The hESC culture protocol (HYI-17-137-6) was approved by the Institutional Review Board of Hanyang Uni-versity. The following hPSCs were used: CHA-hES15 (hESCs; CHA Stem Cell Institute); CHA-hiPS NT2-S4 (hiPSCs; CHA Stem Cell Institute); heterozygous CHA-FT-NT17 (hetero-SCNT-hESCs; Research Institute for Stem Cell Research, CHA Health Systems); homozygous CHA-FT-NT18 (homo-SCNT-hESCs; Research Institute for Stem Cell Research, CHA Health Systems). hPSCs were cultured in TeSR^TM-E8^TM basal medium (Stemcell Technologies) with TeSR^TM-E8^TM 25X supplement (Stemcell Technologies) on Vitronectin XF^TM (Stemcell Technologies)-coated dishes under standard culture conditions (37℃, 5% CO₂). The culture medium was changed daily, and the cells were passaged using ReLeSR (Stemcell Technologies).

hPSC colonies were detached with 1 mg/ml collagenase type IV (Sigma-Aldrich), and colonies were transferred onto uncoated bacterial dishes in Essential 6^TM medium (Sigma-Aldrich) with 1% penicillin-streptomycin (10,000 U/ml, Thermo Fisher Scientific). The culture medium was changed every two days for five days.

Embryoid bodies (EBs) were transferred to culture dishes coated with 15 μg/ml poly-L-ornithine (PLO; Sigma-Aldrich) and 1 μg/ml fibronectin (FN; Sigma-Aldrich). The growth medium for the neural rosettes (1/2 N2 medium) was composed of serum-free medium supplemented with 0.5% 100X N2 supplement (Thermo Fisher Scientific). The 1/2 N2 medium was changed every two days for five days. After this period, the 1/2 N2 medium was replaced with another serum-free medium (N2bF medium) supplemented with 1% 100X N2 supplement and 40 ng/ml basic fibroblast growth factor (R&D System). The culture medium was changed every two days.

The generated neural rosettes were detached using a flame-pulled Pasteur pipette to maintain their mass form. The floating spherical neural masses (SNMs) were transferred to uncoated culture dishes containing the N2bF medium. The N2bF medium was changed every two days. For the purification of SNMs that grew 5∼7 days later, the SNMs were mechanically cut into 10∼15 pieces using a flame-pulled Pasteur pipette while removing the cystic portion within several minutes. Approximately 50 pieces of cut SNMs were transferred to uncoated culture dishes in N2bF medium. After 5∼7 days, the same method was repeated for passaging. The homogeneity of the SNM increased after to 3∼4 purification passages.

Approximately 50∼60 pieces of purified SNMs were collected and fragmented using 100 mesh (φ0.03×100×100 mm; The Nilaco Corporation) prior to being transferred onto PLO/FN-coated culture dishes in N2bF medium. The fragmented SNMs were expanded for several days to allow for adaptation. A few days later, to separate single cells the fragmented SNMs were dissociated using AccutaseTM (Sigma-Aldrich) for 5 minutes at 37℃ and centrifuged at 1,500 rpm for 3 minutes. The cell pellets were resuspended in N2bF medium and re-plated onto PLO/FN-coated culture dishes or onto glass coverslips (φ12 mm; Marienfeld Superior).

For spontaneous differentiation, neural precursor cells (NPCs) were seeded onto PLO/FN-coated dishes and cultured in serum-free differentiation medium (N2B27 medium) with 1% 100X N2 supplement, 50X B-27^TM Supplement (Thermo Fisher Scientific), and 200 μM ascorbic acid (AA; Sigma-Aldrich).

For astrocyte differentiation, NPCs were cultured in N2B27 medium containing 10 ng/ml ciliary neurotrophic factor (CNTF; PeproTech).

A previously published protocol was applied (20). NPCs were plated onto PLO/FN-coated dishes in N2B27 medium containing 40 ng/ml triiodothyronine (T3; Sigma-Aldrich), 200 ng/ml sonic hedgehog (Shh; R&D Systems), 100 ng/ml noggin (R&D Systems), 100 ng/ml IGF (R&D Systems), and 50 nM dibutyryl-cAMP (db-cAMP; Sigma-Aldrich). All cell culture media were changed every 2∼3 days.

Cell differentiation was initiated using N2B27 medium to induce dopaminergic (DA) neurons after NPC expan-sion. After two days, the cells were cultured in N2B27 medium with 200 ng/ml Shh and 100 ng/ml fibroblast growth factor 8 (FGF8; PeproTech) for 2 days. For terminal differentiation, 200 μM AA, 50 nM db-cAMP, and 20 ng/ml glial cell-derived neurotrophic factor (GDNF; R&D System) were added to the N2B27 medium with Shh (without FGF8).

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15∼20 minutes at room temperature. Cells were blocked and permeabilized with 10% normal goat serum (Pel-Freez) and 0.3% Triton X-100 (Sigma-Aldrich) in 0.1% bovine serum albumin/phosphate buffered saline (BSA/PBS) for 1 hour. Samples were incubated with primary antibodies at 4℃ overnight. The samples were then washed three times with 0.1% BSA/PBS and incubated with secondary antibodies at room temperature for 1 hour. The cells were counterstained with 4’,6-diamidino-2-phenylin-dole (Vector Laboratories) and mounted using a mounting solution. Images were captured using an epifluorescence microscope (LEICA Microsystems). The primary antibodies used in this study were as follows: anti-NESTIN (1：500; BioLegend), anti-SOX2 (1：1,000; Sigma-Aldrich), anti-ZO1 (1：50; Thermo Fisher Scientific), anti-PAX6 (1：200; Novus Biologicals), anti-TUJ1 (1：2,000; BioLegend), anti-TH (1：2,000; Pel-Freez), anti-glial fibrillary acidic protein (GFAP, 1：2,000; DAKO), anti-OLIG2 antibody (1：1,000; Chemicon), anti-major histocompatibility complex (MHC) Class I+HLA A/B (1：200, MHC-I; Abcam), anti-MHC Class II (1：200, MHC-II; Abcam). The secondary antibodies used were Alexa Fluor 488 (1：500; Ther-mo Fisher Scientific), Rhodamine, or Cy3 (1：500; Jackson ImmunoResearch Laboratories).

Prior to staining, cells were fixed with 4% paraforma-ldehyde for 10 minutes at room temperature. Alkaline phosphatases (AP) staining was performed using an Alkaline Phosphatase Kit (CA# 86R-1KT; Sigma-Aldrich), according to the manufacturer’s protocol.

Total RNA was extracted using TRI REAGENT (Molecular Research Center, Inc.), and cDNA was reverse transcribed from 1 μg of RNA using the Superscript Kit (Sigma-Aldrich). Polymerase chain reaction (PCR) amplification and primer conditions are listed in Table 1.

All experiments were conducted at least three times. Cell counting for quantification was performed in 5∼10 microscopic fields per well with three wells per experimental condition. All quantified data were statistically analyzed and are presented as mean±SEM. Statistical analyses were performed using SigmaPlot for Windows version 10.0 (SystatSoftware GmbH).

We conducted the following experiments to identify the characteristics of hPSCs at the pluripotent stage, including EB formation. First, AP is known to be expressed at high levels in pluripotent stem cells such as hESCs, hiPSCs, and SCNT-hSCs. Similar to previous reports, AP expression in the hPSCs used in this study was confirmed by AP staining. Representative AP staining of hPSCs 5 days after plating is presented. All cell lines tested positive (Fig. 1A). Next, we confirmed the possibility of differentiating the three germ layers. The pluripotency marker OCT4 decreased after EB induction in addition to the expression of the ectodermal marker PAX6, the mesodermal marker LMO2, the endodermal marker GSC, and the housekeeping gene GAPDH as confirmed by reverse transcription (RT)-PCR analysis at 14 days (Fig. 1B, Table 1).

In this study, we developed an efficient protocol to generate NPCs from several hPSCs in a time-efficient manner on a large scale (Fig. 2A). First, multiple detached hPSC colonies were cultured on uncoated bacterial dishes to form EBs that are the three-dimensional aggregates formed in suspension of hESCs, hiPSCs, and SCNT-hESCs. (Fig. 2B-2I). To generate neural rosettes, EBs were transferred onto PLO/FN-coated culture dishes, cultured in 1/2 N2 medium for 5 days, and then cultured in N2bF medium for an additional 5 days (Fig. 2A, 2J, 2J-2M). After neural rosette selection, the SNMs were isolated via mechanical dissection and cultured in suspension in culture dishes. Following 3∼4 purification passages, the neural rosettes disappeared, and an increased number of SNMs resembling neurospheres were formed (Fig. 2A, 2N, 2N-2Q). To differentiate the SNMs into neurons, astrocytes, and oligodendrocytes, the SNM fragments were transferred to PLO/FN-coated culture dishes. Thereafter, the SNM fragments dissociated into single-cell NPCs that were transferred onto PLO/FN-coated glass coverslips (Fig. 2A, 2R-2U). Thus, this method proved to be equally applicable to all hPSCs with the typical NPC morphology observed in all cell lines.

The characteristics of the NPCs at each stage were identified, including the rosettes, SNMs, and NPCs. Experiments were performed to detect differences in the production efficiency of NPCs from each cell line. Immuno-staining revealed that the neural rosettes expressed a wide range of early neuroectoderm and neural rosette markers such as PAX6 and ZO-1. The rosettes also exhibited a morphological appearance (Fig. 3A). As detected by SNM immunostaining, most NESTIN-expressing cells (green) coexpressed SOX2 (red), a neural precursor cell marker in the central nervous system (CNS) (Fig. 3B). After single-cell dissociated cultures of NPCs, NESTIN, and SOX2 proteins were detected by immunostaining (Fig. 3C), thus suggesting that hPSC-derived NPCs possessed proliferative capacity in all cell lines.

We estimated the differentiation efficiency of the hPSC-derived NPCs. The hPSC-derived NPCs expressed TUJ1, a marker observed spontaneously in early neurons, was detected (Fig. 4A). NPCs were then cultured in a medium containing CNTF and T3 that promoted the formation of astrocytes and oligodendrocytes, respectively. In agreement with this, astrocyte and oligodendrocyte markers such as GFAP and OLIG2 were readily detectable (Fig. 4A). Overall, hPSC-derived NPCs were observed to possess efficient differentiation capabilities for neurons and glial cells; however, oligodendrocyte differentiation efficiencies were slightly lower (Fig. 4B).

The efficient generation of NPCs from hPSCs was observed in all cell lines in addition to their differentiation into neurons, astrocytes, and oligodendrocytes that constitute a large proportion of the CNS. To produce DA neuronal cultures, we optimized a DA neuron differentiation protocol (Fig. 5A) (1). The DA identity of the induced cells was confirmed by the expression of TUJ1 and the rate-limiting enzyme tyrosine hydroxylase (TH; Fig. 5B, 5C). As a result of DA differentiation induction, no difference in differentiation efficiency was observed between homo-SCNT-hESCs and the other cell lines. In conclusion, we expect homo-SCNT-hESCs to be useful, as they can be applied to a greater number of patients.

MHC expression was analyzed by differentiating hPSCs into neurons. In CHA15 hESC, hetero-SCNT-ESC, and homo-SCNT-ESC, almost no MHC expression was observed in the undifferentiated state; however, MHC expression began to increase from the EB stage. In differentiated neurons, MHC I and II expression was maintained (Fig. 6).