Derivation efficiency, cell proliferation, freeze–thaw survival, stem-cell properties and differentiation of human Wharton’s jelly stem cells  Chui-Yee Fong, Arjunan Subramanian, Arijit Biswas, Kalamegam Gauthaman, Prarthana Srikanth, Manoor Prakash Hande, Ariff Bongso  Reproductive BioMedicine Online  Volume 21, Issue 3, Pages 391-401 (September 2010) DOI: 10.1016/j.rbmo.2010.04.010 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Growth kinetics of human Wharton’s jelly stem cells (WJSC). (A) Proliferation rates in complex medium for various passages. Note that all passages (early and late) for the five different cords studied showed increased growth rates with time (0–6days) in culture. (B) Proliferation in simple medium for various passages in the same five cords. Note that although growth increased with time (0–6days) in the simple medium, it was not as high as in complex medium. Early passages (P4, P6) performed better than late passages (P10, P20). (C) Population doubling time (PDT) for human WJSC grown in simple and complex media at various passages. Note that PDT was shorter in complex medium compared with simple medium and increased with serial passaging in both media. D1–D6 = days in culture; P = passage. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Phase-contrast inverted optical images of phenotype of human Wharton’s jelly stem cells at various passages in different culture media. Epitheliod-like cell morphology in early passages (P1) transform to fibroblast-like morphology in late passages (P10, P20, P30) in (A) simple and (B) complex media. Magnification 200×. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Cell mounds in confluent human Wharton’s jelly stem cell cultures. (A–C) Smaller cell mounds in simple medium. (D–F) Larger cell mounds in complex medium. Magnification 10×. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Representative CD markers in human Wharton’s jelly stem cells from umbilical cord from a male fetus (W010M) using monoclonal antibody staining and fluorescence-activated cell sorting analysis at two different passages in simple medium, (A) P2–A and (B) P10–B. Each contour map represents the percentage of FITC-positive cells against unstained controls for each CD marker. White peak = isotype control; turquoise peak = experimental sample. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Telomere length analysis of human Wharton’s jelly stem cells from three umbilical cord samples (W0004M, W010M and W009F) using the Southern blot terminal restriction-fragment length assay. Lanes: 1 = standard; 2,3 = W004M (P5); 4,5 = W004M (P10); 6,7 = W010M (P10); 8 = W009F (P5); 9 = W009F (P1); 10 = control DNA. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Alkaline phosphatase assay for human Wharton’s jelly stem cells. Positive stain for alkaline phosphatase (red) at various passages (P2, P5, P10) in (A) simple medium and (B) complex medium. Magnification 10×. P = passage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Chromosome analysis of human Wharton’s jelly stem cells (WJSC). (A) Relationship of fetal sex to human WJSC sex. Note that normal karyotypes were maintained in the cells for up to 30 passages for five different umbilical cords. (B,C) Representative inverted DAPI and DAPI-stained karyotypes of male human WJSC (P10) and female human WJSC (P10), respectively. P = passage; ∗ = from twin pregnancy. Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 8 Neuronal differentiation of human Wharton’s jelly stem cells in (A) simple and (B) complex medium from two different passages (P2, P10). (a,c,e,g) Untreated (controls). (b,d,f,h) Neurons derived from human WJSC (treated). Grey = phase-contrast image; blue = DAPI negative immunostaining; red = glial fibrillary acidic protein (GFAP) positive immunostaining; green = cell-surface ganglioside epitope (A2B5) positive immunostaining; P = passage. Magnification 20×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Reproductive BioMedicine Online 2010 21, 391-401DOI: (10.1016/j.rbmo.2010.04.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions