A randomized double-blind control study of early intra-coronary autologous bone marrow cell infusion in acute myocardial infarction: the REGENERATE-AMI clinical trial?.

AIMS: Clinical trials suggest that intracoronary delivery of autologous bone marrow-derived cells (BMCs) 1-7 days post-acute myocardial infarction (AMI) may improve left ventricular (LV) function. Earlier time points have not been evaluated. We sought to determine the effect of intracoronary autologous BMC on LV function when delivered within 24 h of successful reperfusion therapy. METHODS AND RESULTS: A multi-centre phase II randomized, double-blind, and placebo-controlled trial. One hundred patients with anterior AMI and significant regional wall motion abnormality were randomized to receive either intracoronary infusion of BMC or placebo (1:1) within 24 h of successful primary percutaneous intervention (PPCI). The primary endpoint was the change in left ventricular ejection fraction (LVEF) between baseline and 1 year as determined by advanced cardiac imaging. At 1 year, although LVEF increased compared with baseline in both groups, the between-group difference favouring BMC was small (2.2%; 95% confidence interval, CI: -0.5 to 5.0; P = 0.10). However, there was a significantly greater myocardial salvage index in the BMC-treated group compared with placebo (0.1%; 95% CI: 0.0-0.20; P = 0.048). Major adverse events were rare in both treatment groups. CONCLUSION: The early infusion of intracoronary BMC following PPCI for patients with AMI and regional wall motion abnormality leads to a small non-significant improvement in LVEF when compared with placebo; however, it may play an important role in infarct remodelling and myocardial salvage. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov NCT00765453 and EudraCT 2007-002144-16.

Plasticity of fetal cartilaginous cells.

Tissue-specific stem cells found in adult tissues can participate in the repair process following injury. However, adult tissues, such as articular cartilage and intervertebral disc, have low regeneration capacity, whereas fetal tissues, such as articular cartilage, show high regeneration ability. The presence of fetal stem cells in fetal cartilaginous tissues and their involvement in the regeneration of fetal cartilage is unknown. The aim of the study was to assess the chondrogenic differentiation and the plasticity of fetal cartilaginous cells. We compared the TGF-β3-induced chondrogenic differentiation of human fetal cells isolated from spine and cartilage tissues to that of human bone marrow stromal cells (BMSC). Stem cell surface markers and adipogenic and osteogenic plasticity of the two fetal cell types were also assessed. TGF-β3 stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens, and variable levels of type X collagen. Although fetal cells showed the same pattern of surface stem cell markers as BMSCs, both type of fetal cells had lower adipogenic and osteogenic differentiation capacity than BMSCs. Fetal cells from femoral head showed higher adipogenic differentiation than fetal cells from spine. These results show that fetal cells are already differentiated cells and may be a good compromise between stem cells and adult tissue cells for a cell-based therapy.

Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts.

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on freshand fresh-frozenbone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Functionalization of microstructured open-porous bioceramic scaffolds with human fetal bone cells.

Bone substitute materials allowing trans-scaffold migration and in-scaffold survival of human bone-derived cells are mandatory for development of cell-engineered permanent implants to repair bone defects. In this study, we evaluated the influence on human bone-derived cells of the material composition and microstructure of foam scaffolds of calcium aluminate. The scaffolds were prepared using a direct foaming method allowing wide-range tailoring of the microstructure for pore size and pore openings. Human fetal osteoblasts (osteo-progenitors) attached to the scaffolds, migrated across the entire bioceramic depending on the scaffold pore size, colonized, and survived in the porous material for at least 6 weeks. The long-term biocompatibility of the scaffold material for human bone-derived cells was evidenced by in-scaffold determination of cell metabolic activity using a modified MTT assay, a repeated WST-1 assay, and scanning electron microscopy. Finally, we demonstrated that the osteo-progenitors can be covalently bound to the scaffolds using biocompatible click chemistry, thus enhancing the rapid adhesion of the cells to the scaffolds. Therefore, the different microstructures of the foams influenced the migratory potential of the cells, but not cell viability. Scaffolds allow covalent biocompatible chemical binding of the cells to the materials, either localized or widespread integration of the scaffolds for cell-engineered implants.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future.

Stimulation of erythropoiesis is one of the most efficient ways of doping. This type of doping is advantageous for aerobic physical exercise and of particular interest to endurance athletes. Erythropoiesis, which takes place in bone marrow, is under the control of EPO, a hormone secreted primarily by the kidneys when the arterial oxygen tension decreases. In certain pathological disorders, such as chronic renal failure, the production of EPO is insufficient and results in anemia. The pharmaceutical industry has, thus, been very interested in developing drugs that stimulate erythropoiesis. With this aim, various strategies have been, and continue to be, envisaged, giving rise to an expanding range of drugs that are good candidates for doping. Anti-doping control has had to deal with this situation by developing appropriate methods for their detection. This article presents an overview of both the drugs and the corresponding methods of detection, and thus follows a roughly chronological order.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Identification by a monoclonal antibody of a glycolipid highly expressed by cells from the human myeloid lineage.

A monoclonal antibody (MAb) HL-C5, which bound selectively to cells of the myeloid lineage tested, was derived from a fusion between P3/NS2/1-AG8 myeloma cells and splenocytes from a mouse immunized with cells of the promyelocytic leukemia line HL-60. Among a panel of 29 human cell lines derived from either hematopoietic or solid tumors, MAb HL-C5 was found to react exclusively with cells from the five differentiated acute myeloid leukemia lines, HL-60, ML1, ML2, ML3, KG-1B and not with the less differentiated myeloid lines. Fluorescence-activated cell sorter analysis of normal bone marrow samples confirmed that the reactivity of MAb HL-C5 was limited to myeloid cells, from the promyelocytic stage of differentiation to the mature granulocytes. Indirect immunoperoxidase staining of cytocentrifuge preparations of normal bone marrow and peripheral blood leukocytes confirmed these results and showed that MAb HL-C5 stained neutrophils but not eosinophils or basophils. The antigen recognized by HL-C5 was recovered in the upper phase of chloroform-methanol-water lipid extracts prepared from HL-60 cells. By competitive binding experiments, it was found that MAb HL-C5 recognizes the same antigenic determinant as MAb WGHS 29-1, which has been reported to react with glycolipids containing the sugar sequence lacto-N-fucopentaose 111. Autoradiographs of thin layer chromatograms of HL-60 glycolipid extracts which were revealed by incubation with MAb HL-C5 or WGHS 29-1 followed by the addition of 125I-labelled rabbit anti-mouse immunoglobulin antibody confirmed that the two MAbs reacted with the same or structurally very similar glycolipids.

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model.

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.

Establishment of models to study mouse and human retinogenesis "in vitro" : isolation and characterization of retinal stem cells

SUMMARY IN FRENCH Les cellules souches sont des cellules indifférenciées capables a) de proliférer, b) de s'auto¬renouveller, c) de produire des cellules différenciées, postmitotiques et fonctionnelles (multipotencialité), et d) de régénérer le tissu après des lésions. Par exemple, les cellules de souches hematopoiétiques, situées dans la moelle osseuse, peuvent s'amplifier, se diviser et produire diverses cellules différenciées au cours de la vie, les cellules souches restant dans la moelle osseuse et consentant leur propriété. Les cellules souches intestinales, situées dans la crypte des microvillosités peuvent également régénérer tout l'intestin au cours de la vie. La rétine se compose de six classes de neurones et d'un type de cellule gliale. Tous ces types de cellules sont produits par un progéniteur rétinien. Le pic de production des photorécepteurs se situe autour des premiers jours postnatals chez la souris. A cette période la rétine contient les cellules hautement prolifératives. Dans cette étude, nous avons voulu analyser le phénotype de ces cellules et leur potentiel en tant que cellules souches ou progénitrices. Nous nous sommes également concentrés sur l'effet de certains facteurs épigéniques sur leur destin cellulaire. Nous avons observé que toutes les cellules prolifératives isolées à partir de neurorétines postnatales de souris expriment le marqueur de glie radiaire RC2, ainsi que des facteurs de transcription habituellement trouvés dans la glie radiaire (Mash1, Pax6), et répondent aux critères des cellules souches : une capacité élevée d'expansion, un état indifférencié, la multipotencialité (démontrée par analyse clonale). Nous avons étudié la différentiation des cellules dans différents milieux de culture. En l'absence de sérum, l'EGF induit l'expression de la β-tubulin-III, un marqueur neuronal, et l'acquisition d'une morphologie neuronale, ceci dans 15% des cellules présentes. Nous avons également analysé la prolifération de cellules. Seulement 20% des cellules incorporent le bromodéoxyuridine (BrdU) qui est un marqueur de division cellulaire. Ceci démontre que l'EGF induit la formation des neurones sans une progression massive du cycle cellulaire. Par ailleurs, une stimulation de 2h d'EGF est suffisante pour induire la différentiation neuronale. Certains des neurones formés sont des cellules ganglionnaires rétiniennes (GR), comme l'indique l'expression de marqueurs de cellules ganglionnaires (Ath5, Brn3b et mélanopsine), et dans de rare cas d'autres neurones rétiniens ont été observés (photorécepteurs (PR) et cellules bipolaires). Nous avons confirmé que les cellules souches rétiniennes tardives n'étaient pas restreintes au cours du temps et qu'elles conservent leur multipotencialité en étant capables de générer des neurones dits précoces (GR) ou tardifs (PR). Nos résultats prouvent que l'EGF est non seulement un facteur contrôlant le développement glial, comme précédemment démontré, mais également un facteur efficace de différentiation pour les neurones rétiniens, du moins in vitro. D'autre part, nous avons voulu établir si l'oeil adulte humain contient des cellules souches rétiniennes (CSRs). L'oeil de certains poissons ou amphibiens continue de croître pendant l'âge adulte du fait de l'activité persistante des cellules souches rétiniennes. Chez les poissons, le CSRs se situe dans la marge ciliaire (CM) à la périphérie de la rétine. Bien que l'oeil des mammifères ne se développe plus pendant la vie d'adulte, plusieurs groupes ont prouvé que l'oeil de mammifères adultes contient des cellules souches rétiniennes également dans la marge ciliaire plus précisément dans l'épithélium pigmenté et non dans la neurorétine. Ces CSRs répondent à certains critères des cellules souches. Nous avons identifié et caractérisé les cellules souches rétiniennes résidant dans l'oeil adulte humain. Nous avons prouvé qu'elles partagent les mêmes propriétés que leurs homologues chez les rongeurs c.-à-d. auto-renouvellement, amplification, et différenciation en neurones rétiniens in vitro et in vivo (démontré par immunocoloration et microarray). D'autre part, ces cellules peuvent être considérablement amplifiées, tout en conservant leur potentiel de cellules souches, comme indiqué par l'analyse de leur profil d'expression génique (microarray). Elles expriment également des gènes communs à diverses cellules souches: nucleostemin, nestin, Brni1, Notch2, ABCG2, c-kit et son ligand, aussi bien que cyclin D3 qui agit en aval de c-kit. Nous avons pu montré que Bmi1et Oct4 sont nécessaires pour la prolifération des CSRs confortant leur propriété de cellules souches. Nos données indiquent que la neurorétine postnatale chez la souris et l'épithélium pigmenté de la marge ciliaire chez l'humain adulte contiennent les cellules souches rétiniennes. En outre, nous avons développé un système qui permet d'amplifier et de cultiver facilement les CSRs. Ce modèle permet de disséquer les mécanismes impliqués lors de la retinogenèse. Par exemple, ce système peut être employé pour l'étude des substances ou des facteurs impliqués, par exemple, dans la survie ou dans la génération des cellules rétiniennes. Il peut également aider à disséquer la fonction de gènes ou les facteurs impliqués dans la restriction ou la spécification du destin cellulaire. En outre, dans les pays occidentaux, la rétinite pigmentaire (RP) touche 1 individu sur 3500 et la dégénérescence maculaire liée à l'âge (DMLA) affecte 1 % à 3% de la population âgée de plus de 60 ans. La génération in vitro de cellules rétiniennes est aussi un outil prometteur pour fournir une source illimitée de cellules pour l'étude de transplantation cellulaire pour la rétine. SUMMARY IN ENGLISH Stem cells are defined as undifferentiated cells capable of a) proliferation, b) self maintenance (self-renewability), c) production of many differentiated functional postmitotic cells (multipotency), and d) regenerating tissue after injury. For instance, hematopoietic stem cells, located in bone marrow, can expand, divide and generate differentiated cells into the diverse lineages throughout life, the stem cells conserving their status. In the villi crypt, the intestinal stem cells are also able to regenerate the intestine during their life time. The retina is composed of six classes of neurons and one glial cell. All these cell types are produced by the retinal progenitor cell. The peak of photoreceptor production is reached around the first postnatal days in rodents. Thus, at this stage the retina contains highly proliferative cells. In our research, we analyzed the phenotype of these cells and their potential as possible progenitor or stem cells. We also focused on the effect of epigenic factor(s) and cell fate determination. All the proliferating cells isolated from mice postnatal neuroretina harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash 1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after the transfer of the cells in different culture media. In the absence of serum, EGF led to the expression of the neuronal marker β-tubulin-III, and the acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating massively cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro. On the other hand, we wanted to find out if the adult human eye contains retina stem cells. The eye of some fishes and amphibians continues to grow during adulthood due to the persistent activity of retinal stem cells (RSCs). In fish, the RSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina. Although, the adult mammalian eye does not grow during adult life, several groups have shown that the adult mouse eye contains retinal stem cells in the homologous zone (i.e. the ciliary margin), in the pigmented epithelium and not in the neuroretina. These RSCs meet some criteria of stem cells. We identified and characterized the human retinal stem cells. We showed that they posses the same features as their rodent counterpart i.e. they self-renew, expand and differentiate into retinal neurons in vitro and in vivo (indicated by immunostaining and microarray analysis). Moreover, they can be greatly expanded while conserving their sternness potential as revealed by the gene expression profile analysis (microarray approach). They also expressed genes common to various stem cells: nucleostemin, nestin, Bmil , Notch2, ABCG2, c-kit and its ligand, as well as cyclin D3 which acts downstream of c-kit. Furthermore, Bmil and Oct-4 were required for RSC proliferation reinforcing their stem cell identity. Our data indicate that the mice postnatal neuroretina and the adult pigmented epithelium of adult human ciliary margin contain retinal stem cells. We developed a system to easily expand and culture RSCs that can be used to investigate the retinogenesis. For example, it can help to screen drugs or factors involved, for instance, in the survival or generation of retinal cells. This could help to dissect genes or factors involved in the restriction or specification of retinal cell fate. In Western countries, retinitis pigmentosa (RP) affects 1 out of 3'500 individuals and age-related macula degeneration (AMD) strikes 1 % to 3% of the population over 60. In vitro generation of retinal cells is thus a promising tool to provide an unlimited cell source for cellular transplantation studies in the retina.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay.

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments.

Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation. The results demonstrate the selective destruction of established human colon carcinoma transplants by intravenous injection of either single or fractionated doses of 131I-MAb F(ab')2.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.