Q: What are stem cells?
The term ‘stem cell’ refers to a progenitor cell that is capable
of transforming, also known as differentiating, into a more
specialized cell with a unique function. Totipotent stem
cells are capable of differentiating into any type of cell in the
body, including cells required during development of the embryonic
membranes. Pluripotent stem cells are also capable of
differentiating into any type of cell in the body, but do not have
the ability to form the embryonic membranes. Multipotent
stem cells are capable of differentiating into specialized cells,
but differentiation is confined within a family of related cells
(Figure 1). Most stem cells currently isolated from adult tissues
for research or clinical use are believed to be of the multipotent
Figure 1: Hierarchical nature of
stem cell (MSCs) differentiation. Totipotent
cells differentiate into all cells of the body including cells of
the fetal membranes. Pluripotent cells differentiate into
all cells of the body, excluding fetal membranes.
Multipotent cells differentiate into a number of
specialized cells within a specific lineage (germ layer). Stem
cells isolated from adult animals are typically considered to be
There are a variety of multipotent stem cells found throughout
the body. Examples include hematopoietic stem cells (HSCs) that
give rise to white and red blood cells, neural stem cells (NSCs)
that generate the cells of the brain, spinal cord, and peripheral
nervous system, and mesenchymal stem cells (MSCs) that
develop into cells of mesenchymal lineage: bone, cartilage, fat,
muscle, tendon, and others. MSCs are spindle-shaped, multipotent
progenitor cells that are found in bone marrow, fat, muscle,
synovium (a tissue that lines joint cavities), periosteum (a tissue
that covers long bones), and many other adult tissues. The
terminology related to MSCs can be confusing, as MSCs have been
given a number of names over the years, including: Mesenchymal Stem
Cells, Multipotent Stromal Cells, Marrow Stromal Cells, and
Medicinal Supply Cells. While many veterinarians, physicians,
scientists, and companies refer to these cells as “stem cells”, the
term MSC is probably a more appropriate term to describe this
diverse population of cells. A preparation of MSCs isolated from
adult tissues is diverse in size, shape, and function. It is
generally accepted that small, spindle-shaped cells (Figure 2A),
present in high numbers in early passages of MSCs, are responsible
for rapid growth and transformation into other tissues (a process
known as differentiation). Large, slowly proliferating cells are
present in low numbers (Figure 2B), but increase in number when
preparations of cells are cultured over extended periods of
Figure 2: Canine stem cells
(MSCs) isolated from bone marrow.
PANEL A: Small, spindle-shaped, rapidly-dividing cells
(arrowheads) predominate fresh cultures of MSCs. A small cell is in
the final stages of division in the bottom left (arrow). Phase
contrast microscopy, 10 X magnification.
PANEL B: Small MSCs (arrowheads) are identified adjacent to a
single large cell (arrow). These large cells are present in low
numbers initially, but increase dramatically when MSCs are cultured
for many weeks.
Under certain circumstances, MSCs are capable of
differentiating into large numbers of specialized cells. In
addition, MSCs appear to produce growth factors and
anti-inflammatory proteins that further contribute to improved
healing and reduced inflammation in injured tissues. For these
reasons, there is tremendous interest in using MSCs for tissue
regeneration. MSCs may serve as powerful cell therapy agents for
use in joint resurfacing, wound healing, and treatment of massive
Canine tissue regeneration and stem cell therapy is currently in
its infancy. To date, a modest number of publications exist
documenting the effectiveness of MSCs in the treatment of small
animal orthopedic disorders. Basic science research and clinical
studies are currently underway at Texas A&M’s Comparative
Orthopedics and Cellular Therapeutics Lab and other veterinary
institutions to determine if MSCs are effective in treating
important orthopedic problems in dogs. Examples include:
osteoarthritis, cruciate ligament rupture, meniscal injury,
non-healing fractures, osteochondrosis, elbow dysplasia, and many
others. While these types of studies take time, it is critically
important that both veterinarians and pet owners are aware of
potential benefits and risks of stem cell therapy.
Q: How are stem cells (MSCs)
characterized and what makes these cells unique?
In the laboratory setting several methods are used to
characterize MSCs. A procedure called flow cytometry is often used
to determine the presence or absence of a specific group of surface
marker proteins (similar to a cell fingerprint) that are often
present on MSCs (Figure 3A). Colony Forming Unit (CFU) assays
determine the ability of individual cells to generate a new colony
of cells (Figure 3B). Differentiation assays are used to determine
the ability of MSCs to transform into cells that resemble bone,
cartilage, and fat forming cells (Figure 4). Interestingly, some
cells that aren’t considered stem cells share many of these same
characteristics. For this reason, the true measure of MSCs that
separates them from other cells is their ability to form new bone,
cartilage, and fat tissue when transplanted to a distant location,
not in a laboratory setting, but in a living patient. Restoration
of bone, cartilage, and fat in the living setting provides
definitive proof that an individual cell is a true stem cell
Figure 3: Characterization of
canine stem cells (MSCs).
PANEL A: Flow cytometry is used to determine a pattern of surface
markers present on a population of MSCs. The histogram on the left
illustrates negative staining for the marker CD34, while the
histogram on the right illustrates positive staining for CD90
(positive cells shown in red, shifted to right).
PANEL B: The Colony Forming Unit (CFU) assay is used to compare
preparations of MSCs. The same number of MSCs were plated on each
dish and cultured for 21 days. Donor 1 has low CFU potential,
whereas Donor 2 has high CFU potential. In general, CFU potential
reflects the percentage of progenitor cells present within a
preparation of MSCs.
Figure 4: In vitro
differentiation of canine stem cells (MSCs).
PANEL A: Osteogenic (bone) differentiation. MSCs are
cultured in media that promotes osteogenesis and assayed for
calcium deposition at 21 days. Alizarin Red Stain binds to calcium,
and is an indicator of bone formation.
PANEL B: Adipogenic (fat) differentiation. MSCs are
cultured in media that promotes adipogenesis and assayed for fat
accumulation at 21 days. Oil Red O stain binds lipid vacuoles, and
is an indicator of fat-like cells.
PANEL C: Chondrogenic (cartilage) differentiation. MSCs
are cultured in small pellets in media that promotes
chondrogenesis. At 21 days, cartilage pellets are photographed,
measured, and, evaluated for cartilage properties.
Q: How are stem cells (MSCs) isolated
from bone marrow?
To isolate MSCs from bone marrow, a procedure referred to as a
bone marrow aspiration is performed. In dogs, this procedure is
typically performed on the humerus (the bone just below the
shoulder) or the iliac crest (point of the hip). Dogs are placed
under heavy sedation and a small patch of hair is clipped over the
donor site. Local anesthetics (as one would receive at the dentist)
are used to block the nerves of the skin and bone and the skin is
prepared for the procedure with a surgical scrub. A needle is
placed through a small skin incision (2 mm in size) into the soft,
spongy bone (called cancellous bone) of the humerus or iliac crest
(Figure 5). This type of spongy bone contains the largest supply of
bone marrow. A small volume of bone marrow is removed with a
syringe, the needle is removed, and the skin incision is closed
with tissue glue. Bone marrow aspiration typically takes 2-5
minutes and, in contrast to what is reported during the procedure
in humans, does not cause observable pain in dogs when performed
with the use of sedation and anesthetics (pain medications).
General anesthesia and surgery are not required.
Figure 5: Needle aspiration of
bone marrow from the humerus (upper arm). Bone marrow MSCs
can be isolated from many locations in dogs. The proximal humerus
is a preferred location because of the large amount of bone marrow
present and the limited soft tissues coverage of the area. Bone
marrow aspiration in the dog usually takes 2-5 minutes using deep
sedation and pain medications. General anesthesia and surgery are
Once the bone marrow sample is isolated, it is taken to the
laboratory where red blood cells are separated from the nucleated
cells using a technique known as Ficoll centrifugation. The
nucleated cell fraction contains marrow-derived MSCs (Mesenchymal
Stem Cells), but also includes white blood cells, fibroblasts,
endothelial cells, hematopoietic stem cells (HSCs), and smooth
muscle cells. All of these nucleated cells are isolated, washed,
counted, and plated onto sterile tissue culture dishes containing
growth media. The following day, the majority of cells are washed
away and a small number of cells remain tightly adhered to the
tissue culture surface. These plastic-adherent, spindle shaped
cells are MSCs. The MSC population is allowed to expand and once
sufficient numbers of cells are present, the MSCs are lifted from
the tissue culture plastic for characterization, long-term storage,
use in cell culture experiments, or use in clinical patients.
Q: How are stem cells (MSCs) isolated
from fat or other tissues?
Stem cells can also be isolated from fat and other tissues. To
obtain MSCs from fat or other tissue, dogs are placed under general
anesthesia and the skin is clipped and prepared for surgery. A skin
incision is made and a small sample of fat or other tissue is
removed. The fat sample is placed in a sterile container and taken
to the laboratory. Once the tissue sample is in the lab, it is
weighed, washed, and mechanically and enzymatically disrupted to
separate fat cells and red blood cells from the Stromal Vascular
Fraction of cells. The Stromal Vascular Fraction, or SVF, is a
mixture of cells including white blood cells, fibroblasts,
endothelial cells, MSCs, and smooth muscle cells. It is
important to note that the SVF is commonly used as a therapeutic
population of cells in human and veterinary cell therapy. In
fact, most commercial vendors that supply stem cells for veterinary
provide a preparation of SVF cells from fat tissue.
When the SVF is plated on tissue culture plastic as described
above, a sub-population of the SVF adheres to the plastic and many
of the SVF cells are washed away during the exchange of culture
media. The remaining cells are fat-derived MSCs. When MSCs are
isolated from fat, they are often referred to as ASCs or Ad-MSCs.
Both SVF cells and ASCs can be used for cell therapy, however,
their properties and regenerative abilities may be different from
MSCs obtained from bone marrow and other tissues.
Q: Are stem cells (MSCs) isolated
from various tissues the same?
Based on current information, it is clear that not all adult
stem cells are identical. Tissue source, patient age, and presence
or absence of disease appear to affect the number and
differentiation capacity of stem cells isolated from adult dogs.
This may be important when considering stem cell therapy for a pet
with severe orthopedic disease. As shown in Figure 6, MSCs are much
more readily isolated from canine fat or synovium as compared to
bone marrow. However, when these same cells are compared using a
functional assay of bone differentiation, synovial and marrow MSCs
exhibit higher levels of ALP activity (a fundamental property of
bone), when compared to fat-derived MSCs. Whether these differences
are relevant in the context of clinical therapy has yet to be
determined, but it appears that cells obtained from different
tissues are not identical, even when obtained from the same
Figure 6: Comparison of cell
yield and bone forming potential for stem cells (MSCs) isolated
from various tissues.
PANEL A: MSCs were isolated from the synovial membrane (joint
lining), bone marrow, and subcutaneous fat from a single donor. MSC
isolation was superior for synovium and adipose (fat) tissue as
compared to bone marrow.
PANEL B: The same cells isolated in panel A were compared in an
ALP activity assay, an assay of osteogenesis (bone
differentiation). Synovium and bone marrow MSCs exhibited elevated
ALP activity in response to increasing doses of the growth factor
BMP-2, whereas little activity was present in adipose-derived
Q: In the context of orthopedic disorders, what is stem
Stem cell therapy is the use of MSCs, ASCs, or SVF cells for the
symptomatic or regenerative treatment of bone and joint disorders.
Either autologous or allogenic stem cell therapy can be used.
Autologous therapy refers to harvesting cells from one dog
and re-administering those cells to the same dog.
Allogenic therapy refers to harvesting cells from one dog
and administering those cells to another dog, similar to a blood
Stem cells potentially treat orthopedic disorders through a
number of mechanisms, two of which are tissue regeneration and
production of anti-inflammatory proteins or growth factors (Figure
7). With a tissue regeneration strategy, cells are induced toward
bone or cartilage cells in the lab in conjunction with
three-dimensional tissue lattices to form tissue engineering
constructs for bone and joint defects. With the anti-inflammatory
protein and growth factor strategy, cells are injected into
diseased joints and tissues to produce proteins and other
substances that suppress inflammation and improve the body’s own
healing mechanisms. These mechanisms are not independent, as
cross-talk between the two strategies exists. For example, cells
delivered in tissue regeneration constructs likely produce specific
growth factors and anti-inflammatory agents to improve healing,
while cells delivered for their anti-inflammatory effects may
undergo some degree of differentiation based on the specific cues
provided by the treated joint or tissue.
Figure 7: Treatment strategies
and potential mechanisms of action of stem cell therapy.
Stem cell therapy is the use of MSCs for the symptomatic or
regenerative treatment of bone and joint disorders. A number of
mechanisms may be involved in this process, two of which are tissue
regeneration and symptomatic therapy. Tissue regeneration
refers to healing or functional repair of an injured organ, such as
a bone, joint, or ligament through differentiation of MSCs into new
tissues. Symptomatic therapy refers to the ability of MSCs
to improve a patient’s ability to heal through the production of
anti-inflammatory agents and growth factors. It is believed that
these factors are produced by the MSCs in response to cues received
from the injured tissue.
The majority of ‘stem cell therapy’ currently being performed
for canine orthopedic disorders relies on the anti-inflammatory
protein and growth factor mechanism. Treatment of osteoarthritis
secondary to hip dysplasia, elbow dysplasia, and cranial cruciate
ligament rupture are common examples. In contrast, orthopedic
disorders such as osteochondrosis (a disorder of joint cartilage)
and non-union fractures (broken bones that fail to heal) are more
likely candidates for tissue regeneration approaches.
Q: Is my dog a candidate for stem cell therapy at Texas
The orthopedic surgeons at Texas A&M are fully committed to
our mission: To develop and advance new treatments that improve
the quality of life of companion animals with orthopedic disease
while providing state of the art surgical care.
We rely on effective, established treatments when they exist,
and offer new therapies in situations in which traditional
treatments have not been effective or are not realistic options. We
are currently offering autologous canine stem cell therapy using
bone marrow MSCs isolated in our Comparative Orthopedics and
Cellular Therapeutics Lab. This lab, located in the adjacent
Veterinary Medical Research Building, is a fully functional cell
biology lab dedicated to canine stem cell research and clinical
cell therapy. The lab is directed by Dr. W. Brian Saunders, and is funded
intramurally, extramurally, and through gifts to the Bone
& Joint Fund.
At the time of your pet’s appointment, we will perform a
complete orthopedic exam, obtain necessary imaging such as
radiographs (x-rays) or Computed Tomography (CT), and consult
extensively with you about your pet’s diagnosis. The vast majority
of orthopedic disorders can successfully be addressed with
traditional treatments. In some cases, however, traditional
treatments will have been attempted and failed or will not be
appropriate treatment options. In these cases, we may discuss cell
therapy as a possible treatment approach. The potential benefits
and risks of autologous cell therapy will be extensively discussed.
If cell therapy is selected, we may perform pre-treatment gait
analysis and other diagnostics, isolate MSCs from your pet’s bone
marrow or other tissues as described above, and subsequently
perform cell therapy. All treatments are administered in-house, and
we follow each patient’s progress through re-check visits to
determine the response to treatment.