Induction of Neural Crest Stem Cells From Bardet-Biedl Syndrome Patient Derived hiPSCs

Abstract

Neural crest cells arise in the embryo from the neural plate border and migrate throughout the body, giving rise to many different tissue types such as bones and cartilage of the face, smooth muscles, neurons, and melanocytes. While studied extensively in animal models, neural crest development and disease have been poorly described in humans due to the challenges in accessing embryonic tissues. In recent years, patient-derived human induced pluripotent stem cells (hiPSCs) have become easier to generate, and several streamlined protocols have enabled robust differentiation of hiPSCs to the neural crest lineage. Thus, a unique opportunity is offered for modeling neurocristopathies using patient specific stem cell lines. In this work, we make use of hiPSCs derived from patients affected by the Bardet-Biedl Syndrome (BBS) ciliopathy. BBS patients often exhibit subclinical craniofacial dysmorphisms that are likely to be associated with the neural crest-derived facial skeleton. We focus on hiPSCs carrying variants in the BBS10 gene, which encodes a protein forming part of a chaperonin-like complex associated with the cilium. Here, we establish a pipeline for profiling hiPSCs during differentiation toward the neural crest stem cell fate. This can be used to characterize the differentiation properties of the neural crest-like cells. Two different BBS10 mutant lines showed a reduction in expression of the characteristic neural crest gene expression profile. Further analysis of both BBS10 mutant lines highlighted the inability of these mutant lines to differentiate toward a neural crest fate, which was also characterized by a decreased WNT and BMP response. Altogether, our study suggests a requirement for wild-type BBS10 in human neural crest development. In the long term, approaches such as the one we describe will allow direct comparison of disease-specific cell lines. This will provide valuable insights into the relationships between genetic background and heterogeneity in cellular models. The possibility of integrating laboratory data with clinical phenotypes will move us toward precision medicine approaches.

Keywords: BBS; Bardet–Biedl Syndrome; hiPSCs; human induced pluripotent stem cells; neural crest.

FIGURE 1

BBS10 variant hiPSCs are pluripotent and can form cilia. (A) Schematic representation of the primary cilium. AX, axoneme; BB, basal body; IFT, intraflagellar transport. BBS1-20 proteins are depicted in their associated complex (BBSome and chaperonin) or by localization to other structures (scheme adapted from Suspitsin and Imyanitov, 2016). (B) Schematics showing BBS10 transcript and protein structure (723AA). Regions exhibiting homology with chaperonin domains are shown in the wildtype protein (scheme adapted from Álvarez-Satta et al., 2017). Variants from two BBS10 mutant lines XIRY (blue) and LAIG (red) are mapped onto protein domains with comparison to control (QOLG or KEGD). (C–E) Cells were immunostained for the pluripotency marker OCT3/4. Control (QOLG) cells and BBS10 mutants (XIRY and LAIG) all had positive staining in the nucleus. Merge with DNA dye Hoechst (C’–E’). All cells exhibit staining although there is some variability with less intense staining being seen in the central regions of the colonies. (F–H) Staining for the ciliary axoneme marker, ARL13B was performed. Control (KEGD) cells show positive staining for ARL13B (F). XIRY cells and LAIG cells (H) both express cilia. Merge with DNA dye Hoechst (F’–H’). Cilia frequency was quantitated by manual counting (I). LAIG cells had a moderate increase in percentage of cells with cilia compared to control cells. XIRY cells showed no significant difference although were more variable. P-values were determined using unpaired Student’s t-tests (^*P ≤ 0.05). Scale bars (E’,H’) = 50 μm.

FIGURE 2

Neural crest induction in vivo and in human induced pluripotent stem cells. (A) In vivo neural crest induction occurs at the border (green) between the neural plate (blue) and the non-neural ectoderm (light pink). As neurulation progresses the neural folds begin to approximate and neural crest delaminate from the epithelium and begin migration. (B) In vitro neural crest induction in hiPSCs following the protocol from Leung et al., 2016. hiPSCs are plated on Matrigel and treated for 48 h in neural crest induction (NCI) media plus Rock inhibitor (RI). This is followed by incubation in neural crest induction media without RI until 120 h of culture. At the start, pluripotency markers (OCT3/4, NANOG, and CMYC) are expressed. By 2 days of culture, neural border markers are expressed (PAX3, PAX7, TFAP2A, ZIC1). By 5 days of culture, cells express markers of neural crest identity (FOXD3, SNAIL2, SOX10).

FIGURE 3

Neural crest induction is delayed in BBS10 mutant cells. Phase contrast images of taken during neural crest induction at specified time points. Control QOLG (A–E), mutant XIRY (F–J), and LAIG (K–O) cells. Discrete iPSC-like colonies can be seen at 24 h in all rows (B,G,L). In control cultures, cells are seen delaminating from colonies at 48 h (blue arrowhead, C); whereas mutant cultures start showing delamination at 96 h (blue arrowheads, I,N). By 120 h there are still some dense, colony-like regions present in all cultures (yellow arrowheads, E,J,O). Scale bars = 50 μm.

FIGURE 4

Delaminated neural crest cells have a mesenchymal morphology. Phase contrast images of differentiating hiPSCs cultures taken during neural crest induction at specified time points. Control QOLG (A–C), mutant XIRY (D–F). Note that control QOLG cells at 48 h (A) exhibit delaminated cells with mesenchymal morphology, in contrast to XIRY cells which do not show cell delamination until 96 h (F). Scale bars = 200 μm.

FIGURE 5

BBS10 mutant cells express lower levels of SOX10 and P75NTR after neural crest induction. Immunostaining for the neural crest markers SOX10 (A–C) and P75NTR (A’–C’). Control QOLG (A) cells had high levels of SOX10 and P75NTR. BBS10 mutant cells [XIRY (B) and LAIG (C)] had significantly lower levels of both markers. Merge with DNA dye Hoechst (A”–C”). No positive staining was observed in the no primary control for SOX10 or P75NTR (D–D”). Scale bars = 50 μm.

FIGURE 6

Dynamics of pluripotency, neural plate border and neural crest marker expression during neural crest induction. Neural crest induction was carried out on control QOLG (blue), mutant XIRY (purple), and LAIG (orange) hiPSC lines. Cells were harvested for RNA before plating (iPSC, time = 0 h), at 48 and 120 h of differentiation. RT-qPCR analysis was carried out for pluripotency markers [CMYC (A), NANOG (B), and OCT3/4 (C)], neural border identity [PAX3 (D), TFAP2A (E), and PAX7 (F)] and subsequently markers of neural crest identity [FOXD3 (G), SNAIL2 (H), SOX10 (I)]. P-values were determined using unpaired Student’s t-tests (^*P ≤ 0.05, ^∗∗P ≤ 0.001, ^∗∗∗P ≤ 0.0001).

FIGURE 7

BBS10 mutant line XIRY is skewed toward the mesodermal fate and has diminished WNT and BMP responsiveness. Neural crest induction was carried out on control QOLG (blue), mutant XIRY (purple), and LAIG (orange) hiPSC lines. Cells were harvested for RNA before plating (iPSC, time = 0 h), at 48 and 120 h of differentiation. (A–D) RT-qPCR analysis for a mesodermal marker, BRY (A), a neural marker, SOX2 (B), neural border/pre-placodal marker, ZIC1 (C) and pre-placodal marker SIX1 (D). Note the increased levels of BRY (A) and decreased levels of ZIC1 (C) and SIX1 (D) in XIRY. (E–F) RT-qPCR analysis for AXIN2 a transcriptional target of WNT signaling (E), MSX1, a transcriptional target of BMP signaling (F) and PTC1, a transcriptional target of Hedgehog signaling (G). (E) Note transient increase in AXIN2 expression in XIRY from 0 to 48 h, but a decrease by 120 h. (F) Note a failure to activate MSX1 expression in XIRY and LAIG compared to control QOLG. (G) Note variability of PTC1 expression in XIRY at 0 h and both mutants at 48 h. P-values were determined using unpaired Student’s t-tests (^*P ≤ 0.05, ^∗∗P ≤ 0.001, ^∗∗∗P ≤ 0.0001).