Different forms of tenascin-C with tenascin-R regulate neural differentiation in bone marrow-derived human mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) are currently thought to transdifferentiate into neural lineages under specific microenvironments. Studies have reported that the tenascin family members, tenascin-C (TnC) and tenascin-R (TnR), regulate differentiation and migration, in addition to neurite outgrowth and survival in numerous types of neurons and mesenchymal progenitor cells. However, the mechanisms by which TnC and TnR affect neuronal differentiation are not well understood. In this study, we hypothesized that different forms of tenascin might regulate the neural transdifferentiation of human bone marrow-derived mesenchymal stem cells. Human MSCs were cultured in media incorporated with soluble tenascins, or on precoated tenascins. In a qualitative polymerase chain reaction analysis, adding a soluble TnC and TnR mixture to the medium significantly enhanced the expression of neuronal and glial markers, whereas no synaptic markers were expressed. Conversely, in groups of cells treated with coated TnC, hMSCs showed neurite outgrowth and synaptic marker expression. After being treated with coated TnR, hMSCs exhibited neuronal differentiation; however, it inhibited neurite outgrowth and synaptic marker expression. A combination of TnC and TnR significantly promoted hMSC differentiation in neurons or oligodendrocytes, induced neurite formation, and inhibited differentiation into astrocytes. Furthermore, the effect of the tenascin mixture showed dose-dependent effects, and a mixture ratio of 1:1 to 1:2 (TnC:TnR) provided the most obvious differentiation of neurons and oligodendrocytes. In a functional blocking study, integrin α7 and α9β1-blocking antibodies inhibited, respectively, 80% and 20% of mRNA expression by hMSCs in the coated tenascin mixture. In summary, the coated combination of TnC and TnR appeared to regulate neural differentiation signaling through integrin α7 and α9β1 in bone marrow-derived hMSCs. Our findings demonstrate novel mechanisms by which tenascin regulates neural differentiation, and enable the use of cell therapy to treat neurodegenerative diseases.

FIG. 1.

Effects of soluble tenascin-C (TnC) and tenascin-R (TnR) in NBR-induced human mesenchymal stem cells (hMSCs). (A) The mRNA levels of neuron markers, MAP2 and NFM; (B) glial cell markers, GFAP and MBP; and (C) the synapse marker, synapsin (SYN) were quantified after 7 days of Tn treatment (0.5–1.0 μg/mL) in hMSCs that were pretreated with NBR for 1 week. Levels were normalized to levels observed in NBR-only treatment (set to 1.0) (presented as a dotted line). The NBR incorporated with the TnC and TnR mixture (TCR) promoted the expression of neuronal and glial cell genes, but not of synaptic genes. The data are presented as the mean±SD of an experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. NBR). DMEM, Dulbecco's modified Eagle medium; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; NFM, neurofilament M; SD, standard deviation.

FIG. 2.

Effects of soluble TnC and TnR on hMSCs without NBR induction. (A) The mRNA levels of neuronal markers, MAP2 and NFM; (B) glial cell markers, GFAP and MBP; and (C) the synapse marker, synapsin (SYN) were quantified after 7 days of Tn treatment (0.5–1.0 μg/mL) in hMSCs. Levels were normalized to levels observed in DMEM with 10% FBS-only treatment (set to 1.0) (presented as a dotted line). The TnC and TnR mixture (TCR) enhanced the expression of neuronal and glial cell genes, but not of synaptic genes. TnC and TnR individually inhibited neuronal and glial marker gene expressions. The data are presented as the mean±SD of one experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. DMEM and 1% FBS). DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum.

FIG. 3.

Effects of TnC and TnR Coating on NBR-induced hMSCs. (A) The mRNA levels of neuronal markers, MAP2 and NFM; (B) glial cell markers, GFAP and MBP; and (C) the synapse marker, synapsin (SYN) were quantified after 7 days of cultivation on surfaces coated with Tns (20 μg/mL) in hMSCs pretreated with NBR for 1 week. Levels were normalized to levels observed in NBR-only treatment (set to 1.0) (presented as a dotted line). NBR-treated hMSCs on TCR-coated dishes promoted neuronal and synaptic gene expression; however, it inhibited GFAP gene expression. Data are presented as the mean±SD of one experiment that was derived from three independent experiments. **p<0.01 (all vs. NBR). nd, not determined.

FIG. 4.

Immunofluorescence of TnC and TnR coating on NBR-induced hMSCs. NBR-stimulated hMSCs cultured on Tn-coated surfaces is shown as (A) MAP2 and NFM; (B) glial cell markers, GFAP and MBP; (C) the synaptic marker, synapsin (SYN) and the cytoskeletal marker, β-catenin. The immunoreaction was examined after 7 days of cultivation on Tn-coated surfaces (20 μg/mL) in hMSCs pretreated with NBR for 1 week. DAPI (blue) was used as a counterstain. The DMEM group served as a control. When cells were cultivated on TnC- and TCR-coated surfaces, hMSCs exhibited neurite outgrowth. The white bar represents 50 μm. (D) Percentages of MAP2-positive cells and NFM-positive cells within all DAPI-positive cells; (E) percentages of GFAP-positive cells and MBP-positive cells within all DAPI-positive cells; (F) percentages of SYN-positive cells and neurite-positive cells within all DAPI-positive cells. All data are presented as the mean±SD. *p<0.05, **p<0.01 (all vs. NBR). (G) The cell areas, calculated by the β-catenin-positive cells. DAPI, 4′, 6-diamidino-2-phenylindole.

FIG. 5.

Effects of TnC and TnR coating on hMSCs without NBR induction. (A) The mRNA levels of neuronal markers, MAP2 and NFM; (B) glial cell markers, GFAP and MBP; and (C) the synapse marker, synapsin (SYN) were quantified after 7 days of cultivation on Tn-coated surfaces (20 μg/mL) in hMSCs. Levels were normalized to levels observed in DMEM 10% FBS-only treatment (set to 1.0) (presented as a dotted line). The hMSCs on TCR-coated dishes promoted neuronal and synaptic gene expression; however, they inhibited GFAP gene expression. TnC and TnR individually inhibited neuronal and glial marker gene expression. The data are presented as the mean±SD of one experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. DMEM 1% FBS). (D) Summary of Tn coating on hMSCs. In neuronal differentiation, TnC triggers SYN expression with NBR treatment. TnR shows promotion for neuronal differentiation with or without NBR, but totally inhibits neurite formation. The combination of TnC and TnR not only promotes neuronal differentiation but also induces SYN expression and neurite outgrowth. Either TnC or TnR alone inhibits astrocyte or oligodendrocyte differentiation when incorporated with NBR. When TnC is combined with TnR, it significantly promotes oligodendrocyte differentiation, although it blocks astrocyte differentiation.

FIG. 6.

Effects of varied doses and ratios of TnC and TnR mixture (TCR) coating on NBR-induced hMSCs. (A) The mRNA levels of neuronal markers, MAP2 and NFM; (B) glial cell markers, GFAP and MBP; and (C) the synapse marker, synapsin (SYN), were quantified after 7 days of cultivation on TCR-coated surfaces (10–40 μg/mL) in hMSCs pretreated with NBR for 1 week. Levels were normalized to levels observed in NBR-only treatment (set to 1.0) (presented as a dotted line). NBR-treated hMSCs on TCR-coated dishes promoted neuronal and synaptic gene expressions, but inhibited GFAP gene expression in a dose-dependent manner. Data are presented as the mean±SD of one experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. NBR). (D) The mRNA levels of neuronal markers, MAP2 and NFM; (E) glial cell markers, GFAP and MBP; and (F) the synaptic marker, synapsin (SYN), were quantified after 7 days' cultivation on TCR-coated dishes (different ratios: 3:1, 2:1, 1:1, 1:2, and 1:3, total 20 μg/mL) in NBR-induced hMSCs. Levels were normalized to levels observed in NBR-only treatment (set to 1.0) (presented as a dotted line). Data are presented as the mean±SD of one experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. NBR). ^#p<0.05, ^##p<0.01 (all vs. TCR 1:1).

FIG. 7.

Effects of a TnC and TnR mixture (TCR) coating on NBR-induced hMSCs through the integrins, α7 and α9β1. The mRNA levels of integrins (A) were analyzed after 7 days of cultivation on TCR-coated surfaces (20 μg/mL) in hMSCs pretreated with NBR for 1 week. After adding integrin-blocking antibodies (5 μg/mL), mRNA levels of neuronal markers, (B) MAP2 and NFM; glial cell markers, (C) GFAP and MBP; and the synapse marker, (D) synapsin (SYN), were quantified after 7 days of cultivation on Tn-coated surfaces (20 μg/mL) in hMSCs pretreated with NBR for 1 week. Levels were normalized to levels observed in NBR only treatment (set to 1.0) (presented as a dotted line). Data are presented as the mean±SD of one experiment that was derived from three independent experiments. *p<0.05, **p<0.01 (all vs. NBR). ^#p<0.05, ^##p<0.01 (all vs. NBR+TCR). (E) Inhibition percentages were calculated from (B–D).