Generation and prolonged culture of NSCs

To derive NSCs, hESC colonies were harvested using a scraper with care taken to avoid high colony fragmentation. Colonies were cultured in suspension as EBs on Petri dishes with agitation for 8 days in StemPro defined media minus FGF2. EBs were then cultured for additional 2–3 days in suspension in neural induction media containing DMEM/F12 with Glutamax, 1×NEAA, 1×N2 and FGF2 (20 ng/ml) prior to attachment on cell culture plates coated with CellStart. Numerous neural rosettes were formed 2–3 days after adherent culture. To obtain a pure population of NSCs, rosettes were manually isolated using stretched glass Pasteur pipette and placed in fresh culture dishes. The rosettes were then dissociated into single cells using accutase and replated onto culture dishes to obtain a homogeneous population of NSCs. The NSCs population was expanded in Neurobasal media containing 1×NEAA, 1×L-Glutamine (2 mM), 1×B27, LIF, FGF2 20 ng/ml.

To confirm that NSCs can differentiate into astrocytes, NSCs were cultured in DMEM/F12 medium supplemented with 1×NEAA, L-Glutamine (2 mM), 1×N2 and 1×B27 for two weeks and processed for immunostaining. To initiate oligodendrocyte differentiation, NSCs were cultured in medium containing DMEM/F12, 1×NEAA, 1×L-Glutamine (2 mM), 1×N2, 1×B27, Shh (200 ng/ml, R&D Systems) and retinoic acid (RA, 2 µM) for 10 days, and then with T3 (30 ng/ml, R&D Systems) and PDGFα (10 ng/ml, R&D Systems) but without Shh and RA for an additional 2 weeks.

Dopaminergic differentiation of NSCs

Dopaminergic differentiation was obtained by culturing NSCs in medium conditioned on PA6 cells (PA6-CM) [23] for 3–6 weeks, or in xeno-free defined media for 4–5 weeks on culture dishes or glass cover slips coated with Poly-L-ornithine (20 ug/ml) and laminin (10 ug/ml). Dopaminergic differentiation in defined media was initiated by culturing NSCs for 10 days in Neurobasal medium supplemented with 1×NEAA, 1×L-Glutamine (2 mM), 1×B27 (Gibco), Shh (200 ng/ml, R&D Systems) and FGF8 (50–100 ng/ml, R&D Systems). Shh and FGF8 were then withdrawn and replaced with BDNF (20 ng/ml, R&D Systems) and GDNF (20 ng/ml, R&D Systems), TGF-β3 (1 µM, R&D Systems) and dcAMP (1 mM, Sigma) for 10–25 days.

Immunocytochemistry

Immunocytochemistry and staining procedures were as described previously [24]. Briefly, hESCs at different stages of dopaminergic differentiation were fixed with 2% paraformaldehyde for half an hour. Fixed cells were blocked in blocking buffer (10% goat serum, 1% BSA, 0.1% Triton X-100) for 1 hour followed by incubation with the primary antibody at 4°C overnight in 8% goat serum, 1% BSA, 0.1% Triton X-100. Appropriately coupled secondary antibodies (Molecular Probes) were used for single and double labeling. All secondary antibodies were tested for cross reactivity and non-specific immunoreactivity. The following primary antibodies were used: Oct4 (ab19857 AbCam) 11000; β-Tubulin isotype III clone SDL.3D10 (T8660 Sigma) 1500; GalC (MAB342 Millipore) 150; GFAP (M0761 Chemicon) 150; Nestin (611658 BD Transduction laboratories) 1500; Sox1 (AB5768 Chemicon) 1200, TH (Pel-Freeze P40101) 1500, TH clone TH-16 (T2928 Sigma) 11000; Lmx1a (a kind gift from Dr. Michael German, UCSF) 11500, Girk2 (AB5200 Chemicon) 1250; Vmat2 (PPS089 R&D) 11000, GABA (ab8891 Abcam) 1500, L-Glutamate (ab8889 Abcam) 1500, DBH (AB1585 Chemicon) 11000, HB9 (81.5C10 DSHB) 150, and as secondary antibodies: Alexa Fluor 488 Goat Anti-Mouse, Alexa Fluor 594 Goat Anti-Mouse, Alexa Fluor 488 Goat Anti-Rabbit, Alexa Fluor 594 Goat Anti-Rabbit. Hoechst 33342 (Molecular Probes H3570) 11000 was used for nuclei identification. Images were captured on a Nikon fluorescence microscope.

The quantification of TH⁺ cells in culture was performed by analyzing fluorescent images using Photoshop on a minimum of 5000 cells of at least 10 randomly chosen fields derived from 3 or more independent experiments. The number of Hoechst labeled nuclei on each image was referred as total cell number (100%).

Gene expression and PCR analysis

RNAs isolated from NSCs and dopaminergic populations were hybridized to Illumina HumanRef-8 BeadChip (Illumina, Inc, performed by Microarray core facility at the Burnham Institute for Medical Research). The Illumina array data were normalized by the quantile method, and then transformed log2 ratio values for a zero mean for expression values of each gene across all samples. The bioinformatics and statistical analyses were conducted by using R and the bioconductor package (www.bioconductor.org).

cDNA was synthesized by using a reverse transcription kit SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's recommendations. Real-time PCR was used to quantify the levels of mRNA expression of 10 genes in NSC and day 16 and 31 dopaminergic populations. PCR reactions were carried out by ABI 900HT instrument according to the manufacturer's instructions. All qRT-PCR primer sequences are shown in supplementary Table S1.

Transplantation of hESC-derived dopaminergic neurons into rat brains and histological analysis

Adult female Sprague-Dawley rats (200–230 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). Four rats were injected with day 20 (after the NSC stage) hESC-derived neuronal populations (approximately 500,000 cells in 5 µl of cell preparation medium) or vehicle with a Hamilton syringe into the striatum (−0.3 mm anterior to bregma, 3.0 mm lateral to midline, and 5.0 mm beneath dura). Cyclosporine A (10 mg/kg; Novartis Pharmaceuticals) was intraperitoneally injected 24 hours before transplantation and every day afterwards until the rats were sacrificed. Experimental protocols were in accordance with the National Institutes of Health Guidelines for Use of Live Animals and were approved by the Animal Care and Use Committee at the Buck Institute. Rats were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde.

For histological analysis, brains were removed and immersion-fixed overnight at room temperature. Brains were then dehydrated in graded ethanols, cleared in xylene, and paraffin-embedded [25]. Seven µm-thick serial coronal sections were cut and mounted on glass slides, which were dried overnight at 42°C. Sections were deparaffinized, rehydrated through a graded series of ethanols, and washed in water. For immunostaining [26], sections were incubated with blocking solution (2% horse serum, 1% bovine serum albumin, and 0.1% Triton X-100 in phosphate-buffered saline, pH 7.5) and then with primary antibodies at 4°C overnight followed by secondary antibodies in blocking solution at room temperature for 2 h. The primary antibodies used were rabbit anti-TH polyclonal antibody (Chemicon, 1500) and mouse polyclonal anti-human nuclear antigen (Chemicon, 1300). The secondary antibodies were rhodamine-conjugated rat-absorbed donkey anti-rabbit IgG (Jackson ImmunoResearch; 1200) and fluorescein isothiocyanate-conjugated pig anti-mouse IgG (Vector Laboratories; 1200). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) using proLong Gold anti-fade reagent (Invitrogen). To quantitatively analyze double labeled neurons in the striatum, fluorescence signals were detected with an LSM 510 NLO Confocal Scanning System mounted on an Axiovert 200 inverted microscope (Carl Zeiss Ltd.) equipped with a two-photon Chameleon laser (Coherent Inc.). Three-color images were scanned using Argon and 543 HeNe lasers. IMARIS (Bitplane AG.) imaging software was used for three-dimensional image reconstruction. Images were acquired using LSM 510 Imaging Software (Carl Zeiss Ltd.) as described previously [27]. The specificity of each label was first verified using single-channel scans that were then merged into multiple-channel views. Neurons were considered double-labeled if co-labeling with relevant morphology was seen throughout the extent of the nucleus for nuclear markers or if a cytoplasmic marker surrounds a nuclear marker when viewed in x-y cross section as well as in x-z and y-z cross-sections produced by orthogonal reconstructions from z-stacks taken at 400× magnification. Human antigen single-labeled neurons and neurons double labeled for TH and human antigen were recorded in every seventh section per animal (n=3). The area of each transplanted region was simultaneously determined for each of the scored sections.

Prolonged culture of NSCs in defined conditions does not diminish their ability to differentiate

To test whether NSCs could be propagated in a defined medium for a prolonged period and retain the capacity to differentiate into neurons and glia, we continuously passaged the cells in the defined medium (NSC medium) on defined substrate for over 2 months. As seen in Fig. 2A, NSCs propagated in defined conditions for 15 passages expressed Sox1 and nestin, and differentiated into β-III-tubulin⁺ neurons, GFAP⁺ astrocytes and GalC⁺ oligodendrocytes in conditions promoting differentiation (Fig. 2B–C). In addition, these cells could be frozen and thawed with greater than 95% viability and without losing the ability to differentiate into neurons (including dopaminergic neurons) and glia.

Open in a separate window

Figure 2

NSCs cultured in xeno-free defined medium for a prolonged period remain to be multipotent.

(A) NSCs propagated in defined conditions for 15 passages retained NSC identity as evident by expressing Sox1 and nestin. (B–C) NSCs can be frozen and thawed and maintained the capacity to differentiate into neurons (B), oligodendrocytes (C) and astrocytes (D).

NSCs cultured for prolonged periods retain their ability to differentiate into dopaminergic neurons upon exposure to PA6-CM or in a xeno-free defined medium

Our laboratory has reported dopaminergic inducing activity of medium conditioned on stromal cell line PA6 (PA6-CM) and shown that exposure to PA6-CM at a defined time period (NSC stage) was sufficient for a dopaminergic fate [23]. We therefore tested whether NSCs grown to passages 15–25 in defined conditions could differentiate into dopaminergic neurons in PA6-CM. As shown in Fig. 3, dopaminergic neuronal induction by PA6-CM was efficient, as numerous β-III tubulin⁺ neurons (A) and TH⁺ cells (B) were present in the cultures after 3 weeks of differentiation. The average percentage of TH⁺ neurons was 35±5% at day 20.

Open in a separate window

Figure 3

NSCs from prolonged culture in defined conditions can be efficiently differentiated into midbrain dopaminergic neurons.

(A–E) Efficient differentiation into midbrain dopaminergic neurons by PA6-CM as shown by immunocytochemistry. The majority of the cells expressed β-III-tubulin and TH after 4 weeks of differentiation in PA6-CM (A–B). Co-expression of midbrain and A9 markers in TH⁺ dopaminergic neurons: Lmx1a (C), VMAT (D) and Girk2 (E). (F) Differential expression of dopaminergic markers in several stages of differentiation (NSC, dopaminergic precursors and dopaminergic neurons) by quantitative PCR. All the examined markers were up-regulated in dopaminergic populations compared to NSCs.

To further test if cells generated under these conditions were authentic midbrain dopaminergic neurons of the A9 type, we first examined the expression of additional midbrain dopaminergic markers in our cultures by immunocytochemistry. After 5 weeks of differentiation, a high percentage of the cells expressed Lmx1a, a transcription factor involved in dopaminergic development. Many TH⁺ neurons co-expressed Lmx1a (Fig. 3C). VMAT was also co-expressed in the majority of TH⁺ neurons (Fig. 3D). Importantly, Girk2, a marker of nigral neurons, was co-expressed in many TH⁺ neurons (Fig. 3E). The expression of a few other neuronal markers in our culture was also analyzed by immunostaining. We did not see DBH-positive cells in our culture and found a very small percentage of the cells (<1%) expressed GABA (Supplementary Figure S1). No glutamate or HB9 immunoreactive cells were found in the culture.

We then used real time PCR to examine the expression of a panel of 10 markers (En1, Otx2, Msx1, Nurr1, Lmx1b, AADC, Aldh1a, VMAT, DAT and Girk2) during dopaminergic differentiation (NSCs, day 16 and day 31). As seen in Fig. 3F, many of these genes (En1, Msx1, Nurr1, Lmx1b, AADC, VMAT, DAT and Girk2) were differentially upregulated in both day 16 and day 31 dopaminergic populations compared to the NSC population.

Finally we performed a large-scale microarray for gene expression profiling of the day 32 dopaminergic population. As summarized in Table 1, many midbrain and dopaminergic neuronal markers such as EN1, Pax2, Otx2, Msx1, Lmx1a, Ngn2 and Nurr1, transcription factors that were believed to be important in regulating dopaminergic differentiation, were detected in the day 32 dopaminergic population. These results indicate that our culture conditions can efficiently generate authentic A9 type neurons.

Since PA6 is a mouse stromal cell line and PA6-CM is not defined, in a separate set of experiments we identified a candidate set of factors that may substitute for PA6-CM in cultures (unpublished data). Given the present results, we tested whether the effects of PA6-CM could be mimicked with a combination of growth factors in a defined medium base. Our gene expression data and preliminary experiments indicated that Shh and FGF8 may substitute for PA6-CM as a source of dopaminergic inducing activity (Step 3: NSC→dopaminergic precursor) and that TGFβ, GDNF and BDNF may mimic the effect of dopaminergic neuron maturation and survival (Step 4: dopaminergic precursor→dopaminergic neuron). To test this, NSCs were exposed to Shh and FGF8 in a defined medium (NSC defined medium minus FGF2 with the addition of Shh and FGF8) for 10 days followed by 18 days of culture in the presence of TGFβ, GDNF and BDNF without Shh and FGF8, in the same basal serum-free defined medium (Fig. 4A). Under these conditions, approximately 25% of the cells expressed TH (Fig. 4B). The efficiency of this dopaminergic differentiation is comparable to that observed with exposure to PA6-CM for 3 weeks. These results indicate that Shh and FGF8 could replace PA6-CM in the step of dopaminergic induction from the NSC stage and that BDNF, GDNF and TGFβ were sufficient to maintain the differentiated population.

Open in a separate window

Figure 4

Four-step differention into midbrain dopaminergic neurons in a completely defined medium.

(A) A schematic diagram of the four-step (ESC→propagation and storage of NSC→induction of a midbrain dopaminergic precursor population→maturation of the precursor to dopaminergic neurons) differentiation protocol for generation of dopaminergic neurons in completely defined media. (B–D) Defined medium adapted NSCs differentiated into dopaminergic neurons in defined media.

Survival of dopaminergic neurons in vivo

To examine the functional properties of dopaminergic neurons derived from ESCs/NSCs cultured in defined medium, we transplanted cells that had been differentiated for 20 days (after the NSC stage) into the striatum of rats. Immunohistochemical analysis was performed 2 and 7 weeks after grafting and donor cells of human origin (positive for human nuclear antigen) were found in all brains throughout the graft sites (Fig. 5A–D). A small number of TH⁺ human cells were identified in the brain at the graft site 2 weeks post transplantation, and the numbers of TH⁺ human neurons were increased in brains 7 weeks after grafting (Fig. 5E–H). These results indicate that dopaminergic neurons generated from ESCs/NSCs cultured in defined medium could survive and integrate into the brain in vivo for at least 7 weeks, the duration of the present study. At 7 weeks post-transplantation, histological analyses showed that 2,579±581 cells/mm³ were TH-positive neurons among the 110,193±19,746 cells/mm³ in these grafts.

Open in a separate window

Figure 5

NSCs differentiated into functional dopaminergic neurons in vivo.

Day 20 cells (after the NSC stage) were transplanted into the striatum of rats. Differentiation and survival of donor-derived dopaminergic neurons are seen in striatum two (A–D) and seven (E–H) weeks post of transplantation. Transplantation of day 20 cells in 6-OHDA rats showed a subset of human dopaminergic neurons, which survived eight weeks post of graft (I–L). Rotary tests showed amelioration of behavioral deficits in PD rats transplanted with donor-derived dopaminergic neurons (M).

To further demonstrate that dopaminergic neurons grown in our culture conditions contained functional dopaminergic neurons, we transplanted the cells (20 days after the NSC stage) into eighteen 6-OHDA rats and showed that they can ameliorate behavioral deficits in PD rats. As seen in Fig. 5M, eight control rats that were transplanted with medium showed no attenuation of rotary behavior over the course of the experiment, whereas rats that were transplanted with grafts demonstrated significant rotational improvement at 12 and 16 weeks after transplantation (p<0.05). Human antigen-immunopositive cells co-expressing TH were found in all brains throughout the graft sites at 8 weeks following the transplantation (Fig. 5I–L).

Completely serum- and xeno-free defined culturing of hESCs and NSCs

Examining our protocols we noted that while our simplified protocols for hESC culture, deriving NSCs, and inducing and maintaining dopaminergic neurons worked well, both the ESC medium and NSC medium used contained one xeno component, BSA. We therefore tested if cells could be grown in a completely humanized base medium with hKSR and the growth factors (Invitrogen) present in the defined medium, and could be differentiated appropriately. Fig. 6A shows an Oct4⁺ hESC colony cultured in this completely xeno-free defined medium for 7 passages. Similar to ESCs cultured in ESC defined medium with BSA, NSCs could be effectively generated from ESCs adapted to this completely xeno-free medium (Fig. 6B–C). Moreover, these NSCs retained the capacity to differentiate into dopaminergic neurons when exposure to PA6-CM or Shh and FGF8 (Fig. 6D). No apparent differences were found between NSCs generated under this xeno-free condition and the defined conditions described above in terms of dopaminergic differentiation efficiency. These results suggest that NSCs and dopaminergic neurons can be generated under xeno-free defined conditions from hESCs adapted to xeno-free defined medium and substrate, an important step for potential clinical applications of hESC-derived cells.

Open in a separate window

Figure 6

Completely serum- and xeno-free defined culturing of hESCs and NSCs.

Morphology and expression of Oct4 (A) in hESCs that were cultured in xeno-free medium (hKSR with growth factors) for 7 passages. NSCs derived from xeno-free cultured hESCs expressed nestin and Sox1 (B–C) and could differentiate into dopaminergic neurons (D).