DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT

Embryonic stem (ES) cells, the undifferentiated cells of early embryos are established as permanent lines and are characterised by their self-renewal capacity and the ability to retain their developmental capacity in vivo and in vitro. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains with inactivated genes by homologous recombination. There are several methods to induce the formation of EBs. One of them the formation by aggregating ES cells in hanging drops, using gravity as an aggregation force. This method presents the advantage of obtaining well-calibrated EBs almost identical in size. We used at our experiment the mouse ES cell line KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical examination was aimed to identify tissue-restricted proteins for the two differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A (TROMA) for the presence of mesenchymal progenitor cells, Oct-4 for the presence of the undifferentiated ES cells. The beating cardiac muscle clumps showed more synchronous rhythm than those seen in EBs obtained from suspension culture method, where the beating cardiac muscle clumps appeared later, had a lower frequency and were uneven. The synaptic networks of neuronal cells were best developed in EBs from suspension, compared to those observed in EBs from hanging-drop method

Pluripotent stem cells in rabbits

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MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989), but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass), it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1) mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP), because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air). The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females

Examination the expression pattern of HSP70 heat shock protein in chicken PGCs and developing genital ridge

Chicken Primordial Germ cells (PGCs) are emerging pioneers in the field of applied embryology and stem cell technology. Now-a-days transgenic chickens are promising models to study human disease pathophysiology and drug designing. However, most of the molecular mechanism, which govern the stemness and pluripotency of chicken PGCs, not known in details. Recent studies have indicated the role of HSP70 in early embryonic development in many vertebrate species. Exposure of chicken to heat stress result in activation of heat shock factors which activate the transcription of HSP70. Exposure chicken eggs to acute heat stress effects HSP70 expression in PGCs and gonads. HSP70 helps in maintaining the integrity of chicken PGCs. A new emerging role of HSP70 in apoptosis has emerged. In our lab, we aim to characterize the expression of cHsp70 in chicken PGCs and gonads during embryonic development by subjecting the parents to acute levels of heat stress. Chickens whose parents subjected to heat stress showed varied expression of cHsp70 and also improved thermo tolerance. In the future we plan to study other factors and miRNAs, which is characterized as an emerging player in regulating heat shock protein response in chicken and also plays an important role in apoptosis

TRIAL TO CULTIVATE AND ISOLATE NEURONAL LIKE CELLS FROM 7 DAYS OLD MICE BRAIN

role to replace lost cells due to physiological turnover, injury, or disease and tosupport cell genesis contributing to the cell number homeostasis. Long time it wasthought that adult mammalian central nervous system doesn't possess any or fewregenerative capacity. Nowadays it was demonstrated that also in the brain thereare stem cells which have the capacity to differentiate into astocytes,oligodendrocytes and neurons.In few degenerative diseases the stem cells lose the regenerative capacity withconsequences in diminishing and loss of functional capacity. Stem cell therapyrepresents a novel and promising therapeutic approach to treatment of a variety ofdegenerative disease as multiple sclerosis. For this it is necessary that a efficientstem cell source can be found and secondary to be proven that these transplantablecells have differential potential into neural tissue.In order to be able to possess a stem cell source capable to build an implant it isnecessary to know the cultivation technology and also the instruments to prove theircapacity to differentiate into specific cells of the nervous system. These were themotives that enabled us to to try to harvest, cultivate and differentiate stem cellsfrom the murine central nervous system

PLURIPOTENT STEM CELLS FROM THE ADULT MOUSE UTRICLE

Researchers discovered that cochlear epithelia in mice especially the vestibular one, contains stem cells that have the capacity to differentiate in sensorial auditory hair cell progenitors specific to the organ. They are reduced in number as the animal progresses in age. This process leads to a loss in the regenerative and proliferative potential of sensorial inner ear epithelia secondary to different injuries. Isolation, cultivation and than in vitro differentiation of vestibular stem cells could become a regenerative implant for acquired hearing loss. These were the motives that determined us to try to isolate, cultivate and finally differentiate vestibular stem cells from vestibular epithelia.Utricles from 7 days old mice NMRI were harvested, the otolites were removed, the utricles were trypsinized in order to isolate cells. Obtained cells were cultivated at 37ºC and 5% CO2 in DMEM with F12 Nutrient mixture, B27, N2 supplement. Pluripotency of obtained spheres was established with the help of stem cell markers Nanog and Oct-4. For identification of progenitor cells we used the marker, which reveals the gene which encodes the protein nestin. In all experiments we obtained floating colonies called spheres, formed by mitotic multiplying. For testing the pluripotency of spheres we used Nanog and Oct-4, two transcription factors that are expressed at high levels in stem cells and that we found to be expressed in our spheres. The presence of nestin mRNA in cells composing the spheres showed that these progressed to a progenitor cell stage.We concluded that utricular epithelia in 7 days old mice contains sufficient stem cells that can be cultivated and that can be later differentiated

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits

Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs

Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency