Germ Line Stem Cells
Week 4
Sources of Stem Cells
EG Cells Discovered
• 1998: Gearhart and colleagues derive human embryonic germ cells from the gonadal ridge and
mesenchyma of 5- to 9-week old fetal
tissue that resulted from elective
abortions
• Embryonic Germ (EG) cells are derived from the primordial germ cells of
developing fetuses.
Development of Mouse Embryonic Primordial Germ Cells (PGCs)
http://stemcells.nih.gov/info/scireport/
Primordial Germ Cells (PGC)
• Primordial Germ Cells are diploid germ cell precursors that
transiently exist in the embryo before they enter into close
association with the somatic cells of the gonad and become irreversibly committed
as germ cells Carlson 1999
PGC: Overview
• Early in development, the cells of the germ cell lineage segregate from the somatic cells. This protects the
developing PGCs from signaling factors and
morphogenic movements in the growing blastocyst.
Independent repression mechanisms provide protection by shutting down gene expression in order to keep the germ cell lineage pluripotential
• Segregation is controlled by maternal factors and signaling factors from extraembryonic cells.
PGC: Overview
• These PGCs proliferate and migrate through the endoderm, hindgut, and mesentery to their final destination in the genital ridges. There, with close association to gonad and somatic cells
(intermediate mesoderm derivatives) the PGCs differentiate into germ
cells Hogan 2001
PGC: Overview
• In the female, these germ cells enter into the prophase of meiosis and represent the maximum number of germ cells ever found in the ovaries. Males go into mitotic arrest and do not resume mitosis until puberty and maintain the ability to divide
throughout life
PGC: Germ-Line-Inducing Factors
• Germ-line-inducing factors present near the junction between the
trophoblast and ICM play a role in germ-line development.
• Lack of TGFß and BMP4 cause the absence of PGCs in
developing embryo.
• BMP4, secreted by
extraembryonic mesoderm, induces the fate of pluripotent proximal epiblast cells to become PGCs.
Carlson 1999
PGC: Pluripotency and Proliferation
Pluripotency:
• Oct4 appears to be a key regulator of the pluripotential phenotype. Originally it is expressed in all cells of the
cleavage-stage embryo, but it is then down regulated and eventually is only expressed in the PGCs. It is finally
extinguished in the germ line when the PGCs begin to differentiate in the gonad, and are only reactivated once the gametes reach maturity.
• Oct4 is the guardian of the pluripotential phenotype and prevents cells from becoming restricted in their
developmental potential. It maintains the undifferentiated state by regulating gene transcription.
PGC: Pluripotency and Proliferation
Proliferation:
• Three different binder/receptor signaling systems
promote the survival and proliferation of PGCs. They are:
1) stem cell factor, a growth factor; 2) the bFGF gene, expressed along the PGC migration route; and 3)
cytokines of the interleukin/LIF family; each with their own respective receptor.
• Leukemia inhibitory factor (LIF) promotes the
undifferentiated, pluripotent phenotype of ES cells. In the absence of LIF and feeder cells, ES cells rapidly
differentiate. LIF functions in combination with a specific level of OCT4 to maintain the undifferentiated phenotype.
PGC proliferation requires fibroblast cell feeder layers.
EGCs Derivation Techniques
• To derive human EGCs, cultures of PGCs (obtained from the gonadal ridge and mesentery) are grown.
• The PGCs were plated on a feeder layer of non-dividing, STO fibroblasts in a growth medium including the cytokine, leukemia inhibitory factor (LIF), and mitogen (basic
fibroblast growth factor, bFGF).
• After three weeks, the PGCs form dense, multilayered colonies of cells resembling EG cells
Establishment of cultures from
PGCs
EG Cells Discovered
• They grow EG cells in vitro for approximately 20 passages, and the cells maintain normal
karyotypes.
• The cells spontaneously form aggregates that differentiate spontaneously, and ultimately
contain derivatives of all three primary germ layers.
• Other indications of their pluripotency include the expression of a panel of markers typical of
mouse ES and EG cells.
• The EG cells do not form teratomas when injected into immune-deficient mice
Shamblott 1998
Shamblott 1998
Shamblott 1998
Shamblott 1998
Shamblott 1998
Molecular pathways of GSC maintenance
Gilboa 2004
Molecular pathways of GSC differentiation
• Please read Gilboa & Lehmann 2004
EGC/ESC Comparison
• The pluripotent cells generated in vitro from human ESCs and human EGCs are not
equivalent in their potential to proliferate or differentiate.
• ESCs are derived from the inner cell mass (ICM) of the blastocyst whereas EGCs are derived
from the PGCs.
• Although ESCs seem to have a broader ability to differentiate, EGCs mature into the mature
gametes.
Comparisons Between Human Embryonic Stem Cells and Embryonic Germ Cells
• In both cases, the cells replicate for an extended period of time, show no chromosomal
abnormalities, generate both XX (female) and XY (male) cultures, and express a set of
markers regarded as characteristic of pluripotent cells.
• When the culture conditions are adjusted to permit differentiation, both ES and EG cells
spontaneously differentiate into derivatives of all three primary germ layers—endoderm,
mesoderm, and ectoderm
Comparisons Between Human Embryonic Stem Cells and Embryonic Germ Cells
• They vary with respect to their growth
characteristics in vitro, and their behavior in vivo
• Human ES cells can proliferate for two years through 300 population doublings or even 450 population doublings.
• Cultures derived from embryoid bodies
generated by human embryonic germ cells have less capacity for proliferation. Most will
proliferate for 40 population doublings; the maximum reported is 70 to 80 population doublings
