hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease

Abstract

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.

Keywords: GDF11; Hirschsprung's disease; axial patterning; axial progenitors; cell therapy; directed differentiation; enteric nervous system; nervous system disorder; neural crest development; pluripotent stem cells; regenerative medicine; sacral neural crest.

Figure 1. |. Derivation of sacral NC from hPSCs

(A) Diagram of the FGF and CHIR titration experiment. (B) qRT-PCR of neural crest genes at D20 under all conditions compared with LSB control, which generates neurectoderm. CNC, cranial neural crest. N= 3 biological replicates. (C) qRT-PCR of HOX genes that indicate regional identity corresponding to distinct axial levels at D20. HOXB4: vagal level; HOXC9: trunk level; HOXD13: sacral level. N= 4 biological replicates (D) qRT-PCR of HOX genes with or without GDF11 at D20. N= 3 biological replicates. (E) Immunocytochemistry of sacral NC at D20. Co-staining for SOX10 with posterior HOX proteins HOXC9 and HOXD13 shows most cells become sacral NC. Scale bars, 50 μm. (F) Flow cytometry of sacral NC for CD49D and p75NTR at D20. Left: representative plot. Right: quantitative data of CD49D+ cell percentage. N= 7 biological replicates. (G) Summary of protocols that generate NC cells at different axial levels. Data are present as Mean ± SEM; Statistical analysis was performed using the Unpaired t-test or ANOVA with Dunnett test.

Figure 2. |. GDF11-mediated expression of 5’ HOX genes via modulation of RA signaling

(A) qRT-PCR analysis showing the progressive expression of HOX genes from 3’ to 5’. N= 4 biological replicates. (B) Diagram of RNA seq and ATAC seq experiments. (C-D) PCA plots of the RNA and ATAC sequencing data respectively. (E) Heat map of HOXA genes, showing increased expression of HOXA genes over time and greater expression of posterior HOXA genes under GDF11 treatment. Presented as normalized counts scaled by row. (F) Expression of GRHL factors from RNA seq. N= 3 biological replicates. (G) Chromatin accessibility status of GRHL3 locus from ATAC seq. N= 3 biological replicates. (H) Expression of RA binding protein CRABP2. N= 3 biological replicates. (I) Schematic drawing of proposed mechanism where GDF11 promotes the generation of sacral NC by RA inhibition. (J) Expression of stem cell marker SOX2. N= 3 biological replicates. (K) Experimental design to test the RA hypothesis. (L-O) qRT-PCR data showing expression of various genes related to RA signaling and AP identity for the experiment depicted in (K), N= 3 biological replicates. (L) Expression of CRABP2, confirming the effect of RA and RA inhibitor AGN. (M-O) HOXB2 was promoted by RA and HOXC9 and HOXC10 was promoted by RA inhibition. RNA sequencing data presented as counts normalized using the Median of Ratios method (DESeq2). Data are presented as mean ± SEM; Statistical analysis was performed using the unpaired t-test or ANOVA with Dunnett test.

Figure 3. |. Sacral NC cells are derived from an NMP-like posterior precursors

(A-C) Analysis of D3 cells for expression of NMP key markers: SOX2, T and CDX2, by IF (A), qRT-PCR (B) and flow cytometry (C), showing most cells are triple-positive NMPs. N= 3 biological replicates. Scale bars, 50 μm. (D) Venn diagram of differentially expressed genes (|log2(FC)|>1) representing D3 cells of our trunk differentiation protocol (BMP D3), sacral differentiation (GDF D3) and D3 NMP cells from Frith et al., study (NMP-Trunk D3) . (E) GO analysis of GDF D3 cells. (F) Top 25 most up and down regulated genes from the common genes in the Venn diagram. (G) Experimental design depicting use of SOX2::TdTomato and T::GFP dual reporter hESC line to test if a pure NMP-like population can give rise to sacral NC. (H) Flow cytometry data of D3 cells using dual reporter line or H9 WT control (left). Purity of sorted NMPs is confirmed with immunostaining for SOX2 and T (right). Scale bars, 50 μm. (I) Flow cytometry data of D20 cells from unsorted and sorted NMPs (left). The sacral NC identity from pure NMPs is confirmed with immunostaining of SOX10 and HOXD13 (right). Scale bars, 50 μm. (J) Summary of anterior and posterior NC domains originating from different precursors. Data are presented as mean ± SEM; Statistical analysis was performed using the unpaired t-test.

Figure 4. |. Sacral NC can be directed to diverse enteric and non-enteric fates

(A) Diagram of protocols to specify sacral NC cells towards enteric neurons (ENS, upper), sympathetic neurons (SNS, middle) and melanocytes (MN, bottom). (B-D) Immunostaining of cells at D30 (B), D40 (C) and D80 (D), indicating cells transit from sacral precursors (B) to mature neurons that contains different subtypes (D). Scale bars, 50 μm. (E-G) Gene expression of NC marker SOX10, enteric NC precursor marker EDNRB and neuronal marker SOX2 over time. N= 3 biological replicates. (H) Neuron subtype composition within the culture at D80. N= 2 biological replicates. (I) Immunostaining of SNS at D80. Neurons form bundle-like structures composed of long neurites. Scale bars, 100 μm (left) and 50 μm (the rest). (J) Gene expression of sympathetic neuronal markers over time. N= 4 biological replicates. (K) Live imaging of MN at D60 using a SOX10::GFP reporter line under florescent microscope (left 3 panels) and BF microscope (right panel), showing continuous expression of SOX10 and pigmentation. Scale bars, 100 μm. (L) Expression of melanocyte markers: SOX10, hMITF, c-KIT and pigment-related genes TYPL1 PMEL over time. N= 3 biological replicates. Data are presented as mean ± SEM; Statistical analysis was performed using the ANOVA with Dunnett test.

Figure 5. |. Vagal and Sacral NC exhibits distinct behavior in vitro

(A) Schematic drawing of experimental design. RFP-tagged hPSC line was used for vagal lineages and GFP-tagged line for sacral lineages. (B) qRT-PCR of SOX10 and HOX genes to confirm the VNC and SNC identity. N= 6 biological replicates. (C) Invasion assay of VNC and SNC. Cells crossed the membrane are visualized (left) and quantified using plater reader (right). Scale bars, 50 μm. N= 3 biological replicates. (D) Migration assay of VNC and SNC on PO/LM/FN surface. Cells migrated out of spherical aggregate were imaged (left) and migration distance were quantified (right). Scale bars, 500 μm. N= 3 biological replicates. (E) 3D Matrigel embedded migration assay of co-cultured VNC and SNC. Fluorescence image of cells at 24 hours (upper) and 96 hours (lower), show self-sorting activity. Scale bars, 500 μm. (F) Co-cultured VNC (red) and SNC (green) cells undergoing ENS differentiation with replating at D10. Scale bars, 100 μm. (G) Heat map of cadherins in NC samples of different axial levels. Presented as normalized counts. (H) Representative traces of electrical activity in NC-derived neurons as recorded by MEA system in over a period of 1 second. (I) Spike raster gram showing 1 m of activity. (J-K) Quantification of mean firing rate and number of bursting electrodes. (L) Immunostaining of synaptic markers in enteric neurons derived from VNC and SNC. Data are presented as mean ± SEM; Statistical analysis was performed using the unpaired t-test.

Figure 6.. Vagal and Sacral NC exhibits distinct behavior in vivo

(A-B) Schematic drawing of mice transplantation experiments. NC cells are cultured under non-adherent conditions to form small spheres composed of either vagal, sacral, or combined vagal/sacral NC and injected into the mouse cecum. (C) Fluorescent images of mouse gut that were transplanted with different axial types of NC cells: VNC (left), SNC (middle), VNC+SNC (right). The images are taken at sequential time points after transplantation: 1 Hour (upper), 2 weeks (middle), 4 weeks (bottom). (D) Immunostaining of 9-month-old KO mouse that received VNC+SNC transplantation. RFP for VNC, indicated by white solid arrows and GFP for SNC, indicated by open arrows. Small intestine (upper); Cecum (middle), Distal colon (bottom). Scale bars, 100 μm.

Figure 7.. Developing a cell-based therapy for HSCR disease in Ednrb KO mouse model

(A) Staining with TUJ1 in distal colon of WT and Ednrb KO (HSCR) mice. Scale bars, 50 μm. (B) Comparison of life spans of Ednrb KO mice in different genetic backgrounds. N= 10 different mice. (C-D) Gut wall thickness in distal colon of 4-week-old WT and HSCR mice without any treatment. Scale bars, 100 μm. (E) Survival curve of NSG/Ednrb KO mice that received various transplantation paradigms. N=12 for KO grafted with VNC+SNC and N= 7 mice for all other groups. (F-G) Gut wall thickness of 9-month-old WT and HSCR mice with VNC+SNC transplantation. Scale bars, 100 μm. (H) Body weight of WT mice and HSCR mice with VNC+SNC transplantation post transplantation. N= 3 different mice. (I-J) Gastric emptying and SI transit measured by rhodamine dextran dye gavage. N= 3 different mice for WT. N= 2 different mice for Ednrb KO. (K-M) Representative spatiotemporal maps and quantification of video imaging of colonic migration motor complexes (CMMCs) in 9 months old WT mice and rescued HSCR mice. Completed CMMCs in rescued mice indicated restored ENS function. N= 3 different mice for WT. N= 2 different mice for Ednrb KO. (N-Q) Wholemount staining for neurons (N) and non-neurons (Q) of 9-month-old rescued HSCR mouse. RFP for VNC (solid arrows) and GFP for SNC (open arrows). Scale bars, 100 μm. (R) Schematic drawing to illustrate combined VNC and SNC transplantation rescues severe HSCR. Data are presented as mean ± SEM; Statistical analysis was performed using the unpaired t-test.