Anat Cell Biol.  2021 Mar;54(1):74-82. 10.5115/acb.19.229.

Expression of neurotrophic factor genes by human adipose stem cells post-induction by deprenyl

• Amiri A
• Kashani MHG
• Ghorbanian MT

• KMID: 2514587
• DOI: http://doi.org/10.5115/acb.19.229

Abstract

Human adipose stem cells (hASCs) were introduced as appropriate candidate due to advantages like ease of isolation, in vitro expansion and lack of immune response. Deprenyl (Dep) was used to induce bone marrow stem cells into neuron-like cells. We investigated the Dep effect on neurotrophin genes expression in hASCs and their differentiation into neuron-like cells. The cells were isolated from small pieces of abdominal adipose tissue and subjected to flow cytometry to confirm purification. The osteogenic and adipogenic differentiation were identified. The proliferation rate and neurotrophin genes expression of treated cells were evaluated by MTT, TH immunostaining and RT-PCR. hASCs had positive response to CD44, CD73, CD90, CD105 markers and negative response to CD34 and CD45 markers and differentiated into adipocytes and osteocytes. Exposure to 10^–7 M of Dep for 24 hours caused a significant increase of viable cells and BDNF, NTF-3 genes expression as compared to cultured cells in serum free medium and had no effect on the expression of NGF and GDNF genes. Based on our results, Dep is able to induce BDNF, NTF-3 and NTF-4 genes expression and neroun-like morphology in hASCs.

Keyword

Human adipose tissue derived stem cells; Deprenyl; Neurotrophins; Polymerase chain reaction

Figure

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  Fig. 1 Identity of isolated hASCs by flow cytometery. Isolated hASCs were treated with fluorescent antibodies against CD44, CD90, CD73, CD105, CD45, and CD34. The isolated hASCs highly expressed (about 99%) CD44, CD90, CD73, and CD105, but there was no expression of CD45 and CD34 markers. FITC, fluorescein isothiocyanate; FL2-H, fluorescence 2-height; hASCs, human adipose stem cells; PE, phycoerythrin.

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  Fig. 2 hASCs isolation and differentiation into osteoblasts and adipocytes. (A) P1 of isolated hASC. There are stem cells and floating blood cells in culture. In P4, hASCs are the only cells in culture (B). Unstained hASCs differentiated into adipocytes (C), Oil Red staining of differentiated cells (D). Unstained hASCs differentiated into osteocytes (E), Alizarin staining of the cells after 21 days of induction proved their differentiation into osteoblasts (F). hASCs, human adipose stem cells.

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  Fig. 3 Evaluation of Dep toxicity for hASCs. Cells were treated with various concentrations of Dep for 24 and 48 hours. After staining with Trypan blue, the live cells were counted by a hemocytometer. There was a significant increase in the proliferation rate of cells treated with 10–7 M of Dep for 24 hours compared with the same group treated for 48 hours. No significant difference was seen at this concentration in the absence and presence of serum. Dep, deprenyl; Dep 10–7+FBS, cultured cells in medium+10% FBS containing 10–7 M Dep; Dep 10–6–Dep 10–10, cultured cells in medium containing 10–6–10–10 M Dep without serum; FBS, fetal bovine serum; Medium, cultured cells in serum-free medium. *P<0.05.

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  Fig. 4 Evaluation of cell division rate by the MTT assay. To evaluate the division rate of hASCs, MTT assay was performed. hASCs division rate was increased dose dependently. Dep at the concentration of 10–7 M showed a significant increase in division rate as compared to various Dep concentrations and media. Absence and presence of serum did not have any effect at this dose. Dep, deprenyl; Dep 10–7+FBS, cultured cells in medium+10% FBS containing 10–7 M Dep; Dep 10–6–Dep 10–10, cultured cells in medium containing 10–6–10–10 M Dep without serum; FBS, fetal bovine serum; Medium, cultured cells in serum-free medium. *P<0.05.

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  Fig. 5 TH immunostaining of differentiated hASCs. Differentiation of hASCs into neuron-like cells after treatment with 10–7 M of deprenyl for 24 hours (A), TH immunopositive cells were observed (B). The intensity of BDNF, NTF-3, and NTF-4 genes bands was higher after deprenyl treatment, whereas NGF and GDNF genes bands were identical with those of untreated cells. GAPDH gene was used as control (C). hASCs, human adipose stem cells; TH, tyrosine hydroxylase.

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  Fig. 6 Evaluation of neurotrophin genes expression in induced hASCs. The intensity of PCR product bands was assessed by the Image J software. The results showed a significant increase in BDNF, NTF-3 (all doses of Dep) and NTF-4 (Dep 10–8 M) genes expression as compared to medium, whereas no significant difference in NGF and GDNF genes expression was seen. Dep, deprenyl; FBS, fetal bovine serum; hASCs, human adipose stem cells; Medium, cultured cells in serum-free medium. *Significant increase versus the Medium group, #Significant increase versus the Dep 10–6 and 10–7 groups, &Significant decrease versus the Dep 10–8 group.

Reference

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