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demonstrated that somatic cells could be reprogrammed
into a totipotential state.
86
However, the most important
development for stem cell biology as applied to a human
application was the ability to initiate reprogramming
of somatic cells to totipotential hES-like cells by ectopic
expression of a mixture of transcription factors known to
be active in cultured embryonic stem cells.
87,88
As these
factors are expressed, the cells gradually change their
morphology from fibroblastic monolayers to clusters of
ES-like cells, which if handled correctly can contribute to
forming all the tissues of a developing embryo including
germ cells capable of generating subsequent progeny.
Cells with these properties are termed induced pleu-
ripotential stem (iPS) cells and conditions for generat-
ing these cells are well established for both mouse and
human cells. A major concern that initially troubled
the use of these cells as a potential pleuripotential cells
source was the viral vectors that deliver the transcrip-
tion factors. The possibility of integration-induced onco-
genesis or unregulated gene expression preclude cells
made in this manner from human use. However, newer
technologies for transiently expressing the transcription
factors without having to require host chromosome inte-
gration have been developed that should overcome the
safety concerns of viral-mediated gene delivery.
89,90
The primary advantage of iPS cells is the ability to
generate an OI patient-specific cell source that will retain
its pleuripotential properties while tolerating the genetic
engineering steps required for gene correction (
Figure
57.5
). Both zinc-finger and TALEN technologies have
FIGURE 57.5
Bone formed by human iPS cells within a mouse calvarial defect. (A) Site-directed insertion of a bone-restricted GFP reporter
construct to human iPS cells used a zinc-finger nuclease strategy. (B) The targeted fragment has homologous ends that match the sites that lank
the cleaved site in the iPS cells. After selection of iPS clones containing insert, a pre-differentiation culture protocol was performed and the
resulting cell implanted into an immune-compromised NSG mice. (C, D) Fluorescent and hematoxylin images of a region where human bone
was formed. The finding of bone-restricted GFP surrounding newly formed bone (red label) is evidence that the iPS cells did form human bone
in vivo
.
