SCIENTIFIC PROGRAM
“Stem Cell Differentiation Training Course”
5th edition
Napoli, November 9-12, 2010
Organized by:
Stem Cell Fate Lab
in collaboration with:
with the support of:
1
1st Day: Tuesday, November 9, 2010
Seminar Room
9:00 - 9:30
Registration
9:30-10:30
Opening remarks - A.Baldini, Director
Plenary lecture: Giuseppe Testa, “Histone methylation in genome
programming and reprogramming”
10:30-11:00
Introducing the SCF Lab – E. Patriarca
Meeting with participants
11:00 11:30 Coffee Break
Meeting Room at II floor
11:30-12:30
Seminar
D. De Cesare: “Derivation, growth and applications of mouse and
human embryonic stem cells”
Propedeutic Lessons
S. Comes (expansion of undifferentiated mouse ESCs)
G. Manganelli (mono-step protocol for neural differentiation and EB
formation for cardiac differentiation)
Cell Culture rooms
12:30-13:30
Experimental Work
Morphological observation (MEFs, undifferentiated mouse/human
ESCs)
Material preparation (coating, mitomycin treatment)
13:00-14:00
Lunch
14:00-17:00
Experimental Work
Maintenance of feeder-independent mESCs
Maintenance of feeder-dependent mESCs (feeder preparation/ESC
splitting and freezing)
Colony Formation Assay
17:00-17:30
Coffee Break
17:30-19:00
Experimental Work
Neural Differentiation: mono-step protocol
Cardiac Differentiation: step I
1. Hanging drops
19:00-19:30
Round Table
2
2nd Day: Wednesday, November 10, 2010
Meeting Room at II floor
9:30-10:30
Seminars
S. De Falco: “New experimental approach for the identification of
modulators of ESC differentiation: combinatorial chemistry”
L. Casalino: “HTS and automation: a new frontier in Stem Cell Biology”
10:30-11:00
Coffee Break
11:00-11:40
Propedeutic Lessons
C. D’Aniello (biochemical analysis of pluripotency/differentiation
markers by RT-PCR)
S. Iaconis (phenotypic analysis of pluripotency/differentiation markers
by IF)
D. De Cesare (maintenance and expansion of human ESCs)
Cell Culture rooms
11:40-13:00
Experimental Work
Morphological observation (mouse ESCs at 24 hours after plating, cells
from colony and differentiation assays)
Cardiac differentiation protocol (Steps 2 and 3)
2. Suspension culture of EBs
3. Adherent culture of EBs
13:00-14:00
Lunch
Cell Culture rooms
14:00-15:00
Experimental Work
Maintenance of feeder-dependent hESCs (feeder preparation/ESC
splitting and freezing)
Laboratories
15:00-17:00 Experimental Work
Phenotypic analysis of the expression of pluripotency/differentiation
markers
IF, phase I
Rehydratation of fixed samples
Blocking, incubation with primary Abs: SSEA1, Oct4, MF20, NFM,
GFAP.
3
Alkaline Phosphatase Assay
17:00-17:30
Coffee Break
Laboratories
17:30-19:00
Experimental Work
Biochemical analysis of the expression of pluripotency/differentiation
markers
RT-QPCR, phase I
Analysis of cDNAs from: undifferentiated ESCs, differentiating ESCs
(4-day old EBs or 8-day old neural precursor cells) and terminally
differentiated cells (12-day old cardiac or neural differentiated ESCs).
cDNA amplification by PCR: Octamer/Nanog, Nestin, NFM/βIIITubulin, Brachyury, alpha-MHC and GAPDH)
II floor Meeting Room
19:00-19:30
Round Table
4
3rd Day: Thursday, November 11, 2010
Laboratories
9:30-10:30
Experimental Work
Phenotypic analysis of the expression of pluripotency/differentiation
markers
IF, phase II
Washing
Incubation with secondary Abs
10:30-11:00
Coffee Break
11:00-12:00
Experimental Work
IF phase III
Washing
Nuclei counterstaining (Hoechst staining)
Cell Culture rooms
12:00-12:30
Experimental Work
Morphological observation (mouse/human ESCs at 24/48 hours after
plating, cells from colony and differentiation assays at 48 hours after
plating)
II floor Meeting Room
12:30-13:00
Round Table
Biochemical analysis of the expression of pluripotency/differentiation
markers
RT-QPCR, phase II
analysis of QPCR results
13:00-14:00
Lunch
14:00-14:45
Seminars
G. Minchiotti: “Stem cells as an in vitro model to study mechanisms
controlling embryonic development: genes and factors”
S. Filosa: “Stem cells as an in vitro model to study molecular
mechanisms governing cellular differentiation”
FREE TIME
21:00
Social Dinner
5
4th Day: Friday, 12 November, 2010
II floor Meeting Room
9:30-10:30
Experimental Work
Phenotypic analysis of the expression of pluripotency/differentiation
markers
IF, phase IV
Observation under an automated inverted fluorescent microscope.
10:30-11:00
Coffee Break
11:00-12:00
Plenary Lecture:
Silvia Brunelli: “Role and contribution of endothelial progenitors cells in
muscle development and regeneration”.
12:00-12:30
Experimental Work
Phenotypic analysis of the expression of pluripotency/differentiation
markers
IF, phase IV
Observation under an automated inverted fluorescent microscope.
12:30-13:00
Round Table and Concluding Remarks
13:00-14:00
Lunch
6