Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

Results from a horizon scan on risks associated with transplantation of human organs, tissues and cells: from donor to patient.

The successful transplantation of human materials such as organs, tissues and cells into patients does not only depend on the benefits, but also on the mitigation of risks. To gain insight into recent publications on risks associated with the process of transferring human materials from donor to recipient we performed a horizon scan by reviewing scientific literature and news websites of 2011 on this subject. We found there is ample information on how extended donor criteria, such as donor age, affect the survival rates of organs or patients. Interestingly, gender mismatch does not appear to be a major risk factor in organ rejection. Data on risks of donor tumor transmission was very scarce; however, risk categories for various tumor types have been suggested. In order to avoid rejection, a lot of research is directed towards engineering tissues from a patient's own tissues and cells. Some but not all of these developments have reached the clinic. Developments in the field of stem cell therapy are rapid. However, many hurdles are yet to be overcome before these cells can be applied on a large scale in the clinic. The processes leading to genetic abnormalities in cells differentiated from stem cells need to be identified in order to avoid transplantation of aberrant cells. New insights have been obtained on storage and preservation of human materials, a critical step for success of their clinical use. Likewise, quality management systems have been shown to improve the quality and safety of human materials used for transplantation.

A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA-A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection-induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization/mass spectrometry (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA-peptide complexes, but had similar binding capacities to HLA-A*0201. The most abundant viral peptide species, identified as residues 84-92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 10(5) copies per cell) and was immunogenic in HLA-A2/Kb-transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA-A*0201 binding capacity. Thus, here we report for the first time the direct discovery through microLC-ESI/MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.

A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.

A microcapillary column switching high-performance liquid chromatography (HPLC) system was developed for the separation of major histocompatibility complex (MHC) class I associated peptides. Combination of the column switching system with electrospray ionization mass spectrometry (ESIMS) enabled the detection and identification of the peptides at low-femtomole levels. Sample volumes of 30-50 microL were injected and concentrated onto a short, 100-micron-i.d. precolumn. The precolumn was coupled to a 100-micron-i.d. reversed-phase analytical HPLC column via a six-port valve. Peptides were separated on the analytical column using an ESI-compatible mobile phase at a flow rate of 0.5 microL/min. Peptides were eluted directly into the ESI source of either a magnetic sector MS or an ion trap MS. Peptides associated with human leukocyte antigen A*0201 molecules were determined in immunoaffinity-purified extracts from either measles virus infected cells or uninfected cells by microcapillary column switching HPLC-ESIMS. The approach toward detection of virus-specific peptides we used was based on the comparison of ion chromatograms obtained from the LC-MS analysis of extracts from virally infected cells and their uninfected counterparts. In this way, the molecular mass of peptides unique to virus infected cells was obtained. The utility of the system is demonstrated by the identification of a candidate epitope. Microcapillary column switching HPLC was used along with ESI ion trap tandem MS to identify the naturally processed viral peptide KLWESPQEI. This peptide was found to derive from the measles virus nonstructural protein C. The approach described here provides a versatile and sensitive method for the direct identification of viral peptides associated with MHC class I molecules.

Development of methionine synthase, cystathionine-beta-synthase and S-adenosyl-homocysteine hydrolase during gestation in rats.

The developmental onset of three homocysteine metabolizing enzymes in the rat conceptus was investigated. Cystathionine-beta-synthase and methionine synthase were assayed from day 10 to day 20 of gestation in decidual and placental tissue, from day 10 to day 12 of gestation in embryonic tissue, from day 14 to day 20 of gestation in fetal liver and from day 14 to day 20 of gestation in fetal tissue without liver. On each day, material was obtained from at least four conceptuses from two dams. S-adenosylhomocysteine hydrolase was assayed in neurulating conceptuses in decidual tissue, parietal yolksac plus ectoplacental cone, visceral yolksac plus amnion and embryo proper. Conceptuses were pooled from seven (day 9.5 of gestation) or three (days 10.5 and 11.5 of gestation) dams. In embryonic and fetal tissue cystathionine-beta-synthase first occurred in fetal liver. During the organogenic phase it was present only in decidual tissue. Methionine synthase was present in all tissues from the first gestational day investigated and S-adenosylhomocysteine hydrolase was present in all tissues throughout the neurulating period. Our results indicate that the homocysteine-methionine cycle, which is crucial to transmethylation reactions, is functional during the neurulating period in embryonic tissue. Owing to the absence of cystathionine-beta-synthase at this stage of development in embryonic tissue, the homocysteinyl moiety is conserved in the homocysteine-methionine cycle.

Immunolocalization of HSP 70-related proteins constitutively expressed during Xenopus laevis oogenesis and development.

Using immunocytochemical and biochemical methods, we analyzed the localization of HSP 70-related proteins constitutively expressed during oogenesis and embryogenesis in the amphibian Xenopus laevis. Our results provided evidence for a regional localization in oocytes. In embryos, the regional distribution observed in oocytes was found to be maintained from fertilization up to late blastula. It is noteworthy that, at the beginning of gastrulation, nuclear transfer of such proteins had already occurred by the time of internalization in the involuting marginal zone (IMZ), whereas cells of the vegetal area exhibited only a perinuclear localization of these proteins. These results suggest that HSP 70-related proteins might be involved in the control of the process of cellular internalization.

Comparison between in vivo and in vitro heat-induced changes in amphibian lampbrush chromosomes.

At normal breeding temperature (20 degrees C), amphibian lampbrush chromosomes are characterized by the presence of lateral loops which are related to the transcriptional process. Heat treatment induces changes in these loops, but the nature and timing of these modifications depend on hyperthermic stress conditions. Indeed, our data demonstrate that, at the same high temperature (34 degrees C), lampbrush chromosome modifications induced by in vivo and in vitro gradual heat treatments are different from those induced by in vitro heat shock. In vivo and in vitro heat treatments lead to progressive disorganization of landmark loops, whereas in vitro heat shock results in chromosome condensation. The progressive adaptation of lampbrush chromosome structure in response to gradual heat stress is considered and discussed.

Chromosomes of amphibian oocytes as a model for gene expression: significance of lampbrush loops.

Amphibian lampbrush chromosome loops exhibit morphological variability in their RNP matrix. The biological significance of such variability remains unknown. In order to approach this problem, the structural organization of each RNP matrix type was analyzed in relation to transcriptional and post-transcriptional processes. First, autoradiographic and transcription inhibition studies in conjunction with macromolecular spread analysis revealed a particular transcription pattern in the most typical loops, i.e. the globular loops. This pattern was characterized by asynchronous variations in RNA synthesis in the different transcription units present in a given loop. Second, morphological and experimental studies provided evidence that the typical morphologies of different RNP matrices were interconvertible and that the differences between the different RNP matrices resulted from various degrees of tightness in packaging of transcription products. In particular, analysis of thermic-shock-induced changes in the structure of lampbrush chromosomes enabled us to visualize the progressive disorganization of dense RNP matrices into globular, granular and normal matrices. Furthermore, these studies suggested that changes in post-transcriptional processes might play a determining role in the specific morphology of the loops. In particular, the kinetics of each of these different processes, related to one another and/or proteins specific to one or another of these processes, might determine the morphological appearance of the loops. The immunological approach revealed that specific nuclear proteins might therefore interfere with each of these processes. Third, the problem of a possible relationship between the specific morphologies of lateral loops and the expression of particular DNA sequences was approached at the molecular level.

Effects of in vivo heat treatment on lampbrush chromosome structure in amphibian oocytes.

When Pleurodeles (Amphibian, Urodele) females were subjected to high temperatures (32-35 degrees C) for varying periods of time (45 min to 7 days), lampbrush chromosome structure underwent striking modifications. These changes included a numerical reduction in normal loops and progressive disorganization of RNP matrices of various loops. The degree of such disorganization was a function of the intensity and duration of the stress. These modifications were completely reversible when females or oocytes were returned to a normal breeding temperature (20 degrees C). Results are discussed in comparison with previous studies on morphological changes induced by heat shock in lampbrush chromosomes carried out in vitro.

[Protein composition of the hemolymph of Biomphalaria glabrata (Mollusca, Gastropoda). Influence of the method of sampling and relation to the protein composition of different organs]. / Composition protéique de l'hémolymphe de Biomphalaria glabrata (Mollusque Gastéropode). Influence de la méthode de prélèvement et relation avec la composition protéique des différents organes.

The blood composition of B. glabrata is analyzed by electrophoresis. Its proteic composition differs in relation with the blood sampling method. Most of the supplementary fractions also have been found in the gut, some proteins possibly come from the hepatopancreas, whereas others could have their origin in the albumen gland. The sampling method must be selected according to these observations.

[The relationship between host and parasite in the Crustacean Carcinus parasitized by Sacculina carcini: effect of crude extracts of Sacculina and haemolymph of parasitized Crab upon the proteinogramm of experimented healthy crabs (author's transl)]. / Relations hote-parasite entre les crustacés Carcinus et Sacculina carcini: effet d'extraits de parasite et de l'hémolymphe de crabe infesté sur le protéinogramme de crabes sains.

The injection of crude extracts of Sacculina to healthy crabs induces, after a variable lapse of time, qualitative and quantitative changes in their proteinogramm and a delay in their moulting cycle. These changes are the same as those observed in infested specimens. The injection of haemolymph from parasitized crabs gives similar effects. So, the Sacculina could be able to produce effect from afar upon organs not invaded by the roots of the parasite.

[Host-parasite relation between Carcinus mediterraneus and Sacculina carcini; immunochemical analysis and demonstration of antisacculin precipitin]. / Relation hôte-parasite entre Carcinus mediterraneus et Sacculina carcini; analyse immunochimique et mise en évidence d'une précipitine antisacculine.

The hemolymph from both healthy and parasitised Carcinus mediterraneus are immunochemically analysed. The study confirms the presence in the blood of the infested Crab of a supplementary fraction which was already observed by electrophorsis. Moreover, some infested Crabs develop an anti sacculin precipitin reaction, not visible on healthy animals. The relations between these proteic fractions are discussed.

[Protein composition of hemolymph from the crabs Carcinus mediterraneus Czerniavsky, healthy or parasitized by Sacculina carcini Thompson]. / Composition protéique de l'hémolymphe des crabes Carcinus mediterraneus Czerniavsky, sains ou parasités par Sacculina carcini Thompson

The proteic composition of haemolymph from C. mediterraneus varies during the intermoult cycle. Some proteic fractions are occasionally absent in post-ecdysis. At each intermoult stage, parasitized Crabs have generally the same protein pattern as healthy ones, but the presence of the observed fractions is not constant. Compared healthy Crabs, the sacculinized animals show a surnumerary proteic fraction. With very short delay, the injection of ecdysterone puts the proteic composition of haemolymph from sacculinized Crabs in order, this composition becomes comparable to that from healthy individuals.

[The effect of freezing on the electrophoretic separation of hemolymph proteins of several arthropods]. / Effets de la congélation sur la séparation électrphorétique des protéines de l'hémolymphe de quelques arthropodes

Electrophoresis in 6% or gradient polyacrylamide gel were realised with fresh and stored hemolymphs. The experiments state that there are variations in relation to storage duration, in the separation pattern of hemocyanin fractions in Astacus leptodactylus, Palinurus vulgaris and Carcinus mediterraneus. Moreover, important changements in the supplementary factor of crayfish plasma are revealed.