Course Identification

Embryonic and pluripotent stem cells: Differentiation and reprogramming

Lecturers and Teaching Assistants

Dr. Rachel Sarig

Course Schedule and Location

First Semester
30/12/ 2018-3/1/ 2019, 10/1/2019, FGS, Lab

Field of Study, Course Type and Credit Points

Life Sciences: Laboratory; Elective; 0.50 points
Life Sciences (Molecular and Cellular Neuroscience Track): Laboratory; Elective; 0.50 points
Life Sciences (Computational and Systems Biology Track): Laboratory; Elective; 0.50 points


The courses full schedule appears in online course materials


Tissue culture experience is highly recommended



Language of Instruction


Registration by


Attendance and participation


Grade Type

Pass / Fail

Grade Breakdown (in %)


Evaluation Type


Scheduled date 1


Estimated Weekly Independent Workload (in hours)



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.

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.

Learning Outcomes

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.

Reading List