Mesenchymal stroma cells in peritoneal dialysis effluents from patients

Abstract

Mesenchymal stroma cells (MSCs) have potential as an emerging cell therapy for treating many different diseases, but discovery of the practical sources of MSCs is needed for the large-scale clinical application of this therapy. This study was to identify MSCs in peritoneal dialysis (PD) effluents that were discarded after PD. The effluents were collected from patients who were on the dialysis for less than 1 month. Adherent cells from the effluents were isolated by incubation in serum-containing medium in plastic culture dishes. Cell surface markers were determined by a flow cytometric analysis, and the in vitro differentiation to chondrocytes, osteocytes or adipocytes was confirmed by staining with a specific dye. After four passages, these isolated cells displayed the typical morphology of mesenchymal cells in traditional 2-D cultures, and were grown to form spherical colonies in 3-D collagen cultures. Flow cytometric analysis revealed that the unsorted cells from all of seven patient samples showed robust expression of typical mesenchymal marker CD29, CD44, CD73, CD90 and CD166, and the absence of CD34, CD79a, CD105, CD271, SSEA-4, Stro-1 and HLA-DR. In differentiation assays, these cells were induced in vitro to chondrocytes, osteocytes or adipocytes. In conclusion, this preliminary study suggests the presence of MSCs in the “discarded” PD effluents. Further characterization of the phenotypes of these MSCs and evaluation of their therapeutic potential, particularly for the prevention of PD failure, are needed.

In VitroExpansion and Characterization of Mesenchymal Stromal Cells from Peritoneal Dialysis Effluent in a Human Protein Medium

“…PD effluents were collected from anonymized donors as described by Liu et al [ 9 ], which was approved by the Clinical Research Ethics Board at the University of British Columbia (Vancouver, BC) in accordance with the Canadian Tri-council policy statement: ethical conduct for research involving humans (protocol number: H15-02466). A total of ten PD effluent samples were collected from ten donor patients (one from each) who were on PD therapy with either Dianeal or Physioneal PD solution within 4 weeks ( Table 1 ).…”

“…The isolation of both adherent cells and proteins from PD effluent was processed within 12 h after collection from donors. Adherent cells were isolated from the effluents and cultured, as described previously by Liu et al [ 9 ]. In brief, cells in PD effluents were pelleted by centrifugation at 751 ×g at 10°C for 10 min.…”

“…After four passages (P4) of culturing cells in plastic dishes, the expression levels of panels of both classical and nonclassical MSC surface markers were examined using fluorescence-activated cell sorting (FACS) analysis, as described by Liu et al [ 9 ]. In brief, after a short trypsinization of the adherent cells from the plastic dishes using Trypin-EDTA solution (Sigma-Aldrich Canada, Oakville, ON, Canada), a single cell suspension was prepared by suspending cells in a culture medium.…”

“…The chondrogenic differentiation was performed using a high-density cell culture, as described by Liu et al [ 9 ]. In brief, after the cells have been cultured to P4, 10 μ L droplets of cells (1 × 10 6 /mL) were placed on a 10 cm petri dish and incubated at 37°C for 2 h, followed by incubation in high glucose DMEM medium supplemented with 1% (v/v) human proteins, 10 ng/mL transforming growth factor- β 1 (Sigma-Aldrich Canada), 50 μ g/mL ascorbate acid, 0.1 μ M dexamethasone, 100 μ g/mL sodium pyruvate, 40 μ g/mL proline, and 50 mg/mL ITS premix (5 μ g/mL insulin, 5 μ g/mL transferrin, and 5 ng/mL selenious acid).…”

“…Therefore, finding alternative sources of MSC is crucial in the current development of a MSC-based therapy. Our group, for the first time, has identified and isolated MSCs in peritoneal dialysis (PD) effluent from PD patients [ 9 ]. With its large source of otherwise discarded PD effluent, which can easily be collected from PD clinics, the PD effluent-derived MSCs (pMSCs) may be a feasible alternative source of MSCs for the MSC therapy [ 9 ].…”