Int J Stem Cells.  2016 Nov;9(2):213-220. 10.15283/ijsc16031.

Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells

• Najafabadi MM
• Bayati V
• Orazizadeh M
• Hashemitabar M
• Absalan F

• KMID: 2362515
• DOI: http://doi.org/10.15283/ijsc16031

Abstract

BACKGROUND AND OBJECTIVES
Schwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated.
METHODS AND RESULTS
ASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×10³, 4×10³ and 8×10³ cells/cm² an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75(NTR) mRNA, another SC marker, were significantly higher at the density of 8×10³ cells/cm² when compared with the other cell densities groups (p<0.001).
CONCLUSIONS
The results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×10³ cells/cm², possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

Keyword

Adipose-derived stem cell; Schwann cells; Differentiation; Initial cell density

MeSH Terms

Animals
Cell Count*
Coculture Techniques
Flow Cytometry
Fluorescent Antibody Technique
Glial Fibrillary Acidic Protein
Neuregulin-1
Peripheral Nerve Injuries
Rats*
RNA, Messenger
S100 Proteins
Schwann Cells
Stem Cells*
Glial Fibrillary Acidic Protein
Neuregulin-1
RNA, Messenger
S100 Proteins

Figure

• 

  Fig. 1 The mesenchymal nature of isolated ASCs was confirmed by their ability to differentiate into adipogenic (A) and osteogenic (B) pathways and also by evaluating the expression rate of positive (CD44, CD73 and CD90) and negative (CD45) markers on the cells surface by flow cytometry (C). Scale bar=10 μm.

• 

  Fig. 2 Immunostaining of the cells cultured in Schwann cell differentiation medium after 2 weeks. Immunofluorescence demonstrated that some cells displayed bipolar and tripolar morphologies and expressed S100 and GFAP proteins at the center of differentiated cell colonies in all cell density groups. In addition, negative control images showed no expression of S100 and GFAP proteins. Arrow heads point to bipolar cells and asterisks show tripolar cell morphology. Scale bar=30 μm.

• 

  Fig. 3 FACS analysis of differentiated ASCs. (A) After 2 weeks of induction, cultures with different cell densities were analyzed by FACS for S100 and GFAP to indicate the number of differentiated SC-like cells. (Data are mean % cells±SEM). (B) Quantitative analysis of cells expressing S100 and GFAP at different initial cell density groups. *p<0.001 as compared to 2000 cells/cm2; **p<0.001 as compared to 4000 cells/cm2.

• 

  Fig. 4 Comparative analysis of P75NTR marker examined by real time RT-PCR at different initial cell density groups. *p<0.001 as compared to 2000 cells/cm2; **p<0.001 as compared to 4000 cells/cm2.

Reference

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