Scope of tissue engineering - Biomedical Engineering Desk Reference - page 714

endothelial cells and myofibroblasts. Given this level of
excitement, it is hardly surprising that the theory-to-
therapy approach to the use of cells would be thrust
forward into translational human studies at the earliest
possible stage [34] . A prompt action came in 2001 with
the publication of a paper in Nature by Orlic and col-
leagues, suggesting that stem cells derived from bone
marrow can replace heart muscle lost as a result of heart
attack, and can improve cardiac function. Injecting bone-
marrow stem cells into an injured heart potentially
represented a new therapy, triggering the launch of nu-
merous clinical studies to investigate the effect of di-
rectly injecting these cells into the damaged heart muscle
of patients following a heart attack. Amajor barrier to the
long-term success of cellular cardiomyoplasty is the
survival of the transplanted cells.
Recent reports suggest that hematopoietic stem cells
can transdifferentiate into unexpected phenotypes such
as skeletal muscle, hepatocytes, ECs, neurons, endothe-
lial cells, and cardiomyocytes, in response to tissue injury
or placement in a new environment. Although most
studies suggest that transdifferentiation is extremely rare
under physiological conditions, extensive regeneration of
myocardial infarcts was reported after direct stem cell
injection, promoting several clinical trials. Under condi-
tions of tissue injury, myocardial replication and re-
generation have been reported and a growing number of
investigators have implicated adult bone marrow in this
process, suggesting that marrow serves as a reservoir for
cardiac precursor cells. It remains unclear which BMCs
can contribute to myocardium, and whether they do so
by transdifferentiation or cell fusion.
Independent studies by Murry et al. [35] and Balsam
et al. [36] seriously challenged Orlic and colleagues'
initial observations and the scientific underpinnings of
the ongoing human studies [37] . Two strategies were
used to show that bone-marrow stem cells do not take on
the role of damaged heart cells. Murry et al. isolated and
purified genetically modified bone-marrow stem cells
from mice. The modification ''tagged'' the cells (with
LacZ), enabling them to be detected in the recipient
mouse heart, into which the cells were directly injected.
Closer inspection of the recipient heart showed that the
label could not be detected in heart muscle cells. Similar
results were shown by Balsam et al. , although the ap-
proach was slightly different. Donor bone-marrow stem
cells were transfused directly into the circulation of re-
cipient. Again, the tag (GFP) could not be detected in
heart muscle cells of the donor; indeed, the BMCs con-
tinued to differentiate into blood cells while in the heart.
So, scientists are asking why there are wide discrepancies
between the earlier report and the current investigations.
As Murry et al. suggest, the differences may arise from
the difficulty of tracking the in vivo fate of transplanted
cells within an intact organ. Orlic et al. mainly relied on

detecting unique protein constituents of bone-marrow
stem cells using fluorescently tagged antibodies. Murry
et al. and Balsam et al. , however, created intrinsic genetic
markers that can be easily recognized without antibody
staining. Owing to its high density of muscle-specific
contractile proteins, intact heart muscle tends to have
high inherent background fluorescence, and can also
display non-specific antibody binding to the abundant
muscle proteins. This makes it difficult, even for the
most experienced labs using the most specialized mi-
croscopes, to track cell fate by simply using techniques
that rely on fluorescent antibody staining of cardiac
proteins. Various experiments using several types of stem
cells support the view that transdifferentiation occurs
rarely, if at all, in many organ systems, including heart
muscle. Less than 2% of the transplanted or injected cells
take on the in vivo fate of heart cells. If this is true, then
the improvement in cardiac function seen by Orlic et al.
might have arisen not because the stem cells trans-
differentiated, but because new blood vessels were en-
couraged to grow around the injected area. Such growth
of new blood vessels has been consistently found in
transplantation studies of diverse cell types in the heart.
Studies in large-animal models of transplanted bone-
marrow-derived stem cells in the injured heart also failed
to document cardiac regeneration [38] . Again, the im-
plication is that any functional improvement seen may
not be related to an increase in functioning heart muscle
per se. A recent clinical study, in which bone-marrow
stem cells were transplanted into injured hearts, was
terminated because of serious cardiac side effects that
threatened the blood flow to the heart. This again sug-
gests that further experimental testing is warranted in
large-animal model systems.

7.2.8.4 ES cells

The first report of a stable ES cell line derived from
a human blastocyst in 1998 was by Thomson et al. [39] .
This led to a surge of interest in ES cells as a potential cell
source for tissue engineering. The ES cells are derived
from the inner cell mass of the preimplantation blasto-
cyts. They are pluripotent and can be maintained and
expanded in culture in an undifferentiated state. Markers
such as Oct-4, SSEA-4, and Nanog have all been used to
characterize and assess the pluripotent capacity of ES
lines when grown in vitro. However, the precise mech-
anisms by which the culture methods routinely
employed in laboratories enable ES cells to remain plu-
ripotent are still not fully understood and it is likely that
further key genes that prevent the cell from proceeding
with differentiation remain to be identified.
Human ES cell lines (hES) are pluripotent diploid
cells that can proliferate in culture indefinitely and