Protocol for generating human craniofacial cartilage organoids from stem-cell-derived neural crest cells

Summary

Here, we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.

For complete details on the use and execution of this protocol, please refer to Foltz et al.¹

Graphical abstract

Highlights

• •Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)
• •Steps for differentiating NCSCs into craniofacial cartilage organoids
• •Instructions for preparing appropriate media and conditions for differentiation
• •Guidance for assessing changes in cell and organoid morphology during differentiation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.

Before you begin

Here, we present a protocol to generate human craniofacial cartilage from pluripotent stem cells. Human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are first induced to form neuroectoderm using SMAD inhibitors to inhibit BMP and TGF-β signaling. Neural crest specification is subsequently achieved using a GSK3β inhibitor to activate WNT pathway signaling, followed by Shh, FGF8, ascorbic acid, and BDNF addition. Neural crest stem cells (NCSCs) are then isolated by magnetic bead sorting. The organoids are self-organizing, do not require a scaffold or embedding in extracellular matrices, and are amenable to handling and histological techniques. The organoids have a glassy gray appearance with tissue architecture and staining typical of elastic cartilage. This protocol recapitulates the normal developmental pathway of craniofacial cartilage formation during embryogenesis and does not employ the use of embryoid bodies for differentiation. Generating these organoids provides opportunities for studying the human-specific cell signaling mechanisms of craniofacial chondrogenesis, craniofacial disease modeling, and growth of cartilage for therapeutic transplantation.

Institutional permissions

Human embryonic stem cells (hESCs) were commercially obtained and H20961 induced pluripotent stem cells (iPSCs) were acquired from our collaborator, Dr. David Gokhman.

All experiments were carried out in compliance with Institutional Biosafety Committee (IBC) of The University of Montana (Application No: 2024–006).

Culturing stem cells on Matrigel-coated plates

Timing: ∼3–4 days

CRITICAL: All drugs and growth factors that are employed in cell culture (see key resources table below) should be stored frozen in aliquots in sterile tubes, pipetted under sterile conditions in a sterile biosafety cabinet. All cell culture media made in the laboratory should be sterile filtered in a sterile biosafety cabinet.

For all steps below where cells are fed with media, all media should be prepared before initiation of each step and warmed to room temperature before use. Warming media to 37°C is not necessary and may accelerate inactivation of certain key growth factors.

This step describes culturing either hESCs or iPSCs on Matrigel coated plates and makes use of mTeSR1 medium. Add 100 mL of mTeSR1 5X supplement to 400 mL of mTeSR1 basal medium to make mTeSR1 complete medium prior to use. The media is changed every day and warmed to room temperature prior to use.

• 1.
  Preparing Matrigel aliquots.

  • a.
    Prepare Matrigel aliquots in advance, prior to use. Keep Matrigel frozen until preparing aliquots and avoid multiple freeze thaws. (https://www.corning.com/catalog/cls/documents/protocols/SPC-354277.pdf).
  • b.
    The protocol described in this paper uses Matrigel at the concentration of 0.5 mg/mL per one 10 cm dish. Aliquots at the concentration of 1 mg/mL would serve to coat two 10 cm dishes at a time.
  • c.
    It is crucial to maintain cold temperature while aliquoting Matrigel. So freeze the sterile Eppendorf tubes, tube racks, and pipette tips overnight at −80°C before preparing the aliquots.
  • d.
    Matrigel concentration varies in every lot. Thus calculate the volume of Matrigel needed for every 1 mg tube and add it to sterile cold Eppendorf tubes. Make sure that the Matrigel is kept on ice during the process to prevent it from gelling.
  • e.
    For more details, follow the instructions in the following link: https://www.wicell.org/product-files/cultureProtocols/SH-2.pdf.
• 2.
  Preparing Matrigel coated tissue culture plates.

  • a.
    Add 12 mL of cold serum-free DMEM to a 15 mL falcon tube and keep it aside.
  • b.
    Add 1 mL cold serum-free DMEM (from the 12 mL) to one frozen 1 mg/mL Matrigel aliquot. Pipette up and down until the Matrigel thaws and mixes with the medium. Transfer it to the 15 mL falcon tube containing 11 mL of medium and mix well.
  • c.
    Use 6 mL of the Matrigel solution to plate one 10 cm dish such that the concentration of Matrigel per plate is 0.5 mg/mL (You should be able to plate two 10 cm dishes with one aliquot of Matrigel).
  • d.
    Allow the Matrigel to set on plates at room temperature for 1 h.
  • e.
    Remove the un-gelled excess liquid by aspiration and immediately add 5 mL of fresh mTeSR1 medium before plating cells.

CRITICAL: Plates coated with Matrigel should not be allowed to dry. Thus, it is important to add 5 mL of mTeSR1 medium to the plate after the one-hour incubation.

• 3.
  Culturing cells on Matrigel coated plates.

  • a.
    Plate hESCs or iPSCs onto Matrigel coated plates. Add 5 mL of mTeSR1 medium containing the CEPT cocktail and 10 μM Y-27632 to the cells and incubate cells at 37°C, 5% CO₂.
  • b.
    Aspirate old media and add fresh mTeSR1 media every day.
  • c.
    Passage the cells using ReLeSR onto freshly coated Matrigel plates once before proceeding to differentiation.
• 4.
  Passaging stem cells.

  • a.
    Aspirate old media and add 3–4 mL ReLeSR and spread it around to form a thin layer on cells.
  • b.
    Aspirate the ReLeSR and incubate the plates at 37°C for 10 min.
  • c.
    After, add 6 mL of mTeSR1 media to the plate. You should see the cells lifting off. Tap the plate gently on the side if needed.
  • d.
    Collect the cells into a 15 mL falcon tube and centrifuge at 200 g for 5 min at room temperature.
  • e.
    Aspirate the supernatant and resuspend the cells in appropriate volume of mTeSR1 medium containing CEPT cocktail and 10 μM Y-27632 and plate the cells onto freshly coated Matrigel-coated plates at the required ratio.
  • f.
    Incubate the plates at 37°C, 5% CO₂.

CRITICAL: hESCs and iPSCs grow well when they are in clumps and are delicate. Thus, it is important to mix the cells gently while thawing and passaging; do not attempt to vigorously break the clumps too much or cell viability may suffer.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
DMEM F-12 media	Thermo Fisher Scientific	Cat# 12500-096
mTeSR1 media	STEMCELL Technologies	Cat# 85850
mTeSR1 Plus media	STEMCELL Technologies	Cat# 100–0483
ReLeSR	STEMCELL Technologies	Cat# 100–0276
Knockout DMEM	Thermo Fisher Scientific	Cat# 10829018
Knockout serum replacement	Thermo Fisher Scientific	Cat# 108280
Chroman - 1	MedChem Express	Cat# HY-15392, CAS: 1273579-40-0
trans-ISRIB	Tocris	Cat# 5284, CAS: 1597403-47-8
Emricasan (IDN-6556)	Selleckchem	Cat# S7775, CAS: 254750-02-2
Polyamine supplement (1000X)	MilliporeSigma	P8483
Antioxidant supplement (1000X)	MilliporeSigma	A1345
CHIR99021	MilliporeSigma	SML1046, CAS: 252917-06-9
SB 431542 hydrate	MilliporeSigma	S4317, CAS: 301836-41-9
SB 431542	Tocris	Cat# 1614, CAS: 301836-41-9
LDN193189	VWR	Cat# 1995-5, 25, CAS: 1062368-24-4
Poly-L-ornithine hydrobromide	MilliporeSigma	P3655, CAS: 27378-49-0
Laminin mouse protein, natural	Thermo Fisher Scientific	Cat# 23017015
Fibronectin, human	Corning	Cat# 356008
Matrigel growth factor reduced (GFR) basement membrane matrix, LDEV-free	Corning	Cat# 354230
L-glutamine	Thermo Fisher Scientific	Cat# 25030081
MEM Non-essential amino acid solution	Thermo Fisher Scientific	Cat# 11140050
2-Mercaptoethanol	Thermo Fisher Scientific	Cat# 21985023
Insulin from bovine pancreas	MilliporeSigma	I6634, CAS: 11070-73-8
Apo-Transferrin human	MilliporeSigma	T1147, CAS: 11096-37-0
Sodium selenite	MilliporeSigma	S9133, CAS: 10102-18-8
Putrescine dihydrochloride	MilliporeSigma	P5780, CAS: 333-93-7
Progesterone	MilliporeSigma	P6149, CAS: 57-83-0
(+)-Sodium L-ascorbate	MilliporeSigma	A4034, CAS: 134-03-2
Human brain-derived neurotrophic factor (BDNF) recombinant protein	Cell Signaling Technology	Cat# 3897, UniProt ID: #P23560
Recombinant human/murine FGF-8b	PeproTech	Cat# 100-25, UniProt ID: P55075
Recombinant Murine Sonic Hedgehog (Shh)	Peprotech	Cat# 315–22, UniProt ID: Q62226
Y- 27632 (dihydrochloride)	STEMCELL Technologies	Cat# 72304, CAS: 129830-38-2
Recombinant human FGF-basic	PeproTech	Cat# 100-18b, UniProt ID: P09038
Animal-free recombinant human EGF	PeproTech	Cat# AF-100-15, UniProt ID: P01133
Sodium bicarbonate	MilliporeSigma	Cat# S5761
Penicillin Streptomycin solution (100X)	Corning	Cat# 30-002-CI
Experimental models: Cell lines
WA09/H9 human	WiCell	RRID: CVCL_9773
H20961 iPS cells	Weizmann Institute of Science	RRID: CVCL_HA53
Other
Robosep buffer 2	STEMCELL Technologies	Cat# 20164
EasySep magnet	STEMCELL Technologies	Cat# 18000
EasySep Release Human PSC-derived Neural Crest Cell Positive Selection Kit	STEMCELL Technologies	Cat# 100-0047
Anti-PE microbeads	Miltenyi Biotec	Cat# 130-048-܁801
MACS Multistand	Miltenyi Biotec	Cat# 130-042-303
MS columns	Miltenyi Biotec	Cat# 130-042-201

Materials and equipment

CEPT cocktail

Reagent	Stock concentration	Final concentration
Chroman-1	5 mM diluted in DMSO	50 nM
Emricasan	5 mM diluted in DMSO	5 μM
trans-ISRIB	10 mM diluted in warm DMSO	0.7 μM
Polyamine Supplement	1000×	1×
Antioxidant Supplement	1000×	1×

Knockout serum replacement (KSR) medium

Reagent	Stock concentration	Final concentration	Amount
Knockout DMEM	N/A	N/A	820 mL
Knockout Serum Replacement	N/A	N/A	150 mL
L-glutamine	200 mM	2 mM	10 mL
MEM Non-Essential Amino Acids Solution	100×	1×	10 mL
Penicillin Streptomycin Solution	100×	1×	10 mL
2 – Mercaptoethanol	55 mM	0.055 mM	1 mL
Total	–	N/A	1000 mL

Store KSR medium at 4°C and use it within 3 months.

N2 base medium

Reagent	Stock concentration	Final concentration	Amount
DMEM F-12	N/A	N/A	500 mL
Sodium bicarbonate	N/A	3.9 g/L	1.96 g
Glucose	N/A	1.54 g/L	0.77 g
Insulin from bovine pancreas	20 mg/mL diluted in acidified water	0.025 mg/mL	625 μL
apo – Transferrin Human	100 mg/mL diluted in water	0.1 mg/mL	500 μL
Sodium selenite	115.6 μM diluted in water	30 nM	129.75 μL
Putrescine dihydrochloride	310 mM diluted in water	100 μM	150 μL
Progesterone	318 μM diluted in 100% ethanol	20 nM	31 μL
Total	–	–	500 mL

Store N2 Base medium at 4°C and use it within 3 months.

Neural crest stem cell (NCSC) specification medium

Reagent	Stock concentration	Final concentration	Amount
N2 base media	N/A	N/A	500 mL
(+) – Sodium L-ascorbate	N/A	200 μM	15 mg
Human Brain-Derived Neurotrophic Factor (BDNF) Recombinant Protein	100 mg/mL diluted in 0.1% BSA made in 1XPBS	20 ng/mL	250 μL
Recombinant Human/Murine FGF-8b	200 μg/mL diluted in 0.1% BSA made in water	100 ng/mL	500 μL
Recombinant Murine Sonic Hedgehog (Shh)	–	20 ng/mL	100 μL
Y- 27632 (Dihydrochloride)	10 mM diluted in water	10 μM	500 μL
Total	–	–	500 mL

Store NCSC Specification media at 4°C and use it within 3 months.

Neural crest stem cell (NCSC) maintenance medium

Reagent	Stock concentration	Final concentration	Amount
N2 base Media	N/A	N/A	500 mL
Recombinant Human FGF-basic	0.25 mg/mL diluted in 0.1% BSA prepared in 1XPBS	10 ng/mL	20 μL
Human Epidermal Growth Factor (hEGF)	0.5 mg/mL diluted in 0.1% BSA prepared in 1X PBS	20 ng/mL	100 μL
Total	–	–	500 mL

Store NCSC Maintenance medium at 4°C and use it within 3 months.

Step-by-step method details

Differentiation of stem cells into neural crest stem cells

Timing: 9 days

The following steps describe the methods to differentiate stem cells into neural crest stem cells. This is a series of sequential steps that are performed on 9 separate days. Ensure that the confluency of cells is at approximately 30% on Day 1, since the cells tend to proliferate as they differentiate over the course of 9 days. The cell culture dishes are incubated in a 37°C, 5% CO₂ tissue culture incubator.

• 1.
  Differentiation of stem cells into neural crest stem cells.

  • a.
    Day 1: Aspirate the stem cell medium and add 100% of KSR medium containing 0.1 μM LDN193189 and 10 μM SB431542 (10 mL KSR medium for a 10 cm dish). Incubate cells at 37°C, 5% CO₂ tissue culture incubator.
  • b.
    Day 2: Aspirate the KSR medium and add 75% KSR medium and 25% N2 medium containing 10 μM SB431542 and 3 μM CHIR99021 (7.5 mL KSR medium + 2.5 mL N2 medium for a 10 cm dish). Incubate cells at 37°C, 5% CO₂ tissue culture incubator.
  • c.
    Day 3: Aspirate the old media and add 50% KSR medium and 50% N2 medium containing 3 μM CHIR99021 (5.0 mL KSR medium + 5.0 mL N2 medium for a 10 cm dish). Incubate cells at 37°C, 5% CO₂ tissue culture incubator.
  • d.
    Day 4: Aspirate the old media and add 25% KSR medium and 75% N2 medium containing 3 μM CHIR99021 (2.5 mL KSR medium + 7.5 mL N2 medium for a 10 cm dish). Incubate cells at 37°C, 5% CO₂ tissue culture incubator.
  • e.
    Day 5: Aspirate the old media and add 100% N2 medium containing 3 μM CHIR99021.
  • f.
    Days 6–9: Change media every day and replace with fresh NCSC Specification medium containing 10 μM Y- 27632 (troubleshooting 2). Incubate cells at 37°C, 5% CO₂ tissue culture incubator.

    CRITICAL: It is important to start the differentiation when the stem cell confluency is at 30%–40% since the cells proliferate during the differentiation over the course of 9 days. Confluence is assessed by estimating the proportion of the dish covered by cells; see Figure 1B, Day 1. The cells may lift off as a layer, so it is vital that the media changes are carried out gently along the side walls of the plates to avoid disturbing the cells.

  • g.
    Day 10: The cells are differentiated and ready for sorting.

Figure 1.

Neural crest differentiation protocol and cartilage organoids

(A) Neural crest stem cell (NCSC) differentiation protocol. hESCs are differentiated into neural crest stem cells (NCSCs) over the course of 10 days by manipulating different signaling pathways using small molecule inhibitors and several growth factors. NCSCs are sorted via magnetic-activated cell sorting based on low affinity nerve growth factor receptor (p75/NGFR) on day 10 and the sorted cells are maintained in NCSC maintenance medium containing EGF and FGF2.

(B) Representative light microscopy images depicting cell morphology over the course of differentiation. Scale bar: 100 μM.

(C) Representative light microscopy images depicting cell morphology and self-organizing organoids at different time points during differentiation post sorting. The cells start migrating together and form multiple clumps (Day 20). The clumps grow bigger in size and start self-organizing into craniofacial cartilage organoids (Days 25 and 34). The clumps may be connected to each other by tendril-like structures and the cells surrounding the organoids become sparse (Days 41 and 48). Scale bar: 100 μM.

(D) Different views of differentiating craniofacial cartilage organoids. Images taken using dissecting microscope (Day 37) and a handheld camera (Days 46, 98, and 190). The organoids attain the morphology as depicted at Day 98 and Day 190 and continue to grow. Dissecting scope scale bar: 1 mm.

Sorting differentiated neural crest stem cells

Timing: ∼2–3 h

This step describes sorting the differentiated neural crest stem cells and plating them on poly-l-ornithine, fibronectin, and laminin coated plates including prepping the plates prior to the sorting. Please prepare poly-l-ornithine, fibronectin, and laminin coated plates fresh as indicated in the following steps.

• 2.
  Preparing poly-l-ornithine, laminin, and fibronectin coated plates.

  • a.
    On day 8 of differentiation, coat a 10 cm dish with 15 μg/mL Poly-l-ornithine prepared in 1X PBS.
  • b.
    Place the plate in a 37°C, 5% CO₂ incubator overnight to allow the coating to adhere to the tissue culture dish (10 mL for a 10 cm dish).
  • c.
    On day 9 of differentiation, carefully aspirate the Poly-l-ornithine solution and further coat the same plates with 2 μg/mL laminin and 2 μg/mL fibronectin prepared in 1X PBS.
  • d.
    Place the plate with this coating solution in a 37°C, 5% CO₂ incubator overnight for to allow the proteins to bind to the dish (10 mL for a 10 cm dish).
• 3.
  Sorting differentiated NCSCs into poly-l-ornithine, laminin, and fibronectin coated plates on day 10.

  • a.
    Before you begin, take the plates coated with poly-l-ornithine, laminin, and fibronectin on day 8 and 9 out of the incubator and aspirate the remaining 1XPBS solution.
  • b.
    Allow these plates to dry while you sort so that the plates are ready for sorted cells.
  • c.
    Add 3–4 mL of Versene to the plates you have been differentiating for the past 9 days.
  • d.
    Incubate for 8 min at 37°C in the incubator.
  • e.
    Aspirate the versene out and add 6 mL of media. The cells should start lifting off. Tap the plate gently on the side if you have trouble with the cells lifting off.

    Note: We do not use Trypsin or Accutase to prepare cell suspensions. Cells are resuspended by pipetting so that no large clumps are visible by eye, and but clumps of 4–16 cells may be visible upon examination using a hemocytometer for cell counting. If cells are pipetted too aggressively, cell viability may decrease.

  • f.
    Collect the cells into a 15 mL falcon tube and centrifuge at 200 g for 5 min at room temperature.
  • g.
    Use 10 μL of the cell suspension before centrifugation to carry out cell counting using suitable technique available in the lab such as a hemocytometer.
  • h.
    After centrifugation, discard the supernatant and resuspend the cells in up to 2 mL of Robosep buffer 2 depending on the cell concentration and manufacturer’s instructions. (You can have up to 2 mL of cell solution with cell concentrations of 2.5 × 10⁷ cells/mL).
  • i.
    Use EasySep Release Human PSC-derived Neural Crest Cell Positive Selection Kit, Stem cell Technologies, Cat# 100–047 to sort the cells based on cell surface CD271 (NGFR) marker using magnetic beads as described in the following attached kit manufacturer’s protocol: https://cdn.stemcell.com/media/files/pis/10000007951-PIS_00.pdf.
  • j.
    The last step of the manufacturer’s protocol entails collection of CD271 (NGFR) positive cells. Plate these cells directly onto dry poly-l-ornithine, laminin, and fibronectin coated plates (coated on days 8 and 9).
  • k.
    Distribute the cells as widely as possible on the tissue culture plate by pipetting.
  • l.
    Allow the cells to settle down and stick to the coat by transferring the plate to 37°C incubator for 5 min.
  • m.
    After incubation, add 9 mL of NCSC maintenance medium containing CEPT cocktail and 10 μM Y27632 and incubate the plates at 37°C, 5% CO₂. (troubleshooting 3, 4).

    CRITICAL: It is important that the plates are completely dry before plating the sorted cells onto the plates. Therefore, take the plates out of the incubator and aspirate the 1X PBS before beginning the sorting process. Allow plates to dry completely in the biosafety cabinet before (or during) the sorting step, which takes around 60 min, so that no PBS solution remains. This protocol of preparing coated tissue culture dishes fresh just before use should ensure that the protein coat on the dish is functional The plates should appear translucent when held up against the light. Allowing the plates to dry while sorting is often sufficient.

    CRITICAL: After sorting cells onto the plate, place the plate in the incubator for 5 min and allow the cells to settle down before adding 10 mL of NCSC maintenance media. This step is critical because the cells attach the tissue culture dish in a uniform pattern during this time rather than aggregate. Additionally, add the room temperature media along the sides the dish gently to prevent the cells from lifting off.

Viability is vastly improved if cells are allowed to adhere prior to adding media. Even if there are spots of adherent cells amongst regions with no cells on the plate, the NCSCs will begin to migrate all over the plate within a few days.

Alternative: NCS cells can alternatively be sorted using the MACS MS protocol by Miltenyi Biotech by positively selecting p75+ NCSCs with the help of PE anti-human CD271 (NGFR) antibody, BioLegend, Cat# 345105, RRID: AB_2282827, and Anti-PE MicroBeads, Miltenyi Biotec, Cat# 130-048-801 as per the manufacturer’s instructions and researchers’ discretion. (https://static.miltenyibiotec.com/asset/150655405641/document_1opk5oen153mbdcblpibi63q4b?content-disposition=inline).

Differentiation of NCSCs into craniofacial cartilage organoids

Timing: ∼60–90 days

This step describes the differentiation of neural crest stem cells into craniofacial cartilage organoids.

Exact cell number yield depend on the efficiency of sorting the cells. At least 50% yield post sorting ensures efficient differentiation into cartilage organoids. We have found, starting with 2 mL of cell solution with cell concentrations of 2.5 × 10⁷ cells/mL before sorting, good cartilage differentiation is obtained when sorted cells are plated into two 10 cm dishes.

• 4.
  Maintaining NCSC cells.

  • a.
    For the first 2 weeks, post sorting completely changes the medium every other feeding day by removing all the medium and adding 10 mL fresh NCSC maintenance medium.
  • b.
    After 2 weeks, change the media three times a week, e.g., every Monday, Wednesday, and Friday. Completely change the medium every third feeding day, e.g., on Mondays and Wednesdays, remove only 50% of the medium and add 50% of fresh NCSC maintenance medium, and Fridays, remove all the medium and add 10 mL of fresh NCSC maintenance medium.
  • c.
    Continue for the next 60–90 days using this feeding schedule.
  • d.
    During this period, cells migrate together and self-organize into organoids (Figure 1C). Eventually, large organoids become detached from the tissue culture plate surface (Figure 1D).
  • e.
    Once visible, organoids may be harvested as discussed below, but they will keep growing for six months or more (a limit is reached when they are no longer submerged). The largest organoid we have grown was about 1 cm.¹

CRITICAL: It is important to adhere to the feeding schedule above – to not change the media every day – to preserve the autocrine and/or paracrine cell signaling mechanisms generated by the self-organizing organoids.

• 5.
  Harvesting craniofacial cartilage organoids.

  • a.
    Harvest the organoids from culture dishes into a 5 mL tube containing 1X PBS using a spatula (or pipette with a large diameter hole). Cells still attached to the dish can differentiate further and form floating organoids, so they can be left for further growth.
  • b.
    Wash the organoids twice with 1X PBS.
  • c.
    For morphological analysis, after washing, add 5 mL of 10% Neutral Buffered Formalin for fixing overnight at room temperature and proceed to tissue processing and characterization.
  • d.
    Organoids may be characterized by a number of different techniques at this point. See Foltz et al.¹ for example characterizations using immunofluorescence, proteomics, and single-nuclei RNA sequencing.

Expected outcomes

This protocol recapitulates the normal developmental pathway of craniofacial cartilage formation during embryogenesis and does not employ the use of embryoid bodies for differentiation, unlike other studies. Thus, the chances of forming teratomas are drastically reduced. The organoids generated from this protocol show very low expression of stem cell markers while expressing markers indicative of their neural crest heritage.¹

This protocol integrates several previously published protocols with the goal of shortening the stage of differentiation and generating the NCSC intermediate as quickly as possible, while optimizing the yield.

Small molecules, LDN193189 and SB435142 are used in the initial days of differentiation induce formation of neuroectoderm by inhibiting BMP and TGF-β signaling, respectively. This is followed by activating the WNT signaling with the help of the small molecule glycogen synthase kinase-3 (GSK-3) inhibitor CHIR99021, which causes NCSC induction and increases the anterior NCSC yield. Shh, FGF-8b, ascorbic acid, and BDNF added to the last stage of NCSC induction further increase the number of NCSCs over a short period of time. During this stage, the cells attain NCSC morphology (flattened, not clumped; multipolar with dynamic protrusions, see Figure 1B) and migrate outwards from hESC colonies.

The NCSCs also express robust levels of low-affinity nerve growth factor receptor (p75/NGFR marker), which can be used to sort cells using magnetic-activated cell sorting method. Post-sorting, when replated on poly-l-ornithine, laminin, and fibronectin-coated plates as a monolayer, the NCSCs migrate together and start self-organizing. This continues for several weeks, and these self-organizing NCSCs form semi-spherical growths on the surface of the plate that detach and start floating in the cell culture media (Figures 1C and 1D; also see Figure 1B in Foltz et al.¹).

These floating organoids can easily be harvested with a spatula and have the physical characteristics of cartilage, appearing opaque and glassy, robust to handling, and resisting deformation in response to applied pressure. The remaining cells in the dish will continue to produce new organoids for up to a year and will maintain a confluency at around 50%.

Histological analysis of the organoids generally shows a typical morphology of cartilaginous tissue – small nuclei surrounded by lacunae (see Figure 1C in Foltz et al.¹) Organoids also express robust levels of cartilage markers like aggrecan, perlecan, proteoglycans, and various collagens.¹ Markers for hypertrophy that indicate further differentiation into bone are low for the hESC and iPSC lines used thus far with this protocol.¹

The protocol also provides a great model for understanding the cell signaling mechanisms that drive the differentiation into craniofacial cartilage.¹ Characterization of cells at different stages of differentiation can provide insights not only into the intermediate cell populations that form during differentiation like mesenchymal, chondroprogenitors, and nascent chondrocytes, but also into the autocrine and paracrine signaling mechanisms driving the interactions between the different cell types that aid in specifying and cementing the cartilage cell fate.

Patient-derived iPSCs can also be utilized to generate craniofacial cartilage organoids using this protocol, which provides tremendous translational applications. This protocol generates organoids without the need for a scaffold or embedding in extracellular matrices, reducing the risk of introducing exogenous material into the human body that could potentially trigger an immune response. Further studies to evaluate the potential for scalability and transplantation will be required to further translational goals for regenerative medicine applications such as in treating craniofacial abnormalities or injuries.

Limitations

This protocol describes the differentiation of stem cells into self-organizing craniofacial cartilage organoids. NCSCs are multipotent, and transiently adopt other expected neural crest cell fates during this procedure.¹ In our published work¹ we extensively characterized intermediate stages of NCSC differentiation and mature organoids with proliferating chondroprogenitors on their surface. Nascent organoids harvested after 4–8 weeks may contain immature chondroprogenitors (mesenchyme cells and immature chondrocytes); other cell types will be present in the culture dish.¹ For this reason, the organoids must be carefully evaluated for morphology and molecular signatures before being employed in subsequent functional assays.¹

We observed no evidence for chondrocyte hypertrophy (bone formation) in our experiments; however, to examine this possibility, collagen 10 and MMP-13, markers for chondrocyte hypertrophy² should be monitored by immunofluorescence or histological staining.

There may be variability in the response of different cell lines to our differentiation protocol; we have found that some iPSCs from different origins do not respond to this protocol in the same way. (This is not an uncommon issue.) To eliminate concerns about genomic instability, which is a known problem for iPSCs,^3,4,5 genetic analysis kits and karyotyping kits are available (e.g., Stem Cell Technologies, Stemmera Inc.). The ATCC FTA Sample Collection Kit for Human Cell Authentication Service may also be employed to compare cell lines to the public STR database (https://www.atcc.org/str) and Progenitor Cell Biology Consortium data repository.⁶

Troubleshooting

Problem 1

Cultures become contaminated (related to every step).

Potential solution

To avoid contamination, prepare all the growth factor aliquots in a sterile biosafety cabinet while rigorously adhering to sterile cell culture techniques. Filter all medium through a 0.2 μM filter before use. Make use of the sterile biosafety cabinet while culturing, passing, and sorting the cells.

Problem 2

Cells start lifting off as a monolayer (typically during days 6–9 of differentiation, related to step 1e–1f).

Potential solution

This could be because the cell confluency was greater than 40% before starting the differentiation protocol. Use a reduced cell confluency before starting the differentiation protocol. Also, don’t touch the bottom of the well directly with their aspirator tip, as the holes created by this can initiate lifting.

Problem 3

Poor attachment of neural crest stem cells after sorting (related to step 3).

Potential solution

It is highly likely that the cells are stressed due to the sorting process. Carry out the process of mixing gently whenever resuspension of cells required. The plates that the cells are being sorted into should be completely dry and appear translucent due to the presence of the protein coating on the plate. Also, do not add NCSC maintenance medium immediately after seeding cells. Incubate the newly sorted cells on their plates in the incubator for 5 min before gently adding the NCSC maintenance medium.

Problem 4

Low yield of neural crest cells after sorting (less than 50% survival) (related to step 3).

Potential solution

Make sure that the cells are prepped according to the manufacturer’s instructions at the right concentration before sorting. Mix gently during the process of sorting whenever necessary to prevent cell death.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mark Grimes (mark.grimes@mso.umt.edu).

Technical contact

Nagashree Krishna Avabhrath (nagashree.avabhrath@umontana.edu).

Materials availability

This study did not generate any unique reagents.

Data and code availability

This protocol did not generate any new datasets, but there are data associated with Foltz et al.¹

Acknowledgments

M.G. was supported by National Institute Of Dental & Craniofacial Research of the National Institutes of Health under award numbers R03DE034487 and R15DE028434, the NIH LINCS program U54 RFA-HG-14-001, University of Montana Center for Translational Medicine Pilot Grants, and generous donations from Craig Wilkinson. We thank David Gokhman for iPS cells.

Author contributions

N.A., L.F., and M.G.: experimental design; N.A. and L.F. performed the experiments; N.A. and M.G.: writing.

Declaration of interests

The authors declare no competing interests.