Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line

Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45% in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5%, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27%, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

La dérivation de cellules souches embryonnaires chez le cheval

Les cellules souches embryonnaires (ES) sont porteuses de grands espoirs en recherche biomédicale dans le but d’apporter un traitement définitif à l’ostéoarthrose. Parce que certaines articulations des chevaux sont similaires à celles des humains, cet animal représente un modèle important dans l’évaluation de stratégies de régénération du cartilage. Cependant, pour expérimenter un traitement par les cellules ES chez le cheval, des cellules ES équines (eES) n’ont toujours pas pu être dérivées. Dans ce contexte, l’objectif principal de cette étude est de dériver des lignées de cellules eES. Le premier objectif de notre étude consiste à optimiser la technique de dérivation des cellules eES. Nous démontrons que la lignée de cellules nourricières et le stade de développement des embryons influencent l’efficacité de la technique de dérivation tandis que l’inhibition de voies de signalisation menant à la différenciation des cellules ES ne l’influence pas sous nos conditions. Le deuxième objectif de notre étude est de caractériser de façon plus approfondie les lignées de cellules eES obtenues. Nous démontrons que les cellules eES dérivées expriment autant des marqueurs associés aux cellules pluripotentes qu’aux cellules différenciées et que l’inhibition de voies de signalisation menant à la différenciation n’influence pas l’expression de ces marqueurs. Pour conclure, nous confirmons avoir dérivé des lignées de cellules semblables au cellules eES (eES-like) ne correspondant pas complètement aux critères des cellules ES.

Identification de déterminants impliqués dans la différenciation des cellules souches embryonnaires

Les cellules souches ont attiré l’attention du public ces dernières années, grâce non-seulement à leur utilisation comme thérapies visant à s’attaquer à certains types de cancers, mais aussi en relation avec leur potentiel dans le domaine de la médecine regénérative. Il est établi que le destin cellulaire des cellules souches embryonnaires (ESC) est régulé de façon intensive par un groupe de facteur clés agissant sur leur pluripotence. Il est néanmoins envisageable que certains déterminants influençant l’auto-renouvellement et la différenciation de ces cellules soient toujours inconnus. Afin de tester cette hypothèse, nous avons généré, en utilisant une méthode par infections virales, une collection de ESC contenant des délétions chromosomales chevauchantes que nous avons baptisée DelES (Deletion in ES cells). Cette librairie contient plus de 1000 clones indépendants dont les régions délétées couvrent environ 25% du génome murin. À l’aide de cette ressource, nous avons conduit un criblage de formation de corps embryoïdes (EB), démontrant que plusieurs clones délétés avaient un phénotype de différenciation anormal. Nos études de complémentation sur un groupe de clones ont par la suite permis l’identification de Rps14 - un gène codant pour une protéine ribosomale (RP) comme étant haploinsuffisant pour la formation de EB. Dans un deuxième temps, l’analyse approfondie des résultats de notre crible a permis d’identifier un groupe de gènes codants pour des RP qui semblent essentiels pour la différenciation des ESC, mais dispensables pour leur auto-renouvellement. De manière intéressante, les phénotypes anormaux de formation en EB les plus marqués sont associés à des délétions de RP qui se retrouvent au site de sortie des ARN messagers (ARNm) du ribosome, soit Rps5, Rps14 et Rps28. Étonnament, alors qu’un débalancement des RP conduit généralement à une réponse de type p53, l’haploinsuffisance de ces trois gènes ne peut être renversée par une simple réduction des niveaux d’expression de ce gène suppresseur de tumeurs. Finalement, nos études de profilage polysomal et de séquençage à haut-débit montrent une signature spécifique de gènes liés au mésoderme chez un clone hétérozygote pour Rps5, suggérant ainsi une explication au phénotype de différenciation p53-indépendant identifié chez ces ESC. Nos travaux rapportent donc la création d’une ressource intéressante de génomique fonctionnelle qui a permis de mettre à jour le rôle essentiel que jouent les RP dans le processus de formation de EB. Nos résultats permettent aussi de documenter une réponse p53-indépendante suite à un débalancement de RP dans un contexte opposant l’auto-renouvellement et la différenciation des ESC.

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.

NG2 cells differentiate into astrocytes in cerebellar slices

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29-30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a [`]reactive' response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.

Ex Vivo Construction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane. Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibres within the compressed gel were dense, homogeneous and similar in structure to those within denuded amniotic membrane. Limbal epithelial cells were successfully expanded upon the compressed collagen resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology and cell stratification. Similar protein expression profiles for cytokeratin 3 and cytokeratin 14 and no significant difference in cytokeratin 12 mRNA expression levels by real time PCR were also observed. This study provides the first line of evidence that a laminin coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification and differentiation to a degree that is comparable to the leading conventional scaffold, denuded amniotic membrane.

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

The origin, molecular regulation and therapeutic potential of myogenic stem cell populations

Satellite cells, originating in the embryonic dermamyotome, reside beneath the myofibre of mature adult skeletal muscle and constitute the tissue-specific stem cell population. Recent advances following the identification of markers for these cells (including Pax7, Myf5, c-Met and CD34) (CD, cluster of differentiation; c-Met, mesenchymal epithelial transition factor) have led to a greater understanding of the role played by satellite cells in the regeneration of new skeletal muscle during growth and following injury. In response to muscle damage, satellite cells harbour the ability both to form myogenic precursors and to self-renew to repopulate the stem cell niche following myofibre damage. More recently, other stem cell populations including bone marrow stem cells, skeletal muscle side population cells and mesoangioblasts have also been shown to have myogenic potential in culture, and to be able to form skeletal muscle myofibres in vivo and engraft into the satellite cell niche. These cell types, along with satellite cells, have shown potential when used as a therapy for skeletal muscle wasting disorders where the intrinsic stem cell population is genetically unable to repair non-functioning muscle tissue. Accurate understanding of the mechanisms controlling satellite cell lineage progression and self-renewal as well as the recruitment of other stem cell types towards the myogenic lineage is crucial if we are to exploit the power of these cells in combating myopathic conditions. Here we highlight the origin, molecular regulation and therapeutic potential of all the major cell types capable of undergoing myogenic differentiation and discuss their potential therapeutic application.

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.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally-modified calcium alginate hydrogel

Aims: Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for ‘on-demand’ use. Materials & methods: In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Results: Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Conclusion: Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

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.

Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes their neuronal subtype specification

Many in vitro systems used to examine multipotential neural progenitor cells (NPCs) rely on mitogens including fibroblast growth factor 2 (FGF2) for their continued expansion. However, FGF2 has also been shown to alter the expression of transcription factors (TFs) that determine cell fate. Here, we report that NPCs from the embryonic telencephalon grown without FGF2 retain many of their in vivo characteristics, making them a good model for investigating molecular mechanisms involved in cell fate specification and differentiation. However, exposure of cortical NPCs to FGF2 results in a profound change in the types of neurons generated, switching them from a glutamatergic to a GABAergic phenotype. This change closely correlates with the dramatic upregulation of TFs more characteristic of ventral telencephalic NPCs. In addition, exposure of cortical NPCs to FGF2 maintains their neurogenic potential in vitro, and NPCs spontaneously undergo differentiation following FGF2 withdrawal. These results highlight the importance of TFs in determining the types of neurons generated by NPCs in vitro. In addition, they show that FGF2, as well as acting as a mitogen, changes the developmental capabilities of NPCs. These findings have implications for the cell fate specification of in vitro-expanded NPCs and their ability to generate specific cell types for therapeutic applications. Disclosure of potential conflicts of interest is found at the end of this article.

Oxidized alginate hydrogels as niche environments for corneal epithelial cells

Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P