Coculture with Embryonic Stem Cells Improves Neural Differentiation

of Adipose Tissue-Derived Stem CellsArash Javeri MD, PhD, Masoumeh Fakhr Taha PhD, Leila Bahmani MSc

Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Introduction: Adipose tissue has been identified as an accessible and rich source of stem cells with multipotential differentiation capacity. So far, several research groups have

reported neural differentiation of adipose tissue-derived stem cells (ADSCs ) in low-serum or serum-free media with a cocktail of neural inducing factors. In the present study,

we evaluated the effectiveness of a medium containing a synthetic serum replacement (KoSR) for neural differentiation of mouse ADSCs, and compared this medium with low-

serum condition. Moreover, we evaluated neural differentiation of the ADSCs following indirect coculture with ES cells.

Materials and Methods: ADSCs from the inguinal adipose tissue of 8 to10-week old NMRI

mice were isolated using 2 mg/ml collagenase A and were characterized (Fig. 1). At first,

neural differentiation of the ADSCs was induced under two different culture conditions,

DMEM plus 4% FBS and DMEM plus 15% KoSR, with or without β-ME. Then, third-

passaged ADSCs were indirectly cocultured with ES cells, and the expression levels of

pluripotency markers, mesenchymal stem cell markers, and proliferating cell nuclear antigen

were assessed in the cocultured ADSCs. Moreover, the control and cocultured ADSCs were

differentiated to neuron with or without RA treatment.

Conclusion: Our findings are indicating that mouse ADSCs are capable of neural

development in KoSR-containing media. Moreover, coculture with the ES cells efficiently

improves neural differentiation of the ADSCs. Probably, secretion of cytokines,

chemokines, interleukins and some growth factors by ES cells have a positive effect on the

late maturation of ADSC-derived neurons. Non-contact coculture with the ES cells may be

used as an efficient strategy to improve differentiation potential of adult stem cells for

developmental studies and regenerative medicine.

Results: The current study showed that KoSR-containing medium without any additional

factor induces neural differentiation of the ADSCs. Two-week differentiated ADSCs

expressed several neuron-specific markers, and RA treatment improved neural differentiation

of the ADSCs (Fig. 2). The expression levels of OCT4, Sox2 and PCNA were upregulated in

the cocultured ADSCs (Fig. 3). Moreover, coculture with the ES cells significantly improved

neural differentiation of the ADSCs. Treatment of the cocultured ADSCs with RA diminished

the expression of neural maturation markers (Figs. 4 and 5).

Figure 1- Characterization of the ADSCs by flow cytometry analysis. ADSCs expressed CD29, CD44, and CD105 as the mesenchymal stem cell markers, while they were mostly negative for CD31 (endothelial marker), CD45 and CD11b (hematopoietic markers).

Figure 2- The expression of some neuron-specific genes in the freshly isolated stromal

vascular fraction (SVF), third-passaged ADSCs (ADSC P3), brain tissue as the positive

control, and two-week differentiated ADSCs which were treated with different concentrations

of RA.

Figure 3- The effect of two-day coculture with the ES cells on the expression of PCNA,

Sox2, OCT4, CD73 and CD105 mRNAs in the control and cocultured ADSCs (A) . (B) and

(C) QPCR analysis of PCNA, CD73, CD105, Sox2 and OCT4 mRNAs expression in the

control and cocultured ADSCs.

Figure 4- RT-PCR and qPCR

analysis of some neuron-specific

genes in the control and cocultured

ADSCs which were differentiated

at the presence or absence of 10-8

M RA, 2 weeks after neural

induction.

Figure 5- Immunocytochemical and western blot analysis of two-week differentiated

ADSCs. The control and cocultured ADSCs were differentiated in the KoSR-containing

medium at the presence or absence of 10-8 M RA. Scale bar: 25 µm.

Reference: Bahmani L, Taha MF, Javeri A. Neural differentiation of adipose tissue-

derived stem cells is improved following coculture with embryonic stem cells.

Neuroscience 2014; 272: 229-239.