Experimental Protocols

Related to Stem Cell

Research

Ying Jin
Developmental Biology Lab
yjin@sibs.ac.cn
63852591
 Contents of the lecture today
★ Concepts:
Pluripotent stem cells;
Multipotent stem cells.

★ Protocols:
Derive ES cell line;
Characterize ES cell line;
Differentiate ES cells;
Nuclear transfer.
 Contents of the lecture today
★ Concepts:
Pluripotent stem cells;
Multipotent stem cells.

★ Protocols:
Derive ES cell line;
Characterize ES cell line;
Differentiate ES cells;
Nuclear transfer.
 What are stem cells?

◆ Reproduce itself (self-renewal);

◆ Generate one or more differentiated

descendant cell types;

◆ Functionally reconstitute an organ or a

whole organism.
 Stem Cells

Self-renewal Differentiation
 Pluripotent Stem Cells

◆ A single pluripotent stem cell has the

ability to give rise to types of cells that
develop from the three germ layers
( ectoderm, mesoderm and endoderm),
including germ cells.

◆ Includes: embryonic stem cells;

embryonic germ cells;
embryonal carcinoma cells.
Nature 414: 92, 2001
Nature 414: 92, 2001
 Embryonic Stem Cells (ES)
Derived from inner cell mass (ICM) of
blastocyst.
ICM

Human ES colony on feeder layer

The first human ES cell line was established in 1998 by James A. Thoma
Science 282: 1145, 1998
 Embryonic Germ Cells (EG)
Derived from fetal tissue, cultured from the
primordial germ cells (PGC) of the
gonadal ridge of fetus. PGCs are
embyonic precursors of the gametes.
Human EG
Colony

Feeder
layer

The first human EG cell lines was derived by John D. Gearhart in 1998
Proc. Natl. Acad. Sci. USA 95: 13726, 1998
Embryonal carcinoma cells (EC)
Derived from teratocarcinoma, which
contains a wide array of tissues derived
from three primary germ layers.

The differentiated cells of the tumor are

formed from pluripotent EC cells present
in the tumor, which themselves are
derived from PGCs.

Several groups derived human EC cell lines during 1970s

Nature 270:515, 1977
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http://stemcells.nih.gov
 Somatic Stem Cells or
Tissue Stem Cells
An undifferentiated cell that occurs in a
differentitated tissue or organ, renews
itself, and yields specialized cell types.

Origin: fetus or adult (adult stem cell);

Potential: multipotent;

Proliferation: limited.
 Contents of the lecture today
★ Concepts:
Pluripotent stem cells;
Multipotent stem cells.
★ Protocols:
Derive ES cell line;
Characterize ES cell line;
Differentiate ES cells;
Nuclear transfer.
De ri vat io n o f ES Ce ll lin e
 Derivation of ES cell line
Material:
◆ Blastcysts;
Normal fertilization, parthenogenetic activation,
nuclear transfer.
◆ Feeder layer: mouse embryo fibroblast cells
(MEFs); irradiate MEFs enough to stop them
from proliferation, but not enough to kill them.
◆ embryo culture medium;
◆ Finely drawn pasteur pipettes;
◆ ES cell culture medium.
oocyte

In vitro activation
oocyte
 Mouse ES derivation
1. Flush embryos (day 3.5) from the uterine horns; Place them individually
onto feeder layer in ES cell culture medium.
2. The embryos hatch from the zona pellucida and attach to feeder layer by
migration of the trophoblast cells.
3. When ICM –derived clump has enlarged enough, dislodge it from the
underlying sheet of trophoblast cells by drawn pasteur pipette. Use the
pipette to disaggregate ES cell clump into smaller aggregates.
4. Transfer the small clump into a fresh dish containing feeder layer.
5. Generally, after 2 days, primary colonies of cells will visible.
6. Discrete colonies of a stem cell morphology are selectively removed after
up to 7-8 days of culture and then dissociated in microdrops of
trypsin/EDTA, and passaged into fresh feeder well (keep the cell density
high).
7. Small nests of ES cells appear within 2-3 days of subculture and can be
expaned 3-5 days later by trypsinizing the whole well and transferring its
contents onto a larger feeder dish.
8. An established ES cell line require careful subculture at 2-3 day intervals.
9. Mouse ES cell medium is supplemented with leukemia inhibitory factor.
 2-cell embryos 4-cell embryos

Fused embryos blastocyst

Sun, et al.
Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition
Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition
Sun, et al.