Course Identification

Embryonic and pluripotent stem cells: Differentiation and reprogramming

Lecturers and Teaching Assistants

Dr. Rachel Sarig, Dr. R. Haffner, Dr. Berkovitz Alina, Dr. Yael Fried, Dr. Gilad Beck

Course Schedule and Location

First Semester

Field of Study, Course Type and Credit Points

Life Sciences: Laboratory; Elective; Regular; 1.00 points
Life Sciences (Molecular and Cellular Neuroscience Track): Laboratory; 1.00 points
Life Sciences (Brain Sciences: Systems, Computational and Cognitive Neuroscience Track): Laboratory; 1.00 points
Life Sciences (Computational and Systems Biology Track): Laboratory; 1.00 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

Numerical (out of 100)

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.

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


  02.01.22 03.01.22 04.01.22 05.01.22 06.01.22 13.01.22    
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            
  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              

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