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Blood, Vol. 110, Issue 9, 3438-3446, November 1, 2007
Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow)
Blood Beltrami et al.
110: 3438
Supplemental materials for: Beltrami et al
Files in this Data Supplement:
- Figure S5. RT-PCR analysis of differentiated polyclonal hMASCs (JPG, 60.2 KB)
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Representative RT-PCR photographs of differentiation marker mRNA expression on Heart derived(hH1 and hH2), Liver derived- (hL1 and hL2) and BM derived- (hBM1 and hBM2) cells differentiated into myocytes (cardiac actin, myocardin), smooth muscle cells (smooth muscle actin), endothelial cells (VE-cadherin), hepatocytes (cytochrome P2E1 -CytP2E1-) and neural cells (NSE, tubulin  3 -Tub3-).
- Figure S6. DNA content of single cell derived clones (JPG, 52.2 KB)
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Flow cytometry histograms showing DNA content of lymphocytes, hMASCs and a mixed population consisting of the previous two cell suspensions.
- Figure S7. FACS analysis of single cell cloned hMASCs (JPG, 301 KB)
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Representative Flow Cytometry histograms of single cell derived clones. Plots show isotype control IgG staining profile (open histogram) versus specific antibody staining profile (dashed histogram).
- Figure S8. Neuroectodermic differentiation of cardiac and liver derived cell clones in differentiation medium (JPG, 101 KB)
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(A,D) Immunocytochemical identification of glial fibrillary acidic protein positive cells in cardiac and liver derived clones, respectively. (B,E) NeuD expression in cell nuclei of cardiac and liver derived clones (red fluorescence). (C,F) Green fluorescence identifies neuron specific enolase in heart derived and liver derived clones. Blue fluorescence of DAPI staining depicts cell nuclei.
- Figure S9. Mesodermic differentiation of bone marrow derived cell clones in osteogenic medium (JPG, 134 KB)
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(A) Osteocalcin antibody decorates cell cytoplasm in green. (B) Tetracycline incorporation in sites of calcification is identified by their red emission, when excited with a UV laser. Blue fluorescence of DAPI staining depicts cell nuclei.
- Figure S10. Mesodermic differentiation of cardiac clones in myogenic medium (JPG, 143 KB)
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(A) The red fluorescence of  -sarcomeric actin antibody staining identifies myocyte cytoplasm, while green fluorescence identifies connexin 43 on the cell surface. (B) Yellow fluorescence demonstrates L-type calcium channel on the cell surface. Blue fluorescence of DAPI staining depicts cell nuclei.
- Figure S11. Hepatocytic differentiation functional assay (JPG, 113 KB)
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(A) PROD assay. Representative aggregates obtained from one liver derived (upper 2 rows) and one BM-derived single cell clone (lower 2 rows) cultured in differentiation medium. Rows 1 and 3 show, in pseudo-colors (Q-LUT, scale bar), resorufin fluorescence of cell aggregates exposed to pentoxyresorufin (PR), while rows 2 and 4 represent the overlay of phase contrast and red fluorescence images of the same cell aggregates; from the left to the right are depicted: negative control, cells exposed to PR only and cells exposed to Phenobarbital and PR. (B) Albumin production. ELISA determination of albumin production (expressed in pg/hour and normalized for the number of seeded cells) produced between day 17 and day 20 from undifferentiated single cell derived clones (black bar), from cardiac-, liver- and BM- single cell derived clones in differentiation medium, and from human hepatocytes cultured in differentiation medium as well. *, **, *** and ****, p<0.05 vs. hMASC, heart-derived, liver-derived and BM-derived single cell clones, respectively.
- Video 1. Spontaneous Calcium Transients in hMASC cultured in myogenic medium (AVI, 3.98 MB)
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hMASCs cultured in myogenic medium for 1-4 weeks, showed spontaneous calcium transients, as detected by fluo-4 fluorescent dye (green fluorescence). Green flashes indicate binding of calcium to fluo-4 upon release of calcium from the sarcoplasmic reticulum. The cells with detectable calcium oscillations were not visibly contracting, which shows an uncoupling of excitation–contraction, presumably because of an immature contractile apparatus or decreased movement caused by a dense extracellular matrix.
- Video 2. Mature beating hMASC-derived myocytes in culture (AVI, 6.95 MB)
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When differentiation was prolonged for more than 2 months, some cells showed a rhythmic spontaneous contractile activity. Spontaneously beating cells acquired rod-shape morphology. Cells kept in controlled CO2 and temperature environment have been noted to beat for at least one month.
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