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First published online 3 August 2004
doi: 10.1242/jcs.01264

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Neurogenesis of Rhesus adipose stromal cells

¹ Division of Gene Therapy, Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA 70433, USA
² Division of Veterinary Medicine, Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA 70433, USA
³ Center of Gene Therapy, Tulane University, New Orleans, LA 70112, USA
⁴ Department of Pharmacology, Tulane University Health Sciences Center, Tulane University, New Orleans, LA 70112, USA
⁵ Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70112, USA

View larger version (72K): [in a new window]   Fig. 1. Morphology and proliferation and differentiation potential of pATSCs. Culture expanded non-human primate ATSCs show the spindle-shaped fibroblastic morphology (A for low density and B for high density). Compared with pATSCs, pBMSCs are more heterogeneous and they have fibroblastic morphology (C). A single cell can be expanded into a clonal population and can generate colony forming units (CFUs) that are shown by Giemsa staining (D). Passage 3-4 pATSCs retain multilineage differentiation capability undergoing adipogenesis (E), osteogenesis (F) and chondrogenesis (G) in vitro. Cumulative population doublings with respect to passage number in multiple animal samples were measured (H). Bars, 40 µm (A); 30 µm (B).

View larger version (39K): [in a new window]   Fig. 3. Evaluation of pATSCs-derived single cell clones pluripotency. Single clones-derived progenies were plated at 50 cells/cm2 and induced differentiation to adipogenic, osteogenic and neurogenic. After differentiation cells were stained with Oil Red O and Alzarin Red S for detection of fat and mineralization. For neural lineage detection, cells were immunostained with GFAP, Tuj and MBP antibodies. Positive colonies were counted.

View larger version (78K): [in a new window]   Fig. 2. In vitro differentiation of pATSCs (A-D), pBMSCs (E-H) along neuronal lineages. Stromal cell-derived neurospheres (A,E) cultured in media supplemented with B27, bFGF and EGF for 4-5 days. Free-floating cell spheres in the NB medium (B,F small inset panel). pATSC and pBMSC-derived neurosphere induced differentiation by culturing on the PDL-Laminin-coated substrate for 10-15 days in B27 supplemented NB media (B-D, F-H). Neurospheres attached to the bottom of the culture dish and protrude extensive cell processes (B,C,F,G). The processes became longer and formed diverse networks 10 days after plating (D,H). Bars, 30 µm (A,C,E); 60 µm (B,D,F,G,H).

View larger version (95K): [in a new window]   Fig. 4. Characterization of pATSCs-derived neurospheres pATSCs-NS. The pATSC-NS express nestin (A, red), and show strong expression of MAP2ab (B, red), GFAP (C, green) and CD133 (D, red). Blue color is DAPI staining. Bar, 60 µm (A-D).

View larger version (54K): [in a new window]   Fig. 5. Time course of MAP2ab expression of pATSC-NS. pATSC were detached from culture dish and cultured in suspension in NB medium supplemented with B27, bFGF, EGF. Each sample was isolated at a predetermined time period, and expression of MAP2ab was analyzed by western blot and quantitated. Maximum MAP2ab levels were detected at 4 days (96 hours), after which levels were downregulated. Control pATSCs do not express the MAP2ab protein.

View larger version (37K): [in a new window]   Fig. 6. RT-PCR analysis of stem cell and neural lineage marker expression in control pATSCs and differentiated pATSCs. Total cellular RNA (1 µg) was analyzed using the primers against several neural markers. The PCR product was separated on 1.5% agarose gel and visualized by ethidium bromide staining. Upper panel: undifferentiated pATSCs. , molecular marker; Lane A, Oct-4; Lane B, telomerase; Lane C, Hes1; Lane D, ß-actin; Lane E, nestin; Lane F, BDNF; Lane G, MAP2ab; Lane H, GAD65; Lane I, GFAP. Lower panel: Neuronal-lineage differentiated pATSCs. Lane J, Oct-4; Lane K, nestin; Lane L, BDNF; Lane M, MAP2ab; Lane N, GAD65; Lane O, GFAP; Lane P, Trk A; Lane Q, Trk B; Lane R, P75.

View larger version (46K): [in a new window]   Fig. 7. Immunocytochemistry of pATSCs after neurosphere differentiation and culture on PDL-Laminin. Intense MAP2ab (A, red; E and F, yellow) and NF160 (A, green; B, green; C, green) expression and strong nuclear staining for Neu N (B, red; D, green) in pATSCs following neurosphere differentiation. High levels of GFAP (C, red), which is absent in undifferentiated pATSCs, were induced in after neurosphere differentiation. Prominent nestin (G, red) expression in cells derived from pATSC-derived neurospheres. Blue color is DAPI staining. Bars (A-G), 150 µm.

View larger version (51K): [in a new window]   Fig. 8. Comparison of nestin and MAP-2 expression between neural differentiated pATSCs and pBMSCs. (A) Western blot analysis of whole cell lysate of fully differentiated pATSC and pBMSC-derived neurosphere (NS) maintained under neurobasal medium supplemented with B27, bFGF and EGF. Thirty micrograms of protein extract from each cell culture were separated on a 12% acrylamide gel and transferred to polyvinyldiethylfluoride (PVDF) membrane. The blots were probed with antibodies to nestin and MAP2ab followed by HRP-conjugated secondary antibody and developed using enhanced chemiluminescence. (B) Summary of the expression of the MAP2ab mRNA following NS differentiation, as quantified by real-time RT-PCR. The gene expression levels were normalized with respect to endogenous GAPDH.

View larger version (20K): [in a new window]   Fig. 9. Flow cytometry histograms of neural markers in neurospheres differentiated from pATSCs. Neurospheres were differentiated under supplemented NB medium for 10 days. The adherent cells were harvested and stained with monoclonal antibodies against GFAP coupled to fluorescein isothiocyanate (FITC) and nestin or MAP2ab coupled to phycoerythrin (PE). The distribution of pATSC-NS differentiated cells stained for those antibodies are shown. The geometric mean and median values for nestin, MAP2ab and GFAP are also shown.

View larger version (17K): [in a new window]   Fig. 10. Expressed gene profile in pATSC and pATSC-NS are similar. Scatter plot shows gene expression in pATSC and pATSCNS. Template RNA was extracted from 3x106 cells derived from neurospheres. cDNA was prepared, labeled and hybridized to the Affymetrix HGU 95A2 array containing 22,000 human genes. Data analysis was done using Genechip software.

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