MSC Characterization Standards

The ISCT MSC Criteria: What They Are and What They Confirm

In 2006, the International Society for Cell Therapy (ISCT) published the minimal criteria for defining human multipotent mesenchymal stromal cells. These criteria were established to create a common identity standard across laboratories and to differentiate MSCs from other plastic-adherent stromal cell populations. They consist of three categories:

Criterion 1: Plastic Adherence

MSCs must adhere to tissue culture-grade plastic under standard culture conditions. This property distinguishes them from hematopoietic progenitors and most immune cells, which grow in suspension. Plastic adherence is a necessary but not sufficient MSC identity marker — many fibroblast populations, endothelial cells, and other stromal cells also adhere to plastic. It is the combination of plastic adherence with surface marker and differentiation criteria that defines the MSC population.

Criterion 2: Surface Marker Phenotype

By flow cytometry, MSCs must express the following markers at 95% or greater positivity:

• CD73 (Ecto-5'-nucleotidase): Catalyzes conversion of AMP to adenosine. Expressed on lymphocytes and stromal cells. On MSCs, CD73 participates in purinergic immunomodulation, contributing to adenosine-mediated T cell suppression. CD73 positivity confirms stromal progenitor identity but is also expressed by fibroblasts.
• CD90 (Thy-1): GPI-anchored glycoprotein involved in cell adhesion, migration, and differentiation signaling. On MSCs, CD90 expression reflects engagement with fibronectin and integrin signaling. Its functional significance in the MSC therapeutic mechanism is not fully defined, but it is a consistent MSC surface marker across isolation sources.
• CD105 (Endoglin): TGF-beta co-receptor expressed on endothelial cells and mesenchymal progenitors. On MSCs, CD105 participates in TGF-beta signaling relevant to differentiation and immunomodulation. CD105 positivity is particularly associated with early-passage MSCs and may decline with passage accumulation in some culture systems.

The following markers must be negative (<2% positivity):

• CD45: Pan-leukocyte marker (protein tyrosine phosphatase receptor). Negativity confirms absence of hematopoietic cell contamination from the isolation procedure.
• CD34: Hematopoietic progenitor and endothelial cell marker. Negativity at passage confirms separation from hematopoietic stem cell contaminants. Note: some MSC populations, particularly freshly isolated UC-MSCs, may transiently express CD34 at P0 before losing expression at P1.
• CD14: Monocyte/macrophage marker (LPS co-receptor). Negativity confirms absence of monocyte and macrophage contamination — important for preparations intended to have immunomodulatory rather than pro-inflammatory properties.
• HLA-DR: MHC class II antigen presentation molecule. Negativity is central to the MSC immunoprivileged status and low immunogenicity. As discussed in the passage number section, HLA-DR upregulates with passage accumulation in senescent MSC cultures — making this marker particularly important for passage-number-independent quality assessment.

Criterion 3: Tri-Lineage Differentiation Potential

Under appropriate induction conditions, MSCs must demonstrate the capacity to differentiate toward osteogenic (bone-forming), adipogenic (fat-forming), and chondrogenic (cartilage-forming) lineages. Osteogenic differentiation is confirmed by Alizarin Red S staining of calcium deposits. Adipogenic differentiation is confirmed by Oil Red O staining of lipid droplets. Chondrogenic differentiation in pellet culture is confirmed by Alcian Blue or Safranin-O staining of glycosaminoglycan deposition.

Why ISCT Criteria Alone Are Insufficient

The ISCT criteria define minimal identity — they confirm that a cell is an MSC by the broadest accepted definition. They do not confirm:

• The passage number at which cells are characterized
• Whether cells are in a therapeutically active or senescent functional state
• Whether the immunomodulatory secretome is intact
• Whether HLA-DR is increasing at a rate that will compromise the next production lot
• Whether the exosome cargo produced by these cells is anti-inflammatory or pro-inflammatory

A P15 MSC that has been in culture for 20 weeks and has accumulated SASP will score positive for CD73, CD90, and CD105, negative for CD45, CD34, CD14, and HLA-DR (if the SASP-related HLA-DR upregulation has not yet reached the 2% threshold), and will demonstrate reduced but detectable tri-lineage differentiation potential. It passes all ISCT criteria. Its secretome is pro-inflammatory. Its exosome cargo is the opposite of the therapeutic standard.

Beyond ISCT: Functional Characterization of MSCs

Mixed Lymphocyte Reaction (MLR) Immunosuppression Assay

The gold standard functional assay for MSC immunomodulatory capacity. PBMCs from two allogeneic donors are co-cultured to stimulate T cell proliferation via alloreactivity. MSCs (or MSC-conditioned media or MSC exosomes) are added at defined ratios. T cell proliferation is measured by CFSE dilution or [3H]-thymidine incorporation. A therapeutically active MSC preparation at P3 should suppress T cell proliferation by 50-70% at a 1:10 MSC:T cell ratio. This assay captures the functional output of the immunomodulatory secretome — including IDO, PGE2, TGF-beta, HLA-G, and IL-10 axes — and therefore reflects actual therapeutic potential in a way that surface markers cannot.

Colony-Forming Unit Fibroblast (CFU-F) Efficiency

CFU-F assays measure the frequency of self-renewing progenitor cells in the MSC population by plating at low density and counting discrete colony formation after 14 days. High CFU-F efficiency (greater than 30 colonies per 100 cells plated) indicates a population with maintained stemness and proliferative capacity. Declining CFU-F efficiency with passage is an early indicator of population aging before frank SASP emergence. Lot-release CFU-F testing is a practical indicator of the functional state of the cell master bank from which exosomes will be produced.

Senescence Markers

Three assays provide direct evidence of senescent cell burden in the MSC culture:

• Senescence-associated beta-galactosidase (SA-beta-gal) staining: Beta-galactosidase activity at pH 6.0 is elevated in senescent cells due to lysosomal expansion. Less than 10% SA-beta-gal positive cells is the expected standard for P2-P4 MSC cultures. Greater than 25% positivity indicates a significantly senescent population.
• p21/CDKN1A expression: p21 is a CDK inhibitor and p53 transcriptional target that drives G1 cell cycle arrest as part of the DNA damage response leading to senescence. Elevated p21 by western blot or qPCR is an early senescence marker preceding SA-beta-gal positivity. p21 should be low in P2-P4 cultures.
• p16/CDKN2A expression: p16 is an alternate CDK inhibitor that acts through the Rb pathway and is associated with epigenetic senescence (rather than telomere-driven senescence). p16 accumulates with passage and is a more stable marker than p21. Elevated p16 is associated with irreversible growth arrest and correlates with loss of therapeutic function.

Exosome COA Requirements: The Full Minimum Standard

A Certificate of Analysis (COA) for a clinical-grade MSC exosome preparation should include the following elements as lot-release testing:

Identity and Quantity

• Particle count per vial (or per mL) by nanoparticle tracking analysis (NTA), reported with concentration and mode/mean size
• Full NTA size distribution curve (confirms vesicle size range consistent with exosome fraction, typically mode 100-150nm)
• Transmission electron microscopy (TEM) image confirming cup-shaped morphology and size range (not required per lot but required for product characterization)

Surface Marker Confirmation

• CD9 positive — by bead-based flow cytometry (e.g., ExoCheck or equivalent) or western blot
• CD63 positive — by bead-based flow cytometry or western blot
• CD81 positive — by bead-based flow cytometry or western blot
• TSG101 positive — by western blot (confirms ESCRT-pathway biogenesis, distinguishes from non-vesicular co-purified material)
• Alix positive — by western blot (accessory ESCRT protein, confirms MVB origin)
• Calnexin negative — by western blot (absence confirms preparation is not contaminated by ER membrane fragments)

Safety Testing (Lot Release)

• Sterility: USP <71> membrane filtration method or equivalent, 14-day incubation, both aerobic and anaerobic
• Endotoxin: LAL (limulus amebocyte lysate) assay, acceptance criterion stated (typically <1.0 EU/mL), reported numerical result
• Mycoplasma: PCR-based detection (MycoAlert or equivalent), negative result
• Appearance: visible particulates, color, and clarity as specified

Manufacturing Documentation

• Tissue source identified (umbilical cord, bone marrow, adipose, etc.)
• Passage number at harvest stated explicitly (not "early passage")
• Culture system stated (3D spheroid, 2D monolayer, bioreactor — specific)
• Media type stated with xenofree/FBS-containing status explicit
• Isolation method stated (ultracentrifugation, SEC, TFF, precipitation)
• Formulation and storage conditions specified
• Lot number and expiration date

Practitioner Procurement Checklist

Use this checklist when evaluating any MSC exosome preparation from a supplier:

• Particle count confirmed by NTA with size distribution data provided
• CD9, CD63, CD81 positive by COA (specific assay method stated)
• TSG101 or Alix positive by western blot on file
• Sterility testing completed by USP 71 or equivalent — result and acceptance criterion shown
• Endotoxin testing completed by LAL — numerical result and acceptance criterion shown
• Mycoplasma testing completed by PCR — negative result confirmed
• Passage number at harvest stated explicitly (accept P2-P4; question P5+; reject if undisclosed)
• Culture system identified as 3D (spheroid, scaffold, or bioreactor); question 2D monolayer
• Xenofree or chemically defined media confirmed throughout (not just final collection stage)
• Tissue source documented (umbilical cord preferred for youth of passage at isolation)
• Isolation method disclosed (SEC or TFF preferred over simple precipitation)
• Lot-specific COA available (reject generic product characterization presented as lot-specific data)

The Product Specification Table

For reference, the specifications that define the 3DMSCS preparation against which the above checklist applies:

Key References

• Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cell Therapy position statement. Cytotherapy. 2006;8(4):315-317.

  PubMed ID: 16923606 | DOI: 10.1080/14653240600855905

  The original ISCT position statement defining the three minimal criteria for MSC identity: plastic adherence, surface marker phenotype (CD73/CD90/CD105 positive; CD45/CD34/CD14/HLA-DR negative), and tri-lineage differentiation potential. Defines the international reference standard.

• Thery C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018). J Extracell Vesicles. 2018;7(1):1535750.

  PubMed ID: 30637094 | DOI: 10.1080/20013078.2018.1535750

  ISEV community consensus guidelines defining minimum information requirements for EV characterization in research and manufacturing contexts. Basis for COA standards including NTA, tetraspanin confirmation, and negative marker requirements.