Derivation of Functional Oligodendrocyte Progenitor Cells (OPCs) from Human Neural Stem Cell Lines

Human neural stem cells (hNSCs) have been intensely studied for their therapeutic potential in neurodegenerative diseases and spinal injuries. Depending on the microenvironment and growth stimuli, these cells can undergo neurogenesis and replace damaged cells. In the central nervous system, neurogenesis alone is not sufficient to repair neuronal damage. Functional neurons need to be protected by a myelin sheath, which is synthesized by oligodendrocytes, in order to successfully deliver a long-distance action potential. Understanding oligodendrocyte development is a necessary milestone towards developing clinical and pharmaceutical treatments for neurodegenerative pathologies such as multiple sclerosis.

In general, hNSCs generate low and inconsistent numbers of oligodendrocytes in vitro. To develop a model system for cultured oligodendrocytes, three neural stem cell lines were tested for their ability to differentiate into oligodendrocyte progenitor cells (OPCs). Two of the cell lines responded to a differentiation regime that included small molecules and growth factors, PDGF, NT3, thyroid hormone (T3) and FGF2. Both cell lines exhibited a 30 to 50% increase in the OPC populations as measured by immunofluorescent staining for multiple oligodendroglial markers: NG2, O4, Sox10 and GalC. These OPCs could be further differentiated to mature oligodendrocytes under mitogen-free conditions. Our data shows that these cells maintain stable OPC characteristics for at least three passages and expressed multiple oligodendrocyte markers upon differentiation. Using a co-culture system, we show that these human OPCs developed into mature oligodendrocytes that produced MBP around the axon in primary rat neuron cultures.

Methods

Human Neural Stem Cell Culture

Two human immortalized neural stem cell lines, ReNcell™ VM (SCC008) and ReNcell™ CX (SCC007) were used along with the ENStem-A™ human ESC-derived neural progenitor cell line (SCR055). Neural stem cells were cultured as monolayers using ReNcell maintenance medium (SCM005) freshly supplemented with 20 ng/mL FGF-2 or ENStem™-A expansion medium (SCM004) freshly supplemented with 20 ng/mL FGF-2 and 2 mM glutamine. ReNcell lines were cultured on 20 μg/mL laminin (L2020) coated flasks while hESC-NPC cells were cultured on Matrigel coated flasks. All cells were maintained in a 5% CO₂, 37 °C tissue culture incubator, enzymatically passaged with Accutase^® solution (A6964) and seeded at 1X10⁵/cm² density until they reached 70–80% confluency.

Oligodendrocyte Differentiation and Maturation

Human neural stem cells were harvested and resuspened in their respective growth media at 5 x 10⁵ cells/mL on ultra-low adhesion plates (CLS3471) to form neurospheres. The neurospheres were treated with retinoic acid (R2625) before being switched to oligodendrocyte enrichment medium (OEM: DMEM/F12 with 1X NEAA, 2 mM glutamine, N21 medium supplement, a cocktail of growth factors (thyroid hormone (T3, T5516); recombinant human PDGF-AA (SRP3268), recombinant human neuro­trophin-3 (N1905), and recombinant human basic FGF2 (F0291). Neurospheres continued to be cultured in OEM for several passages before being plated on a Matrigel-coated flask for expansion in OPC expansion media (SCM107). Seeding density for enriched OPC cultures was 1–2 x 10⁴ cells/cm².

Oligodendrocyte Progenitor Cell Characterization

10⁴ cells/cm² OPCs were plated on 0.1% poly-L-ornithine (P4957) and 10 μg/mL laminin (L2020) coated Millicell^® EZ SLIDES (PEZGS0416) or T25 flasks and cultured overnight before switching to growth factor-free medium. Oligodendrocytes were allowed to mature for 2 to 3 weeks before being fixed with 2% paraformaldehyde or lysed for RNA extraction.

Proliferating OPCs were characterized with antibodies specific to early oligodendrocyte markers, including anti-Sox10 (AB5727, 1:200), anti-NG2 (AB5320, 1:200), anti-Olig-2 (AB9610, 1:100), and anti-O4 (MAB345, 1:50) while two-week differentiated oligodendrocytes were characterized with antibodies to intermediate to late oligodendrocyte markers, including anti-GalC (MAB342, 1:200), anti-PLP-1/DM20 (MAB388, 1:100), anti-MBP (05-675, 1:25), anti-MOG (MAB5680, 1:25), anti-CNPase (1:100), and anti-RIP (1:25). Astrocytes were labeled with anti-GFAP (MAB3402, 1:250) and neurons were labeled with anti-bIII tubulin (MAB1637, 1:100) or anti-MAP2 (MAB3418, 1:200).

In Vitro Myelination Assay

12 mm cover glasses were acid-treated with 1N HCl (H1758) overnight, rinsed with Milli-Q^® H₂O (MILLIQ) and then sterilized with autoclaving. Cover glasses were air dried before being coated with 0.1% poly-L-lysine (P8920) at room temperature for 1 hour and then washed with sterile distilled water. 1 x 10⁵ primary E19 rat hippocampal neurons (R886N-10) were plated on the poly-L-lysine coated cover glass in each well of a 4-well plate in Dulbecco’s Modified Eagle Medium (DMEM) with 10% heat-inactivated horse serum. 24 to 48 hours after plating, cells were transfected with 1 μg of pCAG-GFP plasmid following the manufacturer’s protocol. 24 hours post-transfection, approximately 10–20% of the primary neurons were GFP-positive. Four days after transfection, 2 x 10⁴ human OPCs were seeded to the plate and the neuronal culture medium was replaced with the Human OPC Spontaneous Differentiation Medium (SCM106). Human OPCs and rat primary neuron co-culture was left at 37 °C in a 5% CO₂ tissue culture incubator for 21 days with medium changes every 2 to 3 days.

Results

Three neural progenitor cell lines were tested for OPC enrichment. Among them, the ENStem-A ESC-derived NPC line and the immortalized human fetal NSC line (ReNcell VM) contained a significant number of A2B5- and GalC-positive cells in OEM culture. In contrast, the immortalized human cortical NSC line (ReNcell CX) did not demonstrate a significant difference in marker expression between the parental NSC cell line and the directed differentiation conditions (data not shown).

Figure 1 shows the quantitative and representative images of human NSCs cultured in original medium (parental) or in oligodendrocyte enrichment medium (OEM). ENStem- A cells (Figure 1A and B) displayed moderate increases in the O2A progenitor marker, A2B5, and a 3-fold increase in the intermediate oligodendrocyte marker, GalC in OEM culture. In contrast, ReNcell VM cells (Figure 1C and D) cultured in OEM demonstrated more than 6-fold increases in A2B5 expression and less than 2-fold increase in the GalC expression. This illustrates that, even when subjected to the same enrichment procedure, each NSC line responds differently to small molecules and growth factors.

To examine whether OPCs could be stably maintained in oligodendrocyte enrichment medium, we monitored cell proliferation rate by measuring doubling time. Cells doubled every 48 hours for at least 7 weeks in culture (Figure 2A) and the bipolar morphology suggested that these cells were in the neural progenitor/oligodendrocyte progenitor stage of differentiation.

We next examined the OPC cells for protein expression by immunofluorescent staining between passage 3 and passage 5. We observed high numbers of cells expressing Olig2 (92%, Figure 3A), Sox10 (81%, Figure 3B), O4 (82%, Figure 3C), NG2 (64%, Figure 3D) and GalC (80%, Figure 3E). After 2 weeks of spontaneous differentiation, cells turned into 50 to 70% of MAP2- or bIII-tubulin-positive neurons (Figure 4) and less than 5% of GFAP-positive astrocytes (Figure 4). 30 to 50% of cells showed robust expression of intermediate to late oligodendrocyte markers MBP, PLP-1, MOG, RIP, GalC and NG2 (Figure 4).

Rat primary neurons isolated from the embryonic hippocampus are mostly pyramidal neurons that have a distinctive morphology composed of single axons and multiple dendrites. To facilitate identification of the rat primary neurons, green fluorescent protein (GFP) was introduced by transient transfection. 10%-20% cells were successfully transfected and GFP expression was stable for the period of observation. Human ES cell-derived OPCs were added to the rat primary neuronal culture at a ratio of 1:5. Three weeks after initiating coculture, we analyzed the relative degree of myelination by immunofluorescent staining. Myelin basic protein (MBP) is a major component of the myelin sheath. In the absence of Human OPCs, rat primary neuronal cultures exhibited little to no staining for MBP (Figure 5A). In contrast, in the coculture containing GFP-labeled axons and human OPCs, significant MBP staining was observed (Figure 5B). The expression of MBP colocalized with nuclei, providing evidence that MBP is expressed from human OPCs. This data suggested that in the presence of mature or developing axons, human ES cell-derived OPCs can be further maturated to functional oligodendrocytes that are capable of producing myelin to protect the axons.

Conclusions

We have developed a stable stem cell line that can maintain a high percentage of oligodendrocyte progenitors in culture and that could potentially yield functional, myelin-producing oligodendrocytes upon spontaneous differentiation. The cells may also be used to screen for molecules that promote or inhibit human OPC survival and differentiation. Our current efforts are in identifying those molecules that would increase OPC differentiation. This convenient, reliable source of oligodendrocytes may help to advance research into neurodegenerative therapies for diseases such as multiple sclerosis.