps embryoïdes (EBS) a permis d’identifier plusieurs clones délétés présentant un phénotype hématopoïétique anormal. Comme cet essai ne vérifie que la présence d'hémoglobine, le but de mon projet est d'établir un essai in vitro de différenciation des ESC permettant de mesurer le potentiel hématopoïétique de clones DELES. Mon hypothèse est que l’essai de différenciation hématopoïétique publié par le Dr Keller peut être importé dans notre laboratoire et utilisé pour étudier l'engagement hématopoïétique des clones DELES. À l’aide d’essais de RT-QPCR et de FACS, j’ai pu contrôler la cinétique de différenciation hématopoïétique en suivant l’expression des gènes hématopoïétiques et des marqueurs de surface comme CD41, c-kit, RUNX1, GATA2, CD45, β-globine 1 et TER-119. Cet essai sera utilisé pour valider le potentiel hématopoïétique des clones DELES candidats identifiés dans le crible principal. Mon projet secondaire vise à utiliser la même stratégie rétro-virale a base de Cre-loxP utilisée pour générer la bibliothèque DELES pour générer une bibliothèque de cellules KBM-7 contenant des suppressions chromosomiques chevauchantes. Mon but ici est de tester si la lignée cellulaire leuémique humaine presque haploïde KBM-7 peut être exploitée en utilisant l'approche DELES pour créer cette bibliothèque. La bibliothèque de clones KBM-7 servira à définir les activités moléculaires de drogues anti-leucémiques potentielless que nous avons identifiées dans le laboratoire parce qu’elles inhibent la croissance cellulaire dans plusieurs échantillons de leucémie myéloïde aiguë dérivés de patients. Elle me permettra également d'identifier les voies de signalisation moléculaires qui, lorsque génétiquement perturbées, peuvent conférer une résistance à ces drogues.

Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism

Adult skeletal muscle possesses a resident stem cell population called satellite cells which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration but is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. Here the process of satellite cell migration has been investigated revealing that they undergo two distinct phases of movement; firstly under the basal lamina and then rapidly increasing their velocity when on the myofibre surface. Most significantly we show that satellite cells move using a highly dynamic blebbing based mechanism and not via lamellopodia mediated propulsion. We show that nitric oxide and non-canonical Wnt signalling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background: Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker. Methodology/Principal Findings: K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker. Conclusions/Significance: K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.

Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.

TRIM32 regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H.

Age related changes in speed and mechanism of adult skeletal muscle stem cell migration

Skeletal muscle undergoes a progressive age-related loss in mass and function. Preservation of muscle mass depends in part on satellite cells, the resident stem cells of skeletal muscle. Reduced satellite cell function may contribute to the age-associated decrease in muscle mass. Here we focused on characterising the effect of age on satellite cell migration. We report that aged satellite cells migrate at less than half the speed of young cells. In addition, aged cells show abnormal membrane extension and retraction characteristics required for amoeboid based cell migration. Aged satellite cells displayed low levels of integrin expression. By deploying a mathematical model approach to investigate mechanism of migration, we have found that young satellite cells move in a random ‘memoryless’ manner whereas old cells demonstrate superdiffusive tendencies. Most importantly, we show that nitric oxide, a key regulator of cell migration, reversed the loss in migration speed and reinstated the unbiased mechanism of movement in aged satellite cells. Finally we found that although Hepatocyte Growth Factor increased the rate of aged satellite cell movement it did not restore the memoryless migration characteristics displayed in young cells. Our study shows that satellite cell migration, a key component of skeletal muscle regeneration, is compromised during aging. However, we propose clinically approved drugs could be used to overcome these detrimental changes.

Towards the use of hydrogels in the treatment of limbal stem cell deficiency

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD.

Targeting stem cell niches and trafficking for cardiovascular therapy.

Regenerative cardiovascular medicine is the frontline of 21st-century health care. Cell therapy trials using bone marrow progenitor cells documented that the approach is feasible, safe and potentially beneficial in patients with ischemic disease. However, cardiovascular prevention and rehabilitation strategies should aim to conserve the pristine healing capacity of a healthy organism as well as reactivate it under disease conditions. This requires an increased understanding of stem cell microenvironment and trafficking mechanisms. Engagement and disengagement of stem cells of the osteoblastic niche is a dynamic process, finely tuned to allow low amounts of cells move out of the bone marrow and into the circulation on a regular basis. The balance is altered under stress situations, like tissue injury or ischemia, leading to remarkably increased cell egression. Individual populations of circulating progenitor cells could give rise to mature tissue cells (e.g. endothelial cells or cardiomyocytes), while the majority may differentiate to leukocytes, affecting the environment of homing sites in a paracrine way, e.g. promoting endothelial survival, proliferation and function, as well as attenuating or enhancing inflammation. This review focuses on the dynamics of the stem cell niche in healthy and disease conditions and on therapeutic means to direct stem cell/progenitor cell mobilization and recruitment into improved tissue repair.

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.

Culture bag systems for clinical applications of adult human neural crest-derived stem cells

Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

Investigating the influence of extracellular matrix and glycolytic metabolism on muscle stem cell migration on their native fibre environment

The composition of the extracellular matrix (ECM) of skeletal muscle fibres is a unique environment that supports the regenerative capacity of satellite cells; the resident stem cell population. The impact of environment has great bearing on key properties permitting satellite cells to carry out tissue repair. In this study, we have investigated the influence of the ECM and glycolytic metabolism on satellite cell emergence and migration- two early processes required for muscle repair. Our results show that both influence the rate at which satellite cells emerge from the sub-basal lamina position and their rate of migration. These studies highlight the necessity of performing analysis of satellite behaviour on their native substrate and will inform on the production of artificial scaffolds intended for medical uses.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.