Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions

Abstract

Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti β1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.

FIG. 1.

Effect of extracellular matrix (ECM) molecules on cell proliferation. Tissue culture plastic (TCPS) was precoated with fibronectin or laminin. Proliferation of differentiated adipose-derived stem cells (dASC) was quantified using the Alamar blue reduction assay and compared with control cultures grown on uncoated TCPS. No significant difference in proliferation between groups was recorded over the 6-day period, when cells reached saturation. Data represent n=3 different cell preparations and are expressed as mean±SEM.

FIG. 2.

Influence of ECM molecules on cell viability. Calcein-AM fluorescein complex, labeling viable cells (green staining), and ethidium homodimer-1 (Eth-D1), labeling dead cells (red staining) were used to assess cell viability and survival. Left column: dASC after 48 h of serum-free medium inducing apoptosis. Right column: dASC after 2 h of treatment by phosphate-buffered saline (PBS) inducing cell detachment and anoikis. Representative images of dASC seeded on ECM molecules-coated surfaces showing a better survival and superior surface adhesion when compared with uncoated cell adherent plastic (TCPS). Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 3.

Quantitative analysis of cell viability. Quantitative analysis of live/dead images was performed from the microscopic captured fields. (A) After serum deprivation for 48 h, a significant increase in viability was observed when cells were seeded on fibronectin and laminin when compared with uncoated cell adherent plastic. (B) After exposure to PBS for 2 h, dASC seeded on fibronectin and laminin showed a significantly better adhesion when compared with uncoated cell adherent plastic. (C) Cell viability of attached cells under PBS treatment confirmed the effect of ECM molecules on survival. n=5 different cell preparations, values shown are mean±SEM. Significance is referred to the TCPS group: *p<0.05 and **p<0.01.

FIG. 4.

The neurotrophic effect of ECM-treated dASC on dorsal root ganglia (DRG) neurite outgrowth: soluble molecules. (A) Immunocytochemistry for βIII tubulin of neurite outgrowth from adult rat neurons cocultured with dASC in a trans-well model. dASC were seeded into inserts (uncoated or coated with either laminin or fibronectin) allowing the passage of soluble factors but not the contact between neurons and stem cells. Top left panel shows baseline DRG neurite outgrowth in the presence of an insert containing stem cell differentiation medium only. (B) The average neurite length was significantly increased when dASC were seeded on ECM-coated inserts. (C) Both dASC seeded on uncoated and ECM-coated inserts showed a significant increase in the percentage of neurons bearing neurites, when compared with controls. n=4 different cell preparations. Values shown are expressed as mean±SEM, and significance is referred to the respective controls (uncoated or ECM molecule-coated inserts alone). *p<0.05, **p<0.01 and ***p<0.001, ns: no significant difference between dASC and Schwann cells (SC) cultured on laminin. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

The neurotrophic effect of ECM-treated dASC on DRG neurite outgrowth: direct contact effects. Immunocytochemistry for βIII tubulin (green) of neurite outgrowth from adult rat sensory neurons cocultured in direct contact with dASC. DRG were seeded on uncoated or ECM-coated coverslips in the absence (left column) or presence of dASC (right column). DAPI staining (blue) shows monolayer of dASC. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

The neurotrophic effect of ECM-treated dASC on DRG and NG108-15 neurite outgrowth: analysis of direct contact effects. (A) DRG neurons in contact with laminin-conditioned dASC showed the greatest average neurite length when compared with all the other dASC groups, but this was significantly less than that evoked by primary SCs cultured on laminin. (B) The percentage of neurons bearing neurites was significantly increased when DRG were cocultured with dASC if compared with DRG alone. There were no significant differences between the effects of stem cells seeded on uncoated or ECM-coated coverslips. To confirm a direct effect of ECM on neurotrophic activity we repeated the coculture model using NG108-15 neurons. (C) NG108-15 neurons in contact with laminin conditioned dASC significantly increased average neurite length not only when compared with respective controls but also when compared with NG in contact with dASC seeded on TCPS. (D) The percentage of neurons expressing neurites was significantly increased when NG108-15 were cocultured with dASC if compared with NG108-15 alone. No significant differences were seen between the effects of stem cells seeded on uncoated or ECM molecule-coated TCPS. n=4 different cell preparations, values shown are expressed as mean±SEM. Significance is generally referred to the respective control group (uncoated or ECM molecule-coated) shown by connecting lines *p<0.05, **p<0.01, and ***p<0.001, ns: not significantly different.

FIG. 7.

β1 integrin-mediated DRG neurite outgrowth. The effect of function-blocking antibodies against β1 integrin was compared with the untreated neurons and to the neurite outgrowth observed in presence of an isotype-matched controlled antibody. (A) The β1 blocking antibody significantly reduced neurite outgrowth when DRG were seeded on laminin or when in contact with dASC; the blocking antibody did not affect the neurite length when DRG were seeded on laminin preconditioned dASC. (B) The percentage of neurons expressing neurites was significantly reduced when DRG were seeded alone on laminin or when in contact with simple dASC in the presence of blocking antibody. The antibody also reduces percentage neurite expressing neurons in the presence of laminin-conditioned dASC but this effect was not significant when compared with the isotype control. n=4 different cell preparations. Values shown are expressed as mean±SEM, and significance is referred to both control groups if not otherwise specified. *p<0.05, and ***p<0.001.

FIG. 8.

The dASC release neurotrophic factors. After 24 h of dASC culture alone or in direct coculture with DRG neurons on uncoated or ECM molecule-coated surfaces, the medium from the wells was collected and analyzed by ELISA for detection of neurotrophic factors (A) nerve growth factor (NGF), and (B) brain-derived neurotrophic factor (BDNF). The presence of dASC in the coculture significantly increased the concentration of both neurotrophic factors in the medium when compared with controls (DRG without dASC). No significant differences (ns) were observed between dASC plated on uncoated surfaces or either of the ECM molecules. n=4 different cell preparations. Values shown are expressed as mean±SEM, and significance is referred to respective control groups. **p<0.01 and ***p<0.001.