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. 2024 Mar 22;9(1):13.
doi: 10.1038/s41536-024-00352-4.

Intrinsic and extrinsic actions of human neural progenitors with SUFU inhibition promote tissue repair and functional recovery from severe spinal cord injury

Yong-Long Chen  1 , Xiang-Lan Feng  1 , Kin-Wai Tam  2 , Chao-Yang Fan  3 , May Pui-Lai Cheung  2 , Yong-Ting Yang  3 , Stanley Wong  1 , Daisy Kwok-Yan Shum  2 , Ying-Shing Chan  2 , Chi-Wai Cheung  1   4 , Martin Cheung  2 , Jessica Aijia Liu  5   6
Affiliations

Intrinsic and extrinsic actions of human neural progenitors with SUFU inhibition promote tissue repair and functional recovery from severe spinal cord injury

Yong-Long Chen et al. NPJ Regen Med. .

Abstract

Neural progenitor cells (NPCs) derived from human pluripotent stem cells(hPSCs) provide major cell sources for repairing damaged neural circuitry and enabling axonal regeneration after spinal cord injury (SCI). However, the injury niche and inadequate intrinsic factors in the adult spinal cord restrict the therapeutic potential of transplanted NPCs. The Sonic Hedgehog protein (Shh) has crucial roles in neurodevelopment by promoting the formation of motorneurons and oligodendrocytes as well as its recently described neuroprotective features in response to the injury, indicating its essential role in neural homeostasis and tissue repair. In this study, we demonstrate that elevated SHH signaling in hNPCs by inhibiting its negative regulator, SUFU, enhanced cell survival and promoted robust neuronal differentiation with extensive axonal outgrowth, counteracting the harmful effects of the injured niche. Importantly, SUFU inhibition in NPCs exert non-cell autonomous effects on promoting survival and neurogenesis of endogenous cells and modulating the microenvironment by reducing suppressive barriers around lesion sites. The combined beneficial effects of SUFU inhibition in hNPCs resulted in the effective reconstruction of neuronal connectivity with the host and corticospinal regeneration, significantly improving neurobehavioral recovery in recipient animals. These results demonstrate that SUFU inhibition confers hNPCs with potent therapeutic potential to overcome extrinsic and intrinsic barriers in transplantation treatments for SCI.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Molecular characterization of SUFU KD hNPCs.
a mRNA and protein expression levels of SUFU in hNPCs analyzed by qPCR and immunoblotting in scramble and SUFU KD treatment groups. b qPCR analysis of SHH and its effectors. One-way ANOVA followed by Tukey’s post-hoc test. c Representative bright-field(BF) images showing the formation and renewal of neurospheres in scramble and SUFU KD treatment groups. Quantification of the number (d) and size (e) of neurospheres from scramble and SUFU KD treatment groups, One-way ANOVA followed by Tukey’s post-hoc test. Scale bar = 100 μm. f Representative immunofluorescence images for OLIG2, SOX2, HuC/D, PAX6, and NKX6.1 in hNPCs from scramble and SUFU KD treatment groups. DAPI was used as a nuclei marker. Scale bar = 50 μm. g Quantification of (f). h qPCR analysis of dorsal-ventral markers (PAX6,OLIG2,PAX7), neural genes (SOX2/1, Nestin) and neuronal markers (MPA2,TUJ1,ISL1/2, and HB9) in hNPCs from scramble and SUFU KD treatment groups, One-way ANOVA followed by Tukey’s post-hoc test. All data are expressed as mean ± SEM. Three independent experiments.
Fig. 2
Fig. 2. hNPCs with SUFU inhibition exert cell intrinsic differentiation capacity in generating neurons and oligodendrocytes.
a Representative immunofluorescence of neurofilament(NF) from scramble and SUFU KDs neurospheres at 7 days differentiation in the absence of neurotrophic factors. Scale bar = 100 μm. b Quantification of relative intensity of axon outgrowth in scramble and SUFU KDs neurosphere. c Representative immunofluorescence images of MAP2, and ISL1/2 in scramble and SUFU KD hNPCs at 7 days differentiation. The white box shows a magnified view with indicated markers. Scale bar = 100 μm. DAPI was used as a nuclei marker. d Percentage of ISL1/2-positive cells in scramble and SUFU KDs hNPCs after 14 days differentiation. e Representative immunofluorescence images of ChAT, HB9, MAP2, and synaptophysin (SYN) in scramble and SUFU KDs hNPCs at 21 days differentiation. The white box shows a magnified view with indicated markers, Scale bar = 100 μm. DAPI was used as a nuclei marker. f Quantification of (e). g qPCR analysis of HH effectors (SUFU, GLI1, and PATCH1), neural genes (SOX2 and PAX6) and neuronal markers (MPA2,TUJ1,ISL1/2, ChAT, and HB9) at 21 days differentiation of scramble and SUFU KDs treatment groups. h Representative immunofluorescence images of GFAP, NG2 and SOX10 after 3 weeks differentiation in scramble and SUFU KDs hNPCs, Scale bar = 100 μm. i Quantification of (h). Student’s t test. All data are expressed as mean ± SEM. *p < 0.01, **p < 0.05, ***p < 0.005 versus scramble. Three independent experiments.
Fig. 3
Fig. 3. Non-cell autonomous effects of SUFU KD cells.
a Representative immunofluorescence images showing increased SHH production from SUFU KDs hNPCs compared to scramble hNPCs. DAPI was used as a nuclei marker, Scale bar = 100 μm b Quantification of (a). c Representative immunofluorescence images of caspase 3, neuronal marker MAP2 and nuclei marker DAPI in Scramble and SUFU KD hNPCs after 14 days treatment of homogenate (100 μg/ml) from the injured spinal cord, Scale bar = 100 μm. d Quantification of caspase 3+ cells from (c). Student’s t test. e Schematic diagram showing co-culture of scramble and SUFU KD hNPCs with and without GFP. f Representative immunofluorescence images of caspase 3, HuC/D and GFP in co-cultures of Scramble+scramble(GFP), Scramble+SUFU KD1(GFP), and scramble+SUFU KD2(GFP), Scale bar = 100 μm. The white box shows a magnified view with indicated markers. g Percentage of caspase 3+ cells in GFP-positive or GFP-negative cells in co-cultures of scramble+scramble(GFP), Scramble+SUFU KD1(GFP), and scramble+SUFU KD2(GFP). One-way ANOVA followed by Tukey’s post-hoc test. h Percentage of HuC/D in total GFP cells in co-cultures of scramble+scramble(GFP), Scramble+SUFU KD1(GFP), and scramble+SUFU KD2(GFP). Student’s t test. All data are expressed as mean ± SEM. *p < 0.01, **p < 0.05, ***p < 0. 005. Three independent experiments.
Fig. 4
Fig. 4. SUFU KD grafts display efficient integration and modulate injured niche in the SCI model.
a Representative images of caspase 3 positive cells in the injured spinal cord without grafting or with scramble and SUFU KD1 grafts at 1 month(1 M) post-graft, Scale bar = 200 µm. b Quantification of the caspase 3 positive cells from (a). One-way ANOVA. *p < 0.05, **p < 0.01. c Representative immunofluorescence images of GFP, MAP2 (red), and GFAP (Blue) in sagittal sections with scramble and SUFU KD1 grafts at 1 month(1 M) post-graft. The cystic lesion cavity (LC) formed with surrounding dense GFAP immunoreactivity (blue), whereas SUFU KD1 grafts (GFP-positive) crossed GFAP barriers. The white box shows a magnified view with indicated markers in lower panels. Scale bar = 200 µm. The dotted line indicates the dense astrocytic glia marked by GFAP. d Representative immunofluorescence images of GFP, CSPG (red), and DAPI (Blue) in spinal cord sagittal sections grafted with scramble and SUFU KD1 hNPCs at 1 month post-graft (1 M). The cystic lesion cavity (LC) formed with surrounding dense CSPG immunoreactivity (red). SUFU KD1 grafts with GFP expression attenuated CSPG graft/host interface. Scale bar = 200 µm. e Fluorescence intensity analysis of CSPG surrounding the lesion cavity (n = 4 rats per group). *p < 0.05. f GFP and Neurofilament(NF) immunolabeling in spinal cord sagittal sections revealed GFP-expressing SUFU KD1 grafts at injured sites generated robust axons extending into the host spinal cord caudally after 2 months post-graft(2 M). insets: a-a” and b-b” indicate higher magnification of NF-positive fibers in the graft at different regions from rostral to caudal. Scale bar = 100 µm. g Quantification of axon intercepts at specific distances from graft-host border in the injured cord grafted with scramble and SUFU KD1 hNPCs. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni. All data are expressed as mean ± SEM.
Fig. 5
Fig. 5. SUFU KD hNPC grafts promote beneficial differentiation and regneration intrinsically and extrinsically in the SCI model.
a Representative immunofluorescence images of GFP, GlyT2(red), and ISLET1/2(blue) in sagittal sections of injured spinal cord with scramble and SUFU KD1 grafts at 2 month(1 M) post-graft. The empty arrow shows the indicated markers expression in GFP- positive cells. The white arrow shows the indicated markers in GFP negative cells. The white box shows a zoomed-in view of the co-localization of indicated markers. Scale bar = 100 µm. b Quantification of the percentage of GlyT2 and ISLET1/2 in grafts or non-grafts cells from(a). c Representative immunofluorescence images of GFP, GABA(red) and CaMKII (purple) in sagittal sections with scramble and SUFU KD1 grafts at 2 month(1 M) post-graft. The empty arrow shows the indicated markers expression in GFP positive cells. The white arrow shows the indicated markers in GFP negative cells. The white box shows a zoomed-in view of the co-localization of indicated markers. Scale bar = 50 µm. n = 4–5 rats per group. *p < 0.05, **p < 0.01, ***p < 0.005. Quantification of the percentage of CaMKII (d) and GABA (e) in grafts or non-grafts cells from(c). n = 5–6 rats per group. *p < 0.05, **p < 0.01, ***p < 0.005. (f) Representative immunofluorescence images of GFP, SOX10(red) and HNFEL(blue) in sagittal sections with scramble and SUFU KD1 grafts at 2 month(1 M) post-graft. The white box shows a zoomed-in view of the co-localization of indicated markers. Scale bar = 50 µm. (g) Quantification of the percentage of SOX10 in GFP-positive or -negative cells in the injured cord grafted with scramble and SUFU KD1 hNPCs. n = 5–6 rats per group, 4–5 sections/rats. Scale bar = 50 µm, **p < 0.01. (h) GFP-positive grafts from scramble and SUFU KD1 immunolabeled with HNEFL and myelination marker (MBP, red) at 3 months post-graft. Insets show the distribution of GFP-positive grafts in the injury site. Scale bar = 50 µm. All data are presented as mean ± SEM.
Fig. 6
Fig. 6. Graft-initiated trans-synaptic AAV virus antegrade labeling of host connectivity.
a Sagittal section showing antegrade, trans-synaptically traced host mCherry-expressing cells in the injured spinal cord with Scramble and SUFU KD1 graft. Scale bar=500 μm. Inset, image showing injection sites in the brain region. Inset (a’-a’’’ and b’-b’’’), high-magnification view of boxed area. b Representative immunofluorescence images showing mCherry+;GFP+ trans-synaptically connection from motor cortex to the grafts in the lesion sites. Scale bar=50 μm. c Quantification of the proportion of mCherry-labeled cells/axons in scramble of SUFU KD1 grafts, normalized to the total number of mCherry-labeled axons located 0.5 mm rostral to the lesion site. n = 7 Scramble recipients and n = 6 SUFU KD1 recipients. One-way ANOVA with Tukey’s multiple comparisons; **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with Scramble grafts. d Transverse sections labeled for mCherry and GFP at L2 host spinal cord levels, showing that host neurons monosynaptically connected to grafts were detected over long lengths of the rat spinal cord. Scale bars, 250 μm. left panel showing synaptically connected mCherry+ host neurons included GABA sensory interneurons and ChAT+ motor neurons at the lumbar level. Scale bars=50 μm. The white arrowheads indicate mCherry traced motoneurons(ChAT+) in SUFU KD1 grafts. e Quantification of mCherry traced neurons in L1-L2 in scramble and SUFU KD1 grafts. n = 7 Scramble recipients and n = 6 SUFU KD1 recipients. ***p < 0.001. f Triple labeling for GFP, 5-HT, and human synapsin(hSYN) revealed colocalization of regenerating 5-HT axon terminals with hSYN, suggesting synaptic connectivity. Scale bar=25μm. All data are presented as mean ± SEM.
Fig. 7
Fig. 7. Significant functional improvement after transplantation of SUFUKD1 hNPC grafts into contusive SCI.
a BBB scores of lesion control, and pre-and post-grafting with scramble and SUFU KD1 hNPCs. Two-way repeated-measures ANOVA followed by post-hoc Fisher’s exact test. *p < 0.05, **p < 0.01 SUFU KD1 versus scramble; #p < 0.05, ##p < 0.01 scramble versus lesion control. b Grid walk quantitative analysis measured as a percentage of hind limb placement. One-way ANOVA with Tukey’s post-hoc test; *p < 0.05, **p < 0.01. c Foot fault score analysis of hind limb measured by rating scale for foot placement in the skilled ladder rung walking test (correct placement = 6 points; partial placement = 5 points; placement correction = 4 points; replacement = 3 points; slight slip = 2 points; deep slip = 1 point; and total miss = 0 points). One-way ANOVA with Tukey’s post-hoc test; *p < 0.05, **p < 0.01. d Quantification of stride length in sham, SCI(lesion control) and SCI rats with scramble and SUFU KD1 grafts. Student’s t test. *p < 0.05, **p < 0.01. All data are expressed as mean ± SEM. n = 7(sScramble); n = 5(lesion Control); n = 9(SUFU KD1); n = 6 (Sham).
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