Laminin and Platelet-Derived Growth Factor-BB Promote Neuronal Differentiation of Human Urine-Derived Stem Cells

Abstract

Urine-derived stem cells (USCs) are considered as a promising cell source capable of neuronal differentiation. In addition, specific growth factors and extracellular matrix are essential for enhancing their neuronal differentiation efficiency. In this study, we investigated the possibility of neuronal differentiation of USCs and the role of laminin and platelet-derived growth factor BB (PDGF-BB) as promoting factors. USCs were isolated from fresh urine of healthy donors. Cultured USCs were adherent to the plate and their morphology was similar to the cobblestone. In addition, they showed chromosome stability, rapid proliferation rate, colony forming capacity, and mesenchymal stem cell characteristics. For inducing the neuronal differentiation, USCs were cultured for 14 days in neuronal differentiation media supplemented with/without laminin and/or PDGF-BB. To identify the expression of neuronal markers, RT-PCR, flow cytometry analysis and immunocytochemistry were used. After neuronal induction, the cells showed neuron-like morphological change and high expression level of neuronal markers. In addition, laminin and PDGF-BB respectively promoted the neuronal differentiation of USCs and the combination of laminin and PDGF-BB showed a synergistic effect for the neuronal differentiation of USCs. In conclusion, USCs are noteworthy cell source in the field of neuronal regeneration and laminin and PDGF-BB promote their neuronal differentiation efficiency.

Keywords: Laminin; Mesenchymal stem cell source; Neuronal differentiation; Platelet-derived growth factor-BB; Urine-derived stem cells.

Fig. 1

Morphology of USCs and G-banded karyotypes for USCs. A–H The USCs at passage 4 were expanded and were cultured in growth medium. USCs show the cobblestone-shaped morphology. Images are shown at ×200 magnification. I–P Chromosome analysis of USCs at passage 4. Karyotype analysis showed that USCs had a normal size, shape, and number of chromosomes. USCs did not show any chromosomal aberrations

Fig. 2

Analysis of stem cell proliferation. A Comparison of cell proliferation rates at day 3, 5, and 7. USCs showed a time-dependent increase in growth rate. B Quantity of USCs colonies at passage 4. The number of stained colonies from the 8 USC lines was compared. There was variation between the samples, but the number of colonies increased in all the samples

Fig. 3

Comparison of stem cell marker expression in USCs. A–I Specific cell surface markers were assessed by flow cytometry in passage 4 of USCs. USCs were strongly positive for mesenchymal stem cell markers and negative for hematopoietic lineage and immunogenic markers

Fig. 4

Comparison of stem cell marker expression in USCs. A–P Immunofluorescence staining of USCs at passage 4. USCs were labeled with A–H embryonic/mesenchymal stem cell marker (SSEA4), I-P mesenchymal stem cell marker (CD73)

Fig. 5

Comparison of stem cell marker expression in USCs. A–P Immunofluorescence staining of USCs at passage 4. USCs were labeled with A–H hematopoietic stem cell marker (CD34), and I-P immunogenic marker (HLA-DR). No visible labeled cells were observed for CD34 and HLA-DR

Fig. 6

Neurogenic in vitro differentiation. A Experimental design for the induction of neurons. Four different kinds of neural differentiation conditions (differentiation medium with/without 5 ng/ml PDGF-BB and with/without 5 µg/cm² laminin coating) induced USCs into neural like cells in 2 weeks. B-E Morphology of USCs after differentiation with neural differentiation media for 2 weeks. Images shown at × 200 magnification. F-J Determination of neural gene expression in differentiated USCs by RT-PCR. Quantitative real time PCR was performed on total RNA using neuron-specific primers (Nestin, MAP2, β-tubulinIII, NF-M, and NeuN). The values were calculated with reference to GAPDH and the samples from 8 USC lines were run in duplicate. The neural markers, Nestin, MAP2, β-tubulinIII, NF-M, and NeuN were significantly increased in condition IV compared to condition I. Compared with condition II, condition IV showed significantly higher expression of Nestin (p = 0.0003), MAP2 (p = 0.0376), β-TubulinIII (p = 0.0013), NF-M (p = 0.0154) and NeuN (p = 0.0042). Compared with condition III, condition IV showed significantly higher expression of MAP2 (p = 0.0068) and NeuN (p = 0.0213). Compared with condition I, condition II showed significantly higher expression of MAP2 (p = 0.0389) and NeuN (p = 0.0370). Compared with condition I, condition III showed significantly higher expression of Nestin (p = 0.0049), MAP2 (p = 0.0003), β-TubulinIII (p = 0.0328), NF-M (p = 0.0001) and NeuN (p = 0.0242). Compared with condition II, condition III showed significantly higher expression of β-TubulinIII (p = 0.0397). Student t-test was used for gene expression analysis using real –time PCR data. Values are mean ± SD (*p < 0.05), (**p < 0.005), and (***p < 0.0005). Abbreviations: I, DM; II, DM plus PDGF-BB; III, DM plus Laminin; IV, DM plus PDGF-BB plus Laminin

Fig. 7

Determination of neural protein expression in differentiated USCs using flow cytometry. Immunofluorescence of differentiated USCs at passage 4. Upon neural differentiation, USCs were stained for specific markers. A-Y Flow cytometric analysis. USCs were labeled with mesenchymal stem cell markers (CD44 and CD90), neuron specific markers (Nestin, MAP2, and NF-M). Differentiated USCs were strongly positive for neural cell markers and still expressed mesenchymal stem cell markers

Fig. 8

Determination of neural protein expression in differentiated USCs using immunocytochemistry. Immunofluorescence of differentiated USCs at passage 4. Upon neural differentiation, USCs were stained for specific markers. A-T Immunocytochemistry. USCs were labeled with neuron specific markers (Nestin, MAP2, β-tubulin, and NF-M). Un-differentiated USCs do not express neural markers. Images are shown at x 200 magnification