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Human cord blood-derived cells attain neuronal and glial features in vitro

L. Buzanska1, E. K. Machaj2, B. Zablocka1, Z. Pojda2 and K. Domanska-Janik1,*

1 Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego St. 02-106 Warsaw, Poland
2 Institute of Oncology, 5 Roentgena St. 02-781 Warsaw, Poland



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  		Fig. 1. Flow cytometry of fresh CD34+- immunodepleted cells (A) and cells following 6 weeks of selection in vitro (B). (A) Immunomagnetic depletion of the initial mononuclear fraction resulted in approximately 100% elimination of CD34+ cells, whereas up to 80% of cells expressed CD45 antigen. (B) Following 6 weeks of culture of CD34+- depleted cells in conditions described in the Materials and Methods, CD45+ cells were reduced to almost negligible levels (less than 4% of the whole cell population).

 


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  		Fig. 2. Nestin-positive clones obtained from the selected human cord blood subpopulation. Phase-contrast images of CB-derived cells. (A) The CD34+-depleted cells that were characterised by FACS in Fig. 1B. (B) Cells from A after being re-seeded and expanded in DMEM/10% FCS and 10 ng/ml EGF to form a monolayer of homogenous, round, proliferating cells. An example of a typical, single, clonogenic cell from this culture is shown in the insert. (C) A clone growing for 14 days in the presence of EGF, after low-density (10 cells/cm2) suspension of cells from culture presented in B. (D) The same clone as in C after another 7 days in culture displays a 10-fold increase in the number of cells as quantified by cell counting. (E) Immunocytochemical staining with the anti-human nestin polyclonal antibody shows that the majority of cells are immunoreactive in growing the clone. The insert shows a higher magnification (40x) of a nestin-positive cell with a Hoechst 33258-stained nucleus, showing a typical filamentous pattern of immunostaining. (F) RT-PCR analysis of a nestin gene expression in cells growing in clones (lines 1 and 2) contrasted with an almost complete lack of signal in mRNA sample extracted from CB-derived (not EGF expanded) cells growing in a monolayer as shown in A. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in parallel samples served as a semi-quantitative control for RT-PCR products. Bars: A, 50 µm; B-D, 100 µm; E, 20 µm.

 


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  		Fig. 3. Expression of neural marker proteins by the clone-growing cells after 4 days of treatment with retinoic acid. (A,B) Double immunostaining for the presence of ß-tubulin III (FITC-conjugated secondary antibody) and GFAP or GalC (Texas-Red-conjugated secondary antibodies). Cells derived from the same clone can express neuronal and astroglial (A) or neuronal and oligodendroglial (B) markers. All cell nuclei were additionally stained with Hoechst 33258. Bar, 50 µM. (C) Western blots of the untreated (RA-) controls and the RA-treated (RA+) differentiated cells probed with anti-ß-tubulin III (neuronal marker), anti-GFAP (astrocytic marker) and anti-PLP/DM-20 (oligodendrocytic marker). The position of the PLP splicing variant, DM-20, is marked by arrow.

 


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  		Fig. 4. Cell-type-specific immunostaining for neurons (A,B), astrocytes (C,D) and oligodendrocytes (E,F) in CB-derived cells growing in differentiation-promoting media. (A,B) Cells expressing class III ß-tubulin recognised by the TUJ1 antibody and displaying neuron-like morphology with long axonal projections. The filamentous feature of cytoplasmic structures that are immunoreactive with the TUJ1 antibody and corresponding to neuronal microtubules are clearly visible under higher magnification (B). (C,D) Cells immunostained with the anti-GFAP antibody. Some of the cells are round and relatively small, whereas others contain long projections with immunoreactive filamentous structures that are visible in the cytoplasm (D). (E,F) GalC-immunoreactive cells expressing galactosylceramides and displaying morphology characteristic of oligodendrocytes, with flat cell body and short or long branched projections. Smaller, round immunoreactive cells are also occasionally present (see the right upper corner). Bars: A,C,E, 50 µm; B,D,F, 20 µm.

 


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  		Fig. 6. Extent of CB-derived neural cell differentiation. Quantification of the extent of neuronal, astrocytic and oligodendrocytic differentiation in cultures grown for 4 days in differentiation-promoting conditions. White bars: neurobasal medium (NM) supplemented with 10% FCS; dotted bars: NM with 10% FCS plus 0.5 µM of RA; striped bars: NM, 10% FCS, 0.5 µM RA with addition of 10 ng/ml BDNF; grey bars: cells after seeding on monolayer of rat primary cortical culture grown for 4 days in DMEM supplemented with 10% FCS. Note that promotion of differentiation was best in co-culture (grey bars). In pure, CB-derived cell systems, the presence of RA was decisive for neuronal differentiation, whereas additional supplementation with BDNF promoted mainly astrocytes, with a significant retention of oligodendrocytes. A subpopulation of CB-derived cells growing in a monolayer before clone formation was found to be negative for all investigated antigens. The results are expressed as the mean±s.d. of cell number from nine independent cultures (three parallel experiments from three separate cord blood preparations).

 


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  		Fig. 5. Neural differentiation of CB-derived cells after their plating on the monolayer of rat primary cortical culture. The CB-derived cells (coloured green by the `cell tracer' in A,E,I and by Texas Red after phenotype-specific immunoreactions in B,F,J) can be detected in the vicinity of cells originating from rat cortex (only red). Both stains were detected simultaneously (C,G,K) and together with nuclei of all cells forming rat primary cortical monolayer revealed by the use of Hoechst 33258 staining (D,H,L). Colocalisation of red and green labelling in C,G,K and D,H,L appears yellow after overlaying these two images. (B,C,D) Neuron-specific immunostaining for type III ß-tubulin with TUJ1 antibody. (F,G,H) Astrocyte-specific immunostaining with anti-GFAP antibody. (J,K,L) Oligodendrocyte-specific immunoreaction with galactosylceramide recognised by the GalC antibody. Arrows indicate immunopositive CB-derived cells. Scale bars for A to H shown in A and E correspond to 50 µm. The scale bar for I to L shown in I corresponds to 20 µm.

 


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  		Fig. 7. Examples of phenotypic diversity among a progeny of CB-derived neural precursors induced to differentiate in various culture conditions. (A) Overlaying images of expression of two neuron-specific proteins: class III ß-tubulin (immunostained by a Texas-Red-conjugated secondary antibody) and MAP2 (immunostained green by afluorescein-conjugated secondary antibody). The TUJ1-positive structures, which are also immunoreactive with the anti-MAP2 antibody, appeared yellow when detected simultaneously (arrows). Some of these cells display characteristic neuron-like morphology (arrowhead). Cell nuclei are blue contrastained by Hoechst 33258. (B) Distinct distribution of neuron- and astrocyte-specific proteins is indicated by MAP2 (green) and GFAP (red) immunostaining, respectively. Cell nuclei are blue-stained by Hoechst 33258. Very fine, green, anti-MAP2 immunoreactive cytoskeletal filamentous structures can be seen in the cytoplasm of neuron-like cells. (C) CB-derived cells prelabelled green by `cell tracer' reveal the typical morphology of matured oligodendrocytes with long, branched projections expressing GalC-immunoreactive galactosylceramides. In the figure, fragments of green-traced cells positive for GalC (recognised by Texas-Red-conjugated secondary antibody) appear yellow owing to the overlaying of these two colours when detected simultaneously.

 

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