Stem cell horizons in intervertebral disc degeneration
Stem Cells and Cloning: Advances and Applications
Stem cell horizons in intervertebral disc
Joseph Ciacci 2
Allen Ho 1 2
Christopher P Ames 0
Rahul Jandial 3
0 Department of Neurological Surgery, University of California, San Francisco , San Francisco, CA , USA
1 Del E Webb Neurosciences, Aging and Stem Cell Research Center, The Burnham Institute for Medical Research , La Jolla, California , USA
2 Division of Neurosurgery, University of California , San Diego, La Jolla, California , USA
3 Division of Neurosurgery, Department of Surgery, City of Hope Cancer Center , Duarte, CA , USA
Intervertebral disc degeneration remains a pervasive and intractable disease arising from a combination of aging and stress on the back and spine. The growing field of regenerative medicine brings the promise of stem cells in the treatment of disc disease. Scientists and physicians hope to employ stem cells not only to stop, but also reverse degeneration. However, there are many important outstanding issues, including the hostile avascular, apoptotic physiological environment of the intervertebral disc, and the difficulty of obtaining mesenchymal stem cells, and directing them towards chondrocytic differentiation and integration within the nucleus pulposus of the disc. Given the recent advances in minimally invasive spine surgery, and developing body of work on stem cell manipulation and transplantation, stem cells are uniquely poised to bring about large-scale improvements in treatment and outcomes for degenerative disc disease. In this review we will first discuss the cellular and molecular factors influencing degeneration, and then examine the efficacy and difficulties of stem cell transplantation. stem cell transplantation
intervertebral disc degeneration; stem cells; disc disease; mesenchymal stem cells
Two anatomically distinct regions comprise the cartilaginous intervertebral discs (IVD)
of the human spine. Concurrently, the nucleus pulposus and annulus fibrosus provide
both fluid and viscoelastic support within the IVD. First, the central nucleus pulposus
(NP) occupies the internal structure of disc. It is filled with collagen type II extracellular
matrix (ECM) and hydrophilic proteoglycans. The unique extracellular matrix of the
NP provides “shock absorbing” capacity to the IVD derived from the water content of
its components. The NP is enveloped by the second component of the IVD, the annulus
fibrosus (AF). The AF is mainly collagen type I, and forms a fibrotic circumferential
boundary to the more liquid NP
(Paesold et al 2007)
. Consequently, the AF functions to
gird the viscoelastic NP and provide structural integrity and resistance to its extrusion
when compressive forces are applied to the NP.
The cellular composition of the IVD forms three separate sections. The concentric
lamellae of collagen I fibers of the AF surround the NP. The NP contains two unique types
of cells: a population of primitive notochordal cells, and a population of chondrocytic
cells. The former are most likely the vestige of an embryonic notochord cell that directed
the development of the IVD and spine. They disappear in humans after approximately
10 years of age. This may be due to differentiation into chrondrocytic cells or apoptosis.
Both regions of the IVD are bound above and below by endplates of cartilage. Some experts
believe that these cells play a role in the IVD niche that provides for successful
mesenchymal stem cell (MSC) differentiation into the cells of the NP
(Hunter et al 2003)
Mechanisms of disc degeneration
To attain the goal of cellular regeneration of the IVD, the nature of cellular degeneration
within the IVD must be determined. Most accepted cellular mechanism for IVD
degeneration focuses on the NP because of its importance
in maintaining a healthy and functioning IVD (Figure 1).
Specifically, the ECM of the NP fails to maintain homeostasis
for adequate collagen and proteoglycan synthesis
(Sive et al
. The first step in understanding this phenomenon
begins with identifying the molecular phenotype of the
NP cells. No marker currently exists to distinguish these cells
from common hyaline cartilage cells, since both have similar
ECM macromolecules. Furthermore, even if stem cells could
be instructed to differentiate into hyaline cartilage, they
would still lack the essential fluid properties of the NP, and
would fail to recreate normal function of the IVD. Also of
tantamount importance is being able to separate the different
cells. This could be accomplished first by elucidating the ratio
of proteoglycans and collagen within the NP
(Mwale et al
. Eventually, both the repopulation of NP cells and the
concomitant reproduction of adequate ECM, with a normal
proteoglycan/collagen ratio, must be realized for stem cell
regeneration of the IVD to be considered.
Among the culprits to be targeted when considering
IVD degeneration are the issues of diffusion of nutrients,
cell viability, proteoglycan synthesis, and disruptions in
collagen production leading to decreased proteoglycan and
collagen II in the IVD disc matrix. Molecular mediators such
as degrading enzymes, inflammatory mediators, and growth
factors are involved in all the above processes.
Matrix metalloproteinases (MMPs) are the best
characterized matrix degrading enzyme, and are major players in
IVD degeneration. MMPs degrade various collagens, but
their activity is post-transitionally inhibited by the binding
of inhibitor of matrix metalloproteinases (TIMPs). The
contribution of these enzymes to IVD degeneration most
likely results from an imbalance of normal ECM collagen
degradation by MMPs and MMP inhibition by TIMPs
Maitre et al 2004)
. Proteoglycan and cathepsins degrading
enzymes, such as aggrecanase-1 which targets aggrecan,
have also been suspects in IVD degeneration.
Pain manifesting from IVD degeneration may be linked
to participating molecular inflammatory mediators. Though
heterogeneous models have generally been used to
investigate cytokines, they clearly play a role. Some interleukins
identified may even recruit MMPs. In the same way, growth
factors involved in IVD degeneration, such as transforming
growth factor beta (TGF-B), insulin growth factor-1 (IGF-1),
and fibroblast growth factor (FGF), have also been examined.
These factors function as mitogens that increase rates of
mitosis and proliferation of essential IVD components. For
instance, TGF-B induces increased proteoglycan production
and inhibits MMPs (leading to less ECM degradation) in
(Pattison et al 2001)
. Bone morphogenic proteins
(BMPs) produce similar effects as growth factors but are
considered morphogenic since they are highly chondrogenic.
However, though their function has been delineated, no
clear correlation has been seen between factor levels and
IVD degeneration. Effects on degeneration varied widely
in the presence of different factor levels, although some of
this confusion is probably attributable towards the
heterogenous assortment of investigations, some of which included
extruded discs. Definitive studies remain outstanding, but
many hypothesize that increased growth factor expression
is a likely physiological response to disc herniation that
encourages disc repair.
Transcription factors have also been implicated in
degeneration since ECM turnover is also under the direction of
genetic regulation. SMADs and latent membrane protein-1
(LMP-1) are intracellular regulators that stimulate
proteoglycan and collagen II synthesis by upregulating BMP-2
(Yoon et al 2004)
. Sox9 is another factor that
promotes collagen II expression within the disc by increasing
collagen II mRNA transcription
(Li et al 2004)
The molecular pathways involved in IVD degeneration
are not all the natural consequences of stress and aging, but
can also be influenced by genetics. Gene polymorphisms
for ECM proteins, like aggrecan, have been associated with
disc and early multilevel degeneration
(Doege et al 1997)
A polymorphism in cartilage intermediate layer protein
(CLIP) has also been recently correlated with susceptibility
to disc degeneration
(Seki et al 2005)
. Because of the limited
innervations and blood supply of the intervertebral discs, the
pathology of their degeneration hinges upon the interaction
between a wide array of environmental, genetic, and
molecular factors. Therefore, the therapeutic strategy will have to be
a concerted approach that addresses all these issues.
The aforementioned mechanisms for molecular and
cellular degeneration are important components of the
clinical presentation of disc degeneration. However, it should be
noted that not every patient that has degenerative disc disease
has back or leg pain
(Jensen et al 1994)
. This underscores
the fact that the mechanisms of pain are not well
understood. The degenerated IVD is associated with progressive
dessication, loss of biomechanical properties, loss of disc
height, and in some cases disc herniations
(Haefeli et al
. These entities must be clinically appreciated because
stem cell mediated IVD repair would not obviated the need
to remove a herniated disc compressive a spinal root.
Current experience and knowledge focus on stem cell mediated
IVD repair with the objective of returning the disc ECM to
its premorbid state and allowing for the imbibition of water
and subsequent return of disc height. This may or may not
be associated with pain relief, but currently offers the best
cellular strategy for disc repair. In fact, a cellular strategy
could be used for multiple aims, not just disc repair, but also
the delivery of anti-inflammatory agents to help with the
ultimate clinical objective- reduction of pain.
MSCs and the IVD
Originally discovered 30 years ago, mesenchymal stem
cells (MSCs) are the elusive vehicle for cell therapy in
(Friedenstein et al 1976)
. They are valued for their
multipotency, or ability to differentiate into cell types of
mesenchymal origin (fat, bone, cartilage, etc.), and down
the necessary cell lineage for regeneration or replacement
of degenerated disc cells
(Prockop et al 2003)
. There are
two main strategies for acquisition of these desired somatic
stem cells: through manipulation of embryonic stem cells
(ESCs), or through extraction of MSCs from the fat or bone
marrow of the patient.
ESCs are extracted from the inner cell mass of blastocyst
stage embryos. The promise of ESCs lies in their plasticity
and immortality. These cells are pluripotent, that is, through
specialized culture techniques, ESCs can be induced to
differentiate into cells of all three germ layers (endoderm,
mesoderm, and ectoderm). Indeed, the plasticity of these cells
is much greater than any one group of somatic stem cells
such as bone marrow derived MSCs. Furthermore, once in
culture, ESC lines can be maintained indefinitely, providing
an everlasting source of cells for implantation. There is still
much to uncover about how to derive chondrocytes or even
MSCs from human ESC lines, and the danger of teratoma
formation is always present with ESCs
Instead of direct MSCs differentiation, recent investigations
in the field point towards neural crest stem cells (generated
from ESCs) as a source of MSCs, presenting an alternate
possibility of a reliable and efficient method of MSCs derivation
from human ESCs
(Lee et al 2007)
Even though ESCs are uniquely versatile, adult
MSCs remain the ideal candidate for IVD repair. Readily
extracted from adipose tissue or bone marrow, they offer
an autologous source of cells with low risk of infection
and immunogeniticity. In addition, there is a long history
of in vitro manipulation and clinical in vivo investigations
for orthopedic trials with these cells
(Horwitz et al 2002)
Despite the fact that true MSC yield from bone marrow
aspirate is less than 0.01%, their proliferative capacity of
makes this a sufficient amount for MSC or MSC derived
chondrocyte mediated IVD regeneration
(Pittenger et al
. Adipose tissue is also an attractive substitute source of
MSCs because of the relative ease in procuring fat over bone
marrow. However, some studies have found that the gene
expression profiles of bone marrow derived MSCs match
that of native cartilage more closely when differentiating into
(Winter et al 2003)
. Bone marrow and adipose
derived MSCs also have differing properties when evaluated
for cell surface markers
(De Ugarte et al 2003; Huang et al
. Thus, until adipose MSCs are more thoroughly
investigated and understood, bone marrow MSCs remain the
most efficacious option for stem cell IVD therapy.
Preclinical studies of MSC transplantation exist with disc
degeneration typically induced with a needle puncture method,
and subsequent evaluation of degeneration before and after
MSC transplantation. Though this does not accurately represent
the complex disease and degeneration process that normally
occurs in humans, it is successful in demonstrating the
regenerative capabilities of MSCs in the presence of IVD dessication
(Leung et al 2006)
. Sakai and colleagues (2006) used a rabbit
model of nucleus aspiration to induce degeneration. They
injected MSCs embedded in an atelocollagen matrix where they
persisted over a month, amplifying the proteoglycan content
of targeted discs. Implantation of autogenic MSCs was shown
to preserve annular structure, restablishe disc nuclei positive
for glycosaminoglycan and keratin sulfate proteoglycans, and
partially restore disc height and hydration in similar studies
(Leung et al 2006)
The limiting factor for exploiting stem cells for therapeutic
use is obtaining well characterized cells for transplantation.
Directing the appropriate differentiation of MSCs (and ESCs)
is a complex molecular and cellular puzzle that is contingent
upon not only the inherent properties of cells, but also the
environment in which they are cultured.
The soluble factors TGF-B and BMP are necessary
components of culture media used to induce in vitro
chondrogenic differentiation of MSCs. In fact, careful use
of soluble factors in media can lead to chondrogenesis with
a genetic profile more analogous to IVD tissue than articular
cartilage (Figure 2)
(Steck et al 2005)
. Another method of alter
MSC microenvironment to trigger chondrogenic differentiation
involves co-culturing with different cell populations to take
advantage of cell-cell contact and molecular signal activation.
Utilizing the autocrine and paracrine factors secreted by one cell
type leads to the activation of cell surface receptors on MSCs.
Experiments culturing human NP cells and MSCs found that
differentiation was reliant on cell-cell contact by looking at gene
expression of Sox9, type II collagen, and aggrecan (Figure 3)
(Richardson et al 2006)
The three dimensional properties of the culture system
have also shown to exercise substantial influence on the
process of cell fate determination. MSCs are pelleted down
into a dense micromass before addition of soluble factors to
recreate the in vivo state that leads to cartilage formation.
This structure helps direct the chrondrogenic cascade of MSC
differentiation from micromass into cartilage. Mesenchymal
condensation allows for extracellular signaling molecules
such as Wnt glycoproteins and N-cadherin to form cadherin
and connexin adhesion complexes for the beginning stages of
ECM formation. Cartilage then begins to form on this three
dimensional scaffold. Plating density of MSCs prior to soluble
factor addition also influences the efficiency of
differentiation (Figure 4). This is because plating density can change
the cell morphology; specifically, wider spindle shaped cells
corresponding with denser MSCs plating. (Figure 5). Wider
cells also have an increased propensity to differentiate after
exposure to soluble factors in vitro
(Sekiya et al 2002)
. In this
way, employing density dependent culturing techniques can
produce cartilage formation from MSCs in vitro, and increase
the efficiency of MSC differentiation. Another technique
described to direct MSCs toward the desired cell fate has been
with co-culture systems, where the differentiated cell types
provide the autocrine factors to increase the amount of nucleus
pulposus cells in vitro
(Yamamoto et al 2004)
. Clearly, there
are many microenvironmental considerations to be made
when designing an optimal in vitro MSC culturing system
Visage et al 2006)
. Exposure to a particular microenvironment
may result in physiological variations that can be genetically
perpetuated to daughter cells, epigenetically conditioning
them to a particular cell fate
(Gregory et al 2005)
MSC implantation to IVD and its options
Stem cell therapy can easily be adapted in the clinical setting
(with or without MHC typing) once an appropriate delivery
“package” of cells and molecular adjuncts is devised. The
objective, with our present understanding, would be to restore
disc height by decreasing the dessication of degenerated
discs. Although, disc degeneration is associated with back
pain, it is clearly not the only contributor. As such, stem cell
mediated disc repair would aim to remedy on component
of a complex disease entity. Knowledge and experience
gained, along with increasing understanding of
multifactorial mechaisms of back pain would lay the foundation for
further advances. With that noted, the delivery “package”
engrafted stem cells. Ultimately, the best clinical intervention
would incorporate a personalized strategy for each patient
and the degree and type of disc degeneration identified.
The tools for this strategy would include, but not limited
to, cell transplants, biodegradable implants, and internal
fixation as needed.
A large number of NP like cells will need to be generated
for proficient IVD repair. The NP cell type is not well
characterized, but NP cells identified by their production of the
requisite ECM components can suffice. Though autologous bone
marrow derived MSCs are the ideal candidates for therapy,
allogenic MSCs could serve as a more economical substitute.
MSCs lack HLA class II receptors and sibling donors should
not generate an immune response in recipients
A cocktail of MSCs or differentiated NP cells would have to
include molecules that promote the survival and engraftment
of transplanted cells, and be rigorously tested in large animal
models before clinical trials are undertaken. It will also be
imperative to develop approved protocols of MSC culture
and differentiation with strict quality controls to ensure safety
before application in humans. Once the science and methods
of MSC manipulation are better elucidated, IVD regeneration
with stem cell therapy could be a viable treatment option for
patients. In fact, osteogenesis imperfecta is already being
treated effectively with MSCs and presents a model of the
potential of MSCs for regenerative medicine. Developing this
therapy to the unique cartilaginous tissue of the IVD could
be the safest, most efficient application of stem cell biology
to disease processes treated by surgeons.
The authors report no conflicts of interest in this work.
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