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CD146 expression, as a surrogate biomarker for human mesenchymal stromal cell multilineage differentiation, is preserved during cell expansion in an automated hollow-fiber membrane bioreactor

Jones M, Nankervis B, Frank N, Vang B, DiLorenzo T, Hill D & Coeshott C

Background: Mesenchymal stromal cells (also known as mesenchymal stem cells, hMSCs) are currently being evaluated in a number of clinical trials including those for graft-versus-host disease, autoimmune disease, bone regeneration, wound repair, and other cartilage, liver, kidney, and lung disorders. In biopharmaceutical development and production, the ex vivo expansion of human mesenchymal stromal cells is frequently evaluated through trilineage differentiation assays. This approach requires 2 to 4 weeks of manual effort to complete following the induction of adherent heterogeneous cell populations and it lacks quantification. The development of a more efficient and quantifiable measure of potential differentiation can be helpful in characterizing both MSC-based translational and manufacturing processes.

Methods and Findings: We have evaluated the expression of CD146, a surface biomarker, on actively growing hMSCs in the automated Quantum^® Cell Expansion System bioreactor. Specifically, an index of CD146⁺ median fluorescence intensity (MFI), as measured by flow cytometry during automated cell culturing and subsequent differentiation with respect to cell doublings, provides information for the qualification of MSCs. We demonstrate here that CD146 expression is well above the biomarker expression levels observed in lineage-committed, confluent, or differentiated hMSCs by 2- to 34-fold. Our results show that CD146 expression is high at hMSC harvest and then decreases with lineage differentiation. Moreover, we show that CD146 is expressed across multiple (4) passages out to 27 population doublings and gradually decreases. A similar trend in CD146 expression was observed in cells with various types of bioreactor fibronectin coating agents and fetal bovine serum (FBS)-supplemented media across multiple passages.

Conclusion: hMSCs expanded with the combination of a human-platelet-derived cryoprecipitate (hCPPT)-coated bioreactor and human platelet lysate (hPL)-supplemented media show an increase in CD146 expression across 3 passages. In a comparison of conservative, moderate (control), and aggressive feeding schedules, the moderate feeding schedule generated hMSCs with the highest CD146 expression. Additionally, bioreactor coating agents, such as platelet-derived cryoprecipitate, can facilitate the preservation of CD146 expression during hMSC expansion where MFI indexes are increased to 400-fold above unstained cells through 23.8 population doublings. Furthermore, the hollow-fiber membrane (HFM) bioreactor system, with a programmable gas transfer module (GTM), demonstrates a capability to maintain normoxia (5%-18% O₂), which is known to support CD146 upregulation.