Download Prospering on Adipose for regenerative treatment

MMG 445 Basic Biotechnology (2011) 7:24-30
MMG445.5213335
Prospering on the Fat of the Land: Adipose-derived stem
cells as an industrially-viable resource for regenerative
treatment
Alex Hoekstra
Department of Microbiology and Molecular Genetics, Michigan State University
Correspondence: [email protected]
The field of regenerative medicine can be defined by its unique objectives: to restore and
recover – rather than merely repair or preserve – damaged tissue. Stem cells offer a means of
achieving this goal, but broadly-applicable scalability has been limited by numerous
challenges including availability, purity, tissue development potential and transplant-related
immunogenicity. This review will address these issues through a specific examination of a
unique subset of adult stem cells, derived from adipose tissue.
Abbreviations ASC - Adipose-derived Stem Cell, ESC - Embryonic Stem Cell, iPSC - Induced
Pluripotent Stem Cell, MSC - Mesenchymal Stem Cell
Introduction
The application of stem cells represents an
enormous potential for serving a market in
regenerative treatments. The ability to restore full
function to damaged tissue, beyond the normal
capabilities of the body, is a highly-valued
development in the biotechnological realm with
far-reaching implications. The goals of these
regenerative treatments can be achieved through
the application of stem cells [1-12]. Stem cells can
be defined as “primitive” cells that have the
capacity to differentiate into a variety of tissue
types when induced to do so. Stem cells offer the
potential to restore function to damaged tissue by
way of inducing the growth of new tissue which
can be specialized for the function of that which
was damaged.
At present, the widespread availability of stem
cells as an effective treatment for human tissue
damage and disease is hindered by a number of
technical challenges. Industrial viability requires
consistency, purity, efficacy, cost-effective
scalability and non-patient-specificity in order to
address the needs of a significant market. Stem
cell therapy has thus far faced barriers on all of
these fronts. The abundance, availability and
purity, the efficiency and complexity of
differentiation, and the patient-specificity related
to stem cells have been the subject of research
attempting to address these barriers to their
broad commercialization as a therapeutic agent
[7-12].
One variety of stem cells that exhibits a number of
unique features contributing to the practicality of
its use on an industrial scale is derived from
human adipose tissue. Called adipose-derived
stem cells (ASCs), this variety of cells expresses a
number of unique characteristics which qualify
them as an optimal resource for regenerative
tissue applications on an industrial (as
distinguished from an individual, patient-specific)
scale. Included among these unique features are
factors contributing to abundance and availability
of the stem cells [1-8,9•], their capacity to
differentiate into numerous tissue types and their
low immunogenicity [8,9•].
Distinctions between stem cell
Varieties
Stem cells are characterized both by their source
and potency (their ability to differentiate into
multiple tissue types). The varieties that offer the
greatest potential are embryonic stem cells
(ESCs), induced pluripotent stem cells (iPSCs) and
adult stem cells. ESCs and iPSCs have been the
focus of numerous studies [13-15] because both
varieties are pluripotent and abe to selfreproduce. Both, however, are limited in
abundance, availability (involving both biological
and ethical constraints) and patient-specificity
with regard to immunogenicity [9•,16].
Mesenchymal stem cells (MSCs), also known as
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Hoekstra A
“adult stem cells”, on the other hand, offer
availability and immunocompatibility while
maintaining multipotentiality. MSCs have been
isolated from a variety of human tissues, including
bone marrow, dental pulp and adipose tissue
[8,9•]. Adipose-derived stem cells (ASCs) possess
a number of unique advantages that contribute to
their potential for widespread (industrial-scale)
implementation.
Surface marker proteins are used to distinguish
stem cell types. Regardless of the methods for
isolation, culture or Figgrowth time, ASCs express
similar proteins as previously-established bone
marrow-derived MSCs [9•,10].
Stem cell characteristics necessary for
clinical application
In order to be considered a viable
candidate resource for clinical
regenerative therapies, the following
characteristics have been proposed as
criteria for stem cells [8,9•,11•].
1. Able to be collected in abundant
quantities (millions to billions of
cells);
2. Able to be collected with a
minimally invasive procedure;
3. Able to differentiate into a variety
of tissue types in a regulatable and
reproducible manner;
4. Able to be transplanted to either
an autologous (self) or allogenic
(non-self) host in a safe and
effective manner.
Abundance and Availability
Abundance of MSCs derived from bone marrow
and dental pulp is limited. Collection of stem cells
from these tissues can be painful and yield only
relatively small quantities of viable cells. Adipose
tissue, in contrast, is not in short supply. Per gram,
adipose tissue yields a 500-fold greater number of
MSCs than bone marrow [9•]. Further, the
collection of adipose tissue from a human subject in
substantial quantities can be done with a
minimally-invasive, low-risk procedure that offers
little discomfort to patients. Lipoaspiration (or
“liposuction”) is currently performed across the
United States at a frequency of nearly 400,000
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procedures per year. Each lipoaspiration procedure
routinely produces from 100 mL to more than 3 L
of adipose tissue [11•]. Adipose tissue has a density
of approximately 0.9 g/mL [17], thus a single
lipoaspiration procedure can yield from between
90 and 2,700 grams of raw adipose tissue. On
average, each gram of adipose tissue yields
approximately 5 x 103 stem cells [9•]. ASCs can be
characterized as highly abundant relative to
alternative stem cell varieties.
Isolation of MSCs from human adipose tissue
The protocols for isolation of MSCs from human
adipose tissue have been demonstrated and
replicated by numerous laboratories. Briefly, cells
are extracted and isolated from a tissue specimen
using processes that allow for up-scaling. These
techniques involve chemicals that are commonly
available (such as phosphate-buffered saline [PBS],
collagenase, fetal bovine serum [FBS] and modified
Eagle medium [MEM]) [11•]. This process itself
represents a model that appears suitable for largescale, high-yield expansion to meet an industrialscale demand.
Potentiality and Differentiation
ESCs convey totipotency (a capacity to
differentiate into virtually all cell lineages) and
iPSCs convey pluripotency (a more limited, but
still extensive range of potential differentiation,
including
endodermal,
mesodermal
and
exodermal tissues). Adult stem cells such as MSCs
were previously thought to be limited in their
range of development into the cell line of their
origin. This attribute is termed “multipotency”
which had been used to characterize adult stem
cells has since been replaced by pluripotency
[11•]. MSCs have demonstrated the ability to
mature into a variety of tissues but are still
regarded as “multipotent.” Induced differentiation
of MSCs has resulted in osteogenesis (generation
of bone tissue), chondrogenesis (cartilage tissue),
myogenesis (muscle tissue) and adipogenesis (fat
tissue). ASCs have been widely demonstrated to
possess the ability to yield all of these
“mesenchymal cell lineages” [8-10,11•], and have
been further determined to possess the ability to
differentiate into non-mesenchymal tissues such
as
neuron-like,
endothelial,
epithelial,
hematopoetic and pancreatic cells [9•]. These
features are summarized in Table 1.
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Adipose-derived stem cells
Table 1. A Comparison of stem cell varieties
Potency
Totipotent
Stem cell name
Embryonic stem cells (ESCs)
Source
Embryonic tissue
Potential lineages
Endoderm
Ectoderm
Mesoderm
Extra-embryonic
Pluripotent
Induced pluripotent stem cells (iPSCs)
Adult somatic tissue
Bone marrow
Endoderm
Dental pulp
Ectoderm
Cord blood
Mesoderm
Placenta
Adipose tissue
Stem cell varieties, their respective sources and potential lineage pathways. Examples of
ectodermal tissues include nervous tissue, enamel and the epidermis. Endodermal tissue comprises
of the innermost layer of cells in an organ, including the lining of the gastrointestinal, respiratory,
urinary and auditory tracts, as well as the lining of the follicles of endocrine organs. Mesodermal
tissue comprises the middle layer of cells (existing between the endoderm and the endoderm), and
consists of the primary tissues of the muscular, circulatory, skeletal, and excretory, as well as
connective tissues. Extraembryonic tissue comprises of the structure surrounding a developing
embryo, including the placenta [18].
Pluripotent
Mesenchymal stem cells (MSCs)
In vitro differentiation of ASCs can be
accomplished through treatment with induction
factors, outlined in Table 2. In vivo application of
ASCs is accomplished through either autologous
or allogenic transplantation of isolated stem cells
into the damaged tissue.
Scalability
In order to meet the demands of a developed
market for the application of stem cells in
regenerative medicine, stem cell lines must not
only be available in quantities above what is
directly isolatable from tissue specimens, but also
applicable for both autologous (self) and allogenic
(non-self) transplantation. This requires first the
suppression of surface markers and antigens on
cells that would otherwise trigger an immune
response, and finally expansion of stem cell
numbers through large-scale culture.
Purity
Contamination of stem cell lines with unidentified
and undesired cells is another significant challenge
confronting their clinical implementation. The
presence of undefined cells reduces the efficacy of
induced differentiation and further contributes to
unintended differentiation of both ESCs and iPSCs
[14]. Isolation of pure stem cells has proven to be a
challenge for ESCs, iPSCs and adult stem cells. Due to
the relatively high abundance of stem cells in
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adipose tissue and the efficacy of isolation methods,
ASCs represent a unique opportunity for
establishing high-purity cultures. Due to a high
abundance of stem cells within an adipose tissue
sample, the effect of unintended loss of stem cells
resultant of purification processes is minimized.
The purity of ASCs can be determined by fluorescent
microscopy, which requires significantly fewer cells
than the more commonly-employed purity
assessment through flow cytometry [16,17].
Culturing, large-scale expansion and storage
Numerous studies have demonstrated that ASCs
can be propagated in vitro [8,9•,16,17,19-21].
Approximate doubling time of ASCs in vitro has
been reported to be between 2 and 4 days,
depending upon the origin including age, tissue
type (brown vs. white adipose tissue), the methods
of extraction, isolation and culture [9•]. Bioreactor
technology until now has principally been 3D
scaffolding, which is effective but costly [18-21].
Emerging bioreactor technologies involve the use
of homogenous liquid phase culture and may
provide a less expensive means of high-volume
expansion [19-21]. These reactors could provide a
direct means of not only expanding the number of
stem cells, but also a means of large-volume in vitro
differentiation through chemical treatment (as per
the induction factors listed in Table 2).
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Hoekstra A
Table 2. ACS lineage differentiation induction factors
Cell Type
Induction Factors
Adipocyte
Dexamethasone, isobutyl methylxanthine, indomethacin, insulin, thiazolidinedione
Chondrocyte
Ascorbic acid, bone morphogenetic protein 6, dexamethasone, insulin, transforming growth factor-beta
Osteocyte
Ascorbic acid, bone morphogenetic protein 2, dexamethasone, 1,25 dihydroxy vitamin D3
Endothelial
Proprietary medium (EGM-2-MV; Cambrex) containing ascorbate, epidermal growth factor, basic
fibroblast growth factor, hydrocortisone
Myocyte
Dexamethasone, horse serum
Cardiomyocyte
Transferrin, IL-3, IL-6, VEGF
Neuron-like
Butylated hydroxyanisole, valproic acid, insulin
Prolonged cultures (lasting greater than four
months) revealed the preservation of ASC telomere
length, implying that passage-related (age-related)
degeneration is minimal [8,11•]. Collas et al.
reported successful maintenance of pure ASC lines
for a period of over six months without any agerelated defects in cellular activity [22,23]. On the
other hand, at least one laboratory reported ASCs
cultured for more than four months exhibited
chromosomal abnormalities and induced a high
rate of tumors when implanted in immunodeficient
mice [8]. The conflicting results indicate that while
there does appear to be a means of sustaining
cultures long-term without inducing abnormalities
or defects, this method is not yet widely applied
and requires further investigation. Bunnell et al.
[11•] also describe a method of cryopreservation.
The results of these studies together indicate that
cultured cells, under the correct conditions, can be
sustained and preserved for future use.
Also worth noting is the diminished need for
culture of ASCs due to their relatively high
abundance when taken directly from tissue
specimens. As mentioned above, adipose tissue
yields roughly 4500 CFU-F of stem cells per
milliliter of original tissue sample (in contrast, bone
marrow yields as little as 100 CFU-F per milliliter)
[10,17]. This diminishes the need for culturing in
order to establish quantities sufficient for clinical
application.
(Non) Immunogenicity
Unlike both ESCs and iPSCs, which have
demonstrated immunogenicity and rejection in both
autogenic and allogenic transplantations [16], MSCs
exhibit low immunogenicity after allogenic
transplantation. Imanishi et. al [19] showed that the
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level of inflammatory cytokines in living hearts that
received allogenic transplants of MSCs as a treatment
for acute myocardial infarction returned to nearnormal levels only seven days post-transplant and
remained at normal levels over the following weeks
[19].
MSCs express fewer surface marker proteins and
antigens that induce host immune response and
rejection. Passaged ASCs, in contrast to newlyisolated cells, express diminished numbers of surface
antigens and fail to induce immune reactions when
co-cultured with allogenic immune cells [12].
Expression of surface proteins within the major
histocompatibility complex class signifies the
potential of ACSs for allogenic transplantation
without immunogenic response [10].
Successful human treatment for radiation-induced
tissue damage as well as for acute Graft Versus Host
Disease (GVHD) using allogenically-transplanted
ASCs [9•] indicates that these cells convey
immunosuppressive qualities in humans, reaffirming
their suitability in non-self transplantation, free of
immunogenic response or host rejection. In vivo
application of ASCs has demonstrated successful
restoration of tissue function in these patients, who
remained free from side-effects and immunogenic
host response after a series of follow-ups lasting a
period of 40 months [9•].
Such in vivo studies are, as yet, limited in the human
model, but extensive study has taken place to affirm
the allogenic immunocompatibility of adipose stem
cells in murine models [20,21]. Transplantation of
xenogenic (human) ASCs into mice resulted in
suppression of inflammatory and immune responses,
reducing the stimulation of numerous cytokines and
chemokines [20] (Figure 1A) and inhibiting the
activity of host macrophages [20,21] (Figure 1B).
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Adipose-derived stem cells
Similar experiments involved the application of
autogenic or allogenic mouse ASCs, and resulted in
similar suppression of immunogenicity [21] (Figure
2). These studies indicate that ASCs represent high
immunocompatibility (in autologous, allogenic and
xenogeneic [20,21](non-species) transplantation)
and are thus an attractive candidate for broadlyuseable therapeutic application.
Figure 1. González et. al [20,21] demonstrated the inhibition of inflammatory
cytokines (A) and macrophage activity (B) in mice with induced arthritis through the
treatment with human adipose-derived stem cells (hASC).
Figure 2. González et. al [20] demonstrated inhibition of cytokine activity in mice with induced
arthritis when treated with mouse adipose-derived stem cells (mASC) (A). Further, assessment of
arthritic symptoms revealed that similar success was achieved through both autologous and
allogenic transplantation (B).
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Hoekstra A
Conclusion
Stem cells are the principle resource for
regenerative medicine, which has the distinct goal
of restoring full function of damaged tissue using
the native developmental processes of an
individual. The implications of this manner of
treatment are far-reaching, but limitations in stem
cell technology have thus far inhibited industrial
development. Adipose-derived stem cells in
particular demonstrate a variety of highly-valuable
characteristics with respect to the goal of
developing clinical application beyond isolated and
individualized treatments. Available in relatively
high abundance and expandable via sustained
culture, ASCs exhibit some of the properties
necessary for industrial scaling. Further, the tissue
lineages that have been successfully developed
through ASCs represent a group of tissues of
medical importance. Perhaps most significantly,
ASCs express immunosuppressive properties
including host cytokine level reduction and
macrophage inhibition, as well as cell surface
antigen reduction, making them highly suitable for
both autologous and allogenic transplantation
without host immune response or rejection.
Still, challenges remain before large-scale
manufacturing of stem cells can be employed for
widespread clinical use. As with any scaled-up
manufacturing method, purity on numerous levels
must be accounted for (from serum-free culture
media to sterile bioreactor conditions) in order to
ensure quality and consistency of stem cell
production. The optimal bioreactor conditions
must yet be determined before large-scale stem cell
differentiation and expansion can be achieved
without structurally complex and costly
bioprocessing systems. With further use, the effects
of long-term culture and storage will become
apparent, but must be clarified before use in a fullscale clinical setting.
A great many thanks are due to Professors George
Garrity and Clive Waldron, as well as to my fellow
students enrolled in the MMG 445 course who have all
actively contributed to and participated in review and
enlightening discussion of this and other manuscripts.
Acknowledgements
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