Evaluation of neurogenic potential of human umbilical cord mesenchymal cells; a time- and concentration-dependent manner

Abstract

Background: Retinoic acid as one of the most important regulators for cell differentiation was examined in this study for differentiation of human umbilical mesenchymal cells (hUCM).

Methods: After isolation, hUCM were evaluated for mesenchymal stem cell properties by flow cytometry and alkaline phosphatase assay. Also, doubling time of the cells and their differentiation potential into adipogenic and osteogenic cells were tested. hUCM were then cultured with different concentrations of retinoic acid, and on days 1, 7, and 12, the percentage of differentiated cells was determined by immunostaining for nestin, anti-microtubule associated protein 2 (MAP₂), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) markers.

Results: The isolated cells were negative for the hematopoietic markers and positive for the mesenchymal markers. They showed the population doubling time 60 ± 3 hours and differentiated into osteogenic and adipogenic cells. A descending trend in nestin and an ascending trend in MAP₂, GAD, and GABA expression were observed from the first day until the last day between different concentrations of retinoic acid.

Conclusion: hUCM cells may have the potential to differentiate into neural cells in the presence of different incubation period and concentration of retinoic acid.

Keywords: Cell differentiation; Neural stem cells; Retinoic acid.

Fig. 1

Flow cytometry results. The gray and black histograms show the isotype control-stained cells as well as the antibody-stained cells, respectively. Human umbilical mesenchymal cells were negative for hematopoietic markers (CD34 and CD45) and positive for mesenchymal stem cell markers (CD44, CD90, and CD105).

Fig. 2

Alkaline phosphatase activity in hUCM. (A) Arrows show the cell colonies were positively stained with ALP (dark red color). (B) No ALP-positive cells were detectable in hUCM treated with levamisole. Scale bars are 25 and 100 μm for (A) and (B), respectively.

Fig. 3

Osteogenic and adipogenic differentiation of hUCM. (A) Calcium deposition and osteoid formation as shown by Alizarin red. (B) Their adipocytic phenotypes were signaled by the appearance of tiny intracytoplasmic lipid droplets with Oil red O. (C) Negative control showed no evidence of staining. Scale bars, 50 μm.

Fig. 4

Doubling time in hUCM. Accumulated data obtained from cultures showed that the lag phase lasts around the first day, leading to five days of log phase. The sharp cell number was about 26 × 10⁴ in the six^th day and after it, the cell number started to decrease.

Fig. 5

Differentiation of hUCM to neuronal lineage. (A) Nestin-positive cells that located around nuclei of differentiated hUCM; (B) Nestin-negative control; (C) MAP₂-positive cells that appeared as several patch within perikarya in induced cells at later stage; (D) MAP₂ negative control; (E) GAD-positive cells; (F) GAD negative cells; (G) GABA-positive cells; (H) GABA negative cells. Scale bars, 25 μm for B, C, D, E, F and H; 50 μm for A and G.

Fig. 6

Expression of specific neural markers in various times in two concentrations. *P < 0.05, differences among 1, 7, and 12 days in a marker. **P < 0.05, differences between two concentrations in MAP₂, GAD, and GABA markers