Extracellular matrix-regulated neural differentiation of human multipotent marrow progenitor cells enhances functional recovery after spinal cord injury

Abstract

Background context: Recent advanced studies have demonstrated that cytokines and extracellular matrix (ECM) could trigger various types of neural differentiation. However, the efficacy of differentiation and in vivo transplantation has not yet thoroughly been investigated.

Purpose: To highlight the current understanding of the effects of ECM on neural differentiation of human bone marrow-derived multipotent progenitor cells (MPCs), regarding state-of-art cure for the animal with acute spinal cord injury (SCI), and explore future treatments aimed at neural repair.

Study design: A selective overview of the literature pertaining to the neural differentiation of the MSCs and experimental animals aimed at improved repair of SCI.

Methods: Extracellular matrix proteins, tenascin-cytotactin (TN-C), tenascin-restrictin (TN-R), and chondroitin sulfate (CS), with the cytokines, nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF)/retinoic acid (RA) (NBR), were incorporated to induce transdifferentiation of human MPCs. Cells were treated with NBR for 7 days, and then TN-C, TN-R, or CS was added for 2 days. The medium was changed every 2 days. Twenty-four animals were randomly assigned to four groups with six animals in each group: one experimental and three controls. Animals received two (bilateral) injections of vehicle, MPCs, NBR-induced MPCs, or NBR/TN-C-induced MPCs into the lesion sites after SCI. Functional assessment was measured using the Basso, Beattie, and Bresnahan locomotor rating score. Data were analyzed using analysis of variance followed by Student-Newman-Keuls (SNK) post hoc tests.

Results: Results showed that MPCs with the transdifferentiation of human MPCs to neurons were associated with increased messenger-RNA (mRNA) expression of neuronal markers including nestin, microtubule-associated protein (MAP) 2, glial fibrillary acidic protein, βIII tubulin, and NGF. Greater amounts of neuronal morphology appeared in cultures incorporated with TN-C and TN-R than those with CS. The addition of TN-C enhanced mRNA expressions of MAP2, βIII tubulin, and NGF, whereas TN-R did not significantly change. Conversely, CS exposure decreased MAP2, βIII tubulin, and NGF expressions. The TN-C-treated MSCs significantly and functionally repaired SCI-induced rats at Day 42. Present results indicate that ECM components, such as tenascins and CS in addition to cytokines, may play functional roles in regulating neurogenesis by human MPCs.

Conclusions: These findings suggest that the combined use of TN-C, NBR, and human MPCs offers a new feasible method for nerve repair.

Keywords: Chondroitin sulfate; Extracellular matrix; Human multipotent progenitor cells; Neurogenesis; Tenascin-cytotactin; Tenascin-restrictin.

Fig. 1

Multipotent progenitor cell membrane marker identification. HSC, hematopoietic stem cell.

Fig. 2

Expressions of the neural stem/progenitor marker, nestin, the immature neuronal marker, βIII tubulin, and the mature neuronal marker, MAP2. Immunocytochemical detection of neural proteins in multipotent progenitor cells (MPCs). Undifferentiated bone marrow-derived MPCs appeared as large, flat, fibroblast-like cells. (A, F) Four days’ expansion of MPCs seeded at a density of 1 ×10⁵ showed no immunoreactivity of the neuron markers, nestin (green), βIII tubulin (red), or MAP2 (red). (B, G) Within 7 days after adding nerve growth factor (NGF; 20 ng/mL), brain-derived neurotropic factor (BDNF; 10 ng/mL), and retinoic acid (RA; 2 μM), MPCs differentiated in the presence of NGF, BDNF, and RA showed positive staining for the neuronal proteins, nestin (green), βIII tubulin (red), and MAP2 (red). (C, H) After 6 days of incorporation with CS-proteoglycan, MPCs exhibited decreased levels of nestin, MAP2, and βIII tubulin. (D, I, E, J) When TN-C or TN-R was added, cells showed an increase of immunocytochemistry with antibodies to late-stage neuronal markers, βIII tubulin and MAP2, but not nestin. However, TN-C produced more significant expression than TN-R did. Nuclei were stained by DAPI (blue) in all panels. (K) A reverse transcriptase polymerase chain reaction (RT-PCR) for nestin, βIII tubulin, and MAP2 showed similar expressions. (L) For quantification and analysis of variance statistical analysis (n=3, error bars represents standard error of the mean) of the RT-PCR results, nestin, βIII tubulin, and MAP2 were calculated as a ratio to GAPDH. MAP2, microtubule-associated protein 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMEM, Dulbecco modified Eagle medium; NBR, NGF/BDNF/RA; CS, chondroitin sulphate; TN-C, tenascin cytotactin; TN-R, tenascin restrictin; mRNA, messenger-RNA.

Fig. 3

Expression of the neuroglial marker, GFAP, the immature neuronal marker, βIII tubulin, and the mature neuronal marker, MAP2. Immunocytochemical detection of neural proteins in multipotent progenitor cells (MPCs). Undifferentiated bone marrow derived-MPCs appeared as large, flat, fibroblast-like cells. (A, F) Four days’ expansion of MPCs seeded at a density of 1 ×10⁵ showed no immunoreactivity of the neuron markers, GFAP (green), MAP2 (red), or βIII tubulin (red). (B,G) Within 7 days after adding nerve growth factor (NGF; 20 ng/mL), brain-derived neurotropic factor (BDNF; 10 ng/mL), and retinoic acid (RA; 2 μM), MPCs differentiated in the presence of BDNF, NGF, and RA and showed increased levels of the neural proteins, nestin (green), GFAP (green), MAP2 (red), and βIII tubulin (red). (C, H) After 6 days of incorporation with CS-proteoglycan, MPCs exhibited decreased levels of GFAP, MAP2, and βIII tubulin. After adding TN-C or TN-R, cells showed an increase of immunocytochemistry with antibodies to the late-stage neuronal markers, MAP2 and βIII tubulin. (D, E, I, J) However, the neuroglial marker, GFAP, was downregulated. Nuclei were stained by DAPI (blue) in all panels. (K) A reverse transcriptase polymerase chain reaction (RT-PCR) for GFAP, βIII tubulin, and MAP2 showed similar expression levels. (L) For quantification and analysis of variance statistical analysis (n=3, error bars represents standard error of the mean) of the RT-PCR results, GFAP, βIII tubulin, and MAP2 were calculated as a ratio to GAPDH. DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMEM, Dulbecco modified Eagle medium; NBR, NGF/BDNF/RA; CS, chondroitin sulphate; TN-C, tenascin cytotactin; TN-R, tenascin restrictin; mRNA, messenger-RNA.

Fig. 4

The BBB score of rats with spinal cord injury (SCI) receiving different treatments. A repeated-measures analysis of variance, followed by SNK post hoc tests revealed that SCI rats (Group IV) receiving implantation of multipotent progenitor cells incubated with nerve growth factor/brain-derived neurotropic factor/retinoic acid and tenascin cytotactin showed significantly improved functional recovery measured by the BBB score compared with SCI rats receiving vehicle treatment (Group I) 14, 21, 28, 35, and 42 days after SCI (p<.05 for Day 14 and p<.01 for Days 21, 28, 35, and 42). Respective animal numbers for Groups I, II, III, and IV were 6, 5, 6, and 6. *p<.05; **p<.01 compared with SCI rats with vehicle treatment in Group I. BBB, Basso, Beattie, and Bresnahan. SNK, Student-Newman-Keuls.

Fig. 5

Histologic evaluation and quantitative analysis of spinal tissue in lesions of rats with spinal cord injury (SCI). The representative pictures show the spinal lesions of (A–C) SCI rat receiving vehicle treatment (Group I), (D–F) SCI rats receiving treatment of multipotent progenitor cells (MPCs) (Group II), (G–I) SCI rat receiving implantation of MPCs incubated with nerve growth factor/brain-derived neurotrophic factor/retinoic acid (NBR) (Group III), and (J–L) SCI rats receiving treatment of MPCs incubated with NBR/tenascin cytotactin (TN-C) (Group IV) at the similar section level. The Group I animals had a large lesion with minimal tissue repair without evidence of stem cell. Macrophage/hemosiderin infiltration was apparent. Group II and III spinal lesions showed a similar moderate lesion. The MPCs in Group III showed more proliferative spinal tissue and differentiated progenitor cells (arrows) compared with Group II. A small lesion with well-differentiated progenitor cells (arrows) and proliferative spinal tissue were found in Group IV. All four groups contained apparent macrophage/hemosiderin infiltration in the repair area. The remaining spinal tissue in the lesion center of SCI rats in four groups is shown in (M). One-way analysis of variance followed by SNK post hoc tests revealed that SCI rats in Group IV receiving treatment of MPCs incubated with NBR/TN-C had more preserved spinal tissue in the lesion center than SCI rats receiving vehicle treatment (Group I). The animal numbers for Groups I, II, III, and IV were 6, 5, 5, and 6, respectively. NS, p>.05; *, p<.05 when compared with SCI rats with vehicle treatment in Group I. The scale bar is (A, D, G, J) 500 μm, (B, E, H, K) 100 μm, (C, F, I, L) 25 μm. SNK, Student-Newman-Keuls. NS, No Significance.