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First published online 9 August 2005
doi: 10.1242/jcs.02511

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Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time-lapse video and microarray

Nicoletta Bertani^1,, Paolo Malatesta^1,*,,, Giorgia Volpi¹, Paolo Sonego² and Roberto Perris^1,2

¹ Department of Evolutionary and Functional Biology, University of Parma, Viale delle Scienze 11/a, 43100 Parma, Italy
² Division for Experimental Oncology 2, The National Cancer Institute, CRO-IRCCS, Via Pedemontana Occidentale 1, Aviano 33081, Italy

View larger version (90K): [in a new window]   Fig. 1. In vitro characterization of hMSCs. (A) Undifferentiated hMSCs cultured in standard growth medium show a well-spread or spindle-shaped morphology. (B) hMSCs at early passages display an average doubling time of about 72 hours, but their proliferation rate gradually decreased during ex vivo cell expansion with a replication arrest between passages 10 and 12. (C-F) Analysis of osteogenic and adipogenic differentiation. AP activity of hMSCs cultured in standard conditions (C) and after 10 days of osteogenic induction treatment (D). (E) No lipid droplet formation was seen in hMSCs cultured in control medium. (F) Oil Red O staining after 21 days of adipogenic induction. Bar, 50 µm.

View larger version (125K): [in a new window]   Fig. 2. Morphological changes of hMSCs following different treatments. (A) Cultures of untreated hMSCs display a characteristic fibroblastic morphology. (B) Within 6 hours of DMSO/BHA neural induction treatment about 80% of hMSCs undergo morphological changes and acquire a pseudo-neuronal shape with refractile soma and neurite-like processes. (C) A spontaneous reversion to fibroblastic shape (black arrowhead) is evident 48 hours later. (D-F) Representative photomicrographs extracted from time-lapse sequences, each corresponding to the indicated time points. (D) Time-lapse analysis of DMSO/BHA treatment inducing hMSCs to retract their cytoplasm in the perikaryal region leaving only a few cytoplasmic extensions whose tips still adhere to substrate (white arrowheads). No outgrowth of processes from the cell soma was ever observed. (E,F) The morphological rearrangements can be mimicked by incubating hMSCs with 4 mM EDTA (E) or 10 µM cytochalasin D (F). Bar, 50 µm (A-C); 20 µm (D-F).

View larger version (88K): [in a new window]   Fig. 3. Differentiation potential of human fibroblasts. (A) Untreated cells in subconfluent standard culture. (B) Human fibroblasts exposed for 6 hours to the DMSO/BHA neural induction protocol assumed a pseudo-neural morphology. AP staining of human fibroblasts (C,D) and hMSCs as positive control (E,F). Before osteogenic induction, both human fibroblasts and hMSCs showed low levels of AP activity (C,E). After 10 days treatment in appropriate differentiation media hMSCs increased their level of AP as consequence of osteogenic differentiation (F) whereas human fibroblasts did not undergo osteogenic differentiation and failed to upregulate AP (E). Bar, 20 µm.

View larger version (66K): [in a new window]   Fig. 4. Immunostaining of hMSCs. (A-G) Untreated hMSCs. (A'-D') hMSCs treated for 6 hours with DMSO/BHA. The apparent immunoreactivity of neuronal markers (A'-D') increases in parallel to the immunoreactivity of non-neuronal markers (E',F') and for the unrelated bacterial protein ß-galactosidase (G') compared to that in untreated cells (A-G). Adherent cells with fibroblastic morphology do not show any increase in immunoreactivity (arrowheads). Similarly increased immunoreactivity is also shown by hMSCs treated with cytochalasin D (H-I) and EDTA (J-K). Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 5. Western blot analysis of neural and non-neural markers in hMSCs. (A) Neural marker expression in hMSCs cultured in standard growth medium with 10% FBS (Lane 3), in serum-free medium (Lane 4) and with DMSO/BHA for 6 hours (Lane 5) or 48 hours (Lane 6). Protein extracts from human foetal brain (Lane 1) and HEK293 (Lane 2) served as positive and negative controls, respectively. (B) Non-neural marker expression in hMSCs cultured in standard growth medium with 10% FBS (Lane 1), in serum-free medium (Lane 2) and with DMSO/BHA for 6 hours (Lane 3) or 48 hours (Lane 4). ß-actin was used as a loading control (lower panels in A and B).

View larger version (40K): [in a new window]   Fig. 6. Gene profiling by DNA microarray. (A) Loading plot projected on the first two principal components relative to the PCA of data from the three sets of arrays comparing the transcriptome of untreated hMSCs with 6 and 48 hour-treated hMSCs and foetal brain (positive control). (B) Plot of the average fold changes relative to all genes with detectable expression in all three sets of array experiments. The dimmed field comprising the range between -1.5 and 1.5-fold change contains the genes in which expression is not significantly modulated between the samples and the reference (i.e. uninduced hMSCs). (C,D) Intersections between the sets of the 100 genes most significantly modulated by DMSO/BHA treatment at the specified times and the set of genes differentially expressed between untreated hMSCs and foetal brain (positive control, C) or adult liver (negative control, D).