MLL1 knock-down increases the expression of neural markers in SCAPs

To confirm the identity of the SCAPs, flow cytometry analysis was conducted to detect specific markers. The results indicate positive expression of the surface markers CD90 (99.2%), CD105 (92.8%), and CD146 (88.2%) in SCAPs (Supplementary Fig. 1A–C), while CD34 (0.44%) and CD45 (0.041%) exhibit negative expression in SCAPs (Supplementary Fig. 1D, E). To knock down MLL1 in SCAPs, lentivirus transfection was employed, and the knock-down efficiency was detected by Western blot (Fig. 1a). During neuron induction, we observed morphological changes in SCAPs, indicating their differentiation into neuron-like cells. Specifically, we monitored the dynamic changes of neurosphere at 3, 6, and 9 days and compared these parameters between the SCAP/MLL1sh and the SCAP/Scramsh groups. Our results demonstrated that the SCAP/MLL1sh group exhibited a significant increase in neurosphere volume compared to the SCAP/Scramsh group (Fig. 1b, c). Moreover, we evaluated the expression of neural markers using real-time RT-PCR and found that the expressions of NeuroD and NCAM were higher at 3, 6, and 9 days, and TH expression was higher at 6 and 9 days after induction in the SCAP/MLL1sh group than those in the SCAP/Scramsh group (Fig. 1d–f). Additionally, the immunofluorescence staining and quantification analysis revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the SCAP/MLL1sh group compared to the SCAP/Scramsh group (Fig. 1g–j).

Fig. 1

MLL1 knock-down enhanced the expression of neural markers in SCAPs. a The MLL1 knock-down efficiency was tested by Western blot. Histone H3 served as an internal control; b, c MLL1 knock-down increased the neurosphere volume compared to the SCAP/Scramsh group. Scale bar, 100 μm; Real-time RT-PCR analysis revealed that MLL1 knock-down increased the expression of NeuroD (d), NCAM (e), and TH (f) in SCAPs. GAPDH served as an internal control; g–j The immunofluorescence staining revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the SCAP/MLL1sh group compared to the SCAP/Scramsh group. Scale bar, 100 μm. Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01

Knock-down of MLL1 in SCAPs can promote functional recovery in a rat SCI model

We investigated whether the knock-down of MLL1 in SCAPs could result in better functional recovery in SCI model. To evaluate this possibility, we assessed the motor behavioral deficits of SCI rats using the Basso-Beattie-Bresnahan (BBB) scale at 1, 2, 3, 4, and 5 weeks after cell transplantation (Fig. 2a). Our findings indicate that the rats exhibited significant motor behavioral deficits post-SCI. In the 2nd and 3rd weeks following cell transplantation, there was an improvement in motor function in the SCAP/MLL1sh group compared to the SCI group, and this difference was statistically significant. There was no significant difference observed when compared to the SCAP/Scramsh group. However, in the 4th and 5th weeks, both the SCAP/Scramsh group and the SCAP/MLL1sh group exhibited differences in motor function when compared to the SCI group. Notably, the SCAP/MLL1sh group demonstrated significantly superior motor function recovery compared to the SCAP/Scramsh group, with statistically significant differences observed (Fig. 2b). These subsequent observations suggested a potential improvement in spinal cord function with the transplantation of MLL1 knock-downed SCAPs. Compared with those of the SCI and SCAP/Scramsh groups, the spinal cord morphology in the SCAP/MLL1sh group showed better recovery (Fig. 2c). To assess the effect of SCAP transplantation on SCI-related pathological changes, HE staining was performed to observe the overall damage in spinal cord tissue. Compared to the Sham group, the spinal cord in the SCI group exhibited severe structural damage, characterized by the formation of spinal cord cavities, pathological bleeding, necrosis, and scar formation. On the other hand, The SCAP/Scramsh group showed a reduction in cavities and continuity at the injury site of spinal cord. Furthermore, in comparison to both the SCI and SCAP/Scramsh groups, the SCAP/MLL1sh group exhibited more vacuolar cells and fewer cavities and scars (Fig. 3a). The neural progenitor cells in spinal cord defects was evaluated using immunohistochemical staining of Nestin. A small amount of Nestin expression was observed in the SCI group, with significant difference noted between the SCI and Sham groups. The SCAP/Scramsh group exhibited a higher expression of Nestin than the SCI group, yet it remained significantly lower than that observed in the SCAP/MLL1sh group (Fig. 3b, c). To confirm the presence of implanted SCAPs, a red fluorescent signal indicating human mitochondria-positive cells in the injured spinal cord area was visualized using fluorescence microscopy after SCAP/Scramsh and SCAP/MLL1sh group implantation at 5 weeks (Fig. 3d). Meanwhile, due to the specific fluorescence labeling of transplanted cells with human mitochondria, either the Sham group or the SCI group didn’t exhibit any positive cells (Fig. 3d). The quantification analysis results show that there is no statistically significant difference in the number of human mitochondria-positive cells between the SCAP/Scramsh group and the SCAP/MLL1sh group (Fig. 3e). The expression of NEFM may serve as reliable indicators of SCI repair. Furthermore, immunofluorescence staining was conducted to investigate the changes in NEFM expression. The results revealed a significant decrease in NEFM expression in the SCI group compared to the Sham group. Conversely, the SCAP/Scramsh group exhibited pronounced NEFM expression, which differed significantly from the SCI group. And the NEFM expression significantly increased in the SCAP/MLL1sh group compared to the SCI and the SCAP/Scramsh groups, suggesting that MLL1 knock-down in SCAPs may inhibit axonal degeneration (Fig. 3f, g). Overall, our findings suggest that the transplantation of MLL1 knock-downed SCAPs can accelerate the repair of injured spinal cord tissue and promote motor functional recovery in a rat SCI model

Fig. 2

MLL1 knock-downed SCAPs promoted functional recovery after SCI in rat model. a Spinal cord function was observed at 1, 2, 3, 4, and 5 weeks using the BBB scale to assess the limb motor function of rats. b The BBB scale showed that the SCAP/MLL1sh group gradually exhibited better spinal functional recovery at 4 to 5 weeks compared with that in the SCAP/Scramsh group. c Compared with those in the SCI and SCAP/Scramsh groups, the spinal cord morphology of the SCAP/MLL1sh group recovered well. Statistical significance was determined using one-way ANOVA, or the Kruskal–Wallis test. All error bars represent SD. (n = 6). *P ≤ 0.05. **P ≤ 0.01

Fig. 3

Histopathological changes in the SCI model after SCAP transplantation. a HE staining results revealed that the SCAP/MLL1sh group had more vacuolar cells and fewer cavities and scars than the SCI and SCAP/Scramsh groups. Scale bar, 100 μm and 500 μm. b, c The immunohistochemical results indicated that the SCAP/MLL1sh group exhibited significantly higher levels of Nestin expression compared to the SCAP/Scramsh group. Scale bar, 100 μm. d, e The immunofluorescence results indicated that h-mitochondria-positive cells were in the injury area in SCAP/Scramsh and SCAP/MLL1sh groups at 5 weeks after transplantation. Scale bar, 50 μm. f, g Immunofluorescence staining was used to observe the NEFM expression. Scale bar, 50 μm. Statistical significance was determined using one-way ANOVA, or the Kruskal–Wallis test. All error bars represent SD. (n = 6). **P ≤ 0.01

WDR5 interacted with MLL1, and inhibited the expression of neural markers in SCAPs

To identify potential MLL1-binding proteins in SCAPs, we performed a Co-IP assay. Our findings revealed that knock-down of MLL1 in SCAPs led to a reduction in the binding of MLL1 and WDR5 (Fig. 4a). To further validate this observation, WDR5 knock-downed SCAPs were constructed through lentivirus transfection, and the knock-down efficiency was confirmed by Western blot analysis (Fig. 4b). Subsequently, we investigated the effects of WDR5 knock-down on the binding of WDR5 and MLL1 in SCAPs. Our results demonstrated that knock-down of WDR5 led to a decrease in the binding of MLL1 and WDR5, which was consistent with the observations in MLL1 knock-downed SCAPs (Fig. 4b). Next, the Myc-WDR5 sequence was inserted into the retroviral vector, which was used to infect SCAPs. WDR5 over-expression was tested by Western blot (Fig. 4c). Furthermore, WDR5 over-expression significantly increased the binding of MLL1 and WDR5 in SCAPs (Fig. 4c). These findings provide further evidence for the role of WDR5 as a binding partner of MLL1 in SCAPs.

Fig. 4

MLL1 binding with WDR5 in SCAPs. a MLL1 knock-down reduced the binding of MLL1 and WDR5 in SCAPs; b WDR5 knock-down reduced the binding of MLL1 and WDR5 in SCAPs; c WDR5 over-expression significantly increased the binding of MLL1 and WDR5 in SCAPs. Histone H3 served as an internal control. IgG was used as the negative control

Subsequently, WDR5 was knocked down in SCAPs for functional assays. Morphological changes were observed in neuron-like cells during neuron induction. Notably, the SCAP/WDR5sh group exhibited a significant increase in the neurosphere volume compared to the SCAP/Scramsh group (Fig. 5a, b). Real-time RT-PCR analysis revealed that neural markers such as NeuroD, NCAM, and TH were up-regulated in SCAPs following WDR5 knock-down compared to the SCAP/Scramsh group (Fig. 5c–e). Additionally, the immunofluorescence staining and quantification analysis revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the SCAP/WDR5sh group compared to the SCAP/Scramsh group (Fig. 5f–i).

Fig. 5

WDR5 knock-down enhanced the expression of neural markers in SCAPs. a, b WDR5 knock-down increased the neurosphere volume compared to the SCAP/Scramsh group. Real-time RT-PCR analysis revealed that WDR5 knock-down increased the expression of NeuroD (c), NCAM (d), and TH (e) in SCAPs. GAPDH served as an internal control. f–i The immunofluorescence staining revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the SCAP/WDR5sh group compared to the SCAP/Scramsh group. Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01

We further investigate the role of WDR5 in regulating the neurogenic potential of SCAPs via over-expression of WDR5. The dynamic changes in neuron-like cells were monitored for 3, 6, and 9 days, revealing that the WDR5 over-expressed group exhibited a significant decrease in neurosphere volume compared to the SCAP/Vector group (Fig. 6a, b). After 9 days of neuron induction, real-time RT-PCR analysis suggested that the expression levels of NeuroD at 3, and 6 days, NCAM at 3, 6, and 9 days, and TH at 6, and 9 days in the WDR5 over-expressed group were significantly decreased compared to those in the SCAP/Vector group (Fig. 6c, e). Consistent with these findings, immunofluorescence staining and quantification analysis revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly decreased in the WDR5 over-expressed group compared to the SCAP/Vector group (Fig. 6f, i). Together, these results provide further evidence supporting the inhibitory effects of WDR5 over-expression on neurogenesis and suggest a potential therapeutic strategy for promoting neurogenesis by modulating WDR5 levels.

Fig. 6

WDR5 overexpression inhibited the expression of neural markers in SCAPs. a, b WDR5 over-expression decreased the neurosphere volume compared to the Vector group. Real-time RT-PCR analysis revealed that WDR5 over-expression decreased the expression of NeuroD (c), NCAM (d), and TH (e) in SCAPs. GAPDH served as an internal control. f–i The immunofluorescence staining revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly decreased in the WDR5 over-expressed SCAPs compared to the SCAP/Vector group. Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01

MLL1 and WDR5 directly regulate HES1 transcription through H3K4me3 methylation

To further investigate the co-regulatory role of MLL1 and WDR5 in regulating the neurogenic potential of SCAPs, we performed microarray analysis to identify potential target genes associated with this process. Our findings revealed 35 differentially expressed genes in the WDR5 over-expressed group compared to the Vector group, with 9 genes up-regulated and 26 genes down-regulated in SCAPs over-expressing WDR5 (Supplementary Table 2). The up-regulated genes included CYP1B1, PRSS3, and NPTX1, while the down-regulated genes included HES1, NR4A2, EGR1, IL-6, FOS, ATF3, ID4, and GDF15. To further validate these findings, we performed real-time RT-PCR to detect the expression levels of differentially expressed genes after over-expression of WDR5. Our results showed that, compared to the Vector group, the expression of neuro-related genes such as HES1, NR4A2, EGR1, IL-6, FOS, ATF3, ID4, and GDF15 in the WDR5 over-expressed SCAP were significantly reduced (Fig. 7a–h). Further investigation of the co-regulatory role of MLL1 and WDR5 in the regulation of gene expression was performed. Our results indicated that the knock-down of WDR5 or MLL1 led to the up-regulation of HES1, NR4A2, and IL-6 expression in SCAPs (Fig. 7i–k). Our experimental results also demonstrate that knocking down of MLL1 in SCAPs results in a reduction in H3K4me3 level (Supplementary Fig. 2). Furthermore, we confirmed that MLL1 depletion decreased the level of H3K4me3 in the HES1, NR4A2, and IL-6 promoter regions (Fig. 7L). The enrichment of H3K4me3 in the HES1, NR4A2, and IL-6 promoter regions was also significantly reduced in the SCAP/WDR5sh group compared to the SCAP/Scramsh group (Fig. 7m).

Fig. 7

Knocking down MLL1 or WDR5 promotes the expression of HES1, NR4A2, and IL-6 through H3K4me3 methylation. Real-time RT-PCR analysis revealed that the expression of neural-related genes in the SCAP/Myc-WDR5 group was significantly reduced, including HES1 (a), NR4A2 (b), EGR1 (c), IL-6 (d), FOS (e), ATF3 (f), ID4 (g), and GDF15 (h). GAPDH served as an internal control. i–k Real-time RT-PCR analysis revealed that the knock-down of WDR5 or MLL1 up-regulated HES1, NR4A2, and IL-6 expression in SCAPs. GAPDH served as an internal control. l–m The ChIP assay results revealed that depletion of MLL1 or WDR5 decreased the enrichment of H3K4me3 in the HES1, NR4A2, and IL-6 promoters. Statistical significance was determined using one-way ANOVA, or the Kruskal–Wallis test. All error bars represent SD. (n = 3). *P < 0.05. **P ≤ 0.01

To demonstrate the regulatory effect of the MLL1-WDR5 protein complex on downstream target genes such as HES1, IL-6, and NR4A2, we performed ChIP assays. Our results indicated that WDR5 knock-down led to a decrease binding of MLL1 into the HES1 and IL-6 promoters compared to that in the SCAP/Scramsh group (Fig. 8a, b). However, binding of MLL1 was not enriched in the NR4A2 promoter region (Fig. 8c). To further confirm the regulatory role of MLL1 and WDR5 on HES1, IL-6, and NR4A2, we conducted ChIP assays in the WDR5 over-expressed SCAPs. Our findings revealed that compared to the SCAP/Vector group, WDR5 over-expression increased MLL1 enrichment in the HES1 and IL-6 promoter regions (Fig. 8d, e), while the MLL1 enrichment in NR4A2 promoter region was not affected (Fig. 8f). Together, these results provide further evidence supporting the co-regulatory role of MLL1 and WDR5 in the regulation of HES1 and IL-6.

Fig. 8

MLL1 directly regulated the transcription of HES1 and IL-6 by binding with WDR5. The ChIP assay results showed that WDR5 knock-down reduced the binding of MLL1 to the HES1 (a) and IL-6 promoters (b), except for the NR4A2 promoter region (c); The ChIP assay results showed that WDR5 over-expression increased the binding of MLL1 to the HES1 (d) and IL-6 promoters (e), except for the NR4A2 promoter region (f). Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01

HES1 promoted the expression of neural markers in SCAPs

HES1 was knocked down in SCAPs for functional assays, and the knock-down efficiency was detected by Western blot (Fig. 9a). The results indicate that, compared to the SCAP/Consh group, the expression of HES1 was significantly reduced in the SCAP/HES1sh2 group. Then the SCAP/HES1sh2 group exhibited a significant reduction in neurosphere volume compared to the SCAP/Consh group (Fig. 9b, c). Real-time RT-PCR analysis revealed down-regulation of neural markers such as NeuroD, TH, and βIII-tubulin after HES1 knock-down in SCAPs compared to the SCAP/Consh group (Fig. 9d–f). Consistent with these findings, immunofluorescence staining and quantification analysis revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly decreased in the SCAP/HES1sh2 group compared to the SCAP/Consh group (Fig. 9g–j).

Fig. 9

HES1 knock-down reduced the expression of neural markers in SCAPs. a The HES1 knock-down efficiency was tested by Western blot. β-actin served as an internal control; b, c HES1 knock-down reduced the neurosphere volume compared to the SCAP/Consh group; Real-time RT-PCR analysis revealed that HES1 knock-down reduced the expression of NeuroD (d), TH (e), and βIII-tubulin (f) in SCAPs. GAPDH served as an internal control. g–j The immunofluorescence staining revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly decreased in the SCAP/HES1sh group compared to the SCAP/Consh group. Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01

To further evaluate the function of HES1, the Flag-HES1 sequence was inserted into the retroviral vector, which was used to infect SCAPs. Western blot analysis was performed to confirm the HES1 over-expression efficiency (Fig. 10a). We monitored the dynamic changes in neuron-like cells for 3, 6, and 9 days, revealing that the HES1 over-expressed SCAPs exhibited a significant increase in neurosphere volume compared to the Vector group (Fig. 10b, c). Real-time RT-PCR analysis indicated that the expression levels of NeuroD, TH, and βIII-tubulin were significantly higher in the SCAP/Flag-HES1 group than those in the SCAP/Vector group after induction (Fig. 10d–f). Furthermore, immunofluorescence staining and quantification analysis revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the HES1 over-expressed SCAPs compared to the SCAP/Vector group (Fig. 10g–j).

Fig. 10

HES1 over-expression increased the expression of neural markers in SCAPs. a The HES1 over-expression efficiency was tested by Western blot. β-Actin served as an internal control; b, c HES1 over-expression increased the neurosphere volume compared to the SCAP/Vector group; d–f Real-time RT-PCR analysis revealed that HES1 over-expression increased the expression of NeuroD (d), TH (e), and βIII-tubulin (f) in SCAPs. GAPDH served as an internal control. g–j The immunofluorescence staining revealed that the numbers of Nestin-positive and βIII-tubulin-positive neurospheres were significantly increased in the HES1 over-expressed SCAPs compared to the SCAP/Vector group. Statistical significance was determined using Student’s t test. All error bars represent SD. (n = 3). *P ≤ 0.05. **P ≤ 0.01