Anissa S.H. Chan, Electra Coucouvanis, Susan Tousey, Marnelle D. Andersen and Jessie H.T. Ni
ABSTRACT
Mesenchymal stem cells (MSC) hold significant promise for tissue engineering and regenerative medicine. Clinical and pre-clinical applications of MSC require that they be expanded in culture with maximum efficiency while maintaining the ability to differentiate into key lineages. Fetal bovine serum (FBS) is a key factor in the in vitro maintenance of hMSC, but lot-to-lot variability can significantly impact experimental results. In order to produce a growth medium that consistently allows optimal expansion of hMSC, we tested different concentrations of FBS from several sources. Our results indicate that efficiency of expansion varies greatly among FBS samples, with a 1.8 to ~ 4.2 fold increase after a single passage. The FBS lot supporting the largest fold-expansion of hMSC was used in development of StemXVivo™ Mesenchymal Stem Cell Expansion Media. When tested against another commercially available MSC expansion medium, StemXVivo™ Mesenchymal Stem Cell Expansion Media resulted in a 5.6 fold expansion after two passages compared to a 2.8 fold expansion of cells in competitor medium. We then sought to confirm that cells expanded in StemXVivo Mesenchymal Stem Cell Expansion Media maintained their full differentiation potential. We assessed the phenotype of the cells after expansion and found them to be CD105+, CD90+ and CD34-. We tested their ability to differentiate into chondrocytes, osteocytes, and adipocytes. Staining with lineage-specific antibodies confirmed efficient differentiation of the cells into these lineages. Thus, we concluded that pre-screening of several lots of FBS allows production of expansion medium that provides maximum MSC expansion efficiency without compromising pluripotency.
INTRODUCTION
Mesenchymal stem cells or marrow stromal cells (MSCs) are adult stem cells with multi-lineage potential that are easily genetically modified and demonstrate immunosuppressive ability. Their versatility holds promise for tissue engineering, regenerative medicine and immunotherapy. MSCs have been found in numerous tissues including the bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. They are capable of differentiating into bone, cartilage, muscle, marrow, tendon, adipose, and connective tissues (Figure 1). In the hope of furthering in vitro and in vivo studies, it is our interest to optimize culture conditions for the maintenance and expansion of MSC while preserving pluripotency and differentiation capacity. In order to assess the influence of different serum sources and concentrations on MSC expansion, our rigorous testing assays each FBS for maximum expansion potential, maintenance of the phenotypic characteristics of hMSC, and differentiation of the expanded MSC into adipocytes, osteocytes, and chondrocytes using commercially available differentiation kits and components. The results of our screening of FBS led to the development of StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media. Our study highlights the advantages of using commercially available products that have been developed, optimized and tested for the study of hMSC.
The differentiation potential of MSC.
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Figure 1. MSC have the potential to differentiate into a wide range of cell types. |
MATERIALS & METHODS
Human MSC Culture
Human MSCs (hMSCs) were purchased from Cambrex (Lonza, Walkersville) and cultured initially according to the vendor’s instruction and then later with StemXVivo Human/Mouse Mesenchymal Stem Cell Expansion Media (R&D Systems; Catalog # CCM004). To test the expansion potential of various lots of FBS, each was added to a final concentration of 10% or 20% in α-MEM (Invitrogen, Carlsbad) supplemented with glutamine and sodium pyruvate (Irvine Scientific, Santa Ana). 1.25 x 105 hMSCs were seeded into a T75 flask and cells were allowed to grow for 3 – 4 days before becoming confluent. At the end of the culture period, hMSCs cultured in these various conditions were harvested at the same time with trypsin/ EDTA solution (Irvine Scientific). Cells were counted and fold expansions were calculated using the formula listed below. To initiate a new culture, 1.25 x 105 hMSCs were seeded into a new T75 flask and cultured with the appropriate medium. Cell counting was repeated at the end of each passage.
| Fold expansion = | Number of harvested cells
Number of input cells |
Antibodies
Phycoerythrin (PE)-conjugated mouse anti-human CD90 and CD105 monoclonal antibodies (mAbs), unconjugated goat anti-mouse FABP-4 polyclonal Ab, unconjugated mouse anti-human Osteocalcin mAb, unconjugated goat anti-human Aggrecan polyclonal Ab, PE-conjugated and unconjugated isotype controls, and NorthernLights-557 conjugated donkey anti-goat and donkey anti-mouse secondary Ab were all from R&D Systems. PE-conjugated mouse anti-human CD34 mAb was from BD Pharmingen (BD Biosciences, San Jose).
Flow Cytometry
hMSCs were harvested as described above. Cells were washed 2X with cold FACS buffer (PBS supplemented with 2% FBS and sodium azide) and then resuspended at 106 cells per mL in FACS buffer. 1 x 105 hMSCs were incubated with conjugated mAb or isotype control for 45 minutes at 2 – 8°C. Stained cells were washed 2X in ice cold FACS buffer before data acquisition using FACSCalibur (BD Biosciences) and analysis with CellQuest software (BD Biosciences).
Immunocytochemistry
Detailed protocols can be obtained from the insert of the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC006). Briefly, cells cultured on cover slips were fixed with PBS containing 4% paraformaldehyde for 20 minutes at room temperature and blocked with PBS containing 10% normal donkey serum, 0.3% Triton X-100, and 1% BSA for 45 minutes at room temperature. After blocking, cells were incubated with diluted primary Ab overnight at 2 – 8 °C followed by NorthernLights-557 conjugated secondary Ab at room temperature in the dark for one hour. Between each step, cells were washed 3X with PBS containing 0.1% BSA.
Adipogenic Differentiation
hMSCs were induced to differentiate into adipocytes as described in protocols included with the Human Mesenchymal Stem Cell Functional Identification Kit. Briefly, completed adipogenic differentiation medium and a 24-well tissue culture plate with sterile cover slips were prepared and 3.7 x 105 hMSCs in 5 mL of a-MEM basal medium were plated into 10 wells of a 24-well plate containing sterile cover slips. The plate was incubated at 37°C in a 5% CO2 humidified incubator, cells were confluent the next day and the medium was removed and replaced with 0.5 mL of adipogenic differentiation medium.The differentiation medium was replaced every 3 – 4 days and adipocytes could be seen from day 7 onwards. Adipocytes were fixed by day 21 and characterized by immunocytochemistry.
Osteogenic Differentiation
hMSCs were induced to differentiate into osteocytes using StemXVivo Human/Mouse Osteogenic Base Media (R&D Systems, Catalog # CCM007) and StemXVivo Human Osteogenic Supplement (R&D Systems, Catalog # CCM008). 7.4 x 103 hMSCs in 0.5 mL of completed osteogenic base medium were plated into each well of a 24-well plate containing sterile cover slips. The plate was incubated at 37°C in a 5% CO2 humidified incubator for 1 – 2 days until 50 – 70% confluent. The medium in each well was then replaced with 0.5 mL of completed osteogenic differentiation medium. The differentiation medium was refreshed every 3 – 4 days and osteocytes were seen to appear between 14 – 21 days. The differentiated osteocytes were characterized by immunocytochemistry.
Chondrogenic Differentiation
Chondrocyte differentiation was induced using StemXVivo Human/Mouse Chondrogenic Base Media (R&D Systems, Catalog # CCM005) and StemXVivo Human Chondrogenic Supplement (R&D Systems, Catalog # CCM006). Briefly, 2.5 x 105 hMSCs were transferred into a 15 mL conical tube, containing 5 mL of pre-warmed completed chondrogenic base medium and centrifuged at 200 x g for 5 minutes. hMSCs were then resuspended in 0.5 mL of pre-warmed completed chondrogenic differentiation medium and centrifuged at 200 x g for 5 minutes. Without disturbing the cell pellet formed at the bottom of the tube, the cap of the tube was loosened to allow gas exchange and then incubated upright at 37°C in a 5% CO2 humidified incubator. Differentiation medium was replaced every 2 – 3 days and the chondrocyte pellet was harvested after 14 – 21 days. The chondrocyte pellet was fixed, sectioned and then analyzed by immunocytochemistry.
RESULTS
MSC Expansion Varies with Different Serum Samples
Testing of the various FBS combinations in α-MEM basal medium shows that different FBS samples demonstrate a qualitative difference in the expansion of hMSC, with FBS 3 supporting increased expansion over FBS 2 or FBS 1. Increasing the FBS 3 concentration from 10% to 20% further increases the fold expansion from 3.3 to 4.3 at p5 and 4.6 to 5.6 at p6 (Figure 2).
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Figure 2. Effect of 3 different FBS samples on the expansion of human MSCs. 1.25 x 105 hMSC at passage 4 (p4) were cultured with three different FBS samples at a final concentration of 10% or 20% as described in Materials and Methods. Expanded hMSC were harvested on day 3 or 4, counted and seeded into a new flask. Cell expansion is calculated as described in Materials and Methods. Results from two consecutive passages, p5 and p6, are shown. ∗ indicates a low number of cells that were not countable. |
Expanded hMSC Maintain Correct Phenotype
StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media demonstrated a consistently higher cell expansion potential than the competitor medium as shown by the 4-5 fold expansion at passage 5 and 6 in comparison to the ~2 fold expansion in the competitor medium (Figure 3). Phenotypically, the expanded hMSC highly expressed the key MSC markers CD105 and CD90 at passage 6 but not the CD34 surface marker, suggesting that the expanded cells retain hMSC identity.
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Figure 3. Human MSCs growing in StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media demonstrated a higher expansion potential while retaining the MSC characteristics. A. Human MSCs were cultured with either StemXVivo Human/Mouse MSC Expansion Media (R&D Systems, Catalog # CCM004; dark gray bar) or competitor medium (light gray bar) under the same conditions as described in the legend of Figure 2. Representative results from p5 and p6 are shown. B. Flow cytometric analysis of the surface expression of CD90, CD105, and CD34 (filled histograms) on the expanded cells at p6 was determined using a PE-conjugated Mouse Anti-Human CD90/Thy1 Monoclonal Antibody (R&D Systems, Catalog# FAB2067P), PE-conjugated Mouse Anti-Human Endoglin/CD105 Monoclonal Antibody (R&D Systems, Catalog # FAB10971P), and a PE-conjugated mouse anti-human CD34 monoclonal antibody. Staining with a PE-conjugated Mouse IgG1 Isotype Control (R&D Systems, Catalog # IC002P) or a PE-conjugated Mouse IgG2A Isotype Control (R&D Systems, Catalog # IC003P) is shown (open histogram). |
Expanded hMSC Maintain Differentiation Potential
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SUMMARY
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Figure 5. Media guide for the expansion and differentiation of hMSCs along the adipocyte, osteocyte and chondrocyte lineages. Human MSCs are cultured and expanded in StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media (R&D Systems, Catalog # CCM004), and osteogenesis, adipogenesis and chondrogenesis can be induced with the listed media and reagents all of which are included in the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC006). |
Selected R&D Systems Mesenchymal Stem Cell Products
| Catalog Number | Product | Usage |
| SC006 | Human Mesenchymal Stem Cell Functional Identification Kit | For the identification of human bone marrow-derived stem cells / mesenchymal stem cells by in vitro functional differentiation |
| CCM004 | StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media | A complete medium for the expansion of mesenchymal stem cells. |
| CCM005 | StemXVivo Human/Mouse Chondrogenic Base Media | A base medium for the differentiation of mesenchymal stem cells into chrondrocytes. |
| CCM006 | StemXVivo Human Chondrogenic Supplement | A medium supplement for the differentiation of mesenchymal stem cells into chrondrocytes. |
| CCM007 | StemXVivo Human/Mouse Osteogenic Base Media | A base medium for the differentiation of mesenchymal stem cells into osteocytes. |
| CCM008 | StemXVivo Human Osteogenic Supplement | A medium supplement for the differentiation of human mesenchymal stem cells into osteocytes. |
| CCM009 | StemXVivo Mouse Osteogenic Supplement | A medium supplement for the differentiation of mouse mesenchymal stem cells into osteocytes. |
| FAB2067P | Human CD90/Thy1 Phycoerythrin Monoclonal Ab | For the cytometric analysis of CD90 expressing cells |
| FAB10971P | Human Endoglin/CD105 Phycoerythrin Monoclonal Ab | For the cytometric analysis of CD105 expressing cells. |
| MAB1419 | Human Osteocalcin Monoclonal Ab | For the identification of osteoblast lineage cells by immunohistochemistry. |
| AF1220 | Human Aggrecan Affinity Purified Polyclonal Ab | For the identification of chondrocytes by immunohistochemistry. |
| AF1443 | Mouse FABP4 Affinity Purified Polyclonal Ab | For the identification of adipoctyes by immunohistochemistry. |
CONCLUSION
- Our rigorous testing of FBS sources, lots, and concentrations has allowed us to identify a wide range of MSC culture medium performance. We used these data to develop Human/ Mouse StemXVivo Mesenchymal Stem Cell Expansion Media, which outperforms other commercially available expansion media.
- In comparison with a leading competitor’s expansion medium, StemXVivo Mesenchymal Stem Cell Expansion Media resulted in a higher fold expansion of hMSC.
- Human MSCs cultured and expanded with the StemXVivo Mesenchymal Stem Cell Expansion Media retain the MSC phenotype as demonstrated by the presence of key MSC markers CD105 and CD90, and absence of CD34.
- The expanded hMSCs retain their full potential to undergo adipogenesis, osteogenesis and chondrogenesis.
- In combination with our media and reagents for MSC differentiation, StemXVivo Mesenchymal Stem Cell Expansion Media provides a complete research product line that facilitates study of hMSC, adipogenesis, osteogenesis and chondrogenesis.
ACKNOWLEDGEMENTS
We thank Judy Olson for expert technical assistance.
For research use only. Not for use in diagnostic procedures.