1. 1
PROJECT REPORT
MURINE MESENCHYMAL STEM CELL ISOLATION METHOD
COMPARISON:
BONE MARROW FLUSH AND BONE CHIP
Submitted To
The 2009-2010 Academic Year REU Program
Part of
NSF Type 1 STEP Grant
Sponsored By
The National Science Foundation
Grant ID No.: DUE-0756921
College of Engineering and Applied Science
University of Cincinnati
Cincinnati, Ohio
Prepared By
Jacob Turner-Department of Biomedical Engineering, University of Cincinnati,
Cincinnati, OH
June 21, 2010-August 20, 2010
2. 2
ABSTRACT: Using rabbit mesenchymal stem cells (MSCs), our lab has created tissue
engineering constructs (TECs) that match the normal patellar tendon force-displacement 50%
beyond peak in vivo forces recorded during hopping activities on a treadmill. However,
additional improvement may be needed for more strenuous activities. Understanding temporal
and spatial gene expression in developing tissue may provide insight on how to create a better
TEC. Unfortunately the genome is not mapped in the rabbit so normal development studies
would be quite difficult in this model system. Using functional tissue engineering principles our
lab is taking advantage of the genetic power available in the mouse, whose genome is mapped.
Using the mouse model allows us to translate our findings on tendon development to the rabbit.
Isolating a cell source from the mouse that behaves similarly to rabbit MSCs used previously in
the lab would make this transition more efficient. The goal of this project is to compare two
MSC isolation methods in the mouse (bone marrow flush and bone chip) in order to formulate a
method that will produce large homogenous cultures of MSCs that are phenotypically similar to
our rabbit MSC cultures. Both the flush and chip cultures yielded heterogeneous cell populations
with over 90% hematopoietic cells. These cells also produced higher levels of alkaline
phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) than the rabbit MSCs.
Although the cell cultures produced by this project do not appear to be readily applicable to
tendon tissue engineering, many unexpected outcomes have given insight into how to achieve a
conducive tendon tissue engineering murine cell culture. Future studies will investigate rapid
passaging of the cells to reduce the cell-to-cell interactions, reducing ALP production, and
prevent attachment of hematopoietic cells in order to reduce the ALP production and improve the
homogeneity of the culture, respectively. Incorporating these new strategies will bring us another
step closer to achieving our goal of producing a functional repair tissue in the rabbit.
KEY WORDS: tissue engineering; mesenchymal stem cell; murine
1. INTRODUCTION
Annually, more than 32 million traumatic and repetitive motion injuries to tendons and
ligaments place a large burden on the U.S. economy. An estimated $30 billion is spent on
repairs and surgeries every year, many of which yield a suboptimal recovery leading to
diminished functional capacity and a decrease in quality of life (Praemer et al. 1999). The most
frequent and costly soft tissue injuries consist of rotator cuff tendons in the shoulder, anterior
cruciate ligament (ACL) in the knee, and patellar tendon (PT) in the knee(Praemer et al. 1999;
DeFrances et al. 2005). The previously stated deficiencies of current surgical repairs have given
rise to the rapidly developing field of tissue engineering. Combining principles of biology and
engineering allows tissue engineering to address many unanswered questions in tissue
3. 3
development and biomechanics; ultimately bettering repair outcomes. This projects is designed
to understand our murine cell cultures better, making it possible to do in vitro tests and translate
our finding to our rabbit, ultimately increasing our tissue engineered construct’s stiffness and
applicability.
2. LITERATURE REVIEW
Cell-based tissue engineering uses mesenchymal stem cells (MSCs) placed in a
biocompatible scaffold or directly inserted into a soft tissue defect to try and regenerate the
injured soft tissue. Mesenchymal stem cells offer the benefit of being able to differentiate into
native cells in a variety of tissue types including tendon, ligament, cartilage, bone, skin, etc.
Ouyang et al achieved a repair modulus and stiffness of 87% and 63% of normal Achilles tendon
values 12 weeks after surgery by seeding 10M MSCs onto a polylactide-co-glycolide (knitted
biocompatible material) scaffold and inserting into a rabbit Achilles defect (Ouyang et al.
2003a). Awad et al seeded MSCs at different cell densities (1,4, and 8x106 cells/mL) in type 1
collagen gel, allowed contraction around a suture, and inserted the resulting tissue engineered
construct (TEC) into rabbit PT defects. No dose-dependent advantages were found from seeding
at higher cell densities and 28% of the repair sites formed bone rather than soft tissue (Awad et
al. 2003). By implanting MSCs from rabbit bone marrow taps into TECs, our lab has produced
tendon repair tissue that matches the normal PT force-displacement curve up to 150% of peak in
vivo forces and 85% of normal linear stiffness of rabbit PTs (Ouyang et al. 2003b; Juncosa-
Melvin et al. 2007). However, improvements may still be needed to resist more strenuous
activities surpassing the recorded peak in vivo forces for activities of daily living (Juncosa et al.
2003).
4. 4
Although the rabbit model is large enough to allow for repeatable repair surgery, its
genome is not mapped, making it difficult to quantify genetic expression in this model. To
address this issue, we are currently attempting to improve our tissue-engineered repairs by taking
advantage of the genetic tools in the mouse, whose genome is mapped. Even though in vivo
repair is not possible in the mouse due to its small size, the genetic tools available allow us to test
tissue engineering strategies in vitro and translate them to the rabbit, where repeatable repair
studies can be conducted (Butler et al. 2008). Previous experiments performed by Nat Dyment
and Andrea Lalley in our lab have shown that cells flushed from the bone marrow of mice
produce a culture containing approximately 90% hematopoietic cells (white blood cells and red
blood cells), which are not useful for tendon tissue engineering. Having a consistent cell source
between the rabbit and mouse would allow us to translate tissue engineering strategies more
effectively. Therefore, we would like to compare two MSC isolation and culture methods (bone
marrow flush and cortical bone chip) in the mouse in anticipation of producing a cell population
that can be used effectively to test tissue engineering strategies in vitro and potentially be
translated to the rabbit in both in vitro (laboratory) and in vivo (repair) studies.
3. GOALS AND OBJECTIVES
The objectives of this study are to contrast the marrow flush vs. bone chip methods to
determine which method produces populations of murine mesenchymal stem cells that 1) are
homogenous, 2) are large in number, 3) produce type I collagen (Col1) that is normally found in
tendon, 4) express high levels of other tendon markers (Tnmd and TnC), and 5) do not express
high levels of bone markers (ALP and osteocalcin).
5. 5
4. METHODS
All animal protocols were approved by the Instiututional Animal Care and Use
Committee at The University of Cincinnati.
4.1 Experimental Design
Bone marrow flush and bone chip cells were harvested from seven col1/col2 double
transgenic mice. These transgenic mice have been genetically modified to fluoresce green
where a critical tendon gene, type-I collagen (Col1), is being produced and cyan where a
cartilage gene, type-II collagen (Co12) ,is being produced. Observing through a fluorescent
microscope with appropriate filters allows us to see this fluorescence. This ability allows
researchers to observe the spatial and temporal production of Col1 (main structural protein in
tendon and bone) and Col2 (main structural protein in cartilage). After being passed from their
original culture dish, unsorted and sorted (EasySep Mesenchymal Enrichment Kit;
STEMCELL; Vancouver, BC, Canada) cells from each cell line (animal) were cultured in 12-
well plates and assigned to be tested for Relative Fluorescent Units (RFU), and TRACP/ALP
staining (n=7).
4.2 Isolation Methods and Culture
Cells were isolated by slightly modifying the protocols for bone marrow flush (Soleimani
and Nadri 2009) and cortical bone chip (Zhu et al. 2010) found within Nature Protocols. After
isolating the femur and tibia from each murine hind limb, the epiphyses (bone ends) were
removed and the bone cavities were flushed using Mesencult (STEMCELL, Vancouver, BC,
Canada). The cells from the marrow flush were seeded in tissue culture petri dishes. The bones
were then cut into 1-3 mm3chips, digested in type II collagenase to loosen up the bone pieces,
6. 6
and seeded in petri dishes with Mesencult, allowing for cells within the bone to move outside
onto the culture dish. Temporal changes in cell phenotype (physical shape and size) were
observed throughout proliferation and documented by photo-microscopy. Once filling the entire
dish (confluent), the cells were passaged (transplanted from one dish to another) into 12-well
plates. The unsorted cells were seeded at 40K cells/well, while the remaining cells were sorted
using the StemCell EasySep Mouse Mesenchymal Progenitor Cell Enrichment Kit and seeded in
remaining wells. The kit’s antibodies bind to CD45 (cell surface marker for white blood cells)
and TER119 (cell surface marker for red blood cells), allowing a magnet to separate the
hematopoietic cells from the desired mesenchymal stem cells. At approximately 70% confluency
at passage 1, the cells were isolated for each of their designated response measures. Achilles
fibroblast (AFB) and patellar tendon fibroblasts (PTFB) were also harvested from three of the
mice to be used as a positive control for RFUs and a negative control for TRACP/ALP staining.
4.2 TRACP/ALP Staining Methods
A TRACP/ALP double-stain kit (Takara Bio, Shiga, Japan) was used to stain unsorted
and sorted cells. TRACP (tatrate resistant acid phosphatase) is an enzyme that is highly produced
in osteoclasts (bone resorptive cells) and macrophages (type of white blood cell). ALP (alkaline
phosphatase) is an enzyme highly produced in bone-forming cells (osteoblasts) and an early
marker of bone formation. This kit allows for the identifications of these cells in our culture,
which are not wanted based on our objectives for isolating tendon-like mesechymal stem cells.
After being washed, a fixative was applied to keep the cells stationary in the dish. Once fixed,
the cells were stained first for TRACP and second for ALP. After staining, photo-microscopy
was used to document the TRACP/ALP expression in the cells. We performed TRACP/ALP
7. 7
staining on murine AFB, PTFB, and compared these results with those for rabbit mesenchymal
cells.
4.3 RFU Measurement Methods
After being washed and isolated, we placed the bone marrow flush and bone chip cells in
a UV spectrophotometer and measured Col1 RFU intensity. Collagen 1 (Col1) is a protein that
makes up large amounts of connective tissue such as bone, skin, tendons, etc. Measuring the
RFU intensity of the cells will give an idea as to which method is producing more tendon-like
connective tissue for tendon tissue engineering. The RFU measurement was normalized to the
cell count of each cell line. RFU measurements were also taken from AFB and PTFB for
comparison.
5. RESULTS
5.1 Magnetic Sorting
The magnetic sorting kit removed over 90% of the cells for both the bone marrow flush
and bone chip methods, over 80% for the murine AFB and PTFB, and over 40% for the rabbit
mesenchymal stem cells. Due to the removal of such a high percentage of cells, final sorted
cultures took a much longer time to proliferate than cells in unsorted cultures.
5.2 Phenotypical and Col1 Fluorescence Observations
In general, bone chip cultures (Fig. 1: b,d,f,h) appeared to become confluent in a shorter
amount of time than the bone flush cultures (Fig 2: a,c,e,g). Both methods yielded
heterogeneous populations of small round cells, medium spindle-shaped cells, and large flat
spread-out cells. The bone marrow flush and bone chip cell populations were more consistent
following sorting with the magnetic kit. Typically, the cells were larger and elongated. The
medium elongated cells and small rounded cells seen in the unsorted populations were not seen
8. 8
in the sorted populations initially. However, the small rounded cells began to reappear over the
course of 1 week in the marrow flush populations.
a b
c d
e f
9. 9
5.3 TRACP/ALP Staining
Both bone chip and bone marrow flush cells expressed high levels of ALP and TRACP
before sorting (a,c). However, the TRACP-positive cells were removed by sorting but slowly
reappeared in the bone marrow flush culture after a week. Intended as a negative control, both
murine AFB and PTFB unexpectedly showed high expression levels of ALP (e,f).
Fig. 1: Cells from the early bone marrow flush (a) and bone chip (b) methods
initially proliferate many small rounded (hematopoietic) cells. Near confluency,
a larger amount of bone chip cells (f,h) express Col1 (green) than bone marrow
flush (e,g). After sorting, bone marrow flush (c) and bone chip (d) cells
phenotype change to large elongated cells with elevated Col1 expression. The
positive fibroblast controls (Achilles (i) and patellar tendon (j)) show elevated
Col1 expression and large organized elongated cells.
i j
g h
11. 11
5.4 RFU Measurements
Bone chip cells show higher RFU fluorescence than the bone marrow flush cells. After
sorting, a larger number of the cells from both methods appeared to have elevated RFUs. RFU
measurements taken from AFB and PTFB showed comparable levels to the sorted flush and chip
cell lines.
Fig. 2: In both bone chip (a,b) and bone
marrow flush (c,d), unsorted (a) and sorted
(b) culture show elevated levels of ALP
(purple). However, the small round cells
stained for TRACP (red) in the unsorted
cells are removed in the sorted cells. PTFB
(e) and AFB (f) were intended to be used as
a negative control but surprisingly showed
very elevated levels of TRACP/ALP. Rabbit
MSC cultures (g) show low TRACP/ALP
expression which is ideal for tendon tissue
engineering.
g
Fig. 3: As seen in the graph, unsorted bone chip cells show a higher
RFU/Cell Number than marrow flush cells. Sorted marrow flush
and bone chip cells show comparable levels of RFU/Cell Number to
the AFB and PTFB, but with a high standard deviation the sorted
data is statistically unreliable.
12. 12
6. DISCUSION
Although the cell cultures produced by this project do not appear to be readily applicable
to tendon tissue engineering, many unexpected outcomes have given insight into how to achieve
a conducive tendon tissue engineering murine cell culture. The initial intention of using the
magnetic sorting kit was to remove undesirable, hematopoietic cells in an effort to make a more
homogenous culture of mesenchymal stem cells as seen in rabbit MSC cultures. However, the
sorting kit removed far too many cells to allow the sorted population to proliferate in our desired
time frame, one of our primary objectives. We hypothesize that this may be caused by the
hematopoietic cells being clustered with and attached to mesenchymal stem cells; inadvertently
being sorted out of the final culture. Also, contamination was later discovered in the sorting kit.
Because the EasySep sorting kit has some inconsistencies, we propose a follow-up experiment
that will pass the cell lines more rapidly, not allowing the cultures to become confluent, in an
attempt to create a more homogeneous culture. The rationale behind this is that the
mesenchymal cells have a higher affinity for attaching to the cell culture dish than hematopoietic
cells. Therefore, more rapid passaging of the cells may reduce the number of hematopoietic cells
that attach and proliferate.
Intending to use the murine PTFB and AFB cultures as negative controls for
ALP/TRACP, it was very unexpected that over 75% of the fibroblast culture was positive for
ALP. Given the unexpected expression of ALP in the murine fibroblasts, two hypotheses will be
tested to better understand this outcome. The first hypothesis is that the growth media,
Mesencult, used to feed the cultures may have unknown osteogenic growth factors causing the
cells to differentiate away from fibroblasts and toward an osteogenic lineage. Using media
absent of osteogenic growth factors in the future should aid in producing more MSC-derived,
13. 13
fibroblast-like populations as seen in our rabbit MSC cultures. Our second hypothesis is that the
TRACP/ALP staining kit may not be as accurate as anticipated. Future experiments will compare
this kit to other TRACP/ALP antibody kits using murine and rabbit cultures to test its validity.
Elevated Col1 expression in the bone chip cells may be evidence of the isolation
method’s capability to produce a MSC-rich population in the mouse, but further investigation of
reliable response measures is needed before more conclusions are drawn. It appears that
elevated Col1 expression is seen in the sorted samples, but low cell counts from these samples
gives the RFU data for the sorted bone chip and marrow flush cells a very high standard
deviation, making the data statistically unsound. Samples collected for qRT-PCR (quantitative
real time-polymer chain reaction) assays will be tested for expression of Col1 (to verify RFU
results), osteocalcin (bone marker), tenomodulin (highly expressed in tendon), and Tenascin-C
(highly expressed in tendon) in the near future. qRt-PCR is a method used to quantify genes of
interest in our cells, giving insight into what lineage (bone, tendon, cartilage, etc.) the cells are
differentiating into. Using qRT-PCR will give the most insight into what type of cell cultures
these two isolation methods are producing, as well as what steps need to be taken in order to
produce a culture more applicable to tendon tissue engineering.
In conclusion, this study has shown many ways in which our mouse cultures differ from
our rabbit cultures. It has given much insight into how we can modify our mouse cultures to be
more like our rabbit MSC cultures and achieve our ultimate goal of using in vitro mouse
experiments to better understand how to improve the in vivo rabbit repair studies.
6. ACKNOWLEDGEMENTS
This study was funded by the Research Experience for Undergraduates Program for NSF Type 1
STEP Grant DUE-0756921, NIH AR46574--07--10, and AR56943--01.
14. 14
7. BIBLIOGRAPHY
Awad, H. A., Boivin, G. P., Dressler, M. R., Smith, F. N. L., Young, R. G., and Butler, D. L. (2003).
"Repair of patellar tendon injuries using a cell-collagen composite." Journal of Orthopaedic
Research : Official Publication of the Orthopaedic Research Society, 21(3), 420-431.
Butler, D. L., Juncosa-Melvin, N., Boivin, G. P., Galloway, M. T., Shearn, J. T., Gooch, C., and
Awad, H. (2008). "Functional tissue engineering for tendon repair: A multidisciplinary
strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation."
J.Orthop.Res., 26(1), 1-9.
DeFrances, C. J., Hall, M. J., and Podgornik, M. N. (2005). "2003 Summary: National Hospital
Discharge Survey. Advance data from vital and health statistics." No. 359.
Juncosa, N., West, J. R., Galloway, M. T., Boivin, G. P., and Butler, D. L. (2003). "In vivo forces
used to develop design parameters for tissue engineered implants for rabbit patellar
tendon repair." J.Biomech., 36 483-488.
Juncosa-Melvin, N., Matlin, K. S., Holdcraft, R. W., Nirmalanandhan, V. S., and Butler, D. L.
(2007). "Mechanical stimulation increases collagen type I and collagen type III gene
expression of stem cell-collagen sponge constructs for patellar tendon repair." Tissue Eng.,
13(6), 1219-1226.
Ouyang, H. W., Goh, J. C. H., Thambyah, A., Teoh, S. H., and Lee, E. H. (2003a). "Knitted poly-
lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and
regeneration of rabbit Achilles tendon." Tissue Eng., 9(3), 431-439.
Ouyang, H., Goh, J., Thambyah, A., Teoh, S., and Lee, E. (2003b). "Knitted poly-lactide-co-
glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of
rabbit Achilles tendon." Tissue Eng., 9(3), 431-9.
Praemer, A., Furner, S., and Rice, D. (1999). Musculoskeletal conditions in the united states.
American Academy of Orthopaedic Surgeons, Rosemont, IL.
Soleimani, M., and Nadri, S. (2009). "A protocol for isolation and culture of mesenchymal
stem cells from mouse bone marrow." Nature Protocols, 4(1), 102-106.
Zhu, H., Guo, Z. -., Jiang, X. -., Li, H., Wang, X. -., Yao, H. -., Zhang, Y., and Mao, N. (2010). "A
protocol for isolation and culture of mesenchymal stem cells from mouse compact bone."
Nat.Protoc., 5(3), 550-560.
