Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and could also contribute to the accumulation of more toxicity data on compounds to be registered according to the criteria established by the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) programme.

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX-1 and markers of pluripotency.

UCB (human umbilical cord blood) contains cells able to differentiate into non-haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic-like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM-CSF (granulocyte-macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT-PCR and immunocytochemistry. The results show a dramatic expansion (>150-fold) of haematopoietic progenitors (CD45+/CD133+) which also expressed embryo markers of pluripotency (nanog, kfl-4, sox-2, oct-3/4 and c-myc), nestin, and pancreatic markers such as pax-4, ngn-3, pdx-1 and syt-1 (that is regulated by pdx-1 and provides the cells with a Ca++ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.

Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

Microbiological risk assessment in stem cell manipulation.

Cell therapy based on the use of human stem cells is more complicated than transfusion or organ transplantation because cells may undergo many additional manipulations due to different treatments for isolation, expansion, differentiation, and other types of biological changes. These manipulations require the approval of regulatory agencies (other than ethical) and the processes must be monitored with more tests than the ones applied for minimally manipulated cells. The clinical safety and efficacy of transplanted cells depend on several factors such as homologous or non-homologous sources, extent of manipulation, and culture conditions. Moreover, the kind of information needed to address these issues may differ depending on whether the cells are to be used for tissue reconstruction or repair, or to recover metabolic functions. Also anatomical site, functional integration as well as duration of therapy, are crucial points that indirectly can influence safety. Many important assays have been suggested for environmental monitoring as well as to standardize microbiological controls in stem cell banks to prevent contamination. In order to guarantee safety two main aspects must be considered: one is related to the source of cells (the donor) and the other is depending on cell collection and processing. In this review we critically analyze the steps of the processes (from collection to banking) and consider the main factors involved in the clinical research (continuously in evolution) by suggesting a standardized facsimile form to use in the laboratory for the assessment of the microbiological risk related to the cell manipulations.