Harmonisation of education, training and continuing professional development for laboratory animal caretakers, technicians and technologists: Report of the FELASA-EFAT Working Group.

Competent, confident and caring laboratory animal caretakers, technicians and technologists (LAS staff) are vital for good animal welfare, high-quality science and a secure Culture of Care. This requires high-quality education, training, supervision and continuing professional development (CPD) of LAS staff. However, there is a lack of harmonisation regarding how this education and training is conducted among European countries, and nor are there recommendations adapted to Directive 2010/63/EU. Therefore, FELASA and EFAT established a working group with the task of establishing recommendations for education, training and CPD for LAS staff. The working group established five different levels (LAS staff levels 0-4), defining the required level of competence and attitude, as well as suggesting educational requirements for reaching each level. Defining these levels should help to ensure that appropriate educational and CPD activities are in place, and to enable employers and LAS staff to determine the level and career stage attained. Furthermore, proper assessment of competencies and effective CPD schemes for all relevant staff should be established. Regulators should support this by setting standards for competence assessment and ensuring that they are consistently applied. In addition, establishments should involve the LAS staff in defining and developing the Culture of Care. The Animal Welfare Body should be involved and have oversight of education, training and CPD. These recommendations will contribute to harmonisation and increased quality of education, training and CPD, as well as provide clearer career pathways for LAS staff, helping to ensure high standards of animal welfare and science.

Dopamine mobilizes mesenchymal progenitor cells through D2-class receptors and their PI3K/AKT pathway.

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45-CD31-CD34-CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings.

Intra-bone marrow transplantation of human CD34(+) cells into NOD/LtSz-scid IL-2rgamma(null) mice permits multilineage engraftment without previous irradiation.

BACKGROUND AIMS: Non-irradiated immunodeficient recipients provide the best physiologic setting for revealing hematopoietic stem cell (HSC) functions after xenotransplantion. An approach that efficiently permits the detection of human hematopoietic repopulating cells in non-irradiated recipients is therefore highly desired. METHODS: We compared side-by-side the ability to reconstitute hematopoiesis via intra-bone marrow transplantation (IBMT) in three commonly used mouse strains avoiding previous irradiation. RESULTS: Non-irradiated NOD/SCID and NOD/SCID (beta2m-/- mouse strains prevent engraftment even after IBMT. In contrast, combining the robustness of the NOD/SCID IL-2Rgamma-/- recipient with the sensitivity of IBMT facilitates the detection, without previous host irradiation, of human SCID-repopulating cells 10 weeks after transplantation. The level of chimerism averaged 14% and multilineage engraftment (lymphoid dominant) was observed consistently in all mice. Analysis of injected and non-injected bones, spleen and peripheral blood demonstrated that engrafting cells were capable of in vivo migration and expansion. CONCLUSIONS: Combining the robustness of the NOD/SCID IL-2Rgamma-/- mouse strain with the sensitivity of IBMT strongly facilitates long-term multilineage engraftment and migration for human CD34(+) cells without the need for previous irradiation.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II.

A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-beta and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-beta family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-beta and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-beta in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-beta and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Loss of p53 induces tumorigenesis in p21-deficient mesenchymal stem cells.

There is growing evidence about the role of mesenchymal stem cells (MSCs) as cancer stem cells in many sarcomas. Nevertheless, little is still known about the cellular and molecular mechanisms underlying MSCs transformation. We aimed at investigating the role of p53 and p21, two important regulators of the cell cycle progression and apoptosis normally involved in protection against tumorigenesis. Mesenchymal stem cells from wild-type, p21(-/-)p53(+/+), and p21(-/-)p53(+/-) mice were cultured in vitro and analyzed for the appearance of tumoral transformation properties after low, medium, and high number of passages both in vitro and in vivo. Wild-type or p21(-/-)p53(+/+) MSCs did not show any sign of tumoral transformation. Indeed, after short-term in vitro culture, wild-type MSCs became senescent, and p21(-/-)p53(+/+) MSCs showed an elevated spontaneous apoptosis rate. Conversely, MSCs carrying a mutation in one allele of the p53 gene (p21(-/-)p53(+/-) MSCs) completely lost p53 expression after in vitro long-term culture. Loss of p53 was accompanied by a significant increase in the growth rate, gain of karyotypic instability, loss of p16 expression, and lack of senescence response. Finally, these cells were able to form fibrosarcomas partially differentiated into different mesenchymal lineages when injected in immunodeficient mice both after subcutaneous and intrafemoral injection. These findings show that MSCs are very sensitive to mutations in genes involved in cell cycle control and that these deficiencies can be at the origin of some mesodermic tumors.

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

BACKGROUND: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. RESULTS: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. CONCLUSION: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

In vivo site-specific recombination using the beta-rec/six system.

The prokaryotic beta serine recombinase (beta-rec) catalyzes site-specific recombination between two directly oriented six sites (93 bp) in mammalian cells, both in episomal and in chromosomally integrated substrates. The beta-rec/six exclusive intramolecular site-specific recombination (SSR) system has been proposed as a suitable approach when several independently controlled recombination events are needed in a single cell. Here we explored the use of the beta-rec/six system for selective induction of genome-targeted modifications. We generated and analyzed mouse transgenic lines (Tgbeta) expressing beta-rec under the control of the Lck promoter. beta-rec activity was demonstrated, and there was no evidence of alterations to thymic or peripheral T cell development. We developed two transgenic mouse lines harboring different target sequences (Tgrec and KOsix) and analyzed the effect of beta-rec expression on these animals. The results indicate that the beta-rec/six SSR system is functional for in vivo gene-targeting applications.

Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition.

Carcinomas are widely thought to derive from epithelial cells with malignant progression often associated with an epithelial-mesenchymal transition (EMT). We have characterized tumors generated by spontaneously transformed human mesenchymal cells (TMC) previously obtained in our laboratory. Immunohistopathological analyses identified these tumors as poorly differentiated carcinomas, suggesting that a mesenchymal-epithelial transition (MET) was involved in the generation of TMC. This was corroborated by microarray and protein expression analysis that showed that almost all mesenchymal-related genes were severely repressed in these TMC. Interestingly, TMC also expressed embryonic antigens and were able to integrate into developing blastocysts with no signs of tumor formation, suggesting a dedifferentiation process was associated with the mesenchymal stem cell (MSC) transformation. These findings support the hypothesis that some carcinomas are derived from mesenchymal rather than from epithelial precursors.

Molecular characterization of spontaneous mesenchymal stem cell transformation.

BACKGROUND: We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC). METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection. CONCLUSIONS/SIGNIFICANCE: In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.

Refinement of intrathymic injection in mice.

Direct intrathymic injection is a common procedure used in several types of experimental protocols in the mouse. Currently available approaches involve major surgical procedures that expose the thoracic cavity, resulting in an increased risk of poor recovery and postsurgical complications. The authors sought to refine this surgery to reduce animal pain and distress without compromising overall efficiency of the technique. Using a minimally invasive method that does not expose the thoracic cavity, the authors gave accurately placed intrathymic injections, as confirmed by analyses with a reporter dye. They describe this new approach for intrathymic injection in mice that reduces complications associated with lengthy periods of anesthesia and thoracic cavity exposure.

Alteraciones neuroendocrinológicas en bulimia nerviosa / Neuroendocrin alterations in bulimia nervosa

La bulimia nerviosa es un trastorno de la conducta alimentaria que se caracteriza por tener una etiología multidimensional que incluye factores genéticos, biológicos, psicológicos, familiares y socioculturales, cuyas características más importantes son, básicamente, conductas de descontrol alimentario y de purga, alteración del estado de ánimo y anomalías neuroendócrinas. Estas pacientes presentan disfunciones en diferentes sistemas de neurotransmisores, relacionados de forma directa con la modulación del apetito, del humor y de la funciones neuroendócrinas. Dichos trastornos biológicos desempeñan, con toda probabilidad, un importante papel, y, junto con otros factores de tipo psicosocial y de personalidad que contribuyen al desarrollo y mantenimiento del "complejo sintomático" bulimia nerviosa. La bulimia nerviosa se caracteriza, pues, esencialmente por: a) pérdida de control sobre la conducta alimentaria de la que derivan. a.1) los episodios de ingesta voraz y consumo de una gran cantidad de comida en corto espacio de tiempo, seguida de a.2) conductas compensatorias para evitar el aumento de peso: el ayuno, vómitos autoinducidos, abuso de laxantes y diuréticos, ejercicio físico exagerado. b) alteraciones de humor y c) alteraciones neuroendócrinas (a las cuales haremos referencia más adelante)

Alteraciones neuroendocrinológicas en bulimia nerviosa / Neuroendocrin alterations in bulimia nervosa

La bulimia nerviosa es un trastorno de la conducta alimentaria que se caracteriza por tener una etiología multidimensional que incluye factores genéticos, biológicos, psicológicos, familiares y socioculturales, cuyas características más importantes son, básicamente, conductas de descontrol alimentario y de purga, alteración del estado de ánimo y anomalías neuroendócrinas. Estas pacientes presentan disfunciones en diferentes sistemas de neurotransmisores, relacionados de forma directa con la modulación del apetito, del humor y de la funciones neuroendócrinas. Dichos trastornos biológicos desempeñan, con toda probabilidad, un importante papel, y, junto con otros factores de tipo psicosocial y de personalidad que contribuyen al desarrollo y mantenimiento del "complejo sintomático" bulimia nerviosa. La bulimia nerviosa se caracteriza, pues, esencialmente por: a) pérdida de control sobre la conducta alimentaria de la que derivan. a.1) los episodios de ingesta voraz y consumo de una gran cantidad de comida en corto espacio de tiempo, seguida de a.2) conductas compensatorias para evitar el aumento de peso: el ayuno, vómitos autoinducidos, abuso de laxantes y diuréticos, ejercicio físico exagerado. b) alteraciones de humor y c) alteraciones neuroendócrinas (a las cuales haremos referencia más adelante) (AU)