MACS Handbook

Mesenchymal stem cells

1 Introduction

The ability of specific bone marrow cells to slowly proliferate and adhere to plastic was first described in the early 1970s (PMID: 4455512). Named colony-forming-unit fibroblasts (CFU-Fs) because of their spindle-like morphology, these cells showed non-hematopoietic differentiation potential. Eventually, the cells became known as mesenchymal stem cells or mesenchymal stromal cells (MSC) and a set of minimal defining criteria were established in 2006 to accelerate new discoveries, and facilitate data comparability and development of novel cellular therapies (PMID: 16923606).

Minimal criteria for defining multipotent mesenchymal stromal cells:

  • plastic-adherent growth when maintained under standard culture conditions
  • differentiation to osteoblasts, adipocytes, and chondrocytes in vitro.
  • Expression of CD73, CD90, and CD105 and lack of CD14 or CD11b, CD34, CD45, CD79α or CD19, and HLA DR
MSC research focuses primarily on their potential to influence the immune system and their capacity to regenerate tissue. MSC are also one of the most predominant adult stem cells being investigated for cellular therapies, with 757 clinical trials worldwide exploring applications like the treatment of autoimmune diseases and tissue regeneration (source:

2 MSCs from bone marrow, adipose tissue, umbilical cord and amniotic epithelium

Bone marrow was the first established source of MSCs (PMID: 4455512) and has been the primary and therefore most investigated population. Over the last few years, however, additional sources of MSC have been identified, from adipose tissue-derived MSCs (AT-MSC) to birth-associated MSCs, which includes sources like umbilical cord blood (UCB-MSC), umbilical cord tissue (UC-MSC), placenta (PL-MSC) and amniotic epithelium (AM-MSC) (PMID: 23278600, 16410387).

2.1 Tissue-specific cell subsets, frequencies, marker expression

At a glance: MSCs from different tissue types
Tissue sourceFrequencyMarkers
Bone marrow1–10 CFU-F clones per 1x106 mononuclear cellsCD271, Anti-MSCA-1
Adipose tissuen/aCD34, CD146, CD271
Umbilical cordn/an/a
Umbilical cord blood1 CFU-F clone per 1x108 mononuclear cells to 1–3 CFU-F clones per 1x106 mononuclear cellsn/a
Amniotic epitheliumn/aCD271, Frizzled-9
Placentan/aCD271, Frizzled-9
Dental pulpn/aCD271

2.2 Miltenyi Applications for MCSs from bone marrow, adipose tissue and umbilical cord

2.3 Sample preparation of bone marrow, adipose tissue, and umbilical cord

Bone marrow is commonly aspirated from the sternum or iliac crest, or can be taken from other bones during orthopedic surgeries. Adipose tissue can be obtained from cosmetic surgeries as liposuction material or as solid tissue. These tissues may need to be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. For details, see chapters umbilical cord and adipose tissue.

2.4 Magnetic Cell Separation of MSCs from bone marrow, adipose tissue and umbilical cord

Miltenyi Biotec has developed several products for the straightforward magnetic separation of MSCs. In addition, generic protocols are available for indirect separation of MSCs using Anti-biotin or Anti-flurochrome microbeads in combination with biotin- or flurochrome-conjugated antibodies.

For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation.
2.4.1 Isolation of MSCs
At a glance: Kits and reagents for the separation of MCSs from bone marrow and adipose tissue
Starting material Isolation strategy Comments Automation* Product
Bone marrow, adipose tissue Positive selection of target cells High yield and purity of CD271bright population YesCD271 MicroBead Kit, human
Bone marrow, adipose tissue Positive selection of target cells High purity YesAnti-MSCA-1 (W8B2) MicroBead Kit, human
Bone marrow, adipose tissue Positive selection of target cells High yield of whole CD271 population     YesCD271 MicroBead Kit (APC), human
* Automation performed on the autoMACS® Pro Separator 
The CD271 MicroBead Kits were developed for the positive selection or depletion of cells expressing CD271 (LNGFR). They are ideally suited for the isolation of CD271+ MSCs from human tissues, including bone marrow and lipoaspirate, but have also been successfully used to isolate MSCs from bone marrow of monkey, goat, dog, pig, and sheep.
CD271 MicroBead Kit, human
BM-MNCs before separation
CD271+ cells

Enrichment of CD271+ mesenchymal stromal cells (MSCs) from bone marrow mononuclear cells (BM-MNCs).  MSCs were isolated using the CD271 MicroBead Kit, human, an MS Column, and a MiniMACS™ Separator. Cells were stained with CD271 antibodies conjugated to APC or PE, as well as CD45-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer.

The Anti-MSCA-1 (W8B2) MicroBead Kit is based on the antibody clone W8B2 and recognize the MSCA-1, a highly specific marker to isolate MSCs with a high proliferative potential from bone marrow.
CD271 MicroBead Kit, human
Unseparated fraction
MSCA-1+ cells

Separation of MSCA-1 (W8B2)-positive MSCs from human bone marrow mononuclear cells (BM-MNCs). Combining the Anti-MSCA-1 MicroBead Kit, with two MS Columns and a MiniMACS™ Separator, the highly proliferative population of MSCs was isolated from other mononuclear cells. Staining was done with Anti-MSCA-1 (W8B2)-APC and CD45-FITC.

2.5 Characterization of MSCs by flow cytometry

2.5.1 Quantification of MSCs
At a glance: Kits and reagents for the enumeration of MSCs by flow cytometry
Use Comments Product
Standardized identification and quantification of MSCs from fresh bone marrow aspirate More objective method than colony forming unit-fibroblast (CFU-F) assayMSC Enumeration Kit, human
Large-scale isolation and expansion of MSCs from human bone marrow is time and resource intensive. Success depends on the quality of the starting material, making it desirable to quantify MSC content of bone marrow sources. Human blood sources are commonly qualified using the colony forming unit-fibroblast (CFU-F) assay, but this is time-consuming, and susceptible to variation in serum lots, plating densities, and the subjective definition and scoring of colonies. It has been shown that Anti-MSCA-1 (W8B2) exclusively detects CD271 (LNGFR)bright cells, which are the only cells that give rise to CFU-F (PMID: 17395729). There is a close linear relationship between the number of CFU-F colonies manually counted after 14 days of culture and the number of CD271 (LNGFR)bright cells per milliliter of aspirate (PMID: 22268519).
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Linear relationship between counted CFU-F colonies and number of CD271+MSCA-1+ cells per mL of bone marrow aspirate. R2 = 0.753, P = 0.0003, n = 12 donors.

The MSC Enumeration Kit, human is designed for the quantification of human bone marrow MSCs samples, based on the expression of CD271 (LNGFR) and Anti-MSCA-1 (W8B2). The kit allows identification of CD45+ leukocytes and CD271 (LNGFR)+/Anti-MSCA-1 (W8B2)+ MSCs that are also CD45dim. The MSC Enumeration Kit, human standardizes the identification and quantification of human MSCs from fresh bone marrow aspirate (BMA) by flow cytometry and includes details on the appropriate gating strategy.
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Enumeration of MCSs by flow cytometry. Bone Marrow aspirate (100 µL) was labeled using the MSC Enumeration – Staining Cocktail. After red blood cells were lysed, remaining cell were analyzed with the MACS Quant Analyzer 10. Viable leukocytes were gated for CD45 and CD271 expression (A). This population was then further analyzed for Anti-MSCA-1 expression (C). Gating was performed according to the corresponding isotype control in B.

A scientific poster describing the standardized quantification of MSCs by flow cytometry can be downloaded from the Related Resources panel to the right.
2.5.2 Phenotyping MSCs
At a glance: Kits and reagents for phenotyping MSCs by flow cytometry
Use Comments Product
Standardized identification and quantification of cultivated and expanded MSCs Phenotyping is fast, easy, reliable, and based on ISCT-defined standardsMSC Phenotyping Kit, human
Miltenyi Biotec offers a streamlined and standardized method to identify and phenotype cultured MSCs according to standards defined by the International Society for Cellular Therapy (ISCT; PMID: 16923606). The MSC Phenotyping Kit, human includes all reagents necessary for MSC enumeration based on expression of CD73, CD90 and CD105, as well as their exclusion based on CD14, CD20, CD34, and CD45. The kit data sheet also provides details on the appropriate gating strategy. The MSC Phenotyping Kit, human enables fast, easy, and reliable phenotyping of cultured MSCs based on defined ISCT standards.
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Phenotyping of MSCs according to ISCT standards. 
Cultured MSCs were stained with the MSC Phenotyping Cocktail (purple) or with the Isotype Control Cocktail (black). The fractions were analyzed by flow cytometry using the MACSQuant® Analyzer.

2.6 Cell Culture of MSCs

2.6.1 Expansion of MSCs
At a glance: Kits and reagents for the expansion of MSCs
Use Comments Product
Expansion in serum-containing mediumIncreases proliferation with CytoMix – MSCStemMACS™ MSC Expansion Media, human
Robust expansion under xeno- and serum-free conditionsFree of animal components and requires no pre-coating of culture vessels with attachment substratesStemMACS™ MSC Expansion Media Kit XF, human
Expansion in serum-free, GMP-grade mediumGMP-grade, animal component-free medium requiring no pre-coating of culture vessels. Includes MSC-Brew GMP Basal Medium, MSC-Brew GMP Supplement R I, and MSC-Brew GMP Supplement II.MSC-Brew GMP Medium
SupplementIncreases proliferation with StemMACS™ MSC Expansion Media, humanCytoMix – MSC, human

First protocols to isolate and expand MSCs from human tissues were based on FCS-containing media. To meet mounting safety requirements from regulatory authorities, Miltenyi Biotec provides FCS-containing media (StemMACS MSC Expansion Media), serum- and xeno-free media (StemMACS MSC Expansion Media Kit XF), as well as serum- free, GMP- grade media (MSC-Brew GMP Medium). All media enable reliable and reproducible expansion of MSCs from bone marrow, adipose tissue and umbilical cord.

StemMACS™ MSC Expansion Media Kit XF is perfect for the direct isolation and expansion of human MSCs from various sources, including bone marrow and lipoaspirate. Its xeno-free composition enables fast expansion while maintaining differentiation potential and immunomodulation ability. MSCs cultured in StemMACS MSC Expansion Media XF show superior expansion rate compared to other standard MSC culture methods.
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Comparison of expansion media for MSCs. MSCs cultured in SteMACS™ MSC Expansion Media XF showed an increased clonogenic potential (A) as well as proliferation rate (B) compared to MSCs cultured in αMEM + 5 % platelet lysate or when culture in 10 % fetal calf serum (FCS)-containing media.

MACS Handbook:

Cell culture

Related PDFs:
2.6.2 Differentiation of MSCs
At a glance: Kits and reagents for the expansion of MSCs
Use Comments Product
Differentiation of MSC to adipocytesSupports differentiation of MSC derived from various tissue sourcesStemMACS™ AdipoDiff Media, human
Differentiation of MSC to osteoblastsSupports differentiation of MSC derived from various tissue sourcesStemMACS™ OsteoDiff Media, human
Differentiation of MSC to chondrocytesSupports differentiation of MSC derived from various tissue sourcesStemMACS™ ChondroDiff Media, human

Differentiation potential is a meaningful tool to qualify and define a MSC population. Grade of differentiation into adipocytes, chondrocytes and osteoblasts is influenced by MSC origin, so for example, MSCs from bone marrow show greater potential to differentiate into osteoblasts than MSCs from umbilical cord. Increased age of donor and passaging of cultured MSCs also decrease differentiation potential.

StemMACS™ AdipoDiff, OsteoDiff, and ChondroDiff Media are optimized differentiation media for human MSCs from various tissue sources. The media support the analysis or quality control of the differentiation capacity of expanded MSCs, as well as in vitro studies on the processes of MSC differentiation, including gene expression and protein profiling.
StemMACS™ AdipoDiff, OsteoDiff, and ChondroDiff Media

MSCs differentiation into adipocytes, chondrocytes or osteoblasts. (A) Adipocytes were stained with Oil Red O after cultivation of MSCs in StemMACS™ AdipoDiff Medium for 21 days. (B) Chondrocytes were stained for aggrecan (red) and nuclei (blue) after cultivation of MSCs in StemMACS™ ChondroDiff Medium for 21 days. (C) Osteoblasts were stained with NBT substrate after cultivation of MSCs in StemMACS™ OsteoDiff Medium for 10 days.

2.6.3 Suppression assay
At a glance: Kits and reagents for the immunomodulatory analysis of MSCs
Use Comments Product
Functional characterization of human MSCsStandardized characterization of the immunomodulatory function of MSCsMSC Suppression Inspector, human
During the last few years, scientists have directed their attention towards the immunomodulatory potential of MSCs. It has been observed that MSCs derived from bone marrow suppress proliferation of T cells (PMID: 11986244, 11823036). This function of MSCs can be analyzed using the MSC Suppression Inspector, human, which contains T cell stimulation reagents optimized for an MSC suppression assay.
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Principle of the MSC suppression assay. MSCs are co-cultured with CD4+CD25 or CD4+ responder T cells (Tresp) at different ratios and in the presence of the polyclonal stimulus of the MSC Suppression Inspector. When alone, Tresp cells show a proliferative response. Co-culture with MSCs, however, reduces proliferation of Tresp cells. Cell proliferation is determined by 3H-thymidine incorporation but can also be detected by staining with carboxyfluorescein succinimidyl ester (CFSE).

The suppression assay is performed with a dilution series ranging from a ratio of 1:1 to 8:1 of Tresp cells:MSCs. As additional control, Tresp and MSCs are cultured alone with and without the MSC Suppression Inspector.

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Suppression of T cell proliferation by PA-MSCs or CD271+ MSCs isolated by MACS Technology.

Tresp cells labeled with CFSE were cocultured with either CD271+ cells or with PA-MSCs. Cells were stimulated by using the MSC Suppression Inspector or left untreated. (A) Tresp cell proliferation was flow cytometricall measured as CFSE dilution (indicated by M1). (B) Data are presented as percentage of proliferating Tresp cells cultured in the presence of PA-MSCs or CD271+ cells with respect to responder cells cultured alone.

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