Genetically engineered BMSCs promote dopamine secretion and ameliorate motor dysfunction in a Parkinson's disease rat model

Abstract

Regenerative therapy based on mesenchymal stem cells (MSCs) is regarded as a promising strategy for treating Parkinson's disease (PD). Previous studies have shown that mesenchymal stem cell transplantation has the potential to treat Parkinson's disease, but its specific mechanism of action is still unclear. In the present study, we generate genetically engineered bone marrow mesenchymal stem cells (BMSCs) encoding three critical genes (TH, DDC, and GCH1) for dopamine synthesis (DA-BMSCs). The DA-BMSCs maintain their MSCs characteristics and stable ability to secrete dopamine after passage. Moreover, the DA-BMSCs survived and functioned in a rat model of PD treated with 6-OHDA 8 weeks after transplantation. Histological studies showed that DA-BMSCs could differentiate into various functional neurons and astrocytes, and DA-BMSCs derived mature dopaminergic neurons extended dense neurites into the host striatum. Importantly, DA-BMSCs promoted the reconstruction of midbrain dopamine pathways by upregulating striatal dopamine and 5-HT levels and downregulating the levels of inflammatory factors including IL-6, TNF-α, and IL-10. These findings suggest that engineered mesenchymal stem cell transplantation for dopamine synthesis may be an attractive donor material for treating Parkinson's disease.

Keywords: Bone marrow mesenchymal stem cells; Dopamine; Dopamine decarboxylase; GTP cyclohydrolase 1; Parkinson’s disease; Tyrosine hydroxylase.

Fig. 1

Cell morphology, colony-forming potentials, specific markers, and karyotypes of rat BMSCs in vitro. (A) The timeline for the construction of a cell line of DA-BMSCs capable of stable dopamine production. (B) BMSCs morphology and colony forming efficiency of BMSCs at P4 stained with crystal violet. (C) Immunofluorescent staining of specific markers of BMSCs. Nearly all the cells expressed MSCs markers CD29, CD44, CD73, CD90, and stem cells markers OCT4, SOX2 (red), and the cell nucleus was shown in blue by DAPI, but did not express CD34 and CD45 (red). (D) The expression of specific markers CD29, CD44, CD73, CD90, CD45, and CD34 in BMSCs by Flow cytometry analysis. (E) Expression of specific markers genes CD29, CD44, CD73, CD90, CD45, CD34, S100A1 and COL1A1, and epithelial cell markers CDH1, MUC1 were detected by RT-PCR. The original gels are presented in Supplementary Fig. 2 (Fig. S2). (F) Chromosome G‑banding analysis of rat BMSCs at passage 4. Scale bars represent 100 μm.

Fig. 2

Construction of lentiviral expression vectors and the establishment of a cell line of DA-BMSCs. (A) Dopamine biosynthetic processes involvement in DA-ergic presynaptic and postsynaptic neurons. Dopamine receptor subtypes (D1–D5); DAT dopamine transporter. (B) Schematic diagram of lentiviral vectors of LV-TH and LV-DDC-GCH1. (C) Fluorescent protein expression of BMSCs 72 h after lentivirus infection of LV-TH and LV-DDC-GCH1. LV-mCherry and LV-ZsGreen represented the control lentivirus. (D) The infection efficiency of the above lentivirus (n = 6). (E) Morphological characteristics of DA-BMSCs (P1–P4) and the expression of fluorescent proteins after puromycin and Blasticidin S selection. (F) Western blotting of TH, DDC, and GCH1 proteins in different lentivirus-infected cells. (G) RT-PCR analysis of TH, DDC, and GCH1 genes in different lentivirus-infected cells. (H) The relative mRNA expression of TH, DDC, and GCH1 was analyzed by RT-qPCR. (I) DA concentrations in the cells culture supernatant of each group were detected by HPLC. (J) HPLC chromatogram of cells culture supernatant was presented. Scale bars represent 100 μm. All data were presented as mean ± SEM. The P-values are from multiple comparisons in one-way ANOVA with Dunnett’s multiple comparison test (n = 3) ***P < 0.001, ****P < 0.0001. The original blots and gels are presented in Supplementary Fig. 2 (Fig. S2).

Fig. 3

DA-BMSCs significantly ameliorated the behavioral deficits of a rat model of Parkinson’s disease. (A) A flow chart depicting the experimental process containing animal groups and the number of animals in each group. (B) 6-Hydroxydopamine and DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of each model rat at the two coordinates labeled on the coronal and sagittal planes. (C) The PD rats were immobilized on the stereotaxic apparatus, and the cells were injected into the right MFB of the brain. (D) Rota-rod test on the Rota-rod apparatus. (E) APO-induced rotation test. (F) Heat maps of motion trajectory for the open field test. F1: the model group, F2: the BMSCs group, F3: the DA-BMSCs group, and F4: the control group. (G) Behavioral evaluation of the rats in the Rota-rod test. (H) Behavioral evaluation of the rats in the APO-induced rotation test. (I) Behavioral evaluation of the rats in the open field test. All data were presented as mean ± SEM. The p-values are from multiple comparisons in two-way ANOVA with Sidak’s multiple comparative tests (n = 6), *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. no significance: P > 0.05.

Fig. 4

Effects of DA-BMSCs transplantation on the loss of dopaminergic neurons in the right substantia nigra of PD rat models. (A) Representative images of whole-brain with hematoxylin and eosin staining in each group 8 weeks after transplantation. (B) Representative images of whole-brain with TH‑3,3ʹ‑diaminobenzidine staining (TH-DAB) in each group 8 weeks after transplantation. The right image was the higher magnification of the left boxed areas. (C) Number of TH⁺ cells in the right substantia nigra of each group. All data were presented as mean ± SEM. The P-values are from multiple comparisons in one-way ANOVA with Dunnett’s multiple comparison test (n = 4), ****P < 0.0001, n.s. no significance: P > 0.05. (D) DA-BMSCs formed a distinct graft area and migrated from the graft site into the periphery of the injured area, and the migrated distance gradually increased with time. (E) The transplanted DA-BMSCs could differentiate into neural cells in the host brain and exhibit neuronal morphology (white arrows) and dendritic structure (red arrow heads) after 8 weeks of transplantation.

Fig. 5

Survival, differentiation, migration, and integration of transplanted cells in the right hemisphere of PD model rats. (A–D) The transplanted DA-BMSCs survived and differentiated into various functional neurons in vivo 8 weeks after transplantation. The TH (A), GABA (B), GFAP (C), and PSD (D)-positive cells were detected in the graft region. (E) Transplanted DA-BMSCs expressed the marker of mature synapse synaptophysin (SYN) 8 weeks after transplantation. The neuron-like cells derived from the transplanted DA-BMSCs indicated by white arrows formed densely interacting dendritic networks.

Fig. 6

The levels of IL-6, TNF-α and IL-10 in the midbrain, and the levels of DA and 5-HT in the midbrain and serum, and the expression levels of IBA-1, C3 and DRD1 in the midbrain. (A) The expression of inflammatory factors including IL-6, TNF-α, and IL-10, were effectively inhibited in the absence or presence lentivirus-infected of BMSCs, which played an important role in the process of injury repair. (B) The expression levels of IBA-1 and C3 were significantly reduced in the DA-BMSCs group compared to the model group. (C, D) A significant reduction of DA and 5-HT levels in the midbrain of the injured side and the serum was confirmed, and the effect could be significantly restored to normal levels by DA-BMSCs. (E) An HPLC chromatogram of serum was presented. (F) The DA-BMSCs group exhibited significant upregulation of DRD1 expression compared to the model group. All data were presented as mean ± SEM. The P-values are from multiple comparisons in one-way ANOVA with Dunnett’s multiple comparison test (n = 3), *P < 0.05, ****P < 0.0001, n.s. no significance: P > 0.05.