Knee and Peri-Knee Tissues of Post Mortem Donors Are Strategic Sources of Mesenchymal Stem/Stromal Cells for Regenerative Procedures

Abstract

Tissues of post mortem donors represent valuable alternative sources for the isolation of primary cells with mesenchymal stem/stromal cell (MSC)-like properties. However, the properties of primary cells derived from different tissues and at different post mortem times are poorly recognized. Here, we aim to identify the optimal tissue source between three knee and peri-knee tissues for the isolation of primary cells with MSC-like properties, and to define the influence of the time post mortem on the properties of these cells. We harvested tissues from subchondral bone marrow, synovium and periosteum from 32 donors at various post mortem times. Primary cells were evaluated using detailed in vitro analyses, including colony formation, trilineage differentiation, immunophenotyping and skeletal stem cell marker-gene expression profiling. These data show that the primary cells with MSC-like properties isolated from these three tissues show no differences in their properties, except for higher expression of CD146 in bone-marrow cells. The success rate of the primary cell isolation is dependent on the post mortem time. However, synovium and periosteum cells isolated more than 48 h post mortem show improved osteogenic and chondrogenic potential. This study suggests that knee and peri-knee tissues from donors even 3 days post mortem are strategic sources of MSCs for regenerative procedures.

In Vitro Characterization of the Human Skeletal Stem Cell-like Properties of Primary Bone-Derived Mesenchymal Stem/Stromal Cells in Patients with Late and Early Hip Osteoarthritis

“…p values < 0.05 were considered as statistically significant. A heat map was generated, as described previously [16,17] using the online Heatmapper software [24].…”

“…Briefly, quantitative polymerase chain reaction (qPCR) was performed using 5× HOT FIREPol EvaGreen qPCR Supermix (Solis BioDyne), according to manufacturer protocol. The sequences of the primers (Macrogen, Sigma-Aldrich) used to measure genes encoding hSSC markers and osteogenesis-and adipogenesis-related genes were used from our [17], and other, previous studies [18][19][20][21]. All qPCR experiments were performed in triplicates, using a LightCycler 480 II (Roche).…”

“…Trilineage differentiation was performed, as described previously [6,13,16,17]. Briefly, for osteogenesis and adipogenesis, the cells were seeded as four technical replicates.…”

“…Immunophenotyping was performed using flow cytometry, as described previously [6,13,16,17]. Culture-expanded cells between p1-p5 were immunophenotyped using the following antibodies: anti-CD73 (clone AD2, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD164 (clone 67D2), anti-PDPN (clone NC-08), anti-CD146 (clone P1H12), anti-CD45 (clone 2D1), and anti-CD235a clone (clone HI264) (all BioLegend, San Diego, CA, USA).…”

New Sources, Differentiation, and Therapeutic Uses of Mesenchymal Stem Cells 2.0

“…Tissues from postmortem donors were also proposed as valuable alternative sources for the isolation of primary cells with MSC-like properties [ 19 , 20 ]. Haring et al reported the identification of an optimal tissue source among three knee and peri-knee tissues for the isolation of primary cells with MSC-like properties, and described the effect of the postmortem time on the properties of these cells [ 21 ]. In fact, the success rate of primary cell isolation depends on the postmortem time.…”