Course Identification

Tissue culture experience is highly recommended

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.

Modification of ES cells:
ES cells  will be transfected by Lipofection with plasmids expressing  EGFP. Cells will be screened for 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.

	02.01.22	03.01.22	04.01.22	05.01.22	06.01.22	13.01.22
	SUNDAY	MONDAY	TUESDAY	WEDNESDAY	THURSDAY	THURSDAY
9:00:-10:30 AM	Lecture		Change medium on lipofection		check  transfection for GFP
			Preparing: RNA, cDNA and RT-PCR			Immunofluorescence staining of the in vitro differentiation assay
10:30-11:30	Make media (MEF, 2i)				Establishing ES lines from blastocysts
	Thaw MEFs - 6 well, 96 well	MEF PREP	Plates for RT PCR	iPS clones isolation - Stem Cell Unit
	Change media R1
11:30-12:30	Lecture		Lecture	Seeding EBs on cover slips		Lecture
	Collecting pellets from
	ES, iPS-full, iPS-partial, MEFs					Check blastocysts for ICM expansion
12:30-13:30	LUNCH
13:30-14:30	ES induction to differentiation- plating for EB	Lecture		Lecture	Lecture
14:30-15:30	Cardiac cells	Lipofection
	differentiation
	Plate R1to 6 well  for lipofection
		Transfection of ESCs	Isolation of Mouse Embryonic Fibroblasts (MEFs)	Establishing ES lines from blastocysts		Induction to differentiation	Characterization of iPSC
05.01.20	SUNDAY am	Thaw and plate iMEFs on 6 well plates		Thaw and plate iMEFs on 96 well plates		Plating for EBs
	SUNDAY pm	Thaw and plate R1 ESCs on feeder layer					RT preparations
06.01.20	MONDAY am		Preparation of MEFs from embryos
	MONDAY pm	Lipofection with GFP construct
07.01.20	TUESDAY am	Change medium on lipofection					RT - PCR
	TUESDAY pm
08.01.20	WEDNESDAY am				iPS clones isolation	Seeding EBs on cover-slips
	WEDNESDAY pm
09.01.20	THURSDAY am	Check Transfection for GFP expression	Examination of MEF plates	Flush blastocysts from mouse uterus and plate
	THURSDAY pm
……	…….
16.01.20	THURSDAY am			Transfer expanded ICM to 48 well plate with feeders		Immunostainings
	THURSDAY pm

Upon successful completion of this course students should be able to:

1. Demonstrate theoretical and practical knowledge in various aspects of stem cell growth and differentiation procedures, as well as in the reprogramming of somatic cells to induced pluripotent stem (iPS) cells and their characterization.
2. Practice establishment and maintenance of embryonic stem (ES) cells in culture, including preparation of the feeder layer required for their growth.
3. Establish iPS cells from mouse fibroblasts, and characterize them by RT-PCR and Alkaline-phosphatase assays.
4. Identify the fully reprogrammed clones from the general heterogeneous population.
5. Carry out in vitro and in vivo differentiation assays to examine the pluripotency of the cells, and will learn to distinguish between ES and iPS cells.
6. Apply knowledge in tissue culture and molecular biology techniques in the research of ES cell differentiation programs, as well as for the research of somatic cell reprogramming.
7. Distinguish between fully and partially reprogrammed cells and examine the functionality of the isolated clones.
8. Discuss the usage of the outcomes of ES and iPS cells differentiation for both basic science and therapeutic medicine.
9. Have been introduced to CRISPR as a tool for genome modification in ES cells and in mice.
10. Transfect ES cells in culture.