Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Summary

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Abstract

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Introduction

Human embryonic stem cells (hESCs) have the potential to differentiate into the three primary germ layers, which then differentiate into various multipotent progenitor cell lineages. These lineages subsequently give rise to all cell types in the human body. In vitro hESC culture systems have transformed the ability to study human embryonic development and have served as a valuable tool for obtaining new insights into how these processes are regulated at the biological and molecular levels. Similarly, studies of induced pluripotent stem cells (iPSCs) generated from reprogramming somatic cells isolated from human patients provide novel insights into various diseases. In addition, progenitor and differentiated cells derived from hESCs can be useful for research involving stem cell therapy and drug screening^1,2,3,4.

hESCs can be induced to differentiate into neural progenitor cells (NPCs), which are multipotential cells with an extensive self-renewal capacity. Subsequently, these cells can be differentiated into neurons, astrocytes, and oligodendrocytes^5,6. NPCs also offer a cellular system for in vitro studies of neurodevelopmental biology and various neurological diseases. However, current colony type culture methods involving hESCs and their differentiation into NPCs are inefficient and often involve coculture as well as embryoid body (EB) and rosette formation^5,7,8,9. These protocols exhibit lower survival rates and spontaneous differentiation and are more time-consuming.

Presented here is an improved and robust culture system that is easily scalable and uses high density single-cell type culture of hESCs¹⁰. The inclusion of Roh-kinase (ROCK) inhibitor contributed to significantly enhanced survival efficiency during single cell type culture of hESC^{10,11,12,13,14}. In this culture system, hESCs can be easily maintained and expanded. In addition, the protocol presents an efficient method to generate NPCs from single-cell type culture of hESCs, which allows the production of highly pure NPCs. Inhibition of BMP/TGFβ/activin signaling pathways with ALK inhibitors efficiently induce differentiation of single-cell type hESCs into NPCs^15,16, which then can be induced to differentiate into functional neural lineages, such as dopaminergic neurons and astrocytes.

In summary, the single-cell type culture protocol using hESCs offers an attractive model to study the differentiation of these cells into various lineages, including NPCs. This protocol is easily scalable and therefore suitable for generating cells for research involving regenerative therapy and drug screening.

Protocol

1. Preparation of hESC-qualified Basement Membrane Matrix-coated Plates

1. Slowly thaw the hESC-qualified basement membrane matrix (see Table of Materials) solution at 4 °C for at least 2–3 h or overnight to avoid formation of a gel.
2. To prepare basement membrane matrix-coated plates, dilute matrix in cold DMEM/F12 to a 2% final concentration. Mix well and coat each well of a 6 well plate with 1 mL of the diluted matrix solution.
3. Incubate the basement membrane matrix-coated plates at room temperature (RT) for at least 3 h or at 4 °C overnight.
   NOTE: Plates with basement membrane matrix can be stored at 4 °C for 1 week before the matrix solution is removed and plates are used.

2. Adaptation of Colony type hESCs to Single-cell hESC Culture

1. To passage the feeder-free cultures of colony type H9 (WA09) hESCs grown on basement membrane matrix, aspirate the medium from the wells (Figure 1A)⁹.
2. Wash 1x with 1 mL of DPBS. Add 1 mL of dispase solution (1 U/mL) per well and incubate at 37 °C for 20 min.
3. Remove the dispase, wash the cells 1x gently with 2 mL of DMEM/F12, remove the medium, and add 2 mL of DMEM/F12 to each plate.
4. Gently detach colonies by gently pipetting up and down and transfer to a 15 mL tube.
5. Centrifuge the pellets for 2 min at 370 x g and aspirate the medium.
6. To dissociate the cell pellets into single cells, add 2 mL of cell detachment solution (1x concentration, see Table of Materials) and incubate at 37 °C for 10 min.
7. Centrifuge cells for 2 min at 370 x g and remove the detachment solution.
8. Add fresh mTeSR1 human ESC medium and dissociate the cells into single cells by gentle pipetting up and down.
9. To adapt colony type hESCs to a single-cell type culture, plate approximately 1.5–2.0 x 10⁶ hESCs into each well of the basement membrane matrix-coated 6 well plate in 2 mL of mTeSR1 containing 10 μM ROCK inhibitor for 24 h (Figure 1A).
10. After 24 h, replace the hESC medium with fresh mTeSR1 without ROCK inhibitor and allow the hESCs to grow as a single-cell type for 3 days. Change the medium daily.
11. On day 4, when cultures reach nearly 100% confluency, dissociate cells in detachment solution, then replate as described in step 2.6.
    NOTE: The ROCK inhibitor improves cell survival during the initial 24 h of single-cell type hESC culture.

3. Embryoid Body Formation and Differentiation into Three Germ Layers (Figure 2)

1. To form EBs, first resuspend cells in 3 mL of mTeSR1 medium with 10 μM ROCK inhibitor during the first 24 h, then incubate overnight into 60 mm low attachment dishes in a 37 °C incubator to allow aggregation.
2. After 24 h, the small EBs are transferred to a 15 mL tube. Let the EBs settle to the bottom of the tube and gently remove the medium with a pipette. Transfer EBs into EB medium (knockout-DMEM supplemented with 20% knockout serum replacement, 1x glutamine supplement [see Table of Materials], 1% NEAA [non-essential amino acids; see Table of Materials], and 0.2% β-mercaptoethanol) and allow them to expand in low attachment dishes for 7 days. The medium can be changed every other day as described above (Figure 2A).
3. On day 7, collect the EBs from the dishes and transfer them into a 15 mL tube. Gently remove the medium with a pipette and transfer the EBs to a basement membrane matrix-coated 6 well plate.
4. Allow the EBs to attach to the plate and incubate for 12 days in EB medium, during which they will differentiate into the three germ layers (Figure 2B). Change the medium every other day.
   NOTE: During aggregation of cells, the low attachment dish helps avoid EB attachment.

4. Differentiation of Single-cell Type hESCs Into NPCs (Figure 3)

1. To induce NPC differentiation, dissociate single-cell type hESCs with 1 mL of detachment solution (1x) and incubate for 10 min at 37 °C.
2. Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of DMEM/F12 and resuspend cells by gentle pipetting.
3. Plate cells on a basement membrane matrix-coated 6 well plate at a density of 2 x 10⁵ cells/well in 2 mL of mTeSR1 containing 10 μM ROCK inhibitor.
4. After 24 h, replace the culture medium with neural induction medium (DMEM with 1% B27 minus vitamin A) supplemented with 1 μM dorsomorphin and 5 μM SB431542.
5. Change the medium every other day during the first 4 days of neural induction, then every day until reaching confluence at day 7 (Figure 3B).
   NOTE: (1) Dorsomorphin inhibits the BMP pathway by targeting ALK2,3,6 receptors and also inhibits AMPK; SB431542 is an inhibitor of the TGFβ/Activin pathway by targeting ALK5,7. (2) The same protocol was tested with another ESC line (WA01), which yielded similar results as WA09¹⁶. (3) We have generally used Matrigel for the basement membrane matrix; however, cells can be cultured on Geltrex and then differentiated into NPCs with very similar results as indicated by the expression of several NPC markers (Figure 4D,E). Handle Geltrex in the same manner as Matrigel (see section 1).

5. NPC Expansion and Cryopreservation

1. After 7 days of neural induction, dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 °C.
2. Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of NPC expansion medium (see Table of Materials) and resuspend cells by gentle pipetting.
3. Plate 1 x 10⁵ cells in 2 mL of NPC expansion medium on basement membrane matrix-coated 6 well plates.
4. Passage NPCs multiple times, as necessary, when cultures reach nearly 90% confluency. During the first 3–4 passages, add 10 μM ROCK inhibitor during the initial 24 h to prevent cell death. After these passages, cells can be passaged and cultured without ROCK inhibitor. Change the medium every other day during the expansion.
5. Cryopreserve NPCs when cultures reach confluency.
   1. For cryopreservation, dissociate cells in 1 mL of detachment solution (1x) for 5 min at 37 °C, then centrifuge suspension for 2 min at 370 x g to remove the detachment solution.
   2. Add cell freezing solution (see Table of Materials) to dissociated NPCs at 1 x 10⁶ cells/mL and distribute 1 mL aliquots to cryovials.
   3. Place the cryovials into freezing container and store them overnight in a -80 °C freezer.
6. Store the cryovials in the liquid nitrogen.

6. Preparation of poly-L-ornithine (PLO) and Laminin Coated Plates

1. To prepare PLO/laminin coated plates, dilute the PLO stock solution into PBS or water to a final concentration of 10 μg/mL. Mix well and coat each well of a 6 well plate with 1 mL of diluted PLO solution.
2. Incubate for at least 2 h at 37 °C or overnight at 4 °C.
3. Wash each well with PBS.
4. Dilute laminin stock solution in cold PBS or water at 10 μg/mL. Mix the laminin solution well. Remove the PBS and immediately coat each well with 1 mL of the laminin solution. Keep plates at 4 °C and use within 1 week. Wash with PBS and immediately add culture medium.
   NOTE: Do not let the PLO/laminin coated plates dry out.

7. Differentiation of hESC-derived NPCs Into Dopaminergic Neurons (Figure 6A)

1. Plate NPCs in PLO/laminin-coated dishes in NPC expansion medium at a density of approximately 50%.
2. After 24 h, change the medium to DA1 medium (neurobasal medium containing 2 mM glutamine supplement, 1x NEAA, 1x B27, 200 ng/mL SHH, and 100 ng/mL FGF8) and change the medium every other day.
3. Passage cultures when they reach confluency. Dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 °C.
4. Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of DA1 medium and resuspend the cells by gentle pipetting.
5. Plate 1 x 10⁵ cells in 2 mL of DA1 medium on PLO/laminin-coated 6 well plates.
6. After 10 days, change to DA2 medium (neurobasal medium containing 2 mM glutamine supplement, 1x NEAA, 1x B27, 200 μM ascorbic acid, 20 ng/mL BDNF, and 20 ng/mL GNDF) and change medium every other day for an additional 20 days (Figure 6A).
   NOTE: Add fresh ascorbic acid daily to the DA2 medium. Within the first 14 days, the differentiated cells should be passed when they reach 90% confluency, but no longer after that.

8. Differentiation of ESC-derived NPCs into Astrocytes

1. Plate neural progenitor cells on PLO/laminin-coated plates in NPC expansion medium at a density of approximately 50%.
2. After 24 h, change the medium to astrocyte medium (DMEM/F12 medium including 1:100 N2, 1:100 B27, 200 ng/mL IGF, 10 ng/mL activin A, 10 ng/mL heregulin β-1, and 8 ng/mL bFGF) and change the medium every other day.
3. Passage cultures when they reach confluency. Dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 °C.
4. Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of astrocyte medium and resuspend the cells by gentle pipetting.
5. Plate 1 x 10⁵ cells in 2 mL of astrocyte medium on PLO/laminin-coated 6 well dishes.
6. Allow differentiation to continue for a total of 5–6 weeks (Figure 6B).

9. Immunofluorescence Staining

1. Culture cells in 35 mm μ-dishes as described above for each cell type. Aspirate the medium and add 1 mL of 4% PFA solution per dish and incubate for 20 min at RT.
2. Wash 2x for 5 min with PBS.
   NOTE: Once the cells are fixed, the assay plates can be sealed with parafilm and stored at 4 °C. Stain with an antibody within 1 week after fixation.
3. Add 300 μL of blocking solution (goat or donkey serum in PBS) per dish and incubate at RT for 30–60 min.
4. Wash the dish 2x with PBS for 5 min.
5. Add 300 μL of primary antibody (Table 1) solution (diluted in blocking solution) and incubate at RT for 1.5 h or overnight at 4 °C.
6. Wash 2x with PBS for 10 min.
7. Add 300 μL of fluorescent-conjugated secondary antibody (Table 1) in blocking solution and incubate in the dark for 1 h at RT.
8. Wash the cells 2x for 10 min with PBS.
9. Add Hoechst staining solution (1 μM final concentration in PBS) to stain nuclei. Incubate the cells in the dark for 5 min at RT.
10. Wash cells 1x with PBS for 5 min and then add 500 μL of PBS. Keep dishes in the dark, then observe fluorescence with a confocal microscope (Figure 2B, Figure 5C, Figure 6A, and Figure 7B).

Results

Presented here is an improved protocol for the maintenance and expansion of single-cell type culture of hESCs and their efficient differentiation into neural progenitor cells, which subsequently differentiates into various downstream neural lineages, including dopaminergic neurons and astrocytes.

Representative phase contrast images show cell morphology at different steps during the adaptation of colony type hESCs to the single-cell type culture (Figure 1A...

Discussion

Scalable and efficient methods for the differentiation of hESCs into various lineages and the generation of sufficient numbers of differentiated cells are important criteria for drug screening and stem cell therapy. Various single-cell passing methods have been published, in which cells are cultured in the presence of ROCK inhibitor or other small molecules to improve survival, but the final products of these culture methods are colony type hESCs^17,18,...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm m-dishes	ibidi	81156	Cell culture dish
6-well plates	Corning	3516
Accutase	Innovative Cell Technologies	AT104-500	Cell detachment solution
Activin A	R&D system	338-AC-050
Ascorbic Acid	Sigma Aldrich	A4403
B27 supplement	Thermo Fisher	17504044
B27 supplement (-Vit A)	Thermo Fisher	12587010
BDNF	Applied Biological Materials	Z100065
bFGF	Peprotech	100-18C
Centrifuge	DAMON/ICE	428-6759
CO2 incubator	Thermo Fisher	4110
Corning hESC-qulified Matrix (Magrigel)	Corning	354277	Basement membrane matrix (used for most of the protocol here)
Cryostor CS 10	Stemcell Technologies	7930	Cell freezing solution
Dispase	Stemcell Technologies	7923
DMEM	Thermo Fisher	10569-010
DMEM/F12	Thermo Fisher	10565-018
Dorsomorphin	Tocris	3093
EGF	Peprotech	AF-100-16A
Fetal Bovine Serum	Fisher Scientific	SH3007003HI
FGF8	Applied Biological Materials	Z101705
GDNF	Applied Biological Materials	Z101057
Geltrex matrix	Thermo Fisher	A1569601	Basement membrane matrix
GlutaMax	Thermo Fisher	35050061	Glutamine supplement, 100X
H9 (WA09) human embryonic stem cell line	WiCell	WA09
Heregulin b-1	Peprotech	100-3
IGF	Peprotech	100-11
Knockout DMEM	Thermo Fisher	10829018
Knockout Serum Replacement	Thermo Fisher	10828028
Laminin	Sigma Aldrich	L2020
mTeSR1	Stemcell Technologies	85850	hESC culture medium
N2 supplement	Thermo Fisher	17502001
NEAA	Thermo Fisher	11140050
Neurobasal	Thermo Fisher	21103049
Poly-L-ornithine	Sigma Aldrich	P3655
ROCK inhibitor	Tocris	1254
SB431542	Tocris	1614
SHH	Applied Biological Materials	Z200617
Stemdiff Neural Progenitor medium	Stemcell Technologies	5833	NPC expansion medium