Comparison of methods for deriving neural progenitor cells from nonhuman primate embryonic stem cells

rhESCs (obtained ethically and provided by Anthony Chan’s laboratory at the Yerkes National Primate Center, GA, USA; any protocols involving animal care and handling were previously approved by Emory University’s Institutional Animal Care and Use Committee) were maintained in media composed of knockout-Dulbecco’s modified Eagle’s medium (KO-DMEM) supplemented with 20% Knock-out Serum Replacement (KSR; Invitrogen, Carlsbad, CA, USA), 1 mM glutamine, 1% nonessential amino acids and 4 ng/mL of human basic fibroblast growth factor (bFGF; Chemicon, Temecula, CA, USA). Half of the ESC medium was replaced every other day. ESC colonies were passaged by mechanically dissociation into small clumps and plated on a freshly prepared mitomycin C inactivated mouse embryonic fibroblasts (MEFs).

rhESCs were mechanically passaged and grown without feeder cells on 35 mm tissue culture plates coated with 20 μg/mL poly-L-ornithine/1 μg/mL laminin (P/L) in MEF conditioned medium. MEF-conditioned medium was ES medium used for the culture of MEFs for 24 h. After 4 to 5 mechanical passages in the feeder-free culture, ESCs were dissociated using 0.25% trypsin-EDTA and then plated on P/L-coated dishes. Cells were then cultured in rhNPC induction medium with DMEM/F12 supplemented with N2 supplement (Invitrogen), 4 ng/mL bFGF, and 2 mM L-glutamine for 2 weeks. Cultures were fed with fresh medium every day. At day 14, cells were dissociated by Accutase (Life Technology, Carlsbad, CA, USA) for 5 min at 37°C followed by culturing in neural proliferation medium composed of Neurobasal A medium (Invitrogen) supplemented with 20 ng/mL bFGF, 10 ng/mL LIF (Sigma, St. Louis, MO, USA) and B27 supplement (Invitrogen). Spent medium was replaced with fresh medium every other day, and NPCs were passaged at 80%–;90% confluency.

rhESC colonies were mechanically dissected into small clumps and removed from MEF feeders. The rhESCs clumps were then cultured in suspension in 35 mm petri dishes for 6 to 7 days to form EB in ES culture medium without bFGF. Spent medium was replaced with fresh medium every other day. EBs were then transferred into 35 mm culture dishes coated with P/L (10-20 EBs per dish) and cultured in a preinduction medium composed of DMEM/F12 media supplemented with 0.5 × N2 supplement and 2 mML-glutamine for 3 days. On day 4 of the differentiation, the cells were induced by differentiation into rhNPC by using the same induction medium as described for adherent feeder-free culture. After 6 to 7 days, rosette-like structures were manually picked up, transferred onto a P/L-coated dish, and cut into small pieces for culture. For expansion, the rosette-like structures were incubated in Accutase and then cultured in a neural proliferation medium.

To determine the neuronal differentiation potential of rhNPCs derived by the 2 different methods, NPCs (about 3 × 10⁴ cells) were plated onto P/L coated 35 mm dishes for neural differentiation [13]. NPCs were first cultured in neural differentiation medium composed of DMEM/F12 supplemented with N2 (1:100; Invitrogen) and 1 × B27 supplement (1:50; Invitrogen) for 4 days. On day 5, 200 ng/mL SHH (R&D Systems, Minneapolis, MN, USA) and 100 ng/ mL FGF8 (R&D Systems) were added. On day 8, 160 μM ascorbic acid was added into the medium until day 14. Spent medium was replaced with fresh medium every other day.

The cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4, for 15 min. The fixed cells were washed for 5 min, 3 times in PBS and incubated with blocking buffer consisting of 0.2% Triton X-100 (for intracytoplasmic markers) (Sigma), 3 mM sodium azide, 0.1% saponin (Sigma), 2% bovine serum albumin (Sigma), and 5% horse serum (HyClone, Logan, UT, USA) in PBS (–) for 30 min. To detect NPC, the cells were incubated with primary antibody in PBS for 8 h. The NPC markers were nestin (Chemicon), Musashi, and SOX-2. After being washed 6 times for 5 min each with PBS, the cells were incubated with fluorescent-tagged secondary antibodies in PBS for 2 h followed by washes for 5 times with 5 min each, and the cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI). After staining, the specimens were mounted on slides and examined by using fluorescence microscope.

Total RNA was extracted from early passages (P2 and P3) of rhNPCs cell lines using TRIzol reagent (Ambion, Carlsbad, CA, USA) and subsequently treated with Turbo DNase (Ambion). RNA concentration was determined by using a NanoDrop spectrophotometer (ThermoFisher, Waltham, MA, USA). Three micrograms of RNA were reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. The level of expression of 14 genes containing 6 NPC specific genes (Sox2, Pax6, Nestin, Musashi, TLX, and NPDC1), endoderm specific genes (AFP TTR, and HNF1B), mesoderm specific genes (GATA4, VEGFA, and RUNX1), and astrocyte markers (GFAP and S100a) were determined by quantitative real-time polymerase chain reaction (RT-PCR) using Taq Man Gene Expression Master Mix (Applied Biosystems; ABI) and TaqMan gene expression primers on CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). The relative levels of gene expression of the target RNA were normalized against GAPDH expression. The plotted data represented mean values of at least 3 independent replicates ± standard error of the mean (SEM), and the statistical significance was determined by using one-way ANOVA with a generalized linear model procedure by SAS Institute software (Cary, NC, USA).

The NPCs were dissociated into single cells using 0.25% trypsin and washed with PBS without Ca²⁺ or Mg²⁺. The cells were harvested (1 × 10⁶ cells per sample) in a cold fluorescence-activated cell sorting (FACS) wash buffer and were permeabilized using BD-Perm-2 for 10 min in the dark. The samples were stained with anti-Pax6 or anti-nestin antibodies at 1:100 for 30 min at 4°C in the dark followed by submersion in the appropriate Alexa Fluor 488-conjugated secondary antibody for 30 min. The control group was prepared in parallel by staining cells with secondary antibody without primary antibodies. After being washed in FACS wash buffer, the cells were fixed in 1% PFA. Flow cytometric analysis was performed on an BD FACS Calibur (BD Bioscience, Franklin Lakes, NJ, USA), and the data was analyzed using FlowJo software.

To compare the efficiency of deriving NPCs by feeder-free and EB derivation methods, we first examined the emergence of a neuroepithelial structure or neural rosette. In feeder-free adherent culture, the first sign of neural differentiation emerged at day 8 in the presence of N2 supplement and bFGF. The neural rosettes had a radial arrangement of elongated columnar cells as the signature morphology of neuroprogenitor cells in culture (Figure 1D). At day 14 in culture, these cells had developed an NPC-like bipolar structure, a typical morphology of NPCs (Figure 1E), and continued to be cultured for 1 week in a neural proliferation medium. In the EB method, ES cell clumps were cultured in suspension in low-adherent petri dishes to allow the formation of cell aggregates called EB (Figure 2B). The EBs were transferred onto P/L-coated dishes and cultured in induction medium. The development of compact columnar cells emerged from the EBs and formed neural rosette-like structures at 5 to 7 days after induction (Figure 2C). One of the major challenges in deriving NPCs was to eliminate non-NPCs. The rosette-like structures were identified based on morphology and mechanically isolated from the culture (Figure 2D). The rosette-like structures were then cut into small pieces for expansion in neural proliferation medium. More than 30 NPC lines were derived using the 2 approaches. The morphology of NPCs derived by the 2 methods was similar (Figure 3A). This result demonstrated that the 2 NPC derivation methods (feeder-free and EB methods) can derive NPC-like cells with no phenotypic difference in morphology.

Figure 1

Figure 2

Figure 3

Immunostaining confirmed the expression of NPC markers by NPCs derived by either method (Figure 3A). A similar pattern of expression of the NPC markers (Sox2, Pax6, nestin and Musashi1) and neural proliferation differentiation and control-1 (NPDC1), which is specifically expressed in the nervous system and neural cells in culture when they stop dividing and start to differentiate [14] was observed in rhNPCs derived by either method when compared with ESCs (Figure 3B). These results suggested that the 2 derivation methods were sufficient to generate NPCs with similar neuroprogenitor properties.

Flow cytometry revealed 99.1% of NPCs cells derived using the feeder-free method and 99.5% of NPCs cells derived using the EB method were positive for Pax6, while 97.0% of NPCs from the feeder-free method and 99.4% of NPCs cells from the EB method were positive for nestin (Figure 3C). These results suggested that NPCs with high homogeneity can be produced by either method.

To determine whether NPCs can be differentiated to neurons in vitro, FGF2 and LIF were withdrawn from the culture media, while N2, SHH, FGF8, and ascorbic acid were supplemented to enhance neural differentiation. By the end of the second week of in vitro neural differentiation, differentiated cells from EB derived NPCs developed branches and elongated spindle processes (Figure 4B). Cells from either method showed upregulation of neuron-specific microtubule associated protein (MAP2) expression when compared with NPCs (Figure 4E). Immunoreactivity further confirmed the expression of MAP2 (Figure 4C andD). These results suggest that NPCs derived by either method are capable of differentiating into neurons.

Figure 4

To achieve a homogenous culture of NPCs is one of the major challenges for their application. To determine the homogeneity of NPCs and their commitment to neural lineage, we examined the patterns of expression of nonneural lineage markers, which include markers for mesoderm (e.g., GATA4,RUNX1, and VEGFA) and endoderm (e.g., AFP, TTR, and HNF1B).All markers tested were downregulated when compared with their level of expression in undifferentiated ESC. Although, RUNX1in cells from the feeder-free method was upregulated, the upregulation was not significantly different from the level of expression in ESC (Figure 5A). The lack of significant upregulation of nonneuronal genes indicated that these 2 NPC derivation methods are not favoring nonneural cell lineage differentiation. The level of expression of glial fibrillary acidic protein (GFAP) in NPCs derived by the feeder-free method was significantly higher than that in NPCs derived by the EB method at early passages (Figure 5B). Immunoreactivity confirmed the expression of GFAP in some of the feeder-free derived NPCs (Figure 5C). By contrast with GFAP, an alternative astrocytic marker that is a late marker of astrocyte development [15], S100β was not upregulated in NPCs compared with ESCs, and there was no difference between NPCs derived by either method (Figure 5B). These findings indicate that the feeder-free conditions promote early differentiation of astrocytes.

Figure 5

Several studies reported that the nuclear receptor tailless (TLX) plays an important role in vertebrate brain functions [16,17]. TLX belongs to the nuclear receptor, NE2E orphan nuclear receptor family. TLX is expressed in the neuroepithelium of developing central nervous system [16] and acts as a repressor of cell cycle inhibitors to maintain the undifferentiated state, self-renewal, and proliferation of adult progenitor cells from rodents [18]. In addition, TLX suppresses the expression of GFAP to inhibit astrocyte differentiation and to activate neuronal lineage commitment [3,16]. Therefore, we examined if the downregulation of TLXresulted in the upregulation of GFAP expression in NPCs. As shown in Figure 5D, the expression of TLXin the NPCs derived by the feeder free-method was significantly lower than that of those derived by the EB method. This finding was consistent with the higher GFAPexpression in feeder-free group, and further suggested the astrocytic tendency of feeder-free derived NPCs.