Journal: The Journal of Experimental Medicine

Article Title: A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

doi: 10.1084/jem.20040440

Figure Lengend Snippet: In vitro and in vivo differentiation of USSCs into osteoblasts, chondroblasts, and adipocytes. (A) Differentiation to osteoblasts is shown by the ALP assay. The peak of ALP was already achieved on day 7. Both basic media McCoy (filled square) and DMEM (filled triangle) in the presence of DAG were able to support the osteoblast differentiation. USSCs (control: open triangle, DMEM; open square, McCoy) cultured without DAG showed no ALP activity. (B) Quantitative Ca 2+ release assay. Both basic media McCoy (filled square) and DMEM in the presence of DAG (filled triangle) were able to support the osteoblast differentiation. USSCs (control: open triangle: DMEM; open square, McCoy) cultured without DAG showed no Ca 2+ activity. (C and D) Chondrogenic differentiation. (C) Demonstrates an Alcian blue–positive extracellular matrix at day 21 after stimulation toward the chondrogenic pathway, indicating a homogeneous distribution of sulfated proteoglycans within the matrix structure (×10 magnification). (D) Collagen type II staining of pellet microsections analyzed by fluorescence microscopy; the nuclei are stained with DAPI (×40 magnification). (E) Oil Red-O staining of the lipid vesicles performed 2 wk after stimulation demonstrates an ongoing adipogenesis (×40 magnification). In vivo differentiation of USSCs into bone and cartilage. (F–M) Ceramic cylinders were loaded with USSCs and transplanted into nude rat femur critical size defects of 0.5 cm in length. (F) 4 wk after transplantation, human cells were still present in the bone defect as demonstrated by immunohistochemical staining with the human-specific mAb 6E2 (×20 magnification). (G and H) Images of the longitudinal (G) and cross section (H) demonstrate bony healing between the cell-loaded implant and the host bone (HB). Bony integration was established in forms of cancellous bone as detected by Toluidine blue staining (×4 magnification). (I) Faxitron high resolution x-ray scanning of specimens harvested at 12 wk after surgery demonstrates the healing between the cell-loaded implant and the host bone (magnification, original size). (J) Unloaded ceramic cylinders served as negative controls demonstrating a nonhealing between the scaffold and the host bone. Arrows indicate the interface between the scaffold and the host bone (magnification, original size). (K–M) A successful in vivo chondrogenesis of USSC-loaded Gelfoam sponges in a nude mouse model. Human USSCs loaded into gelatin sponges were cultured in vitro for 1 (K) or 2 (L) wk in a chondrogenic medium with TGF-β. These sponges were s.c. implanted into nude mice for another 3 wk before analysis. At 1 (K) or 2 (L) wk of in vitro culture, cells filled the pores of the Gelfoam sponge. Some local spots demonstrate an extracellular matrix formation which indicate chondrogenic lineage differentiation. The implanted cells demonstrate strong chondrogenic differentiation as documented by Toluidine blue staining (M) (×10 magnification).

Article Snippet: Two different media were used to initiate growth of the adherent USSC colonies: myelocult medium (StemCell Technologies) and low glucose DMEM (Cambrex) with 30% FCS, dexamethasone (10 −7 M; Sigma-Aldrich), penicillin (100 U/ml; Grünenthal), streptomycin (0.1 mg/ml; Hefa-pharma), and ultraglutamine (2 mM; Cambrex).

Techniques: In Vitro, In Vivo, ALP Assay, Control, Cell Culture, Activity Assay, Release Assay, Staining, Fluorescence, Microscopy, Transplantation Assay, Immunohistochemical staining