Mesenchymal Stem Cells - Stemcell Technologies · Mesenchymal stem cells (MSCs) are fibroblast-like...

Mesenchymal Stem CellsMesenCult™: Your High-Performance System

for MSC Isolation, Culture & Differentiation

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Product Overviewfor Mesenchymal Stem Cells

IsolateObtain untouched MSCs from human bone marrow or mouse compact bone or select CD271+ MSCs from human bone marrow.• RosetteSep™ Human MSC Enrichment Cocktail: page 4• EasySep™ Human CD271 Positive Selection Kit: page 5• EasySep™ Mouse MPC Enrichment Kit for Compact Bone: page 13

Dissociate and Cryopreserve Dissociate and cryopreserve MSCs with animal component-free media.• MesenCult™-ACF Dissociation Kit: page 6• MesenCult™-ACF Freezing Medium: page 7• CryoStor®CS10, CS5 and CS2: page 7

Support Products• Hypoxia Chamber: page 15• ALDEFLUOR™ for Stem and Progenitor Cell Identification: page 15• Support Reagents, Antibodies and Primary Cells: page 15

STEMCELL Technologies’ products are manufactured under a Quality Management System (QMS) certified to ISO 13485. Unless otherwise indicated, products are provided for research use only, not for human or animal therapeutic or diagnostic use.

Your regulatory authority will provide guidance on the requirements for ancillary materials for cell therapy applications. Depending on the requirements, STEMCELL may be able to assist you in meeting your regulatory and quality requirements.

STEMCELL Technologies stands behind the quality of our products. We welcome onsite audits of our manufacturing facilities to ensure that your quality requirements are met. If you have any questions or would like to discuss the potential use of a product for your application please contact us.

CulturePerform CFU-F assays and expand human and mouse MSCs.• MesenCult™-ACF Medium for Human MSCs: page 6• MesenCult™-XF Medium for Human MSCs: page 8• MesenCult™ Proliferation Kit (Human): page 10• MesenCult™ Proliferation Kit with MesenPure™ (Mouse): page 12

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DifferentiateDifferentiate MSCs to adipocytes, osteoblasts and chondrocytes• MesenCult™ Adipogenic Differentiation Medium (Human): page 11• Adipogenic Stimulatory Supplement (Mouse): page 14• Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF): page 11• Osteogenic Stimulatory Kit (Human): page 11• Osteogenic Stimulatory Kit (Mouse): page 14• MesenCult™-ACF Chondrogenic Differentiation Medium: page 11New

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Mesenchymal Stem Cell Products

For Every Facet of Discovery

Mesenchymal stem cells (MSCs) are fibroblast-like cells isolated from bone marrow, adipose, and other tissues - including cord blood, peripheral blood, fetal liver, skeletal muscle, placenta, amniotic fluid and synovium.1-7 These are all vascularized tissues, and accumulating evidence suggests that MSCs are pericytes8 which closely encircle endothelial cells in capillaries and microvessels of multiple organs.8-15

MSCs are defined by properties exhibited following in vitro culture. Namely the ability to self-renew, differentiate into bone, adipose and cartilage tissue,16 the expression of CD105, CD73 and CD90, and the lack of expression of CD45, CD34, CD11b and HLA-DR. While originally coined mesenchymal stem cells,17 MSCs are also referred to by other terms, such as multipotent mesenchymal stromal cells,16,18 mesenchymal progenitor cells19 or medicinal signaling cells.20 No single term has been uniformly adopted, and as a result, the acronym MSC is commonly used to encompass all terminologies applied to these cells.

MSCs have potential utility in a range of cellular therapies, with applications relating to tissue engineering and regenerative medicine, and as vehicles for gene therapy.21-24 To realize the therapeutic potential of MSCs, studies in animal models are of fundamental importance. Because MSCs are rare, occurring at an estimated frequency of 1 in 100,000 cells in adult human bone marrow, they must be expanded in vitro to obtain sufficient numbers for research and therapeutic applications.25 The ability to expand human MSCs in xeno-free or animal component-free medium may alleviate concerns about immune rejection of transplanted cells or disease transmission, and is therefore an important consideration when the MSCs are to be used therapeutically.25

Advantages of MesenCult™

HIGH-PERFORMANCE CULTURES. Species-optimized media formulations provide superior performance compared to traditional and competitor media.

COMPREHENSIVE. A comprehensive and integrated range of products is available for the isolation, enrichment, expansion and differentiation of human and mouse MSCs.

STANDARDIZED. Standardized media and culture conditions minimize variability and lead to increased reproducibility between experiments.

EASY TO USE. Products are supplied with detailed technical information to guide researchers through the enrichment and culture of MSCs.

MesenCult™Your High-Performance System for MSC Isolation, Culture & Differentiation

Choose from a comprehensive range of specialty products for human and mouse MSCs designed to service scientists along the basic to translational research continuum.

The MesenCult™ product line was developed as a comprehensive and integrated suite of products to help MSC researchers standardize their cell culture system and overcome issues associated with cell culture media variability. Providing products for both mouse and human MSCs enables STEMCELL Techologies to serve scientists along the basic to translational research continuum.

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Human MSCs can be rapidly and easily enriched directly from unprocessed bone marrow using the RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail and a simple density gradient centrifugation (Figure 1, Table 1).

The RosetteSep™ Enrichment Cocktail contains monoclonal antibodies to deplete cells expressing CD3, CD14, CD19, CD38, CD66b and Glycophorin A. The antibodies link unwanted cells to red blood cells present in the bone marrow sample, causing them to pellet together when centrifuged over the density gradient medium. Desired cells are left untouched and highly enriched at the density gradient medium : plasma interface.

Figure 1. Overview of RosetteSep™ Human Mesenchymal Stem Cell Enrichment procedure.

Table 1. CFU-F frequency and fold enrichment obtained for RosetteSep™-enriched human bone marrow samples (n = 9; Mean ± SD).

* Data normalized to density gradient centrifugation-separated bone marrow.§ p < 0.0001 when compared to density gradient centrifugation alone. The CFU-F assay was performed with the MesenCult™ Proliferation Kit (Human; Catalog #05411).

Add RosetteSep™ cocktail to bone marrow

Unwanted cells are linked to red blood cells (rosetted)

Incubate 20 minutesat room temperature

Layer over density gradient medium (e.g. Lymphoprep™)

Density gradient medium

PlasmaEnriched MSCs

Density gradient medium

Red blood cells and unwanted cells (rosetted)

Enriched MSCs are untouched

Label

Spin

Collect

Enrich Human MSCsWith RosetteSep™ Human MSC Enrichment Kit

PRODUCT QUANTITY CATALOG #

RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail

For labeling 40 mL whole bone marrow

15128

For labeling 200 mL whole bone marrow

15168

DENSITY GRADIENT CENTRIGUFATION

ONLYROSETTESEP™

Frequency of CFU-F

1 : 84,000 ± 37,000 1 : 12,500 ± 6,500§

Recovery of CFU-F

100% 64 ± 24%

Fold Enrichment*

1 8 ± 2

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Isolate Human MSCsWith EasySep™ CD271 Positive Selection Kit

CD271 has been identified as a marker of MSCs and CD271+ cells isolated from human bone marrow display high CFU-F activity.26 CD271 is also known as the p75 neurotrophin receptor (p75NTR) or the low-affinity nerve growth factor receptor (LNGFR). The EasySep™ Human CD271 Positive Selection Kit is designed to select CD271+ cells from fresh bone marrow mononuclear cells with high purity and recovery (Figure 2, Table 2).

EasySep™ is a powerful cell separation tool that is fast, easy and gentle on cells. With the EasySep™ Human CD271 Positive Selection Kit, CD271+ cells are targeted for selection by linking CD271+ cells to magnetic particles. The tube containing the magnetically labeled cells is placed into an EasySep™ magnet, and after a brief incubation the unlabeled cells are poured off. The highly purified CD271+ cells remain in the tube. EasySep™ magnetic particles do not interfere with downstream applications such as flow cytometry and do not need to be removed to preserve cell functionality.

Figure 2. Overview of EasySep™ Human CD271 Positive Selection procedure.

Add EasySep™reagents

Incubate 15 minutes

Add EasySep™magnetic particles

Incubate 15 minutes

Place tube in magnet for 5 minutes

Pour off supernatant. Positively selected cells remain in tube.

Bone marrowcell suspension

Collect

Table 2. CFU-F fold enrichment obtained for EasySep™-enriched human bone marrow samples (n = 4; Mean ± SEM).


SELECTION COCKTAIL

EasySep™ Human CD271 Positive Selection Kit

For 1 x 109 cells 18659

REQUIRED EQUIPMENT

EasySep™ Magnet 1 magnet 18000

EASYSEP™ HUMAN CD271+ SELECTION

CFU-F Fold Enrichment 23 ± 6

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New: Culture Human MSCs With Animal Component-Free MesenCult™-ACF Medium

MesenCult™-ACF is a defined, serum- and animal component-free (ACF) culture medium to support efficient derivation and expansion of MSCs from primary human bone marrow (BM) and adipose tissue. When used in combination with MesenCult™-ACF Attachment Substrate, MesenCult™-ACF Dissociation Kit and MesenCult™-ACF Freezing Medium, MesenCult™-ACF Medium is part of a complete, animal component-free culture system which supports efficient attachment, clonogenic growth and long-term expansion of primary human MSCs.

MSCs cultured with MesenCult™-ACF exhibit robust suppression of T cell proliferation and significant reduction in hematopoietic cell contamination at early passages.

Advantages of MesenCult™-ACF

• Defined, animal component-free culture system

• Optimized for isolation of MSCs directly from primary human tissue

• No adaptation required when transitioning MSCs from serum-containing medium

• Significant reduction in hematopoietic cell contamination at early passages

• Supports efficient clonogenic growth and long-term expansion of human MSCs

Figure 3. Human BM-derived MSCs cultured in MesenCult™-ACF display multi-lineage differentiation potential.

Human BM-derived MSCs cultured in MesenCult™-ACF Medium for 3 passages (A) differentiate into adipocytes (B; Oil Red O staining), osteogenic cells (C; Von Kossa/ALP staining) and chondrocytes (D; Alcian Blue + Nuclear Fast Red staining).

BA

C D

Magnification: 40X

Magnification: 40X

Magnification: 40X

Magnification: 40X


MesenCult™-ACF Culture Kit* 1 kit 05449

MesenCult™-ACF Medium§ 500 mL 05440

MesenCult™-ACF Dissociation Kit 1 kit 05426

*MesenCult™-ACF Culture Kit includes MesenCult™-ACF Medium (Catalog #05440) and MesenCult™-ACF Attachment Substrate (Catalog #05444).§MesenCult™-ACF Medium must be supplemented with L-Glutamine (e.g. Catalog #07100), and must be used in conjunction with MesenCult™-ACF Attachment Substrate and MesenCult™-ACF Dissociation Kit.

Figure 5. Human BM-derived MSCs cultured in MesenCult™-ACF display characteristic expression of MSC surface markers.

Human BM-derived MSCs cultured in MesenCult™-ACF were stained at Passage 4 with antibodies to mesenchymal markers (CD90, CD73 and CD105) and hematopoietic markers (CD45, CD11b and HLA-DR).

0

0 102 103 104 105

0

0 102 103 104 105

0

0 102 103 104 105

CD90 PE CD73 PE CD105 PE

0

0 102 103 104 105

0

0 102 103 104 105

0

0 102 103 104 105

CD45 PE CD11b PE HLA-DR APC

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New

Figure 4. Human BM-derived MSCs cultured in MesenCult™-ACF expand faster than cells cultured in serum (FBS)-based or competitor media. (n = 3 except for Competitor 3 where n = 2; Mean ± SEM).

Competitor 2 (xeno-free medium)

P1 P2 P3 P4 P5 P6 P7 P8

Traditional formulation (FBS-based)

MesenCult™-ACF Medium

Competitor 1 (serum-free medium)

1010

109

108

107

106

105

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102

Competitor 3 (chemically-defined medium)

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Cryopreserve MSCs With Animal Component-Free Freezing Media

MesenCult™-ACF Freezing Medium is a defined, serum-free and animal component-free medium for the cryopreservation of MSCs. This complete and ready-to-use medium is recommended for cryopreservation of human MSCs previously cultured in MesenCult™-ACF (Catalog #05440), MesenCult™-XF (Catalog #05420) or MesenCult™ (MesenCult™ Proliferation Kit; Catalog #05411) media.

Advantages of MesenCult™-ACF Freezing Medium

• Defined, serum-free and animal component-free

• Reproducibly high recovery rates

• Preserves human MSC multipotency and expansion capacities

• Convenient, ready-to-use format

Figure 6. Human MSCs cryopreserved in MesenCult™-ACF Freezing Medium show reproducibly high post-thaw cell viability.

Human MSCs cultured in serum-free medium and cryopreserved in MesenCult™-ACF Freezing Medium show reproducibly high viability of cells after thawing. Viability is higher than observed with a competitor ACF freezing medium (n = 6 independent experiments, P = 0.001).

Figure 7. Human MSCs cryopreserved in MesenCult™-ACF Freezing Medium maintain multi-lineage differentiation potential.

Human MSCs cultured in serum-free medium and cryopreserved with MesenCult™-ACF Freezing Medium (A; 3 days after thawing) differentiate to the adipogenic (B; Oil Red O staining) and osteogenic (C; Alizarin Red staining) lineages.

BA C


MesenCult™-ACF Freezing Medium

50 mL 05490

CryoStor®CS10 100 mL 07930

5 x 16 mL 07930



MesenCult™-ACF Freezing Medium

CryoStor®CS10, CS5 and CS2:

CryoStor® cryopreservation media products are formulated with 2, 5 or 10% USP-grade DMSO and are cGMP manufactured. These versatile media are designed to maximize post-thaw viability and recovery of a variety of cell types, including MSCs.

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Reduce variability by culturing your human MSCs in a standardized xeno-free (XF), serum-free medium. MesenCult™-XF Medium is optimized for both the expansion of MSCs and their enumeration using the CFU-F assay.

MesenCult™-XF supports the serum-free isolation of MSCs from primary tissue. Passage 0 cultures obtained from primary human bone marrow (BM) show significantly reduced hematopoietic cell contamination compared to serum-based cultures (Figure 8). Furthermore, MSCs expanded in MesenCult™-XF for two passages showed no detectable hematopoietic cell contamination, indicated by the lack of CD34 and CD45 expression (Figure 9). MSCs cultured in MesenCult™-XF also robustly express the mesenchymal markers CD90, CD105 and CD73 (Figure 9).

MesenCult™-XF supports the long-term culture (>8 passages) of MSCs, with cells expanding faster in MesenCult™-XF than MSCs cultured in serum-based medium (Figure 10). MSCs cultured in MesenCult™-XF maintain the ability to differentiate into adipogenic, osteogenic and chondrogenic lineages (Figure 11). The chondrogenic differentiation potential of MSCs cultured in MesenCult™-XF is enhanced at early passages (e.g. P2) and better maintained after multiple passages (e.g. P4, P8) compared to cells cultured in serum-based medium (Figure 12). MSCs cultured in MesenCult™-XF also more robustly suppress T cell proliferation and reduce cell cycle division (Figure 13).

* MesenCult™-SF Culture Kit includes MesenCult™-XF Medium (Catalog #05420) and MesenCult™-SF Attachment Substrate (Catalog #05424).§ MesenCult™-XF Medium must be supplemented with L-Glutamine (e.g. Catalog #07100), and must be used in conjunction with the MesenCult™-SF Attachment Substrate and the MesenCult™-ACF Dissociation Kit.

Advantages of MesenCult™-XF

• Defined, xeno-free formulation

• Optimized for use with MSCs obtained directly from primary tissue

• Supports faster cell expansion compared to traditional serum-based medium

• Cultured MSCs exhibit robust suppression of T cell proliferation

• Minimal lot-to-lot variability

Figure 8. Primary human bone marrow-derived MSCs show less hematopoietic cell contamination when cultured in MesenCult™-XF (A) compared to a traditional serum-based medium (B).

Magnification: 5X

A

Magnification: 5X

B

Passage 0 Passage 0

REFERENCESMesenCult™-XF is the best defined culture system for umbilical cord MSCs (Hartmann et al. 2010).www.stemcell.com/references

Culture Human MSCsWith Xeno-Free MesenCult™-XF Medium


MesenCult™-SF Culture Kit* 1 kit 05429

MesenCult™-XF Medium§ 500 mL 05420

MesenCult™-SF Attachment Substrate

1 kit 05424

9


Magnification: 20X

E

Magnification: 40X

A

Magnification: 10X

B

Figure 11. Human bone marrow-derived MSCs cultured in MesenCult™-XF display multi-lineage differentiation potential.

A. Oil red O staining of adipocytes generated from passage 1 MSCs.B. Alizarin Red detection of Ca++ deposits indicates the formation of bone structures in cells generated from passage 4 MSCs.C. Collagen II staining of chondrocytes generated from passage 2 MSCs.

Magnification: 10X

C

0 1 2 3 4 5 6 7 8 9 10

MesenCultTM-XF Medium

Traditional formulation

100

102

104

106

108

1010

1012

Figure 10. Human BM-derived MSCs cultured in MesenCult™-XF expand faster than cells cultured in a traditional serum-based medium (n = 3; Mean ± SEM).

Passage Number

Tota

l Cel

ls

Figure 13. MSCs cultured in MesenCult™-XF more robustly suppress T cell proliferation than MSCs cultured in a traditional serum-based medium.

Passage 2 MSCs generated in MesenCult™-XF or a traditional serum-based medium were treated with mitomycin C prior to co-culture with T cells. T cells were purified from peripheral blood using EasySep™ cell separation and fluorescently labeled with CFSE dye. 2 x 105 CFSE-labeled T cells were cultured with 1 x 105 MSCs in serum-free medium supplemented with 100 U/mL IL-2. T cells were stimulated with tetrameric antibody complexes against CD3ε, CD28 and CD2. On days 3 and 7, cells were harvested, stained with anti-CD45 antibody and propidium iodide and the T cell division history measured as CFSE dye dilution analyzed by flow cytometry.

CFSE

Day 3

Day 7

Unstimulated T cells Activated T cells FBS MSCs MesenCultTM-XF MSCs

1% (37%)

3% (95%) 82% (95%)

18% (37%) 1% (37%)

53% (95%)

Magnification: 4X

A

Magnification: 20X

B

Figure 12. MSCs cultured in MesenCult™-XF (A, B, C) better maintain chondrogenic differentiation potential than MSCs cultured in a traditional serum-based medium (D, E, F).

Magnification: 20X

C

Magnification: 4X

D

Magnification: 20X

F

Passage 2 Passage 4 Passage 8

Figure 9. Phenotype of MesenCult™-XF culture-expanded human MSCs.

Cou

nts

CD90 (Thy-1)+

0

10

20

30

40

50

100 101 102 103 104

CD105 (SH2)+

Cou

nts

100 101 102 103 1040

10

20

30

40

50

60

70

CD73 (SH3/SH4)+

Cou

nts

100 101 102 103 1040

10

20

30

40

50

60

CD34-100 101 102 103 104

Cou

nts

0

10

20

30

40

50

60

CD45-100 101 102 103104

Cou

nts

0

10

20

30

40

50

60

10

Standardize your human MSC cultures with the serum-based MesenCult™ Proliferation Kit (Human). The optimized medium formulation enables efficient expansion of MSCs in vitro (representative expansion data are shown in Table 3) and components are prescreened to minimize lot-to-lot variability. The culture-expanded cell population maintains multi-lineage potential and cells express CD105 (SH2), CD73 (SH3/SH4) and CD90 (Thy-1) and lack expression of CD45 and CD34 (Figure 14).

The MesenCult™ Proliferation Kit (Human) is comprised of MesenCult™ MSC Basal Medium (Human) and Mesenchymal Stem Cell Stimulatory Supplement (Human), and can be used for both the expansion of human MSCs and their enumeration using the CFU-F assay.

Table 3. Expansion of MSCs obtained from four consecutive human bone marrow (BM) samples that were cultured with the MesenCult™ Proliferation Kit (Human).

* The MesenCult™ Proliferation Kit (Human) comprises MesenCult™ MSC Basal Medium (Human; Catalog #05401) and Mesenchymal Stem Cell Stimulatory Supplement (Human; Catalog #05402).

Figure 14. Phenotype of culture-expanded human MSCs using the MesenCult™ Proliferation Kit (Human).

CD34-

Cou

nts

100 101 102 103 104

0

10

20

30

40

50

CD90 (Thy-1)+

Cou

nts

1000

10

20

30

40

101 102 103 104

50

Culture Human MSCsUsing MesenCult™ Proliferation Kit (Human)

BM SAMPLE FOLD EXPANSION DAYS IN CULTURE

A 3732X 40

B 6966X 58

C 1770X 41

D 230X 28


MSC CULTURE MEDIUM

MesenCult™ Proliferation Kit (Human)*

500 mL 05411

PROLIFERATION KIT COMPONENTS

MesenCult™ MSC Basal Medium (Human)

450 mL 05401

Mesenchymal Stem Cell Stimulatory Supplement (Human)

50 mL 05402

CD105 (SH2)+

Cou

nts

1000

10

20

30

40

50

101 102 103 104

CD73 (SH3/SH4)+

Cou

nts

100 101 102 103 1040

10

20

30

40

50

CD45-

Cou

nts

100 101 102 103 1040

10

20

30

40

50

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Optimize the differentiation of human MSCs with standardized media and supplements. New animal component-free MesenCult™-ACF Chondrogenic Differentiation Medium delivers efficient and robust chondrogenic differentiation of human MSCs with as few as 3 x 105 cells per 3D pellet and in as little as 14 days (Figure 15).

MesenCult™ Adipogenic Differentiation Medium (Human) provides robust adipogenic differentiation of human BM- and adipose-derived MSCs previously cultured in serum-containing or serum-free media. The easy-to-use protocol does not require cycles of induction and maintenance.

The complete Osteogenic Stimulatory Kit (Human) enables the differentiation of human MSCs into osteogenic progenitors, and the detection and enumeration of human colony-forming unit - osteoblast (CFU-O). The kit also supports CFU-O assays from rat bone marrow cells. For human MSCs previously cultured in MesenCult™-XF, use the Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF) for in vitro differentiation into osteogenic progenitors.

Culture in MesenCult™ Adipogenic Differentiation Medium (Human)

Adiopocytes derived from human MSCs differentiated in vitro with MesenCult™ Adipogenic Differentiation Medium (Human)

AdipogenicDifferentiation

Culture-expanded MSCs

Culture with the Osteogenic Stimulatory Kit.

Figure 16. Adipogenic and osteogenic differentiation of human MSCs.

OsteogenicDifferentiation

Differentiate Human MSCsTo Adipocytes, Osteoblasts and Chondrocytes


ADIPOGENIC DIFFERENTIATION

MesenCult™ Adipogenic Differentiation Medium

250 mL 05412

OSTEOGENIC DIFFERENTIATION

Osteogenic Stimulatory Kit (Human)

1 kit 05404

Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF)

1 kit 05434

CHONDROGENIC DIFFERENTIATION

MesenCult™-ACF Chondrogenic Differentiation Medium

100 mL 05455

Bone nodule derived from human MSCs differentiated in vitro with the Osteogenic Stimulatory Kit (Human).

A. Phase micrograph of human bone nodule

B. Tetracycline labeled human bone nodule

C. SEM of human bone nodule showing mineralized collagen ECM

D. TEM of human bone nodule

SEM: scanning electron micrographTEM: transmission electron

micrographECM: extracellular matrix

A

CD

B

Photos kindly provided by Dolores Baksh, Peter W. Zandstra and John E. Davies, Institute of Biomaterials & Biomedical Engineering, University of Toronto

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Figure 15. MesenCult™-ACF Chondrogenic Differentiation Medium induces robust chondrogenic differentiation of human MSCs with as few as 3 x 105 cells and as early as day 14.

Human BM-derived MSCs were cultured in MesenCult™-ACF Medium then differentiated to the chondrogenic lineage using MesenCult™-ACF Chondrogenic Differentiation Medium. Robust chondrogenic differentiation was observed (A) starting with as few as 3 x 105 MSCs, or (B) when differentiating for just 14 days starting with 5 x 105 MSCs.

A B

200 μm

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Enrich and Culture Mouse MSCsUsing MesenCult™ Proliferation Kit with MesenPure™ (Mouse)

Establishing mouse MSC cultures is inherently difficult due to both the rarity of these cells in bone marrow (BM) and compact bone (CB) and because these cultures often contain a high proportion of hematopoietic cells.27-29

The MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is a standardized medium for the expansion of mouse MSCs as well as their enumeration using the CFU-F assay. This optimized medium enriches for mouse MSCs in culture without serial passaging and frequent medium changes, steps routinely employed to deplete unwanted hematopoietic cells from mouse MSC cultures using other media. Primary MSCs treated with MesenPure™ appear homogeneous and mostly devoid of hematopoietic cells as early as passage 0 (Figure 17). Cultures also contain increased numbers of MSCs that display more robust differentiation (Figure 18 and 19).

Control With MesenPure™

P0

P3

100 µm

Figure 17. Mouse MSC cultures treated with MesenPure™ appear homogeneous and mostly devoid of hematopoietic cells as early as passage 0 (P0).

Primary BM-derived MSCs were cultured under hypoxic conditions in complete MesenCult™ medium without (Control) or with MesenPure™. In control conditions, many hematopoietic cells (viewed as highly refractile cells under phase contrast optics) are present at P0 and still present at P3 (left panels). In MesenPure™-treated cultures, low numbers of hematopoietic cells were observed as early as P0, with subsequent passages reducing the number further (right panels).

Figure 18. Flow cytometry analysis of P0 MSC culture treated with MesenPure™ demonstrates significant enrichment of CD45-/CD29+/Sca1+ cells.

Primary BM-derived MSCs were cultured for 14 days under hypoxic conditions in complete MesenCult™ medium without (Control) or with MesenPure™ prior to flow cytometry analysis being performed. ~9% of the control culture and ~70% of the MesenPure™-exposed culture was CD45-. Analysis of the CD45- population revealed ~75% of the control cells and ~96% of the MesenPure™-exposed cells co-expressed CD29 and Sca1.

SS

C

SS

C

Sca

1-F

ITC

Sca

1-F

ITC


CD45-

9.04%CD45+

90.53%CD45-

70.48%CD45+

28.88%

6.70% 74.88%

5.57%

1.22% 96.11%

1.29%

P0 Cultures

CD45- Selected Cells from P0 Cultures

12.84% 1.38%

CD29-PECY7 CD29-PECY7

CD45-APC CD45-APC

Mouse MSCs expand faster when cultured at 5% O2 compared to 20% O2. Use the hypoxia chamber (Catalog #27310) to generate a hypoxic environment (See page 15).

Advantages of MesenPure™

SAVES TIME. Fast enrichment of mouse MSC cultures without serial passaging.

HIGH-PERFORMANCE CULTURES. Homogeneous mouse MSC cultures display robust self-renewal and differentiation potential.

VERSATILE. Optimized for use with mouse bone marrow- and compact bone-derived MSCs and MEFs.

EASY TO USE. Simply add MesenPure™ to complete MesenCult™ proliferation medium just prior to use.

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EASYSEP™ MOUSE MPC ENRICHMENT FOR COMPACT BONE

CFU-F Fold Enrichment 110 ± 74

Recovery of CFU-F 58 ± 19%

B

A

Figure 19. MSCs exposed to MesenPure™ require fewer cells to demonstrate robust differentiation.

(A) BM-derived MSCs initially cultured in complete MesenCult™ medium without (Control) or with MesenPure™ were plated at three different densities and then differentiated using MesenCult™ Osteogenic Stimulatory Kit (Mouse; Catalog #05504). Differentiation cultures were fixed and stained for Alkaline Phosphatase (ALP) activity (red areas) and mineralization (black areas). In control cultures, modest differentiation into the osteogenic lineage was only observed when cultures were seeded at the highest density. In MesenPure™-treated cultures, robust osteogenic differentiation was observed at all seeding densities. (B) 4.0 x 104 cells/cm2 of P2 CB-derived MSCs were used in osteogenic and adipogenic differentiation. 5.0 x 105 cells of P1 CB-derived MSCs were used for chondrogenic differentiation. MesenPure™-treated cultures displayed strong osteogenic, adipogenic and chondrogenic differentiation.

Number of cells/cm2 (x103)

40 60 120 40 60 120

BM-MSCs

CB-MSCs

Osteogenic Adipogenic

Number of cells/cm2 (x103)


Chondrogenic

*The MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is comprised of MesenCult™ MSC Basal Medium (Mouse; Catalog #05501), Mesenchymal Stem Cell Stimulatory Supplements (Mouse; Catalog #05502) and MesenPure™ (0.5 mL; Catalog #05500)


MesenCult™ Proliferation Kit with MesenPure™ (Mouse)*

1 kit 05512

MesenCult™ MSC Basal Medium (Mouse)

400 mL 05501

Mesenchymal Stem Cell Stimulatory Supplements (Mouse)

100 mL 05502

VIDEOIsolation of MSCs from Mouse Compact Bonewww.stemcell.com/CompactBoneVideo

Mouse MSC Enrichment from Compact Bone Using EasySep™

The EasySep™ Mouse MPC Enrichment Kit for Compact Bone is designed to enrich MSCs from mouse compact bone cell suspensions by depleting undesired cells (Table 4). It can be used as an alternative method for mouse MSC enrichment from compact bone if culture-based enrichment using the MesenCult™ Proliferation Kit with MesenPure™ (Mouse) is not desired.

EasySep™ is a powerful cell separation tool that is fast, easy and gentle on cells. With the EasySep™ Mouse MPC Enrichment Kit for Compact Bone, cells expressing CD45 and TER119 are targeted for depletion by linking them to EasySep™ magnetic particles via tetrameric antibody complexes. The tube containing the magnetically labeled cells is then placed into an EasySep™ magnet and after a brief incubation the unlabeled MSCs are poured off and collected. The unwanted cells remain in the tube.

Table 4. CFU-F fold enrichment and recovery obtained for EasySep™-enriched mouse compact bone samples compared to control samples in complete MesenCult™ medium without MesenPure™ (n = 6; Mean ± SEM).

* Note: The EasySep™ Mouse MPC Enrichment Kit for Compact Bone is optimized for the enrichment of mouse MSCs from compact bone, not bone marrow. A technical video outlining the procedure for the isolation of cells from mouse compact bone is available at www.stemcell.com/CompactBoneVideo


ENRICHMENT COCKTAIL

EasySep™ Mouse MPC Enrichment Kit for Compact Bone*

For 4 x 108 cells

19771

REQUIRED EQUIPMENT

EasySep™ Magnet 1 magnet 18000

100 µm

Learn more about mouse MSC culture by watching the on-demand webinar “Tips & Techniques for Highly Enriched Mouse MSC Cultures As Early As Passage 0” available at www.stemcell.com/MesenPureWebinar.

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Optimize the differentiation of mouse MSCs to Osteoblasts and adipocytes with standardized media and supplements.

Mouse MSCs or mouse embryonic fibroblasts (MEFs) achieve unparalleled levels of osteogenic differentiation with the MesenCult™ Osteogenic Stimulatory Kit (Mouse) compared to competitor formulations. Superior expression of key osteogenic transcripts involved in bone differentiation and maturation is observed for MSCs differentiated with the MesenCult™ Osteogenic Stimulatory Kit (Mouse) compared to cells differentiated with a leading commercially available medium for mouse cells (qPCR analysis, Figure 20a). Cultures also exhibit robust bone matrix deposition (Alizarin Red-staining, Figure 20c).

The Adipogenic Stimulatory Supplement (Mouse) efficiently differentiates mouse mesenchymal stem cells to adipocytes when combined with MesenCult™ MSC Basal Medium (Mouse) (Figure 21).

* The Adipogenic Stimulatory Supplement (Mouse) must be used in conjunc-tion with MesenCult™ MSC Basal Medium (Mouse; Catalog #05501).

Figure 21. Differentiation of mouse compact bone-enriched MSCs to adipocytes using the Adipogenic Stimulatory Supplement (Mouse) combined with MesenCult™ MSC Basal Medium (Mouse).

A. Unstained adipocytes generated from passage 3 cells previously cultured with the MesenCult™ Proliferation Kit (Mouse).

B. Oil red O staining of adipocytes generated from passage 3 cells previously cultured with the MesenCult™ Proliferation Kit (Mouse).

Figure 20. Differentiation of mouse compact bone-derived MSCs with the MesenCult™ Osteogenic Stimulatory Kit (Mouse).

A. Expression of key osteogenic transcripts in mouse compact bone-derived MSCs as measured by qPCR.

B. Undifferentiated mouse compact bone-derived MSCs do not produce bone matrix and appeared negative for Alizarin Red-staining.

C. Differentiated mouse compact bone-derived MSCs display robust bone matrix deposition as detected by Alizarin Red-staining.

100000

10000

1000

100

10

1

Competitor A

MesenCult™ Osteogenic Stimulatory Kit (Mouse)

Differentiate Mouse MSCsTo Adipocytes and Osteoblasts

Bsp

Fol

d-I

ndu

ctio

n (l

og s

cale

)

Osx Alpl Runx2

A


OSTEOGENIC DIFFERENTIATION

Osteogenic Stimulatory Kit (Mouse)

1 kit 05504

ADIPOGENIC DIFFERENTIATION

Adipogenic Stimulatory Supplement (Mouse)*

100 mL 05503

MesenCult™ MSC Basal Medium (Mouse)

400 mL 05501

B C

A B

Mouse MSCs cultured using the MesenCult™ Proliferation Kit with MesenPure™ (see pages 12-13) can be differentiated to chondrocytes with MesenCult™-ACF Chondrogenic Differentiation Medium and culturing for 21 days under normoxic (20% O2) conditions.

15


Stem and Progenitor Cell IdentificationThe ALDEFLUOR™ reagent system detects viable stem and progenitor cells based on aldehyde dehydrogenase (ALDH) enzyme activity. Over 1000 publications have used it to detect viable stem and progenitor cells of various lineages, including human mesenchymal stem/progenitor cells.30

PRODUCT USE QUANTITY CATALOG #

Lymphoprep™

Density gradient separation of MNCs in bone marrow

250 mL 07801

500 mL 07851

PBS + 2% FBSEasySep™ and RosetteSep™ isolations

500 mL 07905

Ammonium Chloride Solution

Lysing red blood cells present in bone marrow

100 mL 07800

500 mL 07850

L-Glutamine Cell culture 100 mL 07100

100 mm Tissue Culture Treated Dishes

Cell culture10/pack 27125

240/case 27127

3% Acetic Acid with Methylene Blue

Nucleated cell counts

100 mL 07060

Trypan Blue Viable cell counts 100 mL 07050

PBS (without Ca++ or Mg++)

Washing cells 500 mL 37350

Trypsin-EDTADissociation; Detachment

500 mL 07901

Human Platelet Lysate

Cell culture supplement

50 mL 06960

100 mL 06961

500 mL 06962

Human Platelet Lysate, Fibrinogen Depleted

Cell culture supplement

50 mL 06963

100 mL 06964

500 mL 06965

Support Reagents for Cell Enrichment and Culture


Hypoxia Chamber 1 chamber 27310

Single Flow Meter 1 meter 27311

References1. Campagnoli C, et al., Blood 98: 2396-2402, 20012. In't Anker PS, et al., Blood 102: 1548-1549, 20033. Zuk PA, et al., Mol Biol Cell 13: 4279-4295, 20024. Erices A, et al., Br J Haematol 109: 235-242, 20005. De Bari C, et al., Arthritis Rheum 44: 1928-1942, 20016. Kuznetsov SA, et al., J Cell Biol 153: 1133-1140, 20017. Tondreau T, et al., Stem Cells 23: 1105-1112, 20058. Caplan AI, Cell Stem Cell 3: 229-230, 20089. Andreeva ER, et al., Tissue Cell 30: 127-135, 199810. Doherty MJ, et al., J Bone Miner Res 13: 828-838, 199811. Bianco P, et al., Stem Cells 19: 180-192, 200112. Zannettino AC, et al., J cell Physiol 214: 413-421, 200813. Shi S, et al., Bone Miner Res 18: 696-704, 200314. Sacchetti B, et al., Cell. 131: 324-336, 200715. Crisan M, et al., Cel lStem Cell 3: 301-313, 200816. Dominici M, et al., Cytotherapy 8: 315-317, 200617. Caplan AI., J Orthop Res. 9: 641-650, 199118. Horwitz EM et al., Cytotherapy 7: 393-395, 200519. Deana S. et al., Stem Cells Int. 2010: 51902820. Caplan AI., Tissue Eng Part A. 16: 2415-2417, 201021. Horwitz EM, et al., Cytotherapy 10: 771-774, 200822. Jones BJ, et al., Exp Hematol 36: 733-741, 200823. Uccelli A, et al., Nat Rev Immunol 8: 726-736, 200824. Caplan AI, J Cell Physiol 213: 341-347, 200725. Thirumala S, et al., Expert Opin Biol Ther 13: 673-691, 201326. Quirici N, et al., Exp Hematol 30: 783-791, 200227. Phinney DG, et al., J Cell Biochem 72: 570-585, 1999 28. Anjos-Afonso F, et al., J Cell Sci 117: 5655-5664, 200429. Peister A. et al., Blood 103: 1662-1668, 200430. Gentry T, et al., Cytotherapy 9: 259-274, 2007


ALDEFLUOR™ 40 tests/kit 01700

STEMCELL Technologies provides high-quality primary and secondary antibodies, as well as a range of primary cell products, for MSC research. For a complete listing visit www.stemcell.com/Antibodies and www.stemcell.com/PrimaryCells.

Support ProductsFor Mesenchymal Stem Cells

Hypoxia Chamber

A hypoxic environment, easily generated using a hypoxia chamber, mimics physiological conditions. Many cell types, such as mouse MSCs, undergo enhanced proliferation when cultured with lowered atmospheric oxygen tension.

Hypoxic Tissue Culture Environment

Copyright © 2015 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies and Design, STEMCELL shield, Scientists Helping Scientists, MesenCult, MesenPure, EasySep, and RosetteSep are trademarks of STEMCELL Technologies Inc. ALDEFLUOR is a trademark of Aldagen, Inc. All other trademarks are the property of their respective holders.

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DOCUMENT #28367 VERSION 3.4.4 JANUARY 2016

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