Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Podsumowanie

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Streszczenie

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Wprowadzenie

Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have shown immense potential, in particular for the investigation and future treatment of human diseases for which neither good animal models nor primary tissues are available. Application examples for the hESC/hiPSC technology are the following: Cells of particular interest can be generated from hESC/hiPSCs for regenerative medicine at unlimited quantity¹. Cells can be produced from patients carrying a specific disease and used to establish in vitro disease models^2,3. Such disease models can then be employed for large-scale drug screening in the quest for new drug compounds⁴ as well as testing of existing drugs for efficacy and toxicity⁵. In vitro disease models can lead to the identification of novel disease mechanisms. For all applications of the hESC/iPSC technology it is important to work with specific, well-defined cell types affected in the disease of interest. Thus, the availability of solid and reproducible in vitro differentiation protocols is crucial for all applications of the hESC/hiPSC technology. Protocols are desirable that show minimal variability, time expense, effort, difficulty and cost as well as maximal reproducibility among hESC/hiPSC lines and different researchers.

Neural crest (NC) cells emerge during vertebrate neurulation between the epidermis and the neural epithelium. They proliferate and migrate extensively throughout the developing embryo and give rise to an impressive diversity of progeny cell types, including bone/cartilage, the craniofacial skeleton, sensory nerves, Schwann cells, melanocytes, smooth muscle cells, enteric neurons, autonomic neurons, chromaffin cells, cardiac septum cells, teeth and adrenal/thyroid glandular cells⁶. Thus, NC cells are an attractive cell type for the stem cell field and important for the modeling of a variety of diseases, such as Hirschsprung's disease⁷, Familial Dysautonomia⁸ as well as cancers such as neuroblastoma⁹. Furthermore, they offer the possibility to study aspects of human embryonic development in vitro.

The currently available and widely applied in vitro differentiation protocol for the derivation of NC cells from hESCs^10,11 requires up to 35 days of differentiation and it involves neural induction on stromal feeder cells such as MS5 cells and is thus performed under poorly defined conditions. While it can be up-scaled to generate large quantities of NC cells, for example required for high-throughput drug screening⁴, this is labor and cost intensive. Furthermore, it involves manual passaging of neural rosettes, which can be difficult to reproduce and thus is subject to overall variability, in particular when it is applied to a large variety of hESC or hiPSC lines. Here, the stepwise derivation of NC cells in an 18-day protocol that is free of feeder cells is shown. This method is shorter and more defined than the currently used protocol. Furthermore, it is very robust in generating NC cells among different hiPSC lines. Importantly, it is shown that the NC cells yielded by both protocols emerge at the border of neural rosettes (hereafter termed rosette-NC or R-NC). The cells derived using either of the two protocols look morphologically identical, they express the same NC markers and cluster together in microarray analysis. NC cells derived using the new protocol (R-NC) are functional, similar to NC cells derived using the old protocol (MS5-R-NC) such that they can migrate and further differentiate into neurons. Therefore, the cells can be used concurrently with the MS5-R-NC cells. The R-NC cell protocol for the derivation of NC cells from hESC/iPSC will be useful for all applications of the hESC/iPSC technology involving the NC lineage.

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Protokół

1. Preparation of Culture Media, Coated Dishes and Maintenance of hPSCs

1.1 Media preparation

Note: Filter all media for sterilization and store at 4 °C in the dark for up to 2 weeks. Reagent names, company and catalog numbers are listed in the Materials Table.

1. DMEM/10%FBS: Combine 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine.
2. HES-medium: Combine 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Glutamine, 5 ml Pen/Strep, 10 ml MEM minimum essential amino acids solution, 1 ml β-Mercaptoethanol. Add 10 ng/ml FGF-2 after filtering the medium.
   CAUTION: β-Mercaptoethanol is toxic, avoid inhalation, ingestion and skin contact.
3. KSR-differentiation medium: Combine 820 ml Knockout DMEM, 150 ml KSR, 10 ml L-Glutamine, 10 ml Pen/Strep, 10 ml MEM minimum essential amino acids solution and 1 ml β-Mercaptoethanol.
4. N2-differentiation medium: Dissolve 12 g DMEM/F12 powder in 980 ml dH₂O, add 1.55 g Glucose, 2 g Sodium Bicarbonate and 100 mg APO human transferrin. Mix 2 ml dH₂O with 25 mg human insulin and 40 μl 1 N NaOH, add the dissolved solution to the medium. Add 100 mM putrescine dihydrochloride, 0.5 mM selenite, 1 mM progesterone and bring the volume up to 1 L with dH2O.

1.2 Coating of culture dishes

1. Matrigel coating: Thaw 1 ml frozen matrigel aliquot by pipetting 19 ml DMEM/F12 over the aliquot until it has dissolved. Remove clumps by passing it through a 40 μm cell strainer and plate 8 ml/10 cm dish. Incubate the dishes for 1 hr at RT. Aspirate the matrigel immediately before plating the cells.
   Note: Work quickly with matrigel because it can clump at temperatures above 4 °C.
2. PO/Lam/FN coating: Add 10 ml of 1x PBS containing 15 μg/ml Poly-L Ornithin hydrobromide to a 10 cm dish. Incubate the dish over night at 37 °C. Wash plates with 1x PBS once and add 10 ml/10 cm dish of 1x PBS containing 2 μg/ml mouse Laminin-I and 2 μg/ml fibronectin. Incubate the dishes over night at 37 °C. Before plating the cells, completely remove the solution and let the plates dry thoroughly at RT for 15-20 min by standing them up onto the tissue culture hood wall without the lid on. 
   Note: The dishes are completely dry and ready for cell plating when one can see crystal structures on the surface (visible by eye). The plates can be kept at RT for a few hours in this dried state. PO/Lam/FN dishes have to be prepared 2 days ahead of time. In emergency cases Lam/FN can be incubated for 2-4 hr only. However, this may risk suboptimal differentiation/survival results.

1.3 Maintenance of hPSCs

Note: hPSCs are maintained on 0.1% gelatin and mitotically inactivated mouse embryonic fibroblasts (MEFs) in HES-medium supplemented with 10 ng/ml FGF-2 as described previously ^10,12. The cells should be split every 6-8 days.

1. Coat a 10 cm dish with 8 ml 0.1% gelatin (in 1x PBS without Magnesium or Calcium) at RT for 5 min.
2. Thaw frozen MEFs quickly in a 37 °C water bath. Add 1 million MEFs to 10 ml DMEM/10% FBS.
3. Aspirate the gelatin and plate the cells. Incubate at 37 °C for at least 6 hr.
4. Aspirate the DMEM/10% FBS from the prepared MEF plate, wash plate with 1x PBS once and add 10 ml HES-medium supplemented with 10 μM Y-27632 dihydrochloride. Allow the medium to warm up at 37 °C for 20 min. 
   Note: For manual splitting of hPSCs a laminar flow hood with an embedded microscope is used. However, the cells can be passaged using appropriate alternative methods.
5. Under a laminar flow hood with an embedded microscope, detach separate colonies using a cell lifter and let them float.
6. Use a 1 ml syringe to aspirate the floating colonies and dispatch them onto the fresh, warm MEF plate. Transfer approximately one fourth of the cells to the new dish. Incubate at 37 °C.
7. Feed cells daily with fresh HES medium.

2. Plating of hPSCs for Differentiation

Note: hPSCs should be split or plated for differentiation when the colonies are large, but still have sharp edges with as little as possible differentiating cells at their borders (see Figure 1B). When the cells are maintained using manual passaging the colonies should be large enough to easily be seen by eye. To get the right feel for this time point one can maintain a separate hPSC dish for two weeks without passaging and watch the cells reach and pass the ideal time point for passaging/differentiating them.

1. Prepare 10 cm dishes with matrigel 1 hr before starting the differentiation. Successful matrigel coating can be verified at 4x magnification. 
2. When the hPSCs are ready to be split, aspirate the medium and add 4 ml of 0.05% trypsin-EDTA to the cells.
3. Shake the dish horizontally for 2 min vigorously until one can see the MEFs as single cells lifting off the plate under the microscope. The hPSC colonies remain attached as colonies.
4. Immediately and thoroughly, aspirate the trypsin and let the plate stand at RT for 2-4 min.
5. Add 2 ml of HES-medium to the plate and using a P1000 pipette, detach the cells.
   Note: If the cells cannot be lifted off the surface easily, the plate can be incubated empty at RT for another 2-3 min.
6. Transfer the cells to 8 ml HES-medium supplemented with 10 μM Y-27632 dihydrochloride.
7. Aspirate the matrigel from a previously prepared 10 cm dish. Plate the cells at a 1:1 or 1:2 ratio (for example: The cells from one 10 cm dish are split into two 10 cm dishes) onto the matrigel plate. Incubate at 37 °C over night.
   Note: This plating should result in approximately 100,000 cells/cm².

3. Induction of Neural Differentiation

Note: The differentiation can be initiated (day 0) when the cells are 90-100% confluent (see Figure 1C), usually the following day. If the accurate confluency is not reached yet, the cells can be fed daily with HES-medium until they are ready for differentiation. Alternatively, the initial number of cells plated can be increased.

1. On day 0 to 3, feed the cells daily with 10 ml/10 cm dish KSR-differentiation medium containing 0.1 μM LDN193189 and 10 μM SB431542.
2. On day 4 and 5 feed the cells with 75% KSR-differentiation medium and 25% N2-differentiation medium both containing LDN193189 and SB431542.
3. On day 6 and 7 feed the cells with 50% KSR-differentiation medium and 50% N2-differentiation medium both containing LDN193189 and SB431542.
4. On day 8 and 9 feed the cells with 25% KSR-differentiation medium and 75% N2-differentiation medium both containing LDN193189 and SB431542.
5. On day 9 and 10 prepare PO/Lam/FN dishes as indicated in 1.2.2 for replating of the cells on day 11.
6. On day 10 feed cells with N2-differentiation medium containing LDN193189 and SB431542.

4. Replating in Droplets for NC Specification

1. On day 11 aspirate Lam/FN from the previously prepared plates and let them dry completely at RT for 20-30 min, as explained in 1.2.2.
2. Remove medium from the differentiating cells, wash the cells once with 1x PBS and add 8 ml accutase/10 cm dish. Incubate for 20 min at 37 °C.
3. Using a cell lifter, detach the cells and resuspend them in accutase with a 5 ml pipette. Transfer the suspension to a 15 ml tube.
4. Add 5 ml of 1x PBS and spin the cells for 5 min at 114 x g.
5. Resuspend the cells in 10 ml 1x PBS. To ensure a single cell suspension, filter them through a 40 μm cell strainer and count the cells using a hemocytometer or equivalent technique.
6. Spin the cells down and resuspend them in N2-differentiation medium containing 200 μM Ascorbic Acid (AA), 20 ng/ml BDNF, 100 ng/ml FGF8, 20 ng/ml SHH, 10 μM Y-27632 dihydrochloride at the concentration of 100,000-150,000 cells/10 μl.
7. Using a repeat-pipettor, plate 10 μl droplets close to each other without them touching onto the dried PO/Lam/FN 15 cm dishes.
   Note: One 10 cm dish of differentiated cells normally results in approximately 2-3 times 15 cm dishes or approximately 100 x 10 μl droplets/15 cm dish.
8. Let the droplets stand at RT for 20-30 min, then very carefully (not to disturb the droplets) add 30 ml of N2/AA/BDNF/FGF8/SHH/Y and incubate at 37 °C.
9. On day 12, carefully feed the cells with 20 ml/15 cm dish N2/AA/BDNF/FGF8/SHH.
10. From day 14-17 feed the cells every second or third day with N2/AA/BDNF/FGF8.
11. On day 16 and 17 prepare PO/Lam/FN plates to replate NC cells after FACS sorting.

5. Fluorescence Activated Cell Sorting (FACS) of NC cells

Note: The preparation of the cells for FACS requires approximately 2 hr.

1. On day 18 remove medium, wash the cells once with 1x PBS in the dish and add 12 ml accutase. Incubate the cells for 20 min at 37 °C.
2. Using a cell lifter, detach the cells from the surface and let them float. Pipette the cells into suspension using a 5 ml pipette, transfer them to a 50 ml tube and add 30 ml of 1x PBS. Spin the cells for 5 min at 114 x g.
3. Using a P1000 pipette, resuspend the cells in 1 ml of 2% FBS/HBSS (the HBSS contains 15 mM HEPES). Add 19 ml of 2% FBS/HBSS and filter the cells through a 40 μm cell strainer to ensure a single cell suspension. Incubate the cells on ice for 15 min for antibody blocking and then count them using a hemocytometer.
4. Spin the cells down and resuspend them at the concentration of 10 million cells/ml in 2% FBS/HBSS.
5. Set aside 1 million cells each for the unstained, single-stained (HNK-1 only and p75 only) and secondary antibody stained only (APC only and 488 only) FACS controls.
6. Add 5 μl of HNK-1 and 5 μl of p75 primary antibody per 10 million cells in 1 ml suspension to the proper samples and incubate for 20 min on ice.
7. Wash cells in 10 ml 1x PBS (centrifuge 5 min at 114 x g) and resuspend the cells in 1 ml 2% FBS/HBSS. Add 2 μl of APC and 1 μl of 488 secondary antibody per 10 million cells in 1 ml suspension to the proper samples and incubate for 20 min on ice in the dark.
   Note: APC and 488 are the secondary antibodies used here for the HNK-1 and p75 staining, respectively.
8. Wash the cells in 10 ml 1x PBS twice, resuspend 10 million cells in 500 μl of 2% FBS/HBSS containing DAPI (at 0.5 ng/μl) or alternative appropriate live cell stain to exclude dead cells from the FACS analysis.
9. Transfer the samples to appropriate FACS tubes.
10. Prepare FACS tubes with 0.5 ml 2% FBS/HBSS for cell collection.
    Note: The cells and collection tubes are kept on ice in the dark during the sorting time.
11. Using a cell sorting machine with lasers that can detect DAPI, APC and 488 sort DAPI-/HNK-1+/P75+ double positive cells.
    Note: Preparation time and handling of the cells leads to a small percentage of cell death, DAPI stains dead cells only and thus allows for these cells to be excluded from the sorted population. Any alternative live/dead stain works equally well for this purpose. DAPI itself does not cause cytotoxicity in the live cell population.

6. Replating of Sorted Cells, NC Maintenance and Expansion

Note: FACS sorted cells should be handled with special care to ensure optimal survival. Keep them on ice until replating them. Do not vortex or pipette them harshly. The cells can be resuspended by flicking the tube.

1. After FACS sorting, count the NC cells, then spin them down and resuspend in N2-differentiation medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 10 μM Y-27632 dihydrochloride in the volume appropriate to replate them at 30,000 cells/10 μl droplets.
2. Thoroughly dry previously prepared PO/Lam/FN plates and plate approximately 50-70 droplets per 10 cm dish. Let the dishes stand at RT for 20-30 min.
3. Very carefully add 20 ml/10 cm dish of N2/FGF2/EGF/Y-27632 without disturbing the droplets. Incubate the cells at 37 °C.
4. Feed the cells every 2-3 days with N2/FGF2/EGF.
   Note: NC cells are expanded or passaged approximately every 4-5 days or when they start piling up within the droplets.
5. To passage the NC cells, remove the medium, wash the cells once with 1x PBS and add 8 ml accutase/10 cm dish. Incubate for 20 min at 37 °C.
6. Pipette the cells off the plate using a 5 ml pipette and transfer them to a 15 ml tube. Add 6 ml 1x PBS.
7. Spin the cells for 5 min at 114 x g.
8. Resuspend the cells in N2-differentiation medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 10 μM Y-27632 dihydrochloride in order to have 20,000 cells/10 μl droplet.
9. Replate the cells in 10 μl droplets onto dried PO/Lam/FN plates. Let the dishes stand at RT for 20-30 min.
10. Carefully add 20 ml/10 cm dish of N2/FGF2/EGF/Y-27632. Incubate at 37 °C.
    Note: NC cells can be maintained or expanded for up to 2 weeks as a relatively homogeneous progenitor population. Longer culture may lead them to differentiate into various progeny cell types.

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Wyniki

The two most important improvements of the R-NC protocol over the MS5-R-NC protocol ¹¹ are the feeder-free, defined differentiation conditions and the overall shortening of the time requirement. MS5 feeder cells ¹³ are murine bone-marrow derived stromal cells that have been shown to support neural differentiation from hESCs ¹⁴. HESCs cultured on MS5 feeder cells at low density form epithelial structures and neural rosettes ¹⁵, at the periphery of which NC ce...

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Dyskusje

For the successful differentiation of R-NC cells from hESC/hiPSCs the following considerations should be made. It is critical to work under sterile culture conditions at all times. In particular, it is important to test hPSC cultures for mycoplasma contamination regularly, since this contamination will hinder successful differentiation, but cannot readily be detected visually in hPSC cultures. The R-NC differentiation should be initiated at 90-100% cell density; lower cell density affects cell survival and efficiency of ...

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Materiały

Name	Company	Catalog Number	Comments
DMEM	Gibco-Life Technologies	11965-092
Fetal Bovine Serum (FBS)	Atlanta Biologicals	S11150
DMEM/F12	Gibco-Life Technologies	11330-032
Knockout Serum Replacement	Gibco-Life Technologies	10828-028	Lot should be tested
L-Glutamine	Gibco-Life Technologies	25030-081
Penicinlin/Streptomycin	Gibco-Life Technologies	15140-122
MEM minimum essential amino acids solution	Gibco-Life Technologies	11140-050
β-Mercaptoethanol	Gibco-Life Technologies	21985-023	toxic
Recombinant human FGF basic (FGF2)	R&D Systems	233-FB-001MG/CF
Knockout DMEM	Gibco-Life Technologies	10829-018
DMEM/F12 powder	Invitrogen	12500-096
Glucose	Sigma	G7021
Sodium Bicarbonate	Sigma	S5761
APO human transferrin	Sigma	T1147
Human insulin	Sigma	I2643
Putriscine dihydrochloride	Sigma	P5780
Selenite	Sigma	S5261
Progesterone	Sigma	P8783
Matrigel matrix	BD Biosciences	354234
Poly-L Ornithin hydrobromide	Sigma	P3655
Mouse Laminin-I	R&D Systems	3400-010-01
Fibronectin	BD Biosciences	356008
Dispase in Hank's Balanced Salt Solution 5 U/ml	Stem Cell Technologies	7013
Trypsin-EDTA	Gibco-Life Technologies	25300-054
Y-27632 dihydrochloride	Tocris-R&D Systems	1254
LDN193189	Stemgent	04-0074
SB431542	Tocris-R&D Systems	1614
Accutase	Innovative Cell Technologies	AT104
Ascorbic Acid	Sigma	A4034
BDNF	R&D Systems	248-BD
FGF8	R&D Systems	423-F8
Mouse recombinant sonic hedgehog (SHH)	R&D Systems	464SH
HBSS	Gibco-Life Technologies	14170-112
HEPES	Gibco-Life Technologies	1563-080
Human recombinant EGF	R&D Systems	236EG
gelatin (PBS without Mg/Ca)	in house
Antibodies:
Anti HNK-1/N-Cam (CD57) mIgM	Sigma	C6680-100TST	lot should be tested
Anti-p75 mIgG1 (Nerve Growth factor receptor)	Advanced Targeting Systems	AB-N07	lot should be tested
APC rat anti-mIgM	BD Parmingen	550676	lot should be tested
AlexaFluor 488 goat anti-mouse IgG1	Invitrogen	A21121	lot should be tested
Anti Oct4 mIgG2b (used at 1:200 dilution)	Santa Cruz	sc-5279	lot should be tested
Material/Equipment:
Mouse embryonic fibroblasts (7 million cells/vial)	GlobalStem	GSC-6105M
Cell culture dishes: 10 cm and 15 cm plates, centrifuge tubes, FACS tubes,  pipettes, pipette tips
Glass hematocytometer
Cell culture centrifuge
Cell culture incubator (CO2, humidity and temperature controlled)
Cell culture laminar flow hood with embedded microscope
Cell culture biosafety hood
Cell sorting machine, i.e. MoFlo
Inverted microscope
1 ml TB syringe 27Gx1/2	BD Biosciences	309623
Cell lifter Polyethylene	Corning Incorporated	3008