The course will focus on ESC (embryonic stem cell) differentiation as well as reprogramming of somatic cells (MEFs, mouse embryonic fibroblasts) into pluripotent stem cells, in a process known to generate iPS cells and transfection of CRISPR reagents. Students will practice various techniques in cell culture and molecular biology, to study the various processes from the very beginning of preparation of MEFs, ES cells growth and induction towards differentiation, both in vitro and in vivo as well as reprogramming of MEFs.
Establishment of mouse embryonic fibroblasts (MEFS) and embryonic stem (ES) cells:
MEFS will be prepared from mid-term mouse embryos. Blastocysts will be isolated from E3.5 pregnant females and ES colonies will be derived from the blastocysts. Colonies will be passaged onto feeder layers.
Embryonic stem cell differentiation:
ES cell line will be grown on irradiated MEFs. Students will practice preparation of MEFs from embryos. ES cells will undergo differentiation towards various fates, by EB formation following attachment and spreading. EB will be placed on cover slips and students will examine the differentiation to various cell lineages using appropriate antibodies. The antibodies will enable to detect endoderm, mesoderm and ectodermal cells. To gain another validation for the pluripotency, cells will be injected into mice to obtain benign teratomas. Cell lineages in the teratomas will be inspected. Discussions will be held as to the possibilities to enrich and direct towards different differentiation programs of interest.
Reprogramming of somatic cells iPS:
In this part, somatic cells of choice will be MEFs. Cells will be transduced in an appropriate facility (on the main campus) with lentiviruses harboring cDNAs encoding for Oct4, Sox2 and Klf4, together with a fluorescent marker. After ~4 weeks colonies will be isolated and expanded. Individual cell lines will be assayed for stemness markers. Positive clones will be further characterized and their pluripotency will be validated by in vitro and in vivo assays as described above for ES cells.
CRISPR modification of ES cells:
ES cells cells expressing GFP will we transfected by Lipofection with plasmids expressing Cas9 and a guide RNA to EGFP. Cells will be screened for loss of fluorescence.
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The course will grant the students theoretical and practical knowledge in various aspects of stem cell establishment, growth, differentiation and transfection procedures, as well as in the reprogramming of somatic cells to induced pluripotent stem (iPS) cells and their characterization. Students will also be given an introduction to CRISPR technology in ES cells.