Biomedical Engineering Reference
In-Depth Information
certain culture conditions, MSCs were induced to
assume morphology and express protein markers typical
of neurons. Adult MSCs were found to migrate into the
brain and develop into astrocytes. These results suggest
that MSCs may be useful also in neural tissue repair and
regeneration. The use of MSCs has significant advantages
over other cell types. First, the use of adult MSCs means
that autologous transplantation is possible, thus avoiding
detrimental immune responses often caused by alloge-
neic transplantation. Second, ethical concerns associated
with the use of ES cells or fetal tissues are eliminated.
Last, unlike neural stem cells (NSCs) or other neural
precursors that need to be obtained via surgery, MSCs
are relatively easy not only to obtain from a small aspirate
of bone marrow, but also to expand in culture under
conditions in which they retain some of their potential to
differentiate into multiple cell lineages. For clinical use, it
might be desirable to expand and differentiate MSCs
into the cell types of choice
in vitro
before trans-
plantation, because this strategy allows reproducible
and reliable generation of well-defined transplants in a
precisely controlled environment. Besides making tissue-
engineered constructs of a single tissue type, an impor-
tant potential advantage in using MSCs is the ability to
design multifunctional or composite tissue constructs,
such as osteochondral grafts, from a single cell source.
Interestingly, there is evidence to suggest that certain
types of bone marrow-derived cells can themselves pro-
duce an angiogenic mediator VEGF. This observation
raises the possibility that these mesenchymal cells could
potentially regulate angiogenesis.
Several clinical trials with human MSCs, or closely
related cells, have started. For most of the human trials
and animal experiments, MSCs are prepared with
a standard protocol in which mononucleated cells are
isolated from a bone marrow aspirate with a density
gradient, and then both enriched and expanded in the
presence of FCS by their tight adherence to plastic tissue
culture dishes. For instance, bone marrow aspirates are
mixed with Hanks' balanced salt solution (HBSS)
containing heparin. The same amount of high-density
Ficoll solution is carefully placed beneath the HBSS
mixture followed by centrifugation. The nucleated cell
layer existing at the interface between the HBSS and the
Ficoll is removed and placed on a dish. After removal of
non-adherent cells and the subsequent continuation of cell
culture, cells form colonies from which MSCs are
obtained. Cultures of human MSCs, unlike murine cells,
become free of hematopoietic precursors after one or two
passages and can be extensively expanded before they
senesce. However, when the cells are expanded under
standard culture conditions, they lose their proliferative
capacity and their potential to be differentiated into
lineages such as adipocytes, tenocytes, and chondrocytes.
Moreover, cultures of human MSCs are morphologically
heterogeneous, even when cloned from single-cell-
derived colonies.
Studies on human MSCs become complicated by the
fact that there is no consensus as to the characteristic
surface epitopes that can be used to identify the cells. A
series of antibodies to surface epitopes have been em-
ployed by several investigators, but none have been into
general use. Sekiya
et al.
screened over 200 antibodies
but did not find any that efficiently distinguish rapidly
self-renewing cells from mature MSCs
[30]
. It is there-
fore difficult to compare the results that different re-
search groups obtained either in animal models for
diseases or in clinical trials. It follows that several pa-
rameters must be considered in preparing frozen stocks
of human MSCs: (1) variations in the quality and number
of MSCs obtained from different bone marrow aspirates,
(2) the yield of cells required as frozen stocks, (3) the
quality of the cultures in terms of their content of early
progenitor cells that replicate most rapidly and have the
greatest potential for multilineage differentiation, and
(4) the number of cell doublings the cells have undergone
before they are harvested and frozen.
Autologous MSCs have advantages over ES cells that
may lead to terato-carcinoma formation. However,
compared with ES cells, which have an unlimited pro-
liferative life span (period before the cells reach growth
arrest in culture) and consistently high telomerase ac-
tivity, MSCs have very poor replicative capacity and
short proliferative longevity. Thus, an important chal-
lenge in tissue engineering is to improve the replicative
capacity of MSCs, thereby to obtain a number of MSC
sufficient to repair large defects. Forced expression of
telomerase in MSCs markedly increased their pro-
liferative life span and MSCs with a high telomerase
activity showed osteogenic potential.
Cell expansion
The most common source of adult-derived stem cells is
the bone marrow. MSC can be obtained easily and re-
peatedly by bone marrow aspiration, but isolation of
marrow aspirates in great volume causes damage and
pain, and it is difficult to isolate from the bone marrow
10
7
-10
8
MSCs that are required for regeneration of large
injured tissues. In addition, the heterogeneous nature of
bone marrow with both hematopoietic and MSCs con-
founds the result of various therapies using bone marrow.
The scarcity of MSC in bone marrow of older donors may
impose additional requirements with respect to cell ex-
pansion and differentiation. Furthermore, a short life
span of MSC and a reduction in their differentiation
potential in culture have limited their clinical applica-
tion. Thus, the ability to rapidly expand MSCs in culture
is of obvious importance in using the cells for tissue en-
gineering. The expansion of MSCs
in vitro
is a pre-
requisite for autologous cell transplantation. In other
