Murine Mesenchymal Stem Cell Isolation Method Comparison
- 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
- 10. 10 a b c d e f
- 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.
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