No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.

No description available.

No description available.

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.

Development of safe and efficient approaches for gene delivery in human embryonic stem cells (hESc) and particularly in human induced pluripotent stem (hiPS) cells, which can be derived in a person-specific manner, is considered to be imperative for harnessing their full potential in both the basic and applied research. The aim of this study was to evaluate the potential of human artificial chromosome (HAC) for gene delivery and expression in hESc and hiPS cells. HAC offers many potential advantages including the provision for carrying large genes with corresponding regulatory elements to obtain long-term regulated gene expression. In addition, they can replicate and segregate independently without integration into the host cell genome. To develop HAC in hiPS cells, the first part of the study was aimed at generating hiPS cells utilising the Herpes Simplex Virus (HSV)-1 amplicon system. With the use of EBNA-1/OriP retention elements incorporated into the HSV-1 amplicon vectors, hiPS cells completely free of vector and transgenes sequences were successfully derived from human embryonic fibroblasts. The hiPS cells exhibited proliferation and differentiation potential similar to that of hESc. In the second part of the study, development of HAC in hESc and hiPS cells was assessed by utilising the HSV-1 amplicon system to deliver the HAC DNA. Analysis of the hESc confirmed the presence of functional HAC which replicated the behaviour of the host chromosomes. Additionally, HAC generation did not lead to impairment in the developmental potential and pluripotency of hESc. The hiPS cells supported HAC at low frequency but DNA also integrated into the host chromosomes. The HAC system, therefore, needs further refinements to improve the frequency of HAC formation and reduce the chromosomal integration of HAC constructs in hiPS cells. Overall, these findings provide a simple and safe way of pluripotency induction and genetic modification of pluripotent stem cells using the HSV-1 amplicon system and represent an important advance towards patient specific gene and cell therapy.

"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maîtrise en droit (L.L.M.) Option recherche" / La découverte des cellules souches embryonnaires et de leur immense potentiel thérapeutique a fait naître de grands espoirs. De nouvelles thérapies révolutionnaires pour traiter certaines des maladies les plus graves dont souffre l'humanité sont désormais envisageables. Le traitement de la vie à son stade le plus précoce est mis en cause. Le statut juridique reconnu au foetus et à l'embryon humain a des répercussions directes sur le domaine de la recherche et sur leur utilisation à des fins thérapeutiques. Nous avons examiné l'état du droit canadien quant au statut juridique du foetus et de l'embryon. De cette étude, nous avons constaté l'incertitude qui prévaut au Canada quant à leur statut. Par la suite, nous avons étudié les différentes normes canadiennes établies pour encadrer l'utilisation des cellules souches embryonnaires à des fins thérapeutiques et nous les avons analysées et comparées pour faire ressortir leurs similitudes et leurs différences. II est ressorti de notre analyse que les textes canadiens se rejoignent généralement sur l'essentiel et qu'il y a eu peu de changements de 1993 à aujourd'hui, en regard des activités de recherche interdites au Canada. Puis, nous avons examiné les systèmes normatifs applicables à ces recherches à l'étranger, soit aux États-Unis et en GrandeBretagne. Nous avons effectué une analyse comparative des trois systèmes normatifs étudiés, en évaluant différents paramètres communs à ces systèmes. Il est ressorti de cette analyse, que la Grande-Bretagne est le pays le plus libéral relativement à ces domaines de recherche, que les États-Unis sont les plus conservateurs sur ces questions et que le Canada se situe entre les deux. / The embryonic stem cells discovery and the immense therapeutic potential glven to them has created big hopes in the world of today. The appearance of new revolutionary therapies to treat sorne of the most serious known diseases are now conceivable. However, the treatment of life to its earliest stage is questionned. The legal status recognized to the foetus and the embryo has, in fact, a direct effect to the research area and industry as weil as to its therapeutic use. Therefore, we have examined and studied the CUITent canadian law with respect to the legal status of the foetus and embryo. Following this study, we have noticed the uncertainty that prevails in Canada concerning the said legal status. Afierwards, we have examined ail the different canadian norms and regulations already established regarding the use of embryonic stem cells for therapeutic ends. We also did the comparaison between those norms and regulations so as to see their differences and similarities. It appears from our analysis that ail the canadian litterature generally treat the subject in the same way and that there have been few changes from 1993 up until now with respect to the forbidden researchs activities in Canada. We also have analysed the foreign law standards and regulations in United States and Great Britain concerning those forbidden researchs activities. We did the exercise of comparing the state of the law in these three countries with different parameters. It emerges from that that Great Britain is the most liberal country, United States being the most conservative and Canada being in between them.

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. / Embryonic stem (ES) cells carry high hopes for biomedical research in order to provide definitive treatment for osteoarthritis. The horse is considered to be an important animal model for examining osteoarthritis treatments. However, despite almost thirty years of research, authentic equine ES (eES) cells have not yet been derived. In this context, the main objective of this study was to derive eES cell lines. The first objective of our study was to optimize the technique for deriving eES cells. We show that different feeder cell lines and embryo development stages influence the effectiveness of this technique while the use of cell signalling inhibitors does not influence eES cell derivation. The second objective was to characterize markers of pluripotency and differentiation in eES cell lines by RT-PCR. We demonstrate that the eES cells express both markers associated with pluripotent cells and differentiated cells and that the presence of cell signalling inhibitors in the culture medium does not influence the expression of these markers. In conclusion, we confirm having derived eES-like cells but these do not meet all the molecular criteria of authentic ES cells.