Real-Time Monitoring of G-Protein Coupled Receptor Activation as a Functional Assessment of Mesenchymal Stem Cells

Cell Therapy Manufacturing Challenges

To generate enough BMSCs for clinical studies, cells must be expanded over several passages in vitro, in addition to testing for functional capacity at multiple stages in the expansion process. Three major challenges in the expansion process include:

  • Functional variability brought on by batches obtained from different donors
  • Decreases in functional capacity after successive passages
  • Lack of rapid and quantitative assays that can simultaneously provide functional characterization and assess the products’ quality parameters including viability, purity, and potency

Benefits of Assessing  the Functional Activation of GPCRs of MSCs with the xCELLigence Real Time Cell Analysis platform:

  • Consistently measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation
  • GPCR stimulation assay is non-invasive, label-free, and provides a quantitative prediction of inter-donor variability and adipogenic differentiation potential
  • Distinct GPCR profiles in undifferentiated versus differentiated BMSCs provides a rapid prediction of functional status
Real-Time Quality Control and Functional Assessment of Mesenchymal Stem Cells for Cellular Therapies

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Overview of GPCR stimulation assay

Figure 1 (A) Example of a growth profile of BMSCs as monitored on xCELLigence. Upon reaching sub-confluency, BMSCs are stimulated with specific GPCR agonists (blue arrow) and the cellular response was detected by the xCELLigence system. (B) A close look at the stimulation response observed in (A) (dotted box). Cell index was normalized against the cell index at the time of treatment (NCI). The response window is calculated as the maximum NCI (NCImax ) reached upon treatment for a given time point, divided by the NCI of the negative control (NCInegcntl ) at that time point.

Distinct GPCR profiles in undifferentiated versus differentiated BMSCs provides a rapid prediction of functional status.

In the example below, BMSCs from different donors were propagated over 12 passages and a subset of cell samples from different passages were seeded on E-plates followed by adipogenic induction and monitoring on xCELLigence.

Unique GPCR profiles

Taking advantage of the unique GPCR profiles in BMSCs, the rapid GPCR stimulation assay performed on the xCELLigence platform can be used as a quality control parameter whilst cells are expanded over multiple passages, from different donors.   The assay can be easily performed while expanding cells as only a small subset of cells is required (≥1100 cells/ well for 96-well), with results within a few days.  For more rapid results, this assay can be performed within 2 days of cell seeding, if enough cells are used (~10-20K per well)

Quantitative Correlation

There is a quantitative correlation between GPCR stimulation (corresponding to receptor activation) upon addition of agonists for the A1AR, P2Y1, and H1 receptors and adipogenic differentiation potential.  As cells are actively proliferating and successfully differentiating (early passage) expression of these receptors is high. When cells enter senescence and concomitantly exhibit loss of differentiation potential (mid to late passage), expression is low.

Figure 2. Donor 2 results (A-I). Twenty-four hours following adipogenic induction, agonists CYM5541and SEW 2871 were added to undifferentiated control (A, D) or BMSCs induced for adipogenesis at early passage (B, E) or late passage (C, F), to stimulate SIP3 and SIP1 GPCRs, respectively. After 14 days of growth in adipogenic differentiation medium, cells were stained with LipidTox Green Lipid Stain (G-I

Stem Cells and Cell Differentiation Supporting Information:

  • Stem Cells and Cell Differentiation Publications
  1. Optimization and scale-up culture of human endometrial multipotent mesenchymal stromal cells: potential for clinical application. Rajaraman G, White J, Tan KS, Ulrich D, Rosamilia A, Werkmeister J, Gargett CE. Tissue Engineering Part C Methods. 2013 Jan;19(1):80–92.
  2. Comparison of long-term retinoic acid-based neural induction methods of bone marrow in human mesenchymal stem cells. Mammadov B, Karakas N, Isik S. In Vitro Cellular and Developmental Biology. 2011 Aug;47(7):484-91.
  3. Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells. Steinbach SK, El-Mounayri O, DaCosta RS, Frontini MJ, Nong Z, Maeda A, Pickering JG,Miller FD, Husain M. Arteriosclerosis, Thrombosis, and Vascular Biol. 2011 Dec;31(12):2938-48.
  4. Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation. Spörl F, Wunderskirchner M, Ullrich O, Bömke G, Breitenbach U, Blatt T, Wenck H, Wittern KP, Schrader A. Journal of Investigative Dermatology. 2010 May;130(5):1268-78.