PROJECT REPORT

MURINE MESENCHYMAL STEM CELL ISOLATION METHOD COMPARISON:

BONE MARROW FLUSH AND BONE CHIP

Submitted To

The 2009-2010 Academic Year REU ProgramPart of

NSF Type 1 STEP Grant

Sponsored By

The National Science FoundationGrant ID No.: DUE-0756921

College of Engineering and Applied ScienceUniversity of Cincinnati

Cincinnati, Ohio

Prepared By

Jacob Turner-Department of Biomedical Engineering, University of Cincinnati, Cincinnati, OH

June 21, 2010-August 20, 2010

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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

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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).

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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).

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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,

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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

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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

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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

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5.3 TRACP/ALP Staining

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

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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).

a b

c d

e f

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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

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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.

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.

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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,

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

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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.

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.

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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.

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