Table 1.
Real time RT-PCR TaqMan primers and their description.
Fig 1.
Characterization of Decellularized Wharton’s Jelly Matrix (DWJM).
A) A fragment of the isolated DWJM. A skin punch biopsy kit, (right lower corner image) was used to obtain 5–7 mm DWJM scaffolds. B) hematoxylin and eosin (H&E) stained sections of the DWJM showing empty spaces. (Scale bar represents 0.1 mm.) C) Collagen I immunohistochemistry of the DWJM (scale bar is 50 μm), D) Trichrome staining images of human umbilical cord, and E) Decellularized Wharton’s jelly matrix. (Scale bar represents 50 μm.) Red color represents blood, light blue collagen, and cells/nuclei are in black/dark blue. F) Immunohistochemical staining of DWJM by anti- hyaluronic acid antibody. The matrix is rich in collagen and there is abundant hyaluronic acid expression at some parts compared to the others. (Scale bar represents 25μm.) G) Scanning electron microscopy images of DWJM. One surface appears flat with compact matrix (left lower image) while, less dense tissue with open spaces is identified in other areas (lower right and middle images). (Scale bar for the full picture is 600 μm.) H) Transmission electron microscopy images of DWJM. More electron-dense areas of DWJM (left upper image) and less electron dense areas (right upper image) are observed. No intact cells were observed in any of the panels. (Scale bar for left upper image is 2 μm, for right upper image 10 μm, and for the two lower images 500 nm.).
Fig 2.
A) DNA quantification study performed on the matrix before decellularization and after decellularization. DWJM showed significantly less DNA compared to the native WJ matrix before decellularization. B) Glycosaminoglycan content assessment of the matrix before and after decellularization. (* Indicates statistical significance (p < .05)).
Table 2.
Proteins identified in Decellularized Wharton’s Jelly Matrix (DWJM) by mass Spectrometry.
Fig 3.
MSC characterization by flow cytometry.
A) Wharton’s jelly mesenchymal stem cell (WJMSCs) and, B) bone marrow mesenchymal stem cell (BMMSCs). All MSCs stained positive for CD90 by fluoroscein isocyanate (FITC), CD105 by phycoerythrin (PE) and CD73 by allophycocyanin (APC); and they were negative for hematopoietic markers CD45, CD34, CD14 or CD11b, and CD20 as analyzed by Cell Profiler (CP) software (Broad Institute).
Fig 4.
Transplantation and culturing of WJMSCs on DWJM.
A) Confocal microscopy images of DWJM and WJMSCs on DWJM after 2 hours (upper panel), 1 day (center panel), and 2 days (lower panel) post- cell seeding. The cells are labeled with calcein acetylmethyl (AM) that stains the live cells in green. Dual beam imaging of B) DWJM and C) DWJM seeded with WJMSCs for 1 week. The Everhart-Thornley detector (ETD) is a standard secondary electron detector used in scanning electron microscopy to study topography, while the circular backscatter (CBS) is a backscatter detector that reveals lipid content when samples are stained with osmium tetroxide (OT) (red/orange). Images have been pseudo-colored to enhance definition proportional to secondary electron signal for ETD. (Scale bar is 20 μm.) DWJM appears to be a fibrous interpenetrating network with varying pore sizes, while WJMSCs were arranged along the fibers of DWJM.
Fig 5.
Assessing WJMSC viability and proliferation when seeded on DWJM.
A) Alamar blue assay to assess the viability of cells seeded on the matrix and B) Cell migration assay performed using trans-wells with cells alone (control) and cells migrating towards DWJM, (* Indicates statistical significance p < 0.05).
Fig 6.
Relative fold change in the mRNA levels of the indicated genes.
Panel A-F are WJMSCs on DWJM with A) Cell adhesion genes, B) Chondrogenic genes, C) Adipogenic genes, D) Myogenic genes E) Osteogenic genes, F) Apoptosis and proliferation genes. Panel G-L are BMMSCs cultured on DWJM with G) Cell adhesion genes, H) Chondrogenic genes, I) Adipogenic genes, J) Myogenic genes K) Osteogenic genes, L) Apoptosis and proliferation genes. Relative fold- change is represented on the y-axis and the genes were represented along the x-axis. The horizontal line represents the gene expression of cells before seeding at Day 0. (* Represents statistical significance p<0.05.)
Fig 7.
WJMSCs transplantation into an in vivo animal model.
A) Mice with cranial defect, B) mice with cranial defect and DWJM, C) mice with cranial defect and DWJM 14 days post-surgery. Arrows in A represent the defect, B shows the DWJM and C is the defect and DWJM 14 days post-surgery. D) IVIS imaging of the mice post—surgery—1) Mice with DWJM 24 hours post- surgery; 2–6) designates mice 14 days after the surgeries. D2 is mice without any intervention, D3 and D4 are mice with the defect alone, and D5—D6 represent mice with defect and DWJM. The red circles indicate the defect sites and the inset images are a higher magnification of the defect site in mice. The green fluorescence signal at the defect site signifies the migration of the GFP positive cells into the defect. Images E-J represent the histology images of bone specimen with DWJM 14 days post-surgery, with image E) hematoxylin-eosin stained (H&E) section of DWJM tissue specimen 24 hours post-surgery, and image F depicts GFP immunohistochemistry staining of the same. Images G-J represent DWJM sample 14 days post-surgery with G, H and I being H&E stained sections of DWJM viewed at different magnifications as indicated in the figure. J represents the GFP immunohistochemistry of the section in image I. The arrows in image F, J represent GFP positive cells.