RESUMO
Mouse embryonic carcinoma cells (P19 line) were studied for both their survival and developmental potential in the intact cerebellum of B6CBA mice. The P19 cells were cultured and labelled with green fluorescent protein using transfection. Cells were used for transplantation either in the undifferentiated stage or after 3 days of neurodifferentiation induced by retinoic acid. The intracerebellar application was performed in 43 mice: group A (N = 21) received neuroprogenitors and group B (N = 22) received undifferentiated cells. The morphology of transplanted cells within the context of the surrounding cerebellar tissue was evaluated after 3 weeks. Naive P19 cells engrafted and survived in the cerebellum of 7 of the 22 adult mice (survival rate 31.8 %). Neuroprogenitors survived in 13 of the 21 mice (survival rate was 61.9 %). Since the cut-off is P < 0.05, the difference is not statistically significant (P = 0.069). An expansive appearance of the graft was significantly more frequent (P = 0.0047) in naive P19 cells than in neuroprogenitors. In mice in which the grafts did not survive, no marks of grafted cells or only fluorescing detritus were found. In conclusion, this is the first study to track the fate and morphology of embryonic carcinoma cells transplanted into the cerebellum, confirming that neuroprogenitors derived from embryonic carcinoma cells can settle in the host tissue and differentiate according to the surrounding conditions. With further validation, the embryonic carcinoma cells could become a valuable model with which to study the impact of cell therapy on neurodegenerative diseases.
Assuntos
Sobrevivência Celular/fisiologia , Cerebelo/citologia , Neurônios/citologia , Células-Tronco/citologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Transplante de Células , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-TroncoRESUMO
The aim of our study was to characterize mouse embryonal carcinoma (EC) cells P19 in different stages of retinoic acid induced neurodifferentiation by two methods, immunocytochemistry and RT qPCR. The characterization of the cells is crucial before any transplantation into any model, e.g. in our case into the mouse brain with the aim to treat a neurodegenerative disease. Specific protein markers (MAP-2, OCT-4, FORSE-1) were detected by immunocytochemistry in the cell cultures. The mRNA expression levels of PAX-6, MASH-1, Brachyury, GATA-4 and AFP were determined by RT qPCR method. HPRT was used as a housekeeping gene. The degree of differentiation can be characterized by expression of analyzed genes. The presence of OCT-4 and FORSE-1 proteins in undifferentiated pluripotent cells and the presence of dendrite specific MAP-2 in neuroprogenitors was detected. The expression levels of PAX-6 and MASH-1 increased and expression of Brachyury decreased during the neurodifferentiation process. The expression levels of GATA-4 and AFP were the highest after induction of differentiation with retinoic acid. Detailed characterization of cells before transplantation experiments can contribute to better understanding of their effect.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Tretinoína/farmacologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Células-Tronco de Carcinoma Embrionário/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The frequency of functionally relevant mutations of the leukaemia inhibitory factor (LIF) gene in infertile women is significantly enhanced in comparison with fertile controls. The objective of this retrospective cohort study was to evaluate the impact of LIF gene mutations on the outcome of the treatment in women with various causes of infertility. Fifteen infertile women with the G to A transition at position 3400 leading to the valine to methionine exchange at codon 64 were analysed. Group A was made up of women with diagnoses that are frequently accompanied by changes in humoral as well as cell-mediated immunity - idiopathic infertility and endometriosis (N = 7). Group B consisted of patients with polycystic ovary syndrome (PCOS), andrological factor, tubal factor and hyperprolactinaemia (N = 8). The control group comprised 136 infertile women with no LIF gene mutation diagnosed with idiopathic infertility and endometriosis (N = 37) (group C) and patients with PCOS, tubal and andrological factor (N = 99) (group D). Seven of the mutation-positive patients were successfully treated by in vitro fertilization (IVF), but nobody in this group was diagnosed with idiopathic infertility and only one with endometriosis, which means that there is a statistically significant difference in the pregnancy rates between groups A and B (P = 0.01, Fisher's 2 by 2 exact test) but no statistically significant difference when comparing patients with the LIF gene mutation (group A+B) to no LIF gene mutation (group C+D). The results suggest that in mutation-positive women the idiopathic infertility and endometriosis have a negative impact on the outcome of IVF treatment.