Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years

Abstract

Introduction: Regenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years.

Methods: After thawing, hC-MSCs were isolated with a high efficiency (12×10⁶) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches.

Results: In early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells.

Conclusions: The efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies.

Figure 1

Human cadaver mesenchymal stromal/stem cell isolation, early characterization and expansion. Representative histological staining of native (A) and digested arterial tissue (B) after enzymatic isolation of human cadaver mesenchymal stromal/stem cells (hC-MSCs) (scale bars =10 μm). (C), (D) After harvesting, hC-MSCs collected from three postmortem artery segments show clonogenic activity (scale bars = 50 μm). (E) Numerous poly-nucleated cells (arrow), spindle-shaped cells, dendritic (arrowhead) cells and rounded cells (scale bar = 20 μm). (F) hC-MSC growth kinetics. After 3 weeks of culture, the cells seeded were expanded approximately 20-fold and yielded 250 × 10⁶ cells. (G) ki-67 nuclear immunoreactivity (scale bar = 75 μm). (H) The hC-MSCs at passage 3 became elongated and spindle-shaped with long and thin cytoplasmic projections (scale bar =10 μm).

Figure 2

Human cadaver mesenchymal stromal/stem cell phenotypic and molecular characterization. (A) Representative flow cytometry analysis of mesenchymal, pericytic, stem cell, hematopoietic and vascular markers. Isotype controls are presented as filled black histograms, the specific cell markers as white histograms. (B) Flow cytometry analysis of hematopoietic, mesenchymal and pericyte marker coexpressions. Percentage and cytograms from a representative experiment. Immunofluorescence staining for Vimentin (C) and Nestin (D) in human cadaver mesenchymal stromal/stem cells (hC-MSCs). Nuclei were counterstained with DAPI (blue) and cell positive in green. Scale bars = 100 μm. (E) Stem cell gene expression analysis of SOX2, c-KIT, OCT-4, NOTCH-1 and KDR. β-Microglobulin was used as the housekeeping gene. The far left lane contains a 100 base pair ladder.

Figure 3

Human cadaver mesenchymal stromal/stem cell stemness property. Clone-forming potential of (A) a single seeded human cadaver mesenchymal stromal/stem cell (hC-MSC) (arrow) that (B) reached the confluence after 30 days of culture (scale bars = 50 μm). (C) Nonclonogenic single cell with a ring-shaped morphology (scale bar = 10 μm). (D) When plated in nonadhesion conditions, free floating spheres are generated (scale bar = 50 μm). (E) Reverse transcriptase polymerase chain reaction analysis performed on spheroids showed expression of SOX2, OCT-4, c-KIT and KDR. β-Microglobulin was used as the housekeeping gene. The far left lane contains a 100 base pair ladder.

Figure 4

Human cadaver mesenchymal stromal/stem cell mesengenic potential. (A) Control human cadaver mesenchymal stromal/stem cells (hC-MSCs) did not display cytoplasm lipid drops. (B) Oil Red O stained adipocytic multivacuolar cells in red. (A), (B) Scale bars = 10 μm. (C) Transmission electron microscopy (TEM) showed multiple lipid vacuoles and small dense mitochondria in the cytoplasm. L, lipid droplets; M, mitochondria. Scale bar = 2 μm. (D) Reverse transcriptase polymerase chain reaction of peroxisome proliferator-activated receptor gamma (PPARγ) expression. β-Microglobulin was used as the housekeeping gene. (E) Control hC-MSCs did not display calcium deposition in the extracellular matrix. (F) Alizarin Red stained calcium deposits. (E), (F) Scale bars = 10 μm. (G) TEM confirmed the presence of osteoid matrix and needle-shaped hydroxyapatite crystals (arrow). Scale bar = 2 μm. (H) Gene expression analysis of Osteocalcin, Osteopontin and RUNX-2. β-Microglobulin was used as the housekeeping gene. (I) Control hC-MSCs did not display proteoglycan-rich extracellular matrix. (J) Alcian Blue stained proteoglycan-rich extracellular matrix. (K) Glycogen inclusions (arrow) stained by PAS staining with and without diastase pretreatment. (I), (J), (K) Scale bars = 10 μm. (L) Human collagen type II immunostaining positive in the extracellular matrix. Scale bar = 100 μm. (M) TEM analysis revealed proteoglycans adherent to the cell membrane (arrows). Scale bar = 2 μm. (N) Molecular analysis of type II collagen transcript expression. β-Microglobulin was used as the housekeeping gene. (O) Control hC-MSCs did not display contractile filaments. (P) TEM analysis revealed peripherally arranged contractile filaments, dense bodies, glycogen deposits (*) and profiles of rough endoplasmic reticulum. (Q) Elastic lamellae in the extracellular matrix (arrow). O), (P), (Q) Scale bars = 2 μm. Matrigel assay in the absence (R) and presence (S) of vascular endothelial growth factor (VEGF; 50 ng/ml for 7 days) after 6 hours. (R), (S) Scale bars = 10 μm. (T), (U) Flow cytometry analysis for von Willebrand factor (vWF) and CD31 expression in hC-MSCs cultured in the absence and in the presence of VEGF. Uninduced cells are presented as filled black histograms, differentiated cells as white histograms.

Figure 5

Human cadaver mesenchymal stromal/stem cell immunomodulatory ability. Human cadaver mesenchymal stromal/stem cell (hC-MSC) immunosuppressive effect on activated peripheral blood mononuclear cells (PBMCs). Analysis of the PBMC distribution in cell cycle phases after coculture with hC-MSCs. Phytohemagglutin (PHA)-activated PBMCs reduced proliferation when co- = cultured with hC-MSCs. Data expressed as a percentage of PBMCs for each cell cycle phase. Mean ± standard deviation; n = 3. *P <0.05; ***P <0.001.