ABSTRACTMesenchymal stem cells (MSC) hold signifi cant promise for tissue engineering and regenerative medicine. Clinical and pre-clinical applications of MSC require that they be expanded in culture with maximum effi ciency while maintaining the ability to diff erentiate into key lineages. Fetal bovine serum (FBS) is a key factor in the in vitro maintenance of hMSC, but lot-to-lot variability can signifi cantly impact experimental results. In order to produce a growth medium that consistently allows optimal expansion of hMSC, we tested diff erent concentrations of FBS from several sources. Our results indicate that effi ciency 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 confi rm that cells expanded in StemXVivo Mesenchymal Stem Cell Expansion Media maintained their full diff erentiation potential. We assessed the phenotype of the cells after expansion and found them to be CD105+, CD90+ and CD34–. We tested their ability to diff erentiate into chondrocytes, osteocytes and adipocytes. Staining with lineage-specifi c antibodies confi rmed effi cient diff erentiation 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 effi ciency without compromising pluripotency.

IINTRODUCTION Mesenchymal stem cells or marrow stromal cells (MSCs) are adult stem cells with multi-lineage potential that are easily genetically modifi ed 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 diff erentiating into bone, cartilage, muscle, marrow, tendon, adipose and connective tissues (Figure 1). In 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 diff erentiation capacity. In order to assess the infl uence of diff erent 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 diff erentiation of the expanded MSCs into adipocytes, osteocytes and chondrocytes using commer-cially available diff erentiation 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 hMSCs.

MATERIALS & METHODSHuman 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 (Catalog# CCM004; R&D Systems, Minneapolis). To test the expansion potential of various lots of FBS, each was added to a fi nal concentration of 10% or 20% in -MEM medium (Invitrogen, Carlsbad) supplemented with glutamine and sodium pyruvate (Irvine Scientifi c, Santa Ana). 1.25 x 105 hMSCs were seeded into a T75 fl ask and cells were allowed to grow for 3 – 4 days before becoming confl uent. At the end of the culture period, hMSCs cultured in these various conditions were harvested at the same time with trypsin/ EDTA solution (Irvine Scientifi c). 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 fl ask 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 buff er (PBS supplemented with 2% FBS and sodium azide) and then resuspended at 106 cells per mL in FACS buff er. 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 buff er before data acquisition using FACSCalibur (BD Biosciences, San Jose) and analysis with CellQuest software (BD Biosciences).

Immunocytochemistry. Detailed protocols can be obtained from the insert of the Human Mesenchymal Stem Cell Functional Identifi cation Kit (Catalog # SC006, R&D Systems). Briefl y, cells cultured on cover slips were fi xed 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 Diff erentiation. hMSCs were induced to diff erentiate into adipocytes, osteocytes, or chondrocytes as described in protocols included with the Human Mesenchymal Stem Cell Functional Identifi cation Kit. Briefl y, completed adipogenic diff erentiation medium and a 24-well tissue culture plate with sterile cover slips were prepared and 3.7 x 105 hMSCs in 5 mL of -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% CO

2 humidifi ed incubator, cells were confl uent the next day and the medium was removed and replaced with 0.5 mL

of adipogenic diff erentiation medium. The diff erentiation medium was replaced every 3 – 4 days and adipocytes could be seen from day 7 onwards. Adipocytes were fi xed by day 21 and characterized by immunocytochemistry.

Osteogenic Diff erentiation. hMSCs were induced to diff erentiate into osteocytes using StemXVivo Human/Mouse Osteogenic Base Media (Catalog# CCM007; R&D Systems) and StemXVivo Human Osteogenic Supplement (Catalog# CCM008; R&D Systems). 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% CO

2 humidifi ed incubator for 1 – 2 days until 50 – 70% confl uent. The medium in each well was then replaced with 0.5 mL of completed osteogenic diff erentiation medium. The diff erentiation

medium was refreshed every 3 – 4 days and osteocytes were seen to appear between 14 – 21 days. The diff erentiated osteocytes were characterized by immunocytochemistry.

Chondrogenic Diff erentiation. Chondrocyte diff erentiation was induced using StemXVivo Human/Mouse Chondrogenic Base Media (Catalog# CCM005; R&D Systems) and StemXVivo Human Chondrogenic Supplement (Catalog# CCM006; R&D Systems). Briefl y, 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 diff erentiation 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% CO

2 humidifi ed incubator. Diff erentiation medium was replaced every

2 – 3 days and the chondrocyte pellet was harvested after 14 – 21 days. The chondrocyte pellet was fi xed, sectioned and then analyzed by immunocytochemistry.

RESULTS & FIGURES

CONCLUSION1. 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. a. In comparison with a leading competitor’s expansion medium, StemXVivo Mesenchymal Stem Cell Expansion Media resulted in a higher fold expansion

of hMSC.b. 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.c. The expanded hMSCs retain their full potential to undergo adipogenesis, osteogenesis and chondrogenesis.

2. In combination with our media and reagents for MSC diff erentiation, StemXVivo Mesenchymal Stem Cell Expansion Media provides a complete research product line that facilitates study of hMSC, adipogenesis, osteogenesis and chondrogenesis.

ACKNOWLEDGEMENTSWe thank Judy Olson for expert technical assistance.

R&D Systems, Inc. | www.RnDSystems.com | 800.343.7475

HUMAN MESENCHYMAL STEM CELL

Pre-AdipocyteOsteoblast

CULTURE WITH CCM004 CD34 -, CD90+, CD105+

Chondroblast

AdipocytesOsteocytes Chondrocytes

Cardiomyoblast,Myoblast

Cardiac MuscleSkeletal MuscleSmooth Muscle

DIFFERENTIATIONINDUCED WITH

CCM005 & CCM006,OR SC006

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CCM007 & CCM008,OR SC006

DIFFERENTIATIONINDUCED WITH

CCM007 & CCM011,OR SC006

IMPROVED EXPANSION OF MSC WITHOUT LOSS OF DIFFERENTIATION POTENTIALAnissa S.H. Chan, Electra Coucouvanis, Susan Tousey, Marnelle D. Andersen and Jessie H.T. Ni Stem Cell and Developmental Biology Department, R&D Systems, Inc., 614 McKinley Pl. NE, Minneapolis, MN, 55413

MSC PROLIFERATION

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Bone Cartilage Muscle Marrow Tendon/Ligament Connective Tissue

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Figure 2. Eff ect of 3 diff erent FBS samples on the expansion of human MSCs. 1.25 x 105 hMSC at passage 4 (p4) were cultured with three diff erent FBS samples at a fi nal 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 fl ask. 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.

Result: Testing of the various FBS combinations in -MEM basal medium shows that diff erent FBS samples demonstrate a qualitative diff erence 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.

MSC Expansion Varies with Diff erent Serum Samples

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 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 (fi lled histogram) on the expanded cells at p6. Isotype control is shown (open histogram).

Result: 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. 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.

Expanded hMSC Maintain Correct Phenotype

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RESUSPEND hMSCs IN ADIPOGENIC

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RESUSPEND hMSCs IN CHONDROGENIC

DIFFERENTIATION MEDIUM

CULTURE FOR 7 – 10 DAYS (CHANGED MEDIUM EVERY 3 – 4 DAYS)

CULTURE FOR 7 – 10 DAYS (CHANGED MEDIUM EVERY 3 – 4 DAYS)

CULTURE FOR 7 – 10 DAYS (CHANGED MEDIUM EVERY 3 – 4 DAYS)

Harvested hMSCs Harvested hMSCs Harvested hMSCs

Figure 4A. The expanded hMSCs display adipogen-ic potential. Top: Schematic representation of ad-ipogenic diff erentiation of expanded hMSCs using the Human Mesenchymal Stem Cell Functional Identifi cation Kit (SC006). The image at bottom shows 21 day diff erentiated adipocytes stained with anti-FABP4 antibody (red) and counterstained with DAPI (blue). Picture shown was taken at 10X.

Expanded hMSC Maintain Diff erentiation Potential

Figure 4B. Osteogenesis of the expanded hMSCs. Top: Schematic representation of osteogenic dif-ferentiation of expanded hMSCs using StemXVivo Human/Mouse Osteogenic Base Media (CCM007) and StemXVivo Human Osteogenic Supplement (CCM008). The image at bottom shows 21 day dif-ferentiated osteocytes stained with anti-osteocal-cin antibody (red) and counterstained with DAPI (blue). Picture shown was taken at 20X.

Figure 4C. Chondrogenesis of the expanded hM-SCs. Top: Schematic representation of chondro-genic diff erentiation of expanded hMSCs using StemXVivo Human/Mouse Chondrogenic Base Media (CCM005) and StemXVivo Human/Mouse Chondrogenic Supplement (CCM006). The image at bottom shows 21 day diff erentiated chondro-cytes stained with anti-aggrecan antibody (red). Picture shown was taken at 20X.

Adipogenesis Osteogenesis Chondrogenesis

Summary

Figure 5. Media guide for the expansion and diff erentiation of hMSCs along the adipocyte, osteocyte and chondrocyte lineages. Human MSCs are cultured and expanded in StemXVivo Human/ Mouse Mesenchymal Stem Cell Expansion Media, 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 Identifi cation Kit.

B.A.

4A. 4B. 4C.

Figure 1. The diff erentiation potential of MSC.

Catalog Product Usage

SC006 Human Mesenchymal Stem Cell Functional Identifi cation Kit For the identifi cation of human bone marrow-derived stem cells / mesenchymal stem cells by in vitro functional diff erentiation

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 diff erentiation of mesenchymal stem cells into chrondrocytes.

CCM006 StemXVivo Human Chondrogenic Supplement A medium supplement for the diff erentiation of mesenchymal stem cells into chrondrocytes.

CCM007 StemXVivo Human/Mouse Osteogenic Base Media A base medium for the diff erentiation of mesenchymal stem cells into osteocytes.

CCM008 StemXVivo Human Osteogenic Supplement A medium supplement for the diff erentiation of human mesenchymal stem cells into osteocytes.

CCM009 StemXVivo Mouse Osteogenic Supplement A medium supplement for the diff erentiation 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 identifi cation of osteoblast lineage cells by immunohistochemistry.

AF1220 Human Aggrecan Affi nity Purifi ed Polyclonal Ab For the identifi cation of chondrocytes by immunohistochemistry.

AF1443 Mouse FABP4 Affi nity Purifi ed Polyclonal Ab For the identifi cation of adipoctyes by immunohistochemistry.

Selected R&D Systems Mesenchymal Stem Cell Products