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clinical outcome.
80,81
However, direct demonstration
that the infused cells became functional osteoblasts is
still lacking, and the potential consequences of the mes-
enchymal cells engrafting non-skeletal sites, such as
brain, needs very rigorous attention.
As more clinical and animal-based research has
encountered poor engraftment rates, the possibil-
ity that the infused cells produce factors that enhance
host-derived repair has been proposed for the observed
positive outcomes of the transplant. This mechanism is
referred to as a non-cell autonomous action
82
and is cur-
rently receiving attention as an immune modulator for
chronic inflammatory disorders such as Crohn's disease
83
and insulin-dependent diabetes.
84
The same mechanism
is now being invoked for the transient improvement
observed in marrow-transplanted OI children and the
effect has been further localized to the non-adherent cells
of a bone marrow cell culture.
11
The entire field of non-
cell autonomous transplant medicine is still highly con-
troversial and controlled clinical trials will be required to
determine if there are short- or longer-term clinical ben-
efits. However, as a therapy for OI, it does not address the
fundamental need for replacing OI bone cells with nor-
mal bone cells and thus is not a viable curative strategy.
Because direct implantation of BMSCs into bone mar-
row or a bone repair defect does lead to engraftment
and donor cell-formed bone, it would appear to be an
effective vehicle to deliver autologous, gene-corrected
bone progenitor cells to an OI subject. Direct transplan-
tation of normal BMSC into an immune-suppressed OI
mouse appears to gradually replace host cortical bone
with normal bone.
85
The difficulty that arises when a
gene correction strategy is applied to BMSC is the exces-
sive number of cell passages needed to isolate the clone
of cells that was correctly engineered. The ability of
BMSCs to maintain their
in vivo
osteogenic properties is
rapidly lost with a few passages
in vitro
well before the
number of cell divisions required for gene inactivation
or correction. Finding a method to preserve progenitor
potential while performing the genetic engineering of
the OI mutation is a major new direction for cell/gene
therapy of the disease.
the bone surface with osteoblasts. When a generic GFP
reporter is used, the donor cells on the bone surface do
not have an underlying mineralization line nor are they
AP positive. Instead, they are frequently TRAP positive
or carry a macrophage epitope
72
and they do not estab-
lish long-term engraftment.
73
In contrast, when a bone
restricted GFP reporter is used to identify donor-derived
osteoblasts, no GFP-positive cells can be identified.42
42
When freshly isolated bone marrow is allowed to set-
tle on a tissue culture plate, a complex mixture of mesen-
chymal and macrophage cells attaches while other cells
remain in the supernatant. Traditionally, the non-adherent
cells are discarded and the attached cells are expanded in
number. This attached population has been demonstrated
to have bone differentiation properties both
in vitro
and
after transplantation. Mouse models most clearly dem-
onstrated the
in vivo
differentiation potential of this cell
source. The experiments can be performed in which the
osteoblast only of the host animal has a green GFP color
and the donor a blue GFP color to easily discern which
source of cells contributed newly formed cells within
the repair.
74
For example, when the cells are directly
implanted into a scaffold and placed into a repairing bone
defect, most of the cells that form bone in the defect area
express the osteoblast-restricted GFP reporter and these
are the cells that overlie a bone mineralization line.
75
Similarly, when injected into the marrow space, new bone
can be demonstrated expressing the donor color that over-
lies a mineralization line.
74
However, when the same cells
are injected intravenously, most are trapped within the
pulmonary vasculature and none are found on the bone
surface. Direct infusion of isolated human bone marrow-
derived stromal cells (BMSCs) into human OI subjects has
also been reported with transient positive effects; how-
ever, no engrafted cells could be demonstrated.
76
An entire medical industry has developed around the
concept that BMSCs, the mixture of cells that grow out
from tissue culture-expanded bone marrow, have medic-
inal value for a wide variety of skeletal maladies when
injected into the systemic circulation.
77
Because there is
no preclinical animal or clinical data to affirm the claim
in adult subjects, the US Food and Drug Administration
has recently begun closing these operations and their
action has been supported by federal courts.
78
The one
potential exception to systemic infusion, which still
needs rigorous experimentation, is systemic transplanta-
tion of the developing fetus. When exposed to allogeneic
cells
in utero
, tolerance is possible, removing some of the
immune barriers of transplantation.
79
Furthermore, pos-
sibly because the lungs are uninflated, the pulmonary
vasculature does not appear to filter out the infused
cells, thus allowing more to reach the systemic circu-
latory targets including bone. Reports of murine and
human intrauterine transfusion/transplantation show
clear evidence of engraftment and suggest an improved
Induced Pleuripotential (iPS) Cells
The identification of totipotential stem cells derived
from the inner cell mass of blastula of a preimplantation
embryo has radically changed the tools and concepts of
stem cell biology and how cell therapy could be applied
to genetic and degenerative diseases. Despite the politi-
cal and ethical controversies that the human embryonic
cell (hES) technology has spawned, the whole concepts of
genomic organization and the molecular basis of cell lin-
eage and differentiation have been completely redefined.
Equally transformative was cloned sheep “Dolly” who
