Proteomic Profiling of Native Unpassaged and Culture‐Expanded Mesenchymal Stromal Cells (MSC)

Abstract

Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45 /CD31 /CD34 /CD73 /CD105 stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.

Permanent Pro-Tumorigenic Shift in Adipose Tissue-Derived Mesenchymal Stromal Cells Induced by Breast Malignancy

“…There is a growing substantial interest in identifying MSC proliferation properties for future application in cell therapy and tissue engineering [25,26]. Herein, the growth kinetics and population doubling-time were influenced by age when younger and older donors were compared, as shown by others [27,28].…”

Identification of senescent cells in multipotent mesenchymal stromal cell cultures: Current methods and future directions

“…Moravcikova et al have recently used a high-throughput cell surface proteomic approach to analyse the changes in cell surface protein expression of CD45-/CD31-/CD34-/CD73+/CD105+ stromal cells in unpassaged BM MSC and subsequently determine the changes in cell surface markers with accompanying passage [103]. The group presented that the adhesion molecule CD106/VCAM1 is highly expressed in unpassaged MSC but decreases drastically with culture.…”

“…Also for migration of MSCs, a metalloprotease associated with cell motility, CD10/neprilysin, has been shown to be rapidly upregulated in culture but decreased with early senescence [103]. Whereas no consensus yet exists in relation to a specific expression of classical MSC surface molecules on senescent MSCs, recent studies have identified new molecules such as cell surface protein DPP4 and membrane-bound vimentin to identify senescent cells [107,108].…”

“…Predictive software algorithms and new machine learning technologies should be on the forefront of this endeavour. Human diploid fibroblast-like cells [59] BM-MSC [15] Human fibroblast [58,61] [43,67] Skin fibroblast cell [69,70] [17,73,[78][79][80] DP-MSCs [76] Human dermal and lung fibroblasts [77] Gene [86] BM-MSC [2,15,17,86] Adipose tissue mesenchymal stromal cells [86] [53,103,104,106] Human diploid fibroblast cells [107] Mouse lung fibroblasts [108] Cell size measurements…”

Identification and Characterization of a Large Source of Primary Mesenchymal Stem Cells Tightly Adhered to Bone Surfaces of Human Vertebral Body Marrow Cavities

“…We are presently exploring the question of why some populations of MSC are easily dislodged or possibly free floating in the BM, while others remain tightly adhered to the bone/connective tissue matrix and can only be liberated by enzymatic digestion. Determining differences, if they exist, is complicated by the relatively low frequency (<0.01%) of these cells, making them problematic to characterize using common analytical tools, such as flow cytometry, without first expanding in culture, which induces phenotypic and functional alterations [38][39][40][41][42][43][44][45][46]. One previous report found that freshly isolated enzymatic digests of pelvic region trabecular bone contained 15-fold higher CFU-F than aspirated BM [24]; however, we did not find a similar difference between freshly isolated vBA-MSC and BM-MSC.…”