Clinical-Grade Mesenchymal Stromal Cells Produced Under Various Good Manufacturing Practice Processes Differ in Their Immunomodulatory Properties: Standardization of Immune Quality Controls

Abstract

Clinical-grade mesenchymal stromal cells (MSCs) are usually expanded from bone marrow (BMMSCs) or adipose tissue (ADSCs) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSCs using a combination of fully standardized in vitro assays. BMMSCs expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments, including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSCs with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSCs inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to interferon-γ-dependent indoleamine 2,3-dioxygenase activity. MSCs did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSCs produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.

Comparative study of immune regulatory properties of stem cells derived from different tissues

“…All SC types neither activated nor promoted IEC growth (data not shown). Then, the role of SCs in IEC replication was assessed by using different IEC:SC ratios, which ranged from 10:1 (high ratio) to 100:1 (low ratio) for T cells, and from 1:1 (high ratio) to 10:1 (low ratio) for NK and B cells, according to the standardized approach previously used with BM-MSCs [25]. At resting and primed conditions, none of the SC types …”

“…Based on this premise, the immunogenicity and immune modulatory properties of SCs have to be carefully characterized to decipher their potential clinical import. MSCs from the bone marrow (BM) and adipose tissue (AT) have been well defined [21][22][23][24][25][26][27] and similar immune regulatory functions have been identified in MSC-like SCs collected from Wharton's jelly, amniotic fluid, and placenta [28][29][30].…”

“…To test this hypothesis, we studied (1) BM-MSCs [25]; (2) olfactory ectomesenchymal SCs (OEMSCs), which are distributed in the olfactory lamina propria and induce neurogenesis, and restore the hippocampal neuronal network [44][45][46]; (3) non-MSC leptomeningeal SCs (LeSCs), described by us first in rats as nestin-positive cells capable of differentiating into neuronal, astrocyte, and oligodendrocyte precursors [47,48] and, more recently, in mice and humans (personal observation); and (4) human c-Kitpositive SCs isolated from the amniotic fluid (AFSCs) [49], from the adult heart (cardiac SCs: CSCs) [50][51][52]; and adult lung SCs (LSCs) [53]. AFSCs are multipotent, nonteratogenic cells with characteristics intermediate between embryonic and adult SCs [49].…”

“…CSCs are multipotent cells capable of differentiating into cardiomyocytes and coronary vessels [50][51][52], while LSCs form lung structures of both endodermal and mesodermal origin [53]. The standardized approach previously introduced to characterize MSCs [25] was applied to all SCs to define their immunological profile.…”

Immune Regulatory Properties of CD117pos Amniotic Fluid Stem Cells Vary According to Gestational Age

“…Thus, we asked whether AFS immune modulatory properties could change along gestational age. To this aim, we used standardized approaches, previously applied to characterize MSCs and other SCs [20,23] and including immunophenotyping and assessment of immunogenicity and immunomodulatory functions toward T, B, and natural killer (NK) cells, to investigate the immunological profile of AFS cells isolated at different gestational age.…”