Mitigating the Challenges of Clinically
Relevant Mesenchymal Stem Cell
Expansion Through Systems Biology
Elizabeth Logan, ID#994145974

Abstract— Cell therapy is an exciting new,
multidisciplinary frontier of medicine. Among the many
hurdles it faces in the transition from laboratory to clinic is
the need to develop appropriate large-scale cell expansion
protocols.
Mesenchymal stem cells (MSCs) show
therapeutic potential for not only mesenchymal tissues, but
also in myocardial and neural tissues. Furthermore, MSCs
can also be used to improve the success rate of bone
marrow transplants, and to facilitate gene delivery.
However, many limitations surround the expansion of cells
for therapeutic purposes. These areas and proposed
solutions, namely a stirred-suspension culture system, are
presented. However, due to changes in cell availability,
new challenges have been encountered. Systems biology
offers a framework for dissecting complex biological
problems. As such, potential systems biology applications
are proposed to help mitigate the challenges of
mesenchymal stem cell expansion and provide a
framework for future work.
Index Terms—cell expansion, cell therapy, stem cells,
mesenchymal stem cells, systems biology
instance, during cancer treatments, such as radiation or
chemotherapy, the hematopoietic cells within the patient’s
bone marrow are damaged or destroyed. By then transplanting
donor marrow, the patient’s system is able to integrate these
new cells and restore normal hematopoietic function with time.
Furthermore, the forefront of scientific activity has resulted in
the promise of using cells as drug-delivery vehicles,
immunotherapies or for engineered tissue constructs [2,3].
This advancement of cellular therapies however, faces
technological, regulatory and ethical hurdles. Nevertheless,
dedicated scientists and the like are working hard to overcome
these challenges. The primary technological hurdle is that of
cell sourcing [1]. There is continuing work done on identifying
appropriate cell sources, understanding proliferation and
differentiation mechanisms, and tissue targeting of stem cells.
However, in order to make the jump from a theoretical dream
to a clinical treatment, cells need to be expanded in vitro,
while maintaining uniform properties, a given phenotypic
expression and remain pathogen-free. Thus, the role of the
bioprocess engineer, to design optimized bioprocess systems
for cell expansion, is a critical component to the actualization
of cell therapies.
II. MESENCHYMAL STEM CELLS
I. INTRODUCTION
C
ell-based therapy is emerging as a new group of
techniques and technologies to treat disease and injury. It
stands as a crossroad of many dynamic scientific fields: stem
cell biology, immunology, tissue engineering, molecular
biology, biomaterials, transplant medicine, regenerative
medicine and clinical research [1]. Specifically, cell therapy
aims to replace diseased or damaged cells with new, healthy
and functioning ones.
Familiar and commonplace examples of cell therapy include
blood transfusions and bone marrow transplantation. For
Manuscript received November 1, 2004. Elizabeth Logan is a MASc
student with the Department of Chemical Engineering and Applied
Biochemistry and the Institute for Biomaterials and Biomedical Engineering
at the University of Toronto, Ontario, Canada. (phone: 416-946-8018 email:
elizabeth.logan@utoronto.ca)
A. Existence of a Mesenchymal Stem Cells
Both in vivo and in vitro studies have demonstrated the
presence of bone marrow stromal cells that can give rise to
mesenchymal tissues, such as bone, cartilage, muscle, etc.
These cells were originally referred to as ‘colony forming unitfibroblast (CFU-F) [4] with the introduction of the term
mesenchymal stem cell (MSC) by Caplan [5]. MSCs are also
referred to throughout the literature as bone marrow stromal
cells, stromal precursors, multipotent adult progenitor cells
(MAPC) and mesenchymal progenitor cells (MPC) [6-8]. The
key thread between these cell types is that they all differentiate
into mesenchymal tissues. However, the diversity in
appearance, phenotype, etc. reported among these seemingly
similar cell types, makes it difficult to confirm the nature of or
even the existence of a true mesenchymal stem cell [2].
MSCs are most commonly extracted from bone marrow,
although they have been identified in other sources such as the
Logan – Mitigating the Challenges of Clinically Relevant Mesenchymal Stem Cell Expansion Through Systems Biology
liver, spleen and cord blood. Unlike hematopoietic stem cells
that are readily identifiable as being CD34+, there is no
distinct marker set to identify a mesenchymal stem cell.
Although there is no universally accepted set of antigenic
determinants for MSCs, typical markers include, Thy-1
(CDw90), VCAM-1 (CD106), and HCAM (CD44) [3,6].
Marker expression varies both within a population and
between cell sources. Thus, none of the above identifiers are
enough to solely determine/identify a MSC and as such
positive, irrefutable confirmation of an MSC is difficult to
attain.
B. MSC Plasticity and Cell Therapy
Conventional thought is that cell fate conversions by adult
cells are infrequent and restricted, such that a tissue-specific
stem cell was only capable of producing cells specific to their
location. However, when exposed to appropriate
environmental conditions, experimental evidence has shown
that stems cells have the ability to transdifferentiate and
express phenotypes atypical to their origin [8].
Mesenchymal stem cells (MSCs) were first recognized for
their ability to form connective tissues, such as bone, cartilage
and tendon. Thus, the most obvious clinical application would
be that of skeletal tissue repair. Substantial experimental and
clinical evidence also supports this. For instance, Horwitz and
colleagues have shown that MSC grafting can be used for the
correction of osteogenesis imperfecta [9]. In addition,
continual evidence is being collected to show the ability of
allogeneic MSCs to repair other bone and cartilage defects and
fractures [3,7-8].
MSCs have also shown the ability to express neural
phenotypes, both in vitro as well as following transplantation
in murine models [10-11]. They have also demonstrated
myogenic capability both in vitro and in vivo, where successful
engraftment of MSCs following ventricle infusion into rat
myocardial tissue was been seen [10-11]. As such, MSC
engraftment for the correction of heart and neural tissue
defects are being explored.
Evidence suggests that MSCs play an important role in
regulating both hematopoietic activity [10-11] and suppressing
immune response [12]. Therefore, the co-infusion of MSCs
with hematopoeitic cells in bone marrow transplantations
could result in improved engraftment and mitigation of graftvs.-donor disease with a lower usage of immunosuppressants
[10-11]. Early evidence also suggests that MSCs may also be a
promising drug delivery vehicle [7,10].
Extensive work is being done to extract the regulatory and
physiological mechanisms that control stem cell plasticity.
This is a complex field requiring its own systems biology
dissection and as such, discussion will be limited to the fact
that stem cell plasticity is a driving force for cell therapies. As
their recognized potential becomes more diverse and
widespread, the need to develop appropriate expansion
2
protocols to achieve quantities of cells for therapeutic use only
remains heightened.
C. Current limitations
The clinical utility of MSC therapy is limited by the ability
to expand these cells in vitro without differentiation to a
specific cell lineage. MSCs are typically grown on tissue
culture plastic. Using this technique the adherent cell
populations can be cultured to achieve large total cell
expansion [11]. However, this cell expansion results in a loss
of both the proliferation rates and multidifferentiative ability
[7,10-11]. This decrease in potential is variable, with the onset
of the diminishing proliferative potential occurring between
the 4th and the 15th doubling [2,12]. Furthermore, populations
cultured under traditional MSC protocols (in an adherent
environment) are very heterogeneous, with a variety of
appearances, surface phenotype expression, cytokine
expression and subsequently, differentiation potential. These
variations appear to be very stochastic, with no correlations
being drawn to age or gender of the donor [2,12]. Thus, the
expansion potential of MSC is unpredictable, as it is difficult
to predict the characteristics of the final cell population.
III. SUSPENSION CULTURE OF MSCS
Cell fate kinetics are poorly understood, however, empirical
evidence serves as the basis for starting to decipher this code.
It is suggested that adhesion of the cells to either tissue culture
plastic or microcarriers activates a given differentiation
lineage, irreversibly changing cell fate [6].
Early work
demonstrated that stirred suspension cultures supported
hematopoiesis and the unexpected growth of fibroblastic, or
mesenchymal like cells. As such, this culture method has been
further explored.
A. New Protocol Development
Baksh [6] has developed a protocol for culturing MSCs
extracted from bone marrow donations in a stirred suspension
environment capable of generating large numbers of MSCs
that retain both their undifferentiated, progenitor identity and
high proliferation rates. Another key element of this work was
the identification of appropriate cytokines for development of
a serum-free media to attain a homogeneous population [6].
This media, developed through factorial analysis, was
optimized for suspension culture only. When adherent cells
were cultured in this media their proliferation was inhibited. It
was concluded that this suspension culture method presents a
realistic approach for MSC expansion for therapies as it results
in a homogeneous population that retains its high proliferation
rate and undifferentiated state [6].
B. New Problems
With this new culture method just presented it would seem as
if all problems related to clinically relevant MSC expansion
have been solved. Unfortunately, that is not the case. The cells
used in the above expansion were obtained from the third filter
residue of bone marrow (BM) that was purified for transplant.
Logan – Mitigating the Challenges of Clinically Relevant Mesenchymal Stem Cell Expansion Through Systems Biology
However, total BM transplants are slowly being replaced by
peripheral stem cell blood transplants in hospitals as they have
the same efficacy levels, are a less invasive donation
procedure, and transplant recipients have faster hospital
recovery times [13-14]. Therefore, BM (and subsequently
MSCs) for experimental research is now obtained on a
voluntary donation basis via small BM aspirations (BMA)
from the iliac crest. Subsequently, the composition of these
cell broths (BM vs. BMA) is different, due to the differences
in acquisition and processing. As such, the inoculation
population properties differ resulting in differences in growth
patterns: the bioreactor medium/system developed does not
appear to promote growth of BMA-derived MSCs as
effectively. Subsequently, there is no published data showing
clinically relevant cell of BMA-derived MSCs, and hence,
more research is required to develop an expansion protocol for
BMA-derived MSCs.
3
to MSC and as such, stem cells, in general, tend to be
identified through their behavior. However, literature reveals
distinct differences in cell shape, cellular responses to various
tests, and fluorescence staining results of seemingly similar
mesenchymal stem cell-types. This diverse heterogeneity in
phenotype complicates the definition of an MSC, or even the
existence of one.
A key area of interest in stem cell research is developing
models to understand the mechanism behind cell fate
decisions. Both deterministic and stochastic mechanisms have
been presented to understand this mysterious field. Cell fate
appears to be a function of autonomous cell factors and
environmental conditions including cytokines, cell-to-cell
signaling, and other physiological cues (e.g. strain, adhesion).
Furthermore, lineage specific gene expression (i.e.
preprogramming of cells) appears to play a significant role in
determining cell fate.
IV. WHERE SYSTEMS BIOLOGY COULD HELP
A. Systems Biology
Systems biology is simply described as an examination of the
responses of genes, proteins, and biological pathways
following the disruption of a system through biological,
genetic or environmental factors [15]. There are major
concepts that make systems biology a unique field: high
throughput data generation (to observe all activities of cells in
their natural and perturbed states), integration of data types,
and formulation of system models [16]. Through systems
biology, it is possible to create a single cohesive unit
encompassing the “complexity of biological interaction at all
levels…including gene networks, cell signaling cascades, and
metabolic pathways” [17].
By looking at the challenges associated with MSC
expansion, it can only be concluded that they represent a
highly complex and still somewhat undefined system: a prime
candidate for ‘rescue’ by systems biology. Systems biology
approaches to cell characterization, cell fate, media
development will be explored. Extensive work is being
conducted on developing stem cell proliferation models,
including those to incorporate terms for proliferative
heterogeneity [18] However, for this discussion, the problems
encountered in achieving effective expansion of MSCs appear
to be a result of initial phenotypic expression (cell
characterization), unspecified differentiation cues (cell fate
analysis) and cytokine influence (media development). Thus,
discussion will be confined to those three areas.
B. Using Gene Mapping to Characterize MSCs and
Analyze Cell Fate Decisions
A key consideration of systems biology is the ability to
collect, sort and handle large amounts of cellular information.
To date, no single set of phenotypic markers has been
identified to adequately characterize a MSC. The expressed
phenotype appears to be highly dependent on lineage, culture
duration and/or plating density [3]. This problem is not unique
As such, it is valuable to examine the metabolic and
signaling pathways within the cell that bring about a specific
cellular behavior or response [19]. One of the hallmarks of
systems biology is genetic mapping [16]. By deciphering the
complex cellular responses, a genetic network can be
established and thereby developmental or phenotypic changes
can be predicted. This approach is being extensively used in
developmental biology, in which it has been predicted that,
from a systems biology approach, development, which could
be equated to differentiation, results as a progression of
spatially defined regulatory gene expressions [20]. If the
genetic state of a cell, through the application of DNA
analysis, etc., can be identified, algorithms to predict genetic
changes (cell fate) can also be developed. As discussed, this
tool would be employed to help with actual identification and
definition of MSCs. Thus, the genetic networks developed for
characterization would already be in place for deciphering
differentiation mechanisms. Specific to the work addressed in
this paper, uncovering any genetic changes which are a result
of inter-cellular interactions (i.e. with hematopoietic cells) and
cell responses triggered by adhesion receptor activation, would
be of great interest, as these two factors are believed to be
large contributors to MSC fate decisions [10,11]. An
understanding in these areas would help in refining suspension
bioreactor protocols, such that clinical expansion could be
attained for BMA-derived cells. Additionally, triggers for
promoting non-mesenchymal differentiation, i.e. neural tissue
development, could be more readily identified.
C. Using Artificial Neural Networks for media development
Media development is commonly executed through the use
of factorial (statistical) design methods. The effects of a
certain number (k) of test parameters (e.g. cytokines) are
selected and their 2k number of combinations are tested
experimentally. In order to achieve statistical significance,
each study must be conducted n number of times, on m
independent cell sources. Although statistical experimental
designs are powerful tools for efficient data collection, they
lack luster when applied to large number of variables.
Logan – Mitigating the Challenges of Clinically Relevant Mesenchymal Stem Cell Expansion Through Systems Biology
Furthermore, the results cannot be as easily generalized and
applied to similar cell types (without additional
experimentation). BMA-derived MSCs have not been shown
to proliferate to the bioreactor medium, nor traditional BM cell
culture medium. While many other factors (i.e. cell extraction
technique) may contribute to this, it is necessary to exclude
contribution by un-optimized cytokine concentration and
media.
Artificial neural networks (ANN) and genetic algorithms
have been used as an alternative method to design and
optimize fermentation mediums for recombinant protein
production (Nagata, 2003). Stochastic methods (e.g. ANN) do
not require unimodality of the response surface (i.e. the cell) or
extensive experimental testing. Although these stochastic
search methods are inefficient for simple problems, they are
promising for the more complex problems with large variable
spaces [21], such as the perceived optimization problem for
BMA-derived MSC. As such, the input space for an ANN can
be optimized through the use of genetic networks to reflect
design parameters such as initial population size, mutation
rations, kinetic proliferation data, lineage and so forth [22].
The large amount of experimental work necessary to train the
networks to achieve a ‘global optimum’ is perhaps as large as
those required for a comparable statistical approach. However,
the systems biology approach will help to narrow down the
region in which this optimum will lie, at which point a
statistical method can be used to complete the remaining
experiments [21].
potential; however, to attain clinically relevant quantities (i.e.
both uncommitted and highly-proliferative) of these cells is
challenging. Systems biology, through the use of genetic and
neural networks, could be the key to removing some of the
repetitive nature of future experimental work while increasing
current understanding of mesenchymal stem cells.
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