HLA-DR expression in clinical-grade bone marrow-derived multipotent mesenchymal stromal cells: a two-site study

Abstract

Background: Contrary to the minimal criteria proposed by the International Society for Cell and Gene Therapy for defining multipotent mesenchymal stromal cells (MSC), human leukocyte antigen (HLA)-DR expression is largely unpredictable in ex vivo-expanded clinical-grade cultures. Although activation of MSC in culture does not appear to affect their functionality, a large study investigating the impact of HLA-DR expression on cell identity and potency is still missing in the literature.

Methods: A retrospective analysis of HLA-DR expression in 130 clinical batches of bone marrow (BM)-MSC from two independent Good Manufacturing Practice-compliant production facilities was performed in order to identify the consequences on critical quality attributes as well as potential activation cues and dynamics of MSC activation in culture.

Results: HLA-DR⁺ cells in culture were confirmed to maintain fibroblastic morphology, mesenchymal phenotype identity, multipotency in vitro, and immunomodulatory capacity. Interestingly, the use of either human sera or platelet lysate supplements resulted in similar results.

Conclusions: HLA-DR expression should be considered informative rather than as a criterion to define MSC. Further work is still required to understand the impact of HLA-DR expression in the context of product specifications on BM-MSC qualities for clinical use in specific indications.

Keywords: Cell culture; Cellular therapy; HLA-DR; Identity; Multipotent mesenchymal stromal cell; Potency; Quality compliance.

Fig. 1

Differentiation and immunomodulation potential of BM-MSC. Selected examples of characterization assays performed on BM-MSC at Barcelona’s and Helsinki’s facilities. a Differentiation potential was confirmed by von Kossa staining after 4-week culture in osteogenic medium (black depositions), Oil Red O/Sudan III staining after 3-week culture in adipogenic medium (lipid droplets in the cytoplasm of adipocytes stained red), and Safranin O staining after 3-week culture in chondrogenic medium (glycosaminoglycans stained red). b Immunomodulatory properties of MSC were tested by means of the lymphocyte proliferation assay, in which MSC demonstrated their capacity to suppress proliferation of polyclonally stimulated lymphocytes. Barcelona scale bars = 200 μm. Helsinki von Kossa scale bars = 500 μm and Oil Red O scale bars = 100 μm

Fig. 2

Analysis of cell culture parameters. Effect of cell density on HLA-DR expression was studied in batches released from both manufacturing sites, namely Barcelona (a) and Helsinki (b). However, high dispersion without statistical correlation was observed in both cases (p value = 0.2287 and 0.2272, respectively)

Fig. 3

Dynamics of BM-MSC activation in vitro. In culture, BM-MSC are activated by the addition of IFN-γ, which is illustrated by increasing concentration up to 200 UI/mL, reaching a peak 48 h after stimulation (a). Interestingly, the reversal of the process for the recovery of HLA-DR expression levels using IFN-γ-activated cells takes longer than the activation time (b)