Journal of

Therap
y

urnal of St
e
Jo

Cell Resea
r

&
ch

m

Stem Cell Research & Therapy

ISSN: 2157-7633

Duntsch et al., J Stem Cell Res Ther 2014, 4:11
DOI: 10.4172/2157-7633.1000246

Research Article

Open Access

Isolation and Characterization of Adult Spinal Disc Stem Cells from
Healthy Human Spinal Disc Tissues
Christopher Duntsch1-3*, Erika Dillard1 and Umar Akbar1-3
1
2
3

University of Tennessee Health Sciences Center, Department of Neurosurgery, USA
Hybrid Bioscience, Inc., USA
Discgenics, Inc., USA

Abstract
This report details the isolation, culture, and characterization of spinal disc stem cells derived from human adult
spinal disc tissue specimens. Using stem cell suspension culture methods and biology, human adult spinal disc stem
cells were isolated and monoclonally cultured into multicellular sphere-like clusters (discospheres). Discospheres from
the first culture series were collected, processed, and replated as single stem cells for serial expansion studies using
suspension culture, demonstrating linear expansion was possible. Discospheres and adult spinal disc stem cells were
plated on matrix coated culture surfaces in stem cell media for several hours to allow fixation, and assayed for the
stem cell biomarkers. Discospheres and adult spinal disc stem cells were plated on laminin-coated culture surfaces
in chondrogenic media and culture conditions for 14 days to differentiate them into NP cells. NP cells cultured from
these experiments demonstrated NP morphology and phenotype; NP biomarker expression, secretion of extracellular
matrix, and the ability to be serially passaged with large volume expansion possible. Tissue engineering studies
using the “burst kinetic assay”, demonstrated that discospheres have remarkable intrinsic developmental and tissue
engineering biology that is robust and organized. In summary, adult disc stem cells and NP cells have been isolated,
cultured, and characterized, from healthy spinal disc tissues. These findings demonstrate the important potential to be
explored for using stem cell based tissue engineering for the treatment of degenerative disc disease (DDD).

Keywords: Intervertebral disc tissue; Nucleus pulpous; Degenerative

disc disease; Adult spinal disc stem cells; Adult stem cell biology;
Matrix biology; Tissue engineering; Regenerative medicine

Introduction
Adult intervertebral spinal discs are the primary joints of the human
spine, providing the critical stability, flexibility, and biomechanical
support needed by bipedal animals [1-4]. The intervertebral spinal disc
joint consists of three major anatomical structures: an outer capsule,
the annulus fibrosis; and the nucleus pulposus; and the vertebral
endplates. The spinal disc of mammals begins to develop early in fetal
gestation, and originates from notochord cells (NC) that coalesce into
cellular clusters containing large vacuolations [5-7]. Early NC cells
are the primary cell population of the developing fetal and neonatal
spinal disc. They have a driving role in disc tissue development and
disc biology. For example, they actively direct the ordered assembly
of disc tissue extracellular matrix, and they secrete connective tissue
growth factors and extracellular matrix that is low in collagen and high
in proteoglycan content [5-7]. In humans and some species the NC
population classically is thought to rapidly disappear around age 3. The
cell population that replaces notochordal cells with aging is nucleus
pulpous cells [NP].
Most adult organs and tissues have some capacity to repair and
regenerate cells, tissues, and their structural and functional biologic
properties. Stem cells and stem cell biology have been demonstrated
in recent years to play a significant role in repair and regeneration
in adult human tissues. Conversely, the adult tissues and cells of the
intervertebral spinal disc are unable to repair and regenerate in a
similar manner in the face of spinal trauma, DDD, and aging. The tissue
biology of the spinal disc is unlike any other in the human body. The
spinal disc is a closed environment that is cell poor, harsh, avascular,
and biologically formidable. Spinal disc tissue is hypoglycemic,
hypoxic, acidic, and hypometabolic. Very few cell types can thrive
in this environment; much less survive [8-13]. Spinal disc tissue is
susceptible to the early onset of degenerative disc disease (DDD),
J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

which is arthritic in nature, and continues to progress in humans as
they age. DDD is poorly understood, ubiquitous in humans, and has
no boundaries, including gender, ethnicity, and demographics. DDD
is one of the most common and morbid human diseases of the last
several decades [14-18]. Indeed, DDD is one of the most common
reasons to see a physician, to be admitted to a hospital, and to have a
spinal surgical procedure [15]. The large and aging patient population
represented, the impact on the work force and quality of life, combined
with the costs of medical care, make DDD a medical and economic
issue on a global scale [15].
In humans and some species the NC population classically was
thought to rapidly disappear around age 3, as above. However, it
is clear at this time that there are likely several different cell types
comprising intervertebral disc tissue. For the purposes of this report,
spinal disc tissue and spinal disc cells are refers to nucleus pulposus
tissue and nucleus pulposus cells, respectively. Some animals do not
lose their notochordal cell population as they age, and do not develop
DDD (for example, nonchondrodystrophic dogs, rats, rabbits, and
mice) [16-19]. This has led to some concern about which animal
models are appropriate for studies of the intervertebral spinal disc, and
the validity of the results of past studies with these models [15,20,21].
The issue at hand is not the quality or validity of the research in

*Corresponding author: Christopher Duntsch, MD, PhD, 1525 E. Jamison Place,
Centennial, CO 80122 USA, Tel: +469-500-3470; Fax: +303-730-1526; E-mail:
hybridbiomed@icloud.com
Received October 20, 2014; Accepted November 19, 2014; Published November
21, 2014
Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of
Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc Tissues. J Stem Cell
Res Ther 4: 246. doi:10.4172/2157-7633.1000246
Copyright: © 2014 Duntsch C, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 2 of 8
general. Instead, it relates to how relevant these models and studies
are to the human condition. Conversely, other animal species (as
described above for humans) including steadily lose their notochordal
cell population early in life [for example, bovine and porcine], and
subsequently develop DDD as they age [21-24]. Recently, it has been
reported that notochordal cells may persist in adult human spinal
tissues [so called notochordal remnants]. These cells are smaller, have
different morphology than their precursors, and otherwise appear to
lack notochordal phenotype in vivo [15,21,22]. Interestingly, MCann
et al. using an elegant transgenic and genetic experimental approach
demonstrated that all adult spinal disc tissue cells may by derived
originally from notochordal cells [17]. This has significance from a
developmental biology and functional biology perspective, given the
role of notochordal cells in the development of the fetal spinal disc, and
their ability to drive healthy NP biology and function in adult spinal
tissue studies in vitro [18-20,25].
Adult stem cell biology is an established biologic paradigm those
decades in the making, with the discovery of rare atypical cells in
bone marrow with an extraordinary phenotype and array of cellular
properties [8-13,25-27]. Hematopoietic Stem Cells (HSCs) can divide
clonally to create identical stem cells, and maintain and/or expand the
stem cell population in response to external biologic cues. Conversely,
HSCs divide asymmetrically and give rise to stem cell progenitors and
their respective terminal lineage blood cell types, as a part of their
normal in vivo biology. HSCs have features of immortality, and can
be serially passaged in vitro or in vivo several times. HSCs represent
a small fraction of the bone marrow and blood cell population (1 in
10,000 cells, or 1 in 100,000 cells, respectively), and are quiescent
relative to cell population and cell proliferation kinetics. HSCs express
stem cell biomarkers that can be used to identify them in bone marrow
aspirates, blood samples, and bone marrow cell cultures (i.e., CD34).
Finally, HSCs are known to be maintained in vivo and in vitro with
specific stem cell growth factors, receptors, and signal transduction
cascades. Neural stem cells (NSC) were isolated and characterized from
murine brain tissue by Reynolds and Weiss, and by Snyder et al. in
the early 1990s [28,29]. NSCs were found to be rare and atypical cells,
with a remarkable phenotype and array of cellular properties (similar
to HSCs) [30-33]. NSCs can divide clonally and/or asymmetrically,
and both are influenced by external environmental cues. NSCs can
be serially passaged and differentiated into neural progenitors and
terminally differentiated lineages of the central nervous system (i.e.,
neurons, astrocytes, oligodendrocytes, and ependymal cells). NSCs
express known biomarkers (i.e., Nestin), represent a small fraction
of neural tissue cells, (<0.1%), and are typically in quiescence, rarely
undergoing cell division [33].
Over the last several decades, stem cell research for the treatment
of DDD has focused on cell and stem cell based therapies derived
from: 1) nonspinal tissue niches (i.e., chondrocytes, adipose stem cells,
mesenchymal stem cells, etc.); or 2) tissue niches in close proximity
spinal disc tissue (i.e., hyaline cartilage, annulus, etc.). Cells and stem
cells from these tissue niches were delivered directly to spine disease
models, or combined with tissue engineering biology and then used
for therapeutic studies (in some examples, reprograming cell biology,
to induce a more NP-like phenotype in the cells being delivered, was
included) [34-38]. Most of these studies demonstrated some degree
of safety and/or efficacy, although the data and results are typically
limited to biochemistry, cell biology, and molecular biology assays [3438]. Conversely, Risbud et al., in 2007, reported for the first time, the
presence of a rare cell population that could be isolated from spinal disc
tissue that was characterized as having a mesenchymal stem cell (MSC)
J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

phenotype [39]. In 2010, Blanco et al. demonstrated the ability to
isolate and characterize cells from hyaline cartilage of the vertebral end
plates with a mesenchymal stem cell phenotype [40]. In these studies,
they assayed this cell population in parallel with bone marrow derived
MSCs, and found that both were similar in phenotype and cellular
properties. The conclusion from these studies was that BM MSCs could
be a source of stem cells for treating DDD, given their abundance and
the less invasive aspects of a bone marrow aspirate. In 2012, Navone
et al. demonstrated that NP cells isolated from degenerated disc tissue
could express neural and neurotrophic markers, suggesting there
was more (stem) cell plasticity to NP cells and spinal disc tissue than
previously thought [41]. In 2013, Erwin et al. isolated, cultured, and
characterized a “stem/progenitor” cell population from the spinal disc
tissue of the adult nonchondrodystrophic canine model, using stem
cell suspension culture methodology and biologics [42].

Methods
Collection of healthy adult human spinal disc tissue
specimens, and preparation of adult spinal disc tissue single
cell suspensions for stem cell culture
Spinal disc tissue specimens were collected from young adults by
a spine surgeon as part of a surgical procedure that involved lumbar
discectomy. All studies were done according to approved IRB protocols
at the Methodist University Hospital (Memphis, TN). The target
patient population of this first research effort was young adults with
healthy spinal disc tissue. Lumbar discectomy in young adults is rare
and most commonly done in the context of lumbar trauma, requiring
surgical fixation with lumbar interbody fusion and instrumentation. At
the time of surgery, a disc tissue specimen was collected by the surgeon
in a sterile manner, transferred to a 50 ml falcon containing 10 ml of
cold PBS, and transported to the lab for processing. Disc tissue was
placed in a 10 cm dish with sterile saline, and carefully dissected with
surgical instruments under magnification with surgical microscopy.
This step was done over several hours to carefully remove cell and
debris contaminant (i.e., fat, ligament, annulus, muscle, etc.). The
remaining disc tissue was washed, weighed, and placed in 10 cm dish
culture flasks supplemented with media and 0.125% trypsin EDTA
(0.25 mM/ml). The culture plate was placed on a slow rotating culture
apparatus for gentle mechanical stimulation, and digested for 3 hours
in a tissue culture incubator at 37°C, with 95% air, 5% CO2, and 100%
humidity. The single cell adult spinal disc tissue suspension resulting
from digestion was washed, triturated with a fire polished Pasteur
pipette, and resuspended in basic media cell for counting with Trypan
Blue. After staining, aliquots were transferred to a heamocytometer,
the cells were visualized with an inverted microscope, and counted (in
triplicate; the average used for plating); to ensure accurate cell counts,
and to assess for the presence of cell debris, apoptotic bodies, and dead
cells.

Plating of spinal disc stem cells in ultra-low density suspension
culture; culture of discospheres from monoclonally plated
adult spinal disc cells; serial passaging of discospheres
Single cell suspensions were resuspended at a cell density
of 20,000 cells per ml in 2 ml of 2X serum free N2 stem cell media
supplemented with 20-ng/mL basic fibroblast growth factor (b-FGF,
Sigma, USA), and 20-ng/mL epidermal growth factor (EGF, Sigma).
The resuspended disc cells were mixed with 2% methylcellulose in a 1:1
ratio. After mixing the stem cell composite and methylcellulose, a final
density of 10,000 cells per ml was achieved. The stem cell suspensions
were then plated in precoated ultra low binding 6-well tissue culture

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 3 of 8
plates (Corning Bioscience) at 2 ml/well, and placed in tissue culture
incubators at standard conditions. Fresh supernatant and growth
factors were added to the cultures every 3 days. Plated stem cells were
observed in culture over time with inverted microscopy to follow the
development of stem cell sphere clusters. The number of spheres per
well were counted at day 12-14 by defining the culture floor into grids
and counting the grids under 20X magnification with an inverted
microscope. To assess the capacity for discospheres from the first
culture series to be serially passaged, 300 spheres were collected from
several dishes, added to 50 ml falcon tubes with media and Trypsin
EDTA (0.25 mM/ml). The tubes were incubated for digestion for 5
minutes in a tissue culture incubator at incubator at 37°C, with 95%
air, 5% CO2, and 100% humidity. The single cell suspension from the
enzymatic digestion was washed, triturated with a fire polished Pasteur
pipette, resuspended in basic media for cell counting with Trypan
Blue. After staining, aliquots were transferred to a heamocytometer.
The cells were visualized with an inverted microscope and counted (in
triplicate; the average used for plating); to ensure accurate cell counts,
and to assess for the presence of cell debris, apoptotic bodies, and
dead cells. Single cell suspensions were resuspended at a cell density
of 20,000 cells per ml in 2 ml of 2X N2 stem cell media supplemented
with growth factors 20 ng/mL basic fibroblast growth factor (b-FGF,
Sigma, USA), and 20 ng/mL epidermal growth factor (EGF, Sigma).
The resuspended disc cells were mixed with 2% methylcellulose in a 1:1
ratio. After mixing the stem cell composite and methylcellulose, a final
density of 10,000 cells per ml was achieved. The stem cell suspensions
were then plated in precoated ultra low binding 6-well tissue culture
plates (Corning Bioscience) at 2 ml/well, and placed in tissue culture
incubators at 37°C, with 95% air, 5% CO2, and 100% humidity. Fresh
supernatant and growth factors were added to the cultures every 3
days. Plated stem cells and emerging discospheres were observed in
culture over time with inverted microscopy. The number of spheres
per well were counted at day 12-14 by defining the culture floor into
grids and counting the grids under 20X magnification with an inverted
microscope. The spheres from the second culture series were again
passaged as above two additional times (3 total passages).

Expression studies of stem cell and NP cell biomarkers
Adult spinal disc stem cells or discospheres were resuspended in
N2 stem cell media supplemented with growth factors as above, and
plated on cover slips coated with 50 μg/ml laminin (Life Technologies,
Gaithersburg, MD) for 8 hours to allow attachment. To differentiate
adult spinal disc stem cells into NP cells, the cells were plated on cover
slips coated with 50 μg/ml laminin (Life Technologies, Gaithersburg,
MD) as discospheres, or as single stem cell suspensions, and cultured in
chondrogenic media and conditions supplemented with 10% fetal calf
serum [29,36,41] for 14 days. Differentiated NP cells were assayed in
vitro in culture for morphology, phenotype, expression of extracellular
matrix, culture cell biology, and expression of biomarkers. Attached
stem cells or spheres were processed, fixed in 4% paraformaldehyde
(in PBS, pH 7.2, for 30 min at room temperature), followed by three
washes [5 min each, in PBS, pH 7.2]. The cells were permeabilized for 5
min in PBS containing 0.3% Triton X-100, rinsed in PBS two times, and
blocked for 20 min in PBS containing 10% normal goat serum (NGS).
Rabbit monoclonal IgG1 anti-CD133 (Sigma-Aldrich, USA), mouse
monoclonal IgG1 anti-Nestin (AbCam, UK), and mouse monoclonal
IgG1 anti-Survivin (AbCam, UK) were diluted in PBS containing
10% NGS overnight at 4°C, at dilutions of 1/200, 1/200, and 1/100,
respectively. Coverslips were rinsed in PBS three times, and incubated
with mouse anti-rabbit (FITC, AbCam, UK) or rabbit anti-mouse
(spectral red, AbCam, UK) secondary antibodies at dilutions of 1/1000.
J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

The coverslips were rinsed three times in PBS, mounted in Fluoro-mount
(BDH laboratory, Poole, UK). The signal  intensity and distribution
was visualized using confocal microscopy with fluorescent filters, and
the digital images were collected using standard confocal imaging
software. NP cells were processed, fixed in 4% paraformaldehyde
(in PBS, pH 7.2, for 30 min at room temperature), followed by three
washes [5 min each, in PBS, pH 7.2]. The cells were permeabilized for
5 min in PBS containing 0.3% Triton X-100, rinsed in PBS two times,
and blocked for 20 min in PBS containing 10% normal goat serum
(NGS) for 30 min at room temperature, followed by three washes [5
min each] in PBS, pH 7.2. Rabbit monoclonal IgG Anti-Collagen2
and mouse monoclonal IgG anti-Vimentin primary antibodies
(Boehringer Mannheim) were diluted at 1:200 in PBS containing 10%
NGS overnight at 4°C. Coverslips were rinsed in PBS three times, and
incubated with mouse anti-rabbit (FITC, AbCam, UK) or rabbit antimouse (Spectral red, AbCam, UK) secondary antibodies at dilutions
of 1/1000. The coverslips were rinsed three times in PBS, mounted in
Fluoro-mount (BDH laboratory, Poole, UK). The signal intensity and
distribution was visualized using confocal microscopy with fluorescent
filters, and the digital images were collected using standard confocal
imaging software.

2-Dimensional tissue engineering with 3-dimensional
discospheres (the burst kinetic assay)
Discospheres were plated on coverslips coated with 0.1% gelatin,
and cultured in chondrogenic media and culture conditions for 5
days. Imaging data was collected at 24 h intervals with inverted light
microscopy at various magnifications, starting on day 1, ending on
day 5 [28,35,40]. Discosphere cultured in the burst kinetic tissueengineering assay were observed in vitro in sphere and cell morphology
phenotype changes, expression of extracellular matrix, and several
culture cell biology parameters were documented.

Results
Isolation, culture, and characterization of adult spinal disc
stem cells from human spinal disc tissue
Human adult spinal disc tissue was collected from patients at the
time of spine surgery according to approved hospital IRB protocols.
The target patient population was restricted to healthy young adults
with clinically normal spinal disc tissue at the time of presentation,
and presumed normal spinal disc tissue biology and function prior to
surgery. Spinal disc tissue specimens were processed through several
steps in the lab to create single cell suspensions of adult spinal disc cells.
Spinal disc cells were plated in suspension culture in supplemented
stem cell media, at ultra-low clonal plating densities (10,000 cells per
ml). Methylcellulose was mixed with the stem cell/media composite in
a 1:1 ratio to restrict cell movement, and “suspend” cells in culture,
followed by plating of on ultra-low binding tissue culture plates. Figure
1A demonstrates the appearance of human adult spinal disc cells plated
in suspension culture at time 0 at ultra-low clonal densities. Several
critical culture biology parameters and paradigms can be observed
in Figure 1A: ultra-low density of cultured cells; relatively even
distribution of cells in the culture; lack of dead cells or debris; and a
space maintained between all cells and the tissue culture vessel walls
and floor. Plated stem cells were observed in culture over time with
inverted microscopy to follow the development of stem cell sphere
clusters. The number of spheres per well were counted at day 12–14
post plating. Figure 1B demonstrates the appearance of monoclonally
derived stem cell clusters with sphere-like morphology (discospheres),
on day 10 post-plating. In the first experimental series, the sphere

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 4 of 8
yields or early passages, but there was significant gain in disc stem
cell and discosphere yields in the latter half of the culture experiment.
The issues and challenges of the first experimental series are basic
and technical and are described in detail in the discussion and the
supplemental data section.

Expression of stem cell biomarkers in adult spinal disc stem
cells; expression of NP biomarkers in differentiated NP
cells, derived from adult spinal disc stem cells cultured in
chondrogenic media and conditions

yield was less than expected (~80 spheres per ml +/- 9; although the
spheres were viable, healthy, and had typical diameters, cell volume,
cell numbers upon further study).

Serially passaging of discospheres in suspension culture
To assess the capacity for discospheres from the first culture series
to be serially passaged, 300 spheres were collected from several culture
dishes, washed with PBS, and added to 50 ml falcon tubes with media
and prepared enzymatic solutions. The tubes were incubated for 5
minutes in a tissue culture incubator to allow enzymatic digestion.
The single cell suspension resulting from the enzymatic digestion
was washed, triturated with a fire polished Pasteur pipette to ensure
no clumps of cells remained, and resuspended in basic media for cell
counting. Single cell suspensions were resuspended at ultra-low cell
density in supplemented stem cell media. The resuspended single disc
stem cell suspensions were then plated on precoated ultra low binding
6-well tissue culture plates and placed in the tissue incubator. Fresh
supernatant and growth factors were added to the cultures every 3
days. Plated stem cells were observed in culture over time with inverted
microscopy to follow the development of stem cell sphere clusters. The
spheres from the second culture series were passaged twice more. At
the end of each culture series, the number of spheres per well were
counted at day 12–14 m as above. Thus, a total of 3 passages were
made from the stem cell culture product of the first culture series, and
this experiment demonstrated that linear expansion of the stem cell
population was possible (Figure 2, black line graph). As shown in the
figure, and discussed below and in the appendix, the overall yield of
spheres initially, and with serial passaging was far less than expected.
The initial yield was quite low when compared to other organ tissue
results. Further, the stem cell and discosphere frequency did not begin
to expand in suspension culture until after the discospheres or the
first culture series had been passaged once. In a second experimental
series, several revisions, additions, and changes to the suspension
culture protocols and bioreagents were made. Again, 300 spheres were
collected from culture plates, on day 14 post plating, washed, prepared
as single cell suspensions, and plated in suspension culture media and
conditions, as above. The spheres from the second culture series were
passaged twice more. Thus, a total of 3 passages were made from the
stem cell culture product of the first culture series, and demonstrated
that linear expansion of the stem cell population was possible (Figure 2,
red line graph). The second experimental series did not improve initial
J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

2-Dimensional tissue engineering with 3-dimensional
discospheres (the burst kinetic assay)
As functional proof of concept, a simple but very relevant tissue
16000
14000

Number of Spheres

Figure 1: Isolation, culture, and characterization of adult spinal disc stem cells
from human spinal disc tissue. A. Photomicrograph taken with light microscopy
at 20X magnification, of the first plating of adult spinal disc cells as a single
cell suspension, at ultra-low densities, in suspension culture (10,000 cells per
ml). B. Photomicrograph taken with light microscopy at 20X magnification of
monoclonally derived sphere-like cellular clusters (discospheres), comprised
of stem cells and early progenitors. Shown are spheres at day 10 of growth
in suspension culture.

Discospheres and single stem cells were resuspended in stem cell
media, plated on laminin-coated cover slips, incubated for 8 hours
to allow attachment, and then fixed and processed for biomarker
expression studies. As shown in Figures 3A-E (and data not shown),
Nestin and CD133 were expressed in discospheres in most cells at high
levels (single stem cell suspensions plated in parallel also had uniform
and high expression, data not shown). Survivin expression was
ubiquitous and strongly expressed in stem cells plated from single cell
suspensions (Figure 3F), but its expression in discospheres was much
lower (data not shown). NP cells were differentiated by plating them
on laminin-coated cover slips, in chondrogenic media and culture
conditions for 14 days. Differentiated NP cells developed appropriate
morphology and phenotypic changes (Figure 4), and secreted significant
extracellular matrix (Figures 4C and D). Differentiated NP cells could
be serially passaged and expanded exponentially in culture (Figure 5).
Finally, differentiated NP cells and expressed NP biomarkers including
Collagen 2 A, and Vimentin at strong levels (Figure 6).

12000
10000
8000
6000
4000
2000
0

P1

P2

P3

P4

Passages (2 weeks between each passage)

Figure 2: Linear graphs of serially passaging of discospheres in suspension
culture in two experimental series. In the first experimental series, 300
spheres were collected from culture plates, on day 14 post plating, washed,
prepared as single cell suspensions, and plated in suspension culture media
and conditions, as above. The spheres from the second culture series were
passaged twice more. Thus, a total of 3 passages were made from the stem cell
culture product of the first culture series, demonstrated that linear expansion
of the stem cell population was possible (Figure 2, black line graph). The
black line graph represents the expansion of stem cells derived from the first
culture series through serial passaging. In the second experimental series,
several revisions, additions, and changes to the suspension culture protocols
were made. Again, 300 spheres were collected from culture plates, on day
14 post plating, washed, prepared as single cell suspensions, and plated in
suspension culture media and conditions, as above. The spheres from the
second culture series were passaged twice more. Thus, a total of 3 passages
were made from the stem cell culture product of the first culture series, and
again demonstrated that linear expansion of the stem cell population was
possible (Figure 2, red line graph).

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 5 of 8

Discussion
The discovery and development of HSC biology, and subsequently
NSC biology, has led to breakthroughs in understanding, sophistication
of methods and biologics, and applications and approaches that can
be directed at other solid organs and tissues. In recent years, pioneers
in the field have pushed back against many fundamental paradigms
of NSC biology [41-44]. Nonetheless, extensive investigation, review,
and discussion, have led to new understanding, and the identification

engineering assay was used to study the developmental biology and
regenerative potential of discospheres (the “burst kinetic assay”).
Discospheres were plated on coverslips coated with gelatin, and cultured
in chondrogenic media and culture conditions for 5 days. Imaging data
was collected at 24 h intervals starting on day one. Figure 7A shows a
discosphere plated on a gelatin coated surface, and attached at 24 hours
post plating. Figures 7B and 7C show so called “burst kinetic” cell blastic
activity that occurs at the periphery of the discospheres at 48 hours
(with the appearance of spindle shaped elongated cells that radiate in
near 90 degree angles from the discosphere). Most often, spindle cells
were found to migrate from the sphere and change phenotype and
morphology around 72 to 96 hours (Figures 7D and 7E), transforming
into small, innocuous, rounded cells that were highly mobile, and
highly proliferative. Long-term studies with these assays demonstrated
that this cell population would continue to migrate and proliferate
until the culture surface was crowded with cells. Cell crowding and
cell contact with other cells and physical structures, resulted in yet
another change in morphology and phenotype (Figure 7F). Mature NP
cells began to appear, ceased proliferation and migration, and secreted
significant extracellular matrix around the cell periphery. The culture
product was a NP confluent culture bed, embedded in extracellular
matrix, with local tissue remodeling (i.e., appearance, properties, and
characteristics of prefabricated engineered tissue) (Figure 7F).
J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

Figure 4: Differentiation of human adult spinal disc stem cells into NP
cells, and characterization of their phenotype. Single stem cell suspensions
derived from discospheres were plated on cover slips coated with 50 μg/ml
laminin in chondrogenic media supplemented with 10% fetal calf serum, and
chondrogenic culture conditions, for 14 days. A. Photomicrograph taken with
light microscopy at 10X magnification of NP cells differentiated from spinal
disc stem cells over 14 days, at 70% confluency. B. Photomicrograph taken
with light microscopy at 10X magnification of NP cells differentiated from
spinal disc stem cells over 14 days at 100% confluency. C. Photomicrograph
taken with light microscopy at 20X magnification of NP cells differentiated from
spinal disc stem cells over 14 days at 100% confluency. D. Photomicrograph
taken with light microscopy at 40X magnification of NP cells differentiated
from spinal disc stem cells over 14 days at 100% confluency. Escalating
photomicroscopic magnifications are shown to demonstrate the significant
amount of secreted extracellular matrix in differentiated mature confluent NP
cells.

300
250
Cells (x 105)

Figure 3: Immunohistochemical assays of stem cell biomarker expression
in adult spinal disc stem cells. Discospheres and single stem cells were
resuspended in stem cell media, plated on laminin-coated cover slips,
incubated for 8 hours to allow attachment, and then fixed and processed for
biomarker expression studies. A. C. E. Negative controls for immunostaining
of CD133 (A), Nestin (C), and Survivin (E). B. D. F. Immunohistochemistry
for the expression of these stem cell biomarkers. B and D. The expression
of CD133 (B) and Nestin (D) in discospheres attached to laminin coated
coverslips for 8 hours. Photomicrographs taken with fluorescent confocal
microscopy at 40X magnification demonstrate uniform and high expression
of both biomarkers throughout the sphere structure. F. The expression
of Survivin in adult spinal disc stem cells plated on laminin coated culture
surfaces as single cell suspensions, as shown in photomicrographs taken
with fluorescent confocal microscopy at 40X magnification, demonstrate
uniform and high expression of Survivin biomarkers in attached spinal disc
stem cells.

200
150
100
50
0

2

6

12

16

Passages (weeks)
Figure 5: Linear graph of NP cells differentiated from spinal disc stem cells,
and serially passaged for expansion. 40 discospheres were plated on a T25
tissue culture flask, coated with laminin, in chondrogenic media and culture
conditions. The T25 culture was passage into a T75 culture flask at confluency.
The T75 culture was expanded into 3 T75 culture flasks at confluency.

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 6 of 8
of several caveats of note. Most of these issues, caveats, and outliers,
can be explained by one of three findings. First, not all adult tissue
stem cells are the same. There is heterogeneity at the tissue level that
carries forward in vitro with R&D and suspension culture methods
and approaches. Second, there is significant biologic rigor and good
research practice required for success with these approaches and it
is not uncommon to find outliers that relate to technical errors and
misconceptions by some researchers. Finally, there is still much stem
cell biology that is simply unknown at this time. Regardless, with these
caveats in mind, adult stem cell biology has stood the test of time, peer
review, and independent challenges. Thus, it is a rationale and proven
paradigm, in the present, to use this knowledge base and methodology
as a platform and approach to new stem cell research programs. Proof
of concept, can be found by noting the many adult stem cell discoveries
over the last few years in various solid tissues, disease models, and solid
cancers (i.e., breast, liver, prostate, neural crest, cardiac, etc.) [45-50].

Figure 6: Immunhistochemical assays of stem cell biomarker expression
in adult NP cells. Single stem cell suspensions derived from discospheres
were plated on cover slips coated with 50-μg/ml laminin in chondrogenic
media supplemented with 10% fetal calf serum and chondrogenic culture
conditions for 14 days. A. and C. Negative controls for immunostaining of
Collagen 2a (A) and Vimentin (C). B. and D. Immunohistochemistry for. B and
D. The expression of the NP cell biomarkers Collagen2a (B) and Vimentin
(D) in NP cells. Shown are photomicrographs taken with fluorescent confocal
microscopy at 60X magnification.

Figure 7: 2-Dimensional Tissue engineering with 3-dimensional discospheres
(the burst kinetic assay) Discospheres were plated on coverslips coated with
0.1% gelatin, and cultured in chondrogenic media and culture conditions
for 5 days. Imaging data was collected at 24 h intervals with inverted light
microscopy at various magnifications, starting on day 1, ending on day 5.
A. Photomicrograph taken with light microscopy at 60X magnification of a
discosphere cultured in the burst kinetic tissue-engineering assay at 24 hours.
B. and C. Photomicrographs taken with light microscopy at 60X magnification
of a discosphere cultured in the burst kinetic tissue-engineering assay at
48 hours (B) and 72 hours (C) demonstrating a dramatic “blastic” phase.
As shown in the figure, the cells at the sphere edge change dramatically in
phenotype, and become elongated spindle shaped cells that migrate from the
sphere and change phenotype and morphology. D. and E. Photomicrographs
taken with light microscopy at 20X magnification at 96 hours (D) and 120 hours
(E) of a discosphere cultured in the burst kinetic tissue engineering assay;
demonstrating the appearance of small rounded cells that migrate out across
the culture surface away from the sphere, and gain and cell migration and
proliferation biology. F. Cultures allowed to continue to grow were followed
over time. Long-term studies with these assays demonstrated that these cells
continue to migrate and proliferate, until the culture surface was crowded
with cells. At this time, the NP cells change morphology and phenotype,
differentiated into mature NP cells, ceased migration and proliferation, and
secreted significant extracellular matrix.

J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

As mentioned above in the introduction, Erwin et al. was able to
isolate and culture disc stem cells from adult canine models. Stem cells
were isolated that could survive harsh suspension culture conditions,
and grow into sphere-like cellular clusters over 2 weeks. Isolated stem
cells expressed a rich array of stem cell markers, and demonstrated
significant pluripotency in MSC (mesoderm) and NSC (ectoderm)
differentiation assays. This study is important and of note as a first
report of its kind. Namely, the author reports the isolation of stem cells
from adult mammalian spinal disc tissue, using stem cell suspension
culture methodology and biologics. However, the study did have
some limitations making the results difficult to interpret and apply
to humans, especially the aging population with DDD. First, by using
the nonchondrodystrophic canine model, the isolated stem cells were
derived from notochordal rich and healthy spinal disc tissue. Second,
the stem cell biomarker assays were done with semiquantitative PCR,
but without other assays to validate, and to correlate the expression of
mRNA with protein expression. Finally, despite excellent pluripotency
in these studies, they did not include differentiation assays and biology
of NP cells (the primary and terminal lineage cells of the tissue from
which the stem cells were isolated). These are caveats that should be
addressed and further investigated, given the potential and importance
of novel stem cell based therapies for treating DDD in the aging human
population.
This report endeavors only to present a new stem cell population,
and to prove the phenotype using classic and proven approaches and
criteria. The survival of adult spinal disc stem cells in these harsh
culture conditions, and their growth over time into discospheres from
adult disc stem cells and early progenitors is major proof of stem cell
phenotype. These findings support the discovery and isolation of a
spinal disc stem cell population in healthy adult spinal disc tissues. In
particular, contact inhibition (an integral part of suspension culture),
derived from 1) plating the cells at ultra-low density; 2) supplementing
stem cell media with methylcellulose; and 3) using ultra low binding
culture plates, is critical for stem cell selection and acts as a filter of
sorts. Late stage progenitors, and adult differentiated spinal disc cells,
cannot survive in these conditions, and undergo apoptosis. The ability
to serial passage NP cells is another proof of stem cell phenotype. In
this first report, only 3 passages were done, with the primary intent
of determining if serial passaging was possible, and if stem cell and
discosphere expansion was possible. Differentiation of adult spinal disc
stem cells into NP cells with appropriate phenotype, and capacity for
expansion is further proof of concept. Additionally, the expression of
stem cell biomarkers in discospheres, and NP biomarkers in NP cells
is strong evidence that a tissue specific stem cell has been isolated, that

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 7 of 8
can be give rise readily to the terminally differentiated lineage cell from
which it was derived. Finally, the robust tissue engineering biology in
the burst kinetic assay is functional evidence of value.
In summary, this report details the isolation, culture, and
characterization of spinal disc stem cells derived from human adult
spinal disc tissue specimens. Using stem cell suspension culture
methods and biology, we were able to isolate spinal disc stem
cells, and monoclonally grow them into multicellular sphere-like
clusters (discospheres). Discospheres from the first culture series
were collected, processed, and replated as single stem cells for serial
expansion studies using suspension culture, demonstrating linear
expansion was possible. Discospheres and adult spinal disc stem cells
were plated on matrix coated culture surfaces in stem cell media for
several hours to allow fixation, and assayed for the stem cell biomarkers.
Discospheres and adult spinal disc stem cells were plated on laminincoated culture surfaces in chondrogenic media and culture conditions
for 14 days to differentiate them into NP cells. NP cells cultured from
these experiments demonstrated NP morphology and phenotype; NP
biomarker expression, secretion of extracellular matrix, and the ability
to be serially passaged with large volume expansion possible. Tissue
engineering studies using the “burst kinetic assay”, demonstrated
that discospheres have remarkable developmental biology potential,
and in vitro tissue engineering biology that is robust and organized.
In summary, adult disc stem cells and NP cells have been isolated,
cultured, and characterized from healthy spinal disc tissues. These
findings demonstrate the exciting potential to be explored for using
stem cell based tissue engineering for the treatment of DDD.
Acknowledgements
This is novel report and first in class, and an exciting time for our research
program. This report is the first of its kind in the academic literature, and is the first
in a series (of many publications to follow) that come from a research program that
now is in its 8th year of activity (founded in 2005 at the University of Tennessee,
Department of Neurosurgery.) This research effort, over the years, has been
collaboration with faculty, neurosurgeons, residents, students, and the rest of the
team. We would like to acknowledge the following for the contributions and efforts
here and over the last many years. Jon Robertson, MD. Rano Chatterjee, MD.
Zoran Pavecevic, MD. Zorica Janjetovic, MD, Mariya Nazarova, PhD., Qihong
Zhou, MD, PhD, Bing Teng, MD, Terreia Jones, PharmD, Jeff Sorenson, MD, Julius
Fernandez, MD. Valery Kukekov, PhD, Gala Dulatova, MD. Raisa Krukalina, MD.

Funding
Methodist Neuroscience Institutional Grant 1.12534 (c)

Disclosures and Conflicts of Interest
There are no conflicts of interest for this submission. I would add for clarification,
that this research was done at the University of Tennessee, Department of
Neurosurgery, in the laboratories of the senior author. Early results and proof
of concept were deemed important for academic research, but also as having
potential for clinical science as well. Thus, early patents were filed to protect the IP
as recommended by leadership in the group. While this and subsequent studies
led to a formal biotechnology group in 2009 (life sciences intellectual property and
patents), the senior author of both patents and manuscripts, maintained at all times
an academic track, and agreed to patent these findings before publishing, only
after it was agreed that there would be no restriction by biotechnology in academic
endeavors, including publishing this research. The senior author was a Professor
in the department from 2000 – 2012, but was also the Chief Science Officer for the
company (Discgenics, Inc.), from 2006–2011 by request. He has no executive role,
no leadership responsibilities, and no control of the interests of this company at this
time. The senior author has been assigned equity in this company, for collective
efforts over 15 years that relate to this research program. However, all research
reported here, was patented many years ago, and has been issued by the USPTO
and by the WPTO. Thus, the effort to publish the same science, publically and
in a peer review context, is a commitment to academics, and to the fundamental
ideology that research and scientific results are public domain and should be
shared.

dysfunction, adaptation, and enhancement. J Spinal Disord 5: 383-389.
[PubMed]
2.  Panjabi M (1992) The stabilizing system of the spine. Part II. Neutral zone and
instability hypothesis. J Spinal Disord 5: 390-396. [PubMed]
3.  Solomonow M, Zhou B, Baratta R, Lu Y, Harris M (1992) Biomechanics of
increased exposure to lumbar injury caused by cyclic loading: Part 1. Loss of
reflexive muscular stabilization. Spine 24: 2426-2434. [PubMed]
4.  Solomonow M, Zhou BH, Harris M, Lu Y, Baratta RV (1998) The ligamentomuscular stabilizing system of the spine. Spine 23: 2552-2562. [PubMed]
5.  Erwin WM, Inman RD (2006) Notochord cells regulate intervertebral disc
chondrocyte proteoglycan production and cell proliferation. Spine 31: 10941099. [PubMed]
6.  Erwin WM, Ashman K, O'Donnel P, Inman RD (2006) Nucleus Pulposus
notochord cells secrete connective tissue growth factor and upregulate
proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum
54: 3859-3867. [PubMed]
7.  Cappello R, Bird JLE, Pfeiffer D, Bayliss MT, Dudhia J (2006) Notochordal cell
produce and assemble extracellular matrix in a distinct manner, which may be
responsible for the maintenance of healthy NP. Spine 31: 873-882. [PubMed]
8.  Erwin WM (2010) The enigma that is the NP cell: the search goes on. Arthritis
Res Ther 12: 118. [PubMed]
9.  Ludwinski FE, Gnanalingham K, Richardson SM, Hoyland JA (2013)
Understanding the native NP cell phenotype has important implications for
intervertebral disc regeneration strategies. Regen Med 8: 75-87. [PubMed]
10. Urban J, Roberts S (2003) Degeneration of the intervertebral disc. Arthritis Res
Ther 5: 120-130. [PubMed]
11. Moore RJ (2006) The vertebral endplate: disc degeneration, disc regeneration.
Eur Spine J 15: S333-S337. [PubMed]
12. Rodriguez AG, Slichter CK, Acosta FL, Rodriguez-Soto AE, Burghardt AJ, et
al. (2011) Human disc nucleus properties and vertebral endplate permeability.
Spine (Phila Pa 1976) 36: 512-520. [PubMed]
13. Butler WF (1989) Comparative anatomy and development of the mammalian
disc.
14. Webb R, Brammah T, Lunt M, Urwin M, Allison T, et al. (2003) Prevalence
and predictors of intense, chronic, and disabling neck and back pain in the UK
general population. Spine 28: 1195-1202. [PubMed]
15. Hestbaek L, Leboeuf YdeC, Manniche C (2003) Low back pain: what is the
long-term course? A review of studies of general patient populations. Eur Spine
J 12: 149-165. [PubMed]
16. Koes BW, van Tulder MW, Ostelo R, Burton AK, Waddell G (2001) Clinical
guidelines for the management of low back pain in primary care. Spine 26:
2504-2514. [PubMed]
17. Smeets RJ, Maher CG, Nicholas MK, Refshauge KM, Herbert RD (2009)
Do psychological characteristics predict response to exercise and advice for
subacute low back pain? Arthritis Rheum 61: 1202-1209. [PubMed]
18. Luoma K, Riihimaki H, Luukkonen R, Raininko R, Viikari-Juntura E, et al. (2000)
Low back pain in relation to lumbar disc degeneration. 25: 487-492. [PubMed]
19. Webb R, Brammah T, Lunt M, Urwin M, Allison T, et al. (2003) Prevalence
and predictors of intense, chronic, and disabling neck and back pain in the UK
general population. Spine 28: 1195-1202. [PubMed]
20. Koes BW, van Tulder MW, Ostelo R, Burton AK, Waddell G (2001) Clinical
guidelines for the management of low back pain in primary care. Spine 26:
2504-2514. [PubMed]
21. Gilson A, Dreger M, Urban JP (2010) Differential expression level of cytokeratin
8 in cells of the bovine NP complicates the search for specific intervertebral disc
cell markers. Arthritis Res Ther 12: R24. [PubMed]
22. Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA (2010)
Transcriptional profiling of bovine intervertebral disc cells: implications
for identification of normal and degenerate human intervertebral disc cell
phenotypes. Arthritis Res Ther 12: R22 [PubMed]

References

23. An HS, Masuda K (2006) Relevance of in vitro and in vivo models for
intervertebral disc degeneration. J Bone Joint Surg Am 88: 88-94. [PubMed]

1.  Panjabi M (1992) The stabilizing system of the spine. Part I. Function,

24. Alini M, Eisenstein SM, Ito K, Little C, Kettler AA, et al. (2008) Are animal

J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

Volume 4 • Issue 11 • 1000246

 Citation: Duntsch C, Dillard E, Akbar U (2014) Isolation and Characterization of Adult Spinal Disc Stem Cells from Healthy Human Spinal Disc
Tissues. J Stem Cell Res Ther 4: 246. doi:10.4172/2157-7633.1000246

Page 8 of 8
models useful for studying human disc disorders/degeneration? Eur Spine J
17: 2-19. [PubMed]
25. Dagenais S, Caro J, Haldeman S (2008) A systematic review of low back pain
cost of illness studies in the United States and internationally. Spine J 8: 8-20.
[PubMed]
26. Dietrich S, Schubert FR, Gruss P (1993) Altered Pax gene expression in
murine notochord mutants: the notochord is required to initiate and maintain
ventral identity in the somite. Mech Dev 44: 189-207. [PubMed]
27. Pourquié O, Coltey M, Teillet MA, Ordahl C, Le Douarin NM (1993) Control of
dorsoventral patterning of somitic derivatives by notochord and floor plate. Proc
Natl Acad Sci USA 90: 5242-5246. [PubMed]
28. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from
isolated cells of the adult mammalian central nervous system. Science 255:
1707-1710. [PubMed]
29. Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, et al. (1992)
Multipotent neural cell lines can engraft and participate in development of
mouse cerebellum. Cell 68: 33-51. [PubMed]
30. Taupin P, Gage FH (2002) Adult neurogenesis and neural stem cells of the
central nervous system in mammals. J Neurosci Res 69: 745-749. [PubMed]
31. Bonnamain V, Neveu I, Naveilhan P (2012) Neural stem/progenitor cells as
promising candidates for regenerative therapy of the central nervous system.
Front Cell Neurosci 6: 17. [PubMed]
32. Lobo MV, Alonso FJ, Redondo C, López-Toledano MA, Caso E, et al. (2003)
Cellular Characterization of Epidermal Growth Factor-expanded Free-floating
Neurospheres. J Histochem Cytochem 51: 89-103. [PubMed]

38. Liu LT, Huang B, Li CQ, Zhuang Y, Wang J, et al. (2011) Characteristics of
Stem Cells Derived from the Degenerated Human Intervertebral Disc Cartilage
Endplate. PLoS One 6: e26285. [PubMed]
39. Risbud MV, Guttapalli A, Tsai TT, Lee JY, Danielson KG, et al. (2007) Evidence
for skeletal progenitor cells in the degenerate human intervertebral disc. Spine
(Phila Pa 1976) 32: 2537-2544. [PubMed]
40. Blanco JF, Graciani IF, Sanchez-Guijo FM, Muntión S, Hernandez-Campo P,
et al. (2010) Isolation and characterization of mesenchymal stromal cells from
human degenerated NP: comparison with bone marrow mesenchymal stromal
cells from the same subjects. Spine (PhilaPa 1976) 35: 2259-2265. [PubMed]
41. Navone SE, Marfia G, Canzi L, Ciusani E, Canazza A, et al. (2012) Expression
of neural and neurotrophic markers in NP cells isolated from degenerated
intervertebral disc. J Orthop Res 30: 1470-1477. [PubMed]
42. Erwin WM, Islam D, Eftekarpour E, Inman RD, Karim MZ, et al. (2013)
Intervertebral disc-derived stem cells: implications for regenerative medicine
and neural repair. Spine (Phila Pa 1976) 38: 211-216. [PubMed]
43. Singec I, Knoth R, Meyer RP, Maciaczyk J, Volk B, et al. (2006) Defining the
actual sensitivity and specificity of the neurosphere assay in stem cell biology.
Nat Methods 3: 801-806. [PubMed]
44. Louis SA, Rietze RL, Deleyrolle L, Wagey RE, Thomas TE, et al. (2008)
Enumeration of Neural Stem and Progenitor Cells in the Neural ColonyForming Cell Assay. Stem Cells 26: 988-996. [PubMed]
45. Coles-Takabe BL, Brain I, Purpura KA, Karpowicz P, Zandstra PW, et al.
(2008) Don't look: growing clonal versus nonclonal neural stem cell colonies.
Stem Cells 26: 2938-2348. [PubMed]

33. Tanja Z, Sanberg PR, Sanchez-Ramos JR (2002) Neural stem cells: methods
and protocols. Humana Press.

46. Cordey M, Limacher M, Kobel S, Taylor V, Lutolf MP (2008) Enhancing the
reliability and throughput of neurosphere culture on hydrogel microwell arrays.
Stem Cells 26: 2586-2594. [PubMed]

34. Leung VY, Chan D, Cheung KM (2006) Regeneration of intervertebral disc by
mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine
J 15: S406-413. [PubMed]

47. Pilon N, Raiwet D, Viger RS, Silversides DW (2008) Novel pre- and postgastrulation expression of Gata4 within cells of the inner cell mass and
migratory neural crest cells. Dev Dyn 237: 1133-4113. [PubMed]

35. Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, et al. (2004)
Differentiation of mesenchymal stem cells towards a NP-like phenotype in
vitro: implications for cell-based transplantation therapy. Spine 29: 2627-2632.
[PubMed]

48. Tamaki T, Akatsuka A, Okada Y, Uchiyama Y, Tono K, et al. (2008)
Cardiomyocyte formation by skeletal muscle-derived multi-myogenic stem cells
after transplantation into infarcted myocardium. PLoS One 3: e1789. [PubMed]

36. Richardson SM, Hughes N, Hunt JA, Freemont AJ, Hoyland JA (2008)
Human mesenchymal stem cell differentiation to NP-like cells in chitosanglycerophosphate hydrogels. Biomaterials 29: 85-93. [PubMed]
37. Henriksson HB, Svala E, Skioldebrand E, Lindahl A, Brisby H (2012) Support
of concept that migrating progenitor cells from stem cell niches contribute to
normal regeneration of the adult mammal intervertebral disc: a descriptive study
in the New Zealand white rabbit. Spine (Phila Pa 1976) 4: 722-732. [PubMed]

J Stem Cell Res Ther
ISSN: 2157-7633 JSCRT, an open access journal

49. Mimura T, Amano S, Yokoo S, Uchida S, Usui T, et al. (2008) Isolation and
distribution of rabbit keratocyte precursors. Mol Vis 14: 197-203. [PubMed]
50. Barraud P, He X, Caldwell MA, Franklin RJ (2008) Secreted factors from olfactory
mucosa cells expanded as free-floating spheres increase neurogenesis in
olfactory bulb neurosphere cultures. BMC Neurosci 9: 24. [PubMed]

Volume 4 • Issue 11 • 1000246