what-when-how
In Depth Tutorials and Information
mesenchymal-derived cell types, this step is not neces-
sarily predictive of
in vivo
bone formation after trans-
plantation. Thus the standard to claim that a progenitor
cell population has the potential for cell therapy of bone
needs to be based on
in vivo
transplantation at sites
where bone is normally made. It should be expected that
the donor osteoblasts would participate with the host in
making and maintaining bone matrix production.
The more widely used immune-compromised mouse
models eventually rejected marrow transplants, which
may explain early studies using human BMSC sources
for human bone formation that did not demonstrate per-
sistence of the transplanted cells. The need for murine
models to study AIDS required a mouse that could
achieve long-term engraftment of human bone marrow.
With the development and distribution of the
NOD/Scid/
IL2rg null
(NSG) mouse strain by Jackson Laboratories,
96
it is now possible to achieve long-term engraftment from
progenitors other than bone marrow.
95,97
Furthermore,
our group has bred an osteoblast/fibroblast-restricted
GFP reporter into the NSG mouse line to more easily dis-
criminate between mouse and human tissue.
There are two techniques for introducing a GFP
reporter into human cells to follow the fate of trans-
planted cells in a murine donor. Viral vectors will deliver
a strongly expressed but unregulated GFP that in most
cases will remain active in BMSC-derived cells after
transplantation. However, when employed in hES or iPS
cells, the activity is unpredictably repressed during dif-
ferentiation
in vitro
and after transplantation. However,
when a GFP gene is introduced into a chromosomal site
that remains active at all stages of the hES cell differen-
tiation, it will remain active and will be regulated in a
cell-specific manner.
98
Thus using zinc-finger-directed
recombination, it is possible to insert an osteoblast-
restricted GFP reporter in hES and iPS cells as a sensitive
marker of human osteoblast differentiation in trans-
planted NSG mice. In both cases, viral transduction of
BMSCs or site-specific recombination in hES/iPS cells in
combination with associated evidence of new bone for-
mation (mineralization dyes and AP staining) provides a
rapid and unequivocal interpretation of a human-based
bone cell transplantation study refer to figure 57.3.
Utilizing these tools, we have become very cautious
not to overinterpret the ability of either BMSCs or hES/
iPS-derived cells to achieve meaningful osteoblast dif-
ferentiation
in vivo
. Both cell sources appear to induce
a vigorous mouse bone ingrowth and it is usually in
those regions that human osteoblasts can be observed.
However, human osteogenesis is being achieved because
groups of cells expressing the bone-restricted GFP
reporter are observed in association with active min-
eral deposition and AP activity. The progress to date
suggests that it is possible to obtain osteoblast differen-
tiation
in vivo
but that significant improvements in the
preimplantation differentiation protocols are needed to
achieve the level of bone formation required for a cell-
based OI therapy.
Build Murine Models of OI as a Platform to
Evaluate Corrected Human iPS Cells
Based on the outcomes of the two major objectives
discussed above, it should be possible to improve the
skeletal strength of a mouse with an OI mutation with
human-derived and gene-corrected OI cells. Thus breed-
ing a murine transgenic phenocopy of a human OI
mutation into the NSG background carrying a bone-
restricted GFP reporter will provide the test OI host into
which human-derived cells will be placed. Comparing
treatment of the OI mouse with human OI-derived pro-
genitors before and after gene correction needs to be per-
formed to validate the importance of the gene correction
step. Still uncertain is whether this is the correct environ-
ment for evaluating human cells since the mouse bone
marrow may not interact appropriately with the trans-
planted osteoblastic cells or the rate of matrix production
by human cells may not match the level required by a
mouse. However, showing some level of success without
evidence of cell transformation or off-site migration may
provide an adequate rationale to progress to closely mon-
itored human trials using patient-specific corrected cells.
In summary, the technologies of cell and gene therapy
are making rapid progress in many other tissue special-
ties and lessons learned from their experiences will be
incorporated into strategies used for bone and other con-
nective tissues. However, there will be issues that are
unique to bone and other skeletal tissues that will require
investigators with intimate knowledge of the devel-
opmental and reparative biology of bone and adjacent
structures. These problems will be solved and eventually
an effective therapy will be developed, but many years
of basic research and closely monitored clinical trials will
be required before the promise of a curative therapy can
be delivered.
Acknowledgement
The author appreciates the many contributions of
his laboratory and collaborators that have been utilized
in this chapter. The individuals include Dr. Alexander
Lichtler, Xiaonan Xin, Xi Jiang and Liping Wang.
