Abstract
Neural crest cells (NCCs) are a multipotent and migratory cell population in the developing embryo that contribute to the formation of a wide range of tissues. Defects in the development, differentiation and migration of NCCs give rise to a class of syndromes and diseases that are known as neurocristopathies. NCC development has historically been studied in a variety of animal models, including xenopus, chick and mouse. In the recent years, there have been efforts to study NCC development and disease in human specific models, with protocols being established to derive NCCs from human pluripotent stem cells (hPSCs), and to further differentiate these NCCs to neural, mesenchymal and other lineages. These in vitro differentiation platforms are a valuable tool to gain a better understanding of the molecular mechanisms involved in human neural crest development. The use of induced pluripotent stem cells (iPSCs) derived from patients afflicted with neurocristopathies has also enabled the study of defective human NCC development using these in vitro platforms. Here, we review the various in vitro strategies that have been used to derive NCCs from hPSCs and to specify NCCs into cranial, trunk, and vagal subpopulations and their derivatives. We will also discuss the potential applications of these human specific NCC platforms, including the use of iPSCs for disease modeling and the potential of NCCs for future regenerative applications.
Introduction
Neural crest cells (NCCs) are transient, migratory stem cells that originate from the neural tube and migrate to different embryonic tissues to give rise to a wide variety of cell types (). They form ectodermal derivatives, such as sensory and enteric neurons, Schwann cells, as well as mesenchymal derivatives (). Thus, NCCs have been widely studied in animal models to elucidate their role in a range of neurocristopathies involving the craniofacial skeleton and the peripheral nervous system. The advent of techniques for the derivation of human neural crest cells (hNCCs) from human pluripotent stem cells (hPSCs), has not only enabled the understanding of NCC development and disease in a human-specific context but also opened new opportunities for therapeutic applications. This review will highlight the state-of-the-art protocols used to derive hNCCs from hPSCs and discuss opportunities and challenges in the applications of hNCCs in disease modeling and tissue regeneration (Figure 1).
FIGURE 1
Derivation of Human NCCs and Its Derivatives From hPSCs
During embryonic development, NCCs are specified at the neural plate border (NPB) region before they undergo an epithelial-mesenchymal transition and migrate out of the neural tube (Figure 1A). It has been shown that NCC induction at the NPB relies on BMP, WNT, Notch/Delta, and FGF signaling emanating from the surrounding embryonic tissues (). Based on these molecular developmental programs, researchers have developed increasingly specific and efficient protocols to derive an NCC population from hPSCs in vitro as highlighted below.
Directed NCC Induction Using Small Molecules
Since NCCs are specified adjacent to neural plate cells in vivo, early strategies sought to derive a mixed neural precursor cell (NPC) population and enrich the NCC subpopulation. NPC induction methods include stromal cell co-culture (; ; ), neurosphere culture (; ) or defined monolayer induction of neural rosettes using dual-SMAD inhibition of BMP and Activin A/Nodal signaling (). Limited p75+/HNK-1+ NCCs could be purified from the periphery of these neural rosettes (). These NPC-based protocols were highly inefficient and variable because NCCs represented a small subset of NPCs, and groups used different combinations of NCC markers such as p75, HNK-1, and SOX10 to isolate NCCs (). Also, the role of the stromal cells/cell aggregates/neural rosettes in inducing NCC formation was unclear, limiting their utility in the study of the molecular mechanisms of NCC development.
To overcome these limitations, researchers sought to achieve directed and specific NCC induction in adherent culture using a defined system. NCC development in vivo requires the activation of canonical WNT signaling and well as intermediate levels of BMP signaling, after an initial inhibition to establish neural plate identity (). Mimicking this, researchers used combinations of small molecule activators of WNT signaling and inhibitors of BMP and Activin/Nodal signaling to achieve directed NCC differentiation from both hESCs and iPSCs (, ; ). While Mica et al. found a short pulse of BMP inhibition to be essential for NCC specification, Menendez et al. found it unnecessary in their hPSC culture system. This controversy on the requirement of BMP inhibition for NCC specification was addressed by Hackland et al., who demonstrated that the levels of endogenous BMP signaling differ across various hPSC cell lines and in vitro culture systems. They could achieve the precise intermediate BMP level required for robust NCC derivation by a top-down inhibition of BMP signaling in a completely defined culture system (). The directed induction approach is the most commonly used method to derive NCCs from hPSCs, and can generally achieve a high derivation efficiency, ranging from 40 to 90%. However, different groups employ different markers (e.g., SOX10+ or p75+/HNK1+) to identify NCCs and may not be isolating identical NCC populations.
Transcription Factor-Based Reprogramming
Neural crest cells have also been directly induced from fibroblasts by reprogramming with a single transcription factor SOX10, in the presence of environmental cues including a WNT activator (). This system could be used to derive induced NCCs (iNCCs) directly from human patient fibroblasts. Direct reprogramming from fibroblasts allows for the generation of patient-specific NCCs as well as potentially reducing the length of in vitro culture before the iNCCs can be administered clinically. Another method to induce NCCs from fibroblasts involves the use of a chitosan substrate to effect the gene transfer of FOXD3, leading to increased NCC marker expression and the ability to rescue impaired neural function in a zebrafish model (). While this system provides a non-viral alternative for reprogramming to iNCCs, the exact nature of the induced cells was not fully defined due to incomplete characterization and lack of in vitro differentiation studies. Further work is required to reprogram iNCCs using non-viral methods to enable their use in clinical applications.
Regional Specification of Derived NCCs
Neural crest cells arise from four distinct regions of the anterior-posterior axis of the neural tube: cranial, cardiac, trunk, and vagal (). These NCC sub-populations express specific markers and have distinct differentiation potentials (Figure 1A). The dual-SMAD inhibition/WNT activation protocols tend to derive a HOX-negative NCC population that is disposed toward anterior (cranial) over posterior (vagal) identity (). While early inhibition of BMP signaling is necessary to establish NCC identity, late supplementation of BMP-4 during NCC derivation can enhance cranial identity, as indicated by the upregulation of cranial-specific DLX genes (). These cranial NCCs also expressed pharyngeal mesenchymal genes and showed osteogenic and chondrogenic differentiation potential in vitro. This can be advantageous as a defined culture system to generate mesenchymal derivatives from hNCCs as compared to the use of undefined serum to drive NCCs into mesenchymal lineages.
Retinoic acid (RA) and FGF-2 are known to be caudalizing factors during neural development (), and thus were utilized to posteriorize hPSC-derived NCCs as well. Vagal NCCs could be specified by the addition of RA or FGF-2 to a NC induction protocol (). Multiple studies then showed that RA could posteriorize hPSC-derived NCCs to enteric NCCs (ENCCs), which expressed vagal-specific HOXB genes and could differentiate into various enteric neuron subtypes (; ). Sequential treatment with RA and BMP could generate PHOX2B+ trunk NCCs, which could differentiate into sympathoadrenal cells (). This mimicked the in vivo induction of sympathoadrenal trunk NCCs by BMP signaling from the dorsal aorta (). The emergence of these protocols to define regional NCC identity will be useful in determining intermediate cell populations during differentiation to NCC derivatives. Currently, it appears that many groups use either FBS/BMP-4 or RA to direct anterior or posterior NCC specification before differentiating NCCs to end-point derivatives (Figure 1B) using protocols optimized for other cell types. Widespread adoption and further development of defined protocols to drive and characterize region-specific NCCs will be beneficial to create more defined culture systems. This will enable the future use of NCC derivatives for tissue regeneration applications.
Applications of hPSC-Derived NCCs for Disease Modeling
Human-specific NCCs provide an invaluable resource to complement animal models in the understanding of neurocristopathies. Therefore, a major application of hPSC-derived NCCs to date is in the modeling of various neurocristopathies (Table 1). The following section highlights notable neurocristopathies whereby the generation of either patient-specific iPSC-derived NCCs or genetically modified hESCs bearing specific genetic mutations have been used to mimic clinically relevant NCC dysfunctions and discover novel molecular mediators.
Table 1
| NCC derivatives | Intended applications | Cell source | NCC derivation method | Key outcomes | Reference |
|---|---|---|---|---|---|
| None | Disease modeling-CHARGE syndrome | CHD7 knockdown H9 hESC cell line | Neurosphere culture followed by isolation of migratory NCCs | Reduced formation of multipotent, migratory TWIST1+ NCCs | |
| iPSCs from CHARGE patient-derived fibroblasts | Methods of and | Defective delamination in vitro and migration in vitro and in vivo by CHARGE NCCs | |||
| Mesenchymal stem cells (MSCs) | Disease modeling-Ewing’s Sarcoma | H9 hESC cell line | Ectopic expression of EWS-FLI1 in p75+ NCCs isolated after PA6 co-culture | Transition of EWS-FLI1 MSCs to a more primitive state, p16 repression | |
| Tendon regeneration | iPSCs from human BMSCs | p75+ migratory NCCs isolated after cell aggregate culture | Enhanced healing by NCCs delivered in fibrin gel compared to control in rat patellar tendon window defect model | ||
| Cartilage regeneration | 414C2 human iPSC cell line | WNT activation and TGF-β inhibition, followed by 10% FBS (MSC induction) | Poor defect repair by NCC cell sheet compared to control BMSC cell sheet in rat femoral osteochondral defect model | ||
| Smooth Muscle Cells (SMCs) | Disease modeling- Bicuspid aortic valve (BAV) | iPSCs from BAV patient-derived PBMCs | p75+/HNK-1+ NCCs isolated after dual-SMAD inhibition | BAV SMCs had impaired contractility and increased mTOR signaling | |
| Enteric neurons | Disease modeling- Hirschsprung’s disease (HSCR) | iPSCs from HSCR patient-derived fibroblasts and RET mutant IMR90 iPSCs | Dual-SMAD inhibition, WNT activation, RA treatment to derive p75+/CD49+ ENCCs | Defective migration and neuronal differentiation in HSCR NCCs; identification of mutations associated with HSCR and correction with CRISPR/Cas9 | |
| Enteric Nervous System (ENS) Regeneration Enteric Nervous System (ENS) Regeneration | H9 hESCs and iPSC cell lines | Dual-SMAD inhibition, WNT activation, RA treatment to derive p75+/CD49+ ENCCs | Extensive migration of grafted ENCCs delivered in 70% matrigel; rescue of disease-related mortality in Hirschsprung disease mice (Ednrbs-l/s-l) | ||
| H9 hESCs and WTC iPSC cell lines | Dual-SMAD inhibition, WNT activation, RA treatment to derive p75+/CD49+ ENCCs | Establishment of ganglia, neuronal repopulation, neuron-dependent contractility by 3-D spheroids of ENCCs implanted in human tissue-engineered small intestine | |||
| iPSCs from human dermal and embryonic fibroblasts | p75+/HNK-1+ migratory NCCs isolated after aggregate culture; co-culture with gut explants to induce enteric neurons | Longitudinal migration in E5 chick hindgut, migration toward myenteric and submucosal regions in SCID mice upon engraftment of NCC spheres | |||
| H1 and H9 hESCs, WTC iPSC cell lines | Neurosphere culture followed by isolation of migratory NCCs | Migration of ENCCs into mesenchyme; neuronal and glial differentiation upon mechanical aggregation of ENCCs with tissue-engineered human intestinal organoids | |||
| Peripheral Neurons and Schwann Cells | Disease modeling- Familial dysautonomia (FD) | iPSCs from FD patient-derived fibroblasts | p75+/HNK-1+ NCCs isolated after MS5 co-culture | IKBKAP splicing defect, reduced migration and neuronal differentiation of FD NCCs | |
| Normal and FD patient fibroblasts | Direct NCC reprogramming by SOX10 overexpression and WNT activation | IKBKAP splicing defect, reduced iNCC induction and migration in FD iNCCs | |||
| Peripheral Nerve Regeneration | Human iPSC cell lines | LNGFR1 (p75) +/THY1+ migratory NCCs isolated after neurosphere culture | Promotion of axonal regrowth and remyelination in silicone nerve conduit in NCC group in mouse sciatic nerve defect model | ||
| hESCs | p75+ NCCs isolated after dual-SMAD inhibition and WNT activation | Robust regeneration throughout the trimethylene carbonate 𝜀-caprolactone nerve conduit in NCC group in rat sciatic nerve injury model | |||
| H9 hESCs | Dual-SMAD inhibition and WNT activation | Therapeutic efficacy of NCC filled poly (𝜀-caprolactone) and ethyl ethylene phosphate nerve conduits reduced with increased passage number in rat sciatic nerve injury model | |||
| Melanocytes | Disease modeling- Hermansky-Pudlak (HP) and Chediak-Higashi (CH) syndromes | iPSCs from HP and CH patient-derived fibroblasts | Dual-SMAD inhibition and WNT activation followed by isolation of SOX10+/cKit+ melanocyte precursors | Loss of pigmentation and reduction in mature melanosomes in different degrees in CH and HP NCC derived melanocyte clones | |
Applications of hPSC-derived NCCs in disease modeling and tissue regeneration.
hESCs: human embryonic stem cells; PBMCs: peripheral blood mononuclear cells.
CHARGE Syndrome
CHARGE syndrome is an acronym for a complex combination of congenital abnormalities including malformations of the craniofacial skeleton, peripheral nervous system, eyes, ears, and heart. It is often associated with mutations in the CHD7 gene, which is postulated to cause NCC dysfunction (). CHD7 knockdown in hESCs affects the formation of multipotent, migratory NCCs by diminishing the expression of NC specifiers TWIST1, SOX9, and SLUG (). Another study derived NCCs from CHARGE patient-derived iPSCs, which also showed defective delamination, migration and motility in vitro and defective migration in vivo when implanted into a chick embryo (). Therefore, CHD7 mutations in CHARGE syndrome result in defects in NCC migration, although the underpinning molecular mechanism remains to be elucidated.
Ewing’s Sarcoma
Ewing’s sarcoma family tumors (ESFT) are common malignant bone and soft tissue tumors, whose genetic hallmark involves the expression of an EWS-FLI1 fusion gene due to chromosome translocation (). While their cellular origin remains elusive, one popular hypothesis implicates NCCs as the source of these cells (). Von Levetzow et al. demonstrated that hESC-derived NCCs and NCC-derived MSCs were permissive for EWS-FLI using lentiviral transduction. Expression of EWS-FLI1 pushed NCC-derived MSCs to a more primitive NCC state and led to the loss of cellular senescence and repression of p16 (). They found that ESFT are genetically closely related to NCCs, supporting the hypothesis that some malignant ESFT cells may develop from NCC-derived cells. The use of this hESC-derived NCC model also helped to delineate the mechanism of oncogene tolerance in these cells.
Hirschsprung’s Disease
Hirschsprung’s disease (HSCR) is caused by the defective migration of ENCCs in the gut, leading to loss of peristaltic activity, causing bowel obstruction and megacolon. The severity of the phenotype is determined by the length of the aganglionic segment- short (S-HSCR), long (L-HSCR) or total colonic aganglionosis (). While HSCR is genetically heterogeneous, mutations in the receptor tyrosine kinase RET are implicated in many cases. Lai et al. generated iPSCs from HSCR patients as well as CRISPR-Cas9 edited RET mutant iPSC lines, and demonstrated that both HSCR and RET-mutant ENCCs showed defective neuronal differentiation and migration compared to control ENCCs (). They identified a novel mutation in the vinculin gene associated with S-HSCR, and corrected this mutation using CRISPR/Cas9 to restore ENCC function. This study demonstrates the great potential of hPSC-based in vitro assays to identify novel disease-associated mutations with high power. This will be useful in the study of HSCR as its genetic etiology is still not completely known.
Familial Dysautonomia
Familial dysautonomia (FD) is a rare but fatal, hereditary sensory and autonomic neuropathy usually caused due to a point mutation in the IKBKAP gene (). FD is known to affect NCCs and cause degeneration of peripheral neurons. Lee et al. first reprogrammed iPSCs from FD patient fibroblasts and differentiated them to NCCs, while Kim et al. directly reprogrammed FD patient fibroblasts to iNCCs by SOX10 overexpression (; ). In both studies, FD NCCs showed lower levels of normal IKBKAP transcripts, reduced migration and lower neuronal differentiation efficiency compared to control NCCs. Lee et al. went on to identify kinetin as a candidate drug to rescue aberrant IKBKAP splicing, while Kim et al. shed light on a previously unknown aspect of FD pathogenesis- aberrant splicing in other genes such as PAX3 and MEF2C in FD iNCCs.
Hermansky-Pudlak Syndrome and Chediak-Higashi Syndrome
Pigment producing melanocytes in the skin arise from NCCs during development. Mica et al. developed a protocol involving timed exposure to WNTs, BMPs, and EDN3s for the sequential specification of NCCs, melanoblasts and mature melanocytes (). They then derived iPSCs from patients with Hermansky-Pudlak syndrome (HP) and Chediak-Higashi syndrome (CH), both of which cause defects in melanocyte vesicle formation and trafficking. Melanocytes derived from HP and CH NCCs showed different degrees of pigmentation loss and reduction in melanosome number and size, corresponding to the expected disease phenotype.
Applications of hPSC-Derived NCCs in Regenerative Medicine
Due to their wide differentiation potential, unlimited numbers, and developmental relevance to many tissues, hPSC-derived NCCs are a promising stem cell source for tissue regeneration and as therapies for neurocristopathies. iPSC-derived NCCs are a potentially autologous cell source that can overcome immune-compatibility issues. The preliminary investigations that have been done to assess the regenerative potential of hPSC-derived NCCs are discussed below (Table 1).
Bone, Cartilage, and Tendon
Currently, mesenchymal stem cells (MSCs) derived from bone marrow and adipose tissues are the paradigm cell source for the regeneration of craniofacial bone and cartilage (; ). However, since a significant portion of craniofacial mesenchymal tissues originate from NCCs, hPSC-derived NCCs are a promising alternative cell source for craniofacial bone and cartilage tissue engineering. hPSC-derived NCCs can be induced into MSCs either by fetal bovine serum (FBS) or by BMP-4 treatment (). Although FBS treatment is more prevalent, the use of undefined serum in MSC induction medium will limit future clinical applications. Mechanical cues such as substrate stiffness have recently been shown to modulate the differentiation potential of NCC-derived MSCs (). To date, only a handful of studies have evaluated the regenerative potential of NCC-derived MSCs. Chijimatsu et al. showed that although MSC-like cells differentiated from iPSC-derived NCCs showed chondrogenic ability in vitro, they had very limited repair efficiency in a rat osteochondral defect model (). On the other hand, Xu et al. demonstrated that iPSC-derived NCCs exhibited enhanced tendon healing compared to the acellular control group in a rat patellar tendon defect model (). Such differences in the regenerative efficiencies of NCC-derived MSCs are likely due to variations in the differentiation protocols for NCCs and MSCs as well as the choice of defect models. In both studies, the use of a single marker p75 to isolate NCCs is problematic, as studies have shown that p75 is not exclusive to NCCs and is widely expressed in the embryonic tissues (). Further work is required to develop chemically defined protocols to drive human NCCs into different mesenchymal lineages, and comprehensively benchmark their regenerative potential to mesodermal MSC sources.
Enteric Nervous System
As the enteric nervous system (ENS) is derived from the neural crest (), NCC-derived enteric neurons are an obvious choice of cell source for ENS regeneration. ENCC precursors derived from hPSCs were able to colonize postnatal and adult mouse colons upon in vivo engraftment and showed extensive migration (). The ENCCs were also able to rescue disease-related mortality in a genetic mouse model of HSCR (Edrnbs-l/s-l mice). Two recent studies demonstrated the use of hPSC-derived ENCCs to populate human intestinal organoids with an ENS (; ). Schlieve et al. demonstrated that the implantation of ENCCs into their tissue-engineered small intestine derived from human intestinal organoids (HIO-TESI) led to the repopulation of an ENS in the HIO-TESI system and the establishment of neuron-dependent motility. Another study showed that upon transplantation of NCC spheres into E5 chick embryonic hindgut, they showed ganglial organization within submucosal and myenteric regions and longitudinal migration (). Taken together, these studies suggest that hPSC-derived ENCCs are a promising cell source for treating human ENS disorders.
Peripheral Nerves
Peripheral nerve regeneration using primary Schwann cells is very difficult due to limited cell numbers, long culturing times and invasive harvesting techniques (). Thus, stem cell sources such as NCCs that can differentiate into Schwann cells have emerged as a promising alternative. Multiple studies have demonstrated the ability of NCCs to differentiate into Schwann cells and repair peripheral nerve defects when implanted as a nerve graft including a scaffold. Using rat or mice sciatic nerve injury models, these studies showed that grafted NCCs survived and promoted axonal regeneration in the artificial nerve conduits (; ; ). While Jones et al. derived NCCs from hESCs that were only p75+, Kimura et al. derived a LNGFR+ (p75), THY1+ (CD90, a common MSC marker) NCC population from iPSCs, likely selecting for an MSC sub-population. Thus, the different markers used in these studies probably led to the isolation of two different cell populations, which may affect regeneration via different mechanisms. The in vitro culture duration of hPSC-derived NCCs, as indicated by the passage number, is also found to impact of their therapeutic efficacy as indicated by Schwann cell differentiation, survival and axonal growth (). This finite expansion window presents a practical constraint in the application of hPSC-derived NCCs in peripheral neuronal regeneration applications.
Conclusion and Future Perspectives
This extensive body of work to induce the formation of NCCs and its derivatives in vitro has enabled the use of hPSC-derived NCCs for applications including disease modeling and tissue regeneration. Studies involving patient-derived and genetically modified NCCs have already broadened our understanding of NCC development and disease. The use of iNCCs reprogrammed directly from patient fibroblasts will likely advance this process further. So far, most of these studies have used 2-D monolayer culture systems. As signaling from surrounding tissues is so critical in NCC development in vivo, the development of 3-D organotypic models containing multiple cell types would better replicate the in vivo environment. This will help us gain a better understanding of NCC disease development in human-specific models. Moving forward, the use of human NCC-based models to test developmental toxicity and screen for possible human teratogens is a likely prospect. This can be enabled by the development of scalable, cost-effective and biomimetic models of NCC development.
The preliminary studies on the use of hPSC-derived NCCs for tissue regeneration show the great promise of these cells due to their wide differentiation potential and large cell numbers. However, more defined differentiation regimes optimized for specifying NCCs into specific lineages are needed to reliably produce purified cell populations. These should be characterized not only by marker expression, but also transplantation to test functional capability. The chief issue that impedes the clinical translation of NCC-derived cells for regeneration is the safety concern with the use of hPSC-derived cells. While a few studies have shown that the implantation of hPSC-derived NCCs in animal models did not cause teratoma formation (; ), thorough strategies to prevent uncontrolled proliferation are required to prevent any risk. Also, there are still unanswered questions regarding the relevance of a cell source’s developmental origin in regenerative medicine. It remains to be seen whether, for example, the use of developmentally relevant NCC-derived MSCs for craniofacial regeneration improves the therapeutic efficacy over other MSC sources, such as mesodermal derived bone marrow MSCs.
Statements
Author contributions
AS and Y-CT conceived and wrote this manuscript.
Funding
This work was supported by National University of Singapore (R-397-000-192-133, R-397-000-211-133, R-397-000-299-114, and R-397-000-242-112), Singapore Ministry of Education (R-397-000-215-112), and Singapore Institute for Neurotechnology (R-719-004-100-305).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
neural crest, disease model, tissue regeneration, pluripotent stem cell, neurocristopathy
Citation
Srinivasan A and Toh Y-C (2019) Human Pluripotent Stem Cell-Derived Neural Crest Cells for Tissue Regeneration and Disease Modeling. Front. Mol. Neurosci. 12:39. doi: 10.3389/fnmol.2019.00039
Received
25 October 2018
Accepted
01 February 2019
Published
22 February 2019
Volume
12 - 2019
Edited by
Sabine Wislet, University of Liège, Belgium
Reviewed by
Virginie Neirinckx, Luxembourg Institute of Health (LIH), Luxembourg; Shyam Gajavelli, University of Miami, United States; Gabriele Zanirati, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil
Updates
Copyright
© 2019 Srinivasan and Toh.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yi-Chin Toh, biety@nus.edu.sg
Disclaimer
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