Differentiation of Human ES Cells into Oligodendrocytes and Their Characterization
We used an EB-based method for in vitro differentiation of oligodendrocytes and their progenitors from human ES cells. Human ES cell colonies were detached by treatment with collagenase type IV. After being transferred onto bacterial culture dishes, detached human ES cell colonies were incubated in EB medium for 4 days to form EBs. The EBs were attached on culture dishes and cultured in the ITSFn medium for 5 days for the selection of neural precursors. For the expansion of neural precursors, the EBs were continuously cultured in the presence of bFGF and N2 supplements for an additional 5 days. After differentiation into neural precursors, structures with neural rosettes (Fig. 1, arrows in 1A and 1B) were formed [20, 21]. These neural-specific structures that expressed neural markers such as nestin (Fig. 2Aa)  were mechanically isolated and attached on matrigel-coated dishes for differentiation into oligodendroglial progenitors. To further expand the neural precursors, the neural rosettes were cultured in suspension with N2 and bFGF supplements in bacterial dishes. The SNMs that resemble so-called “neurospheres” were formed during the incubation of these neural structures (Fig. 1C). The spheres were mechanically dissected and expanded every 5–10 days, depending on size. During this expansion, portions with non-neural morphologies were eliminated by mechanical cutting, which increased the purity of the SNMs. Pure SNMs that were generated after three or four passages could be expanded for a long period of time, thus, continuously increasing the total mass. This means that we could supply neural precursors indefinitely for any experiment that requires mass production.
Figure Figure 1.. Differentiation of human embryonic stem (ES) cells into oligodendrocytes. Phase contrast microscopic morphology of ES cell-derived neural precursor cells (neural rosettes [A] and [B], spherical neural masses [SNMs] [C]), oligodendrocyte precursor cells (D, E), oligodendrocytes (F–I) following in vitro differentiation of human ES cells. (A, B): Neural rosettes (arrows) were generated from attached embryoid bodies. The neural rosettes were mechanically dissociated and replated on matrigel-coated dishes for differentiation or cultured in bacterial dishes to form SNMs (C) for further expansion of neural precursors. (D, E): Cells show the typical bipolar morphology after ∼10 days of differentiation from neural rosettes (D) or SNMs (E). (F, G): Dendrites were spread in several directions. Ten days after basic fibroblast growth factor and platelet-derived growth factor withdrawal. (H, I): Terminal differentiation evidenced by highly branched morphology (arrows). Scale bar, 50 μm.
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Figure Figure 2.. Expression of specific markers for neural precursors, oligodendrocyte precursors, and oligodendrocytes. (A): Immunocytochemical staining of specific markers for neural precursors ([a], nestin), oligodendrocyte precursors ([b], PDGF-R; [c], A2B5; [d], NG2), and oligodendrocytes ([e], O4; [f], O1; [g], MBP). Anti-β-III-tubulin and GFAP antibodies were used for the detection of neurons (h) and astrocytes ([i], inset; positive control-human normal astrocytes [Cambrex, Walkersville, MD, http://www.cambrex.com]). 4′,6-Diamidino-2-phenylindole staining was also shown in blue in b, d, g, h, and i. Scale bar, 50 μm. (B): Semiquantitative reverse transcription-polymerase chain reaction analysis for various markers during in vitro differentiation of human embryonic stem (ES) cells. The expression levels of each gene were normalized to that of GAPDH. Stage I (undifferentiated ES cells); stage II (EB); stage III (neural precursors); stage IV (oligodendrocyte precursor cells); stage V (oligodendrocytes). Nestin (neural marker); PDGF-R and NG2 (oligodendrocyte precursor markers); MBP and PLP (oligodendrocyte markers); GFAP (astrocyte marker); Neurofilament (neuronal marker). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; PDGF-R, platelet-derived growth factor-recpetor; PLP, proteolipid protein.
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Attached neural precursors (from neural rosettes or SNMs) on matrigel-coated culture dishes were incubated in the presence of EGF for 4 days and, after adding PDGF, for an additional 8 days. EGF stimulates the proliferation of neural precursor cells in serum-free cultures , and PDGF regulates the timing of oligodendrocyte differentiation by inducing the proliferation of early oligodendrocyte progenitor cells for several divisions, thereby preventing premature differentiation . The combination of EGF and PDGF was known to promote the proliferation of glial precursor cells [10, 24, –26]. These conditions yielded an isomorphous population of round to bipolar cells characteristic of immature cells (Fig. 1D, 1E). These cells expressed oligodendrocyte precursor markers such as PDGF-R, A2B5, and NG2 (Fig. 2Ab–2Ad). PDGF-R, A2B5, and NG2 are membrane epitopes typically expressed in oligodendrocyte precursor cells [27, 28]. We also used neurotrophin (NT)3 with PDGF or NT3 alone, but no differences in effect were found compared with PDGF alone (data not shown).
For further differentiation into mature oligodendrocytes, the growth factors (EGF, PDGF, and bFGF) were removed, and T3 was added. Thyroid hormone is required for oligodendrocyte survival and activates the effector component of the timer in oligodendrocyte precursor cells to initiate differentiation into mature oligodendrocytes . Upon growth factor withdrawal and the addition of thyroid hormone, the cells differentiated into mature oligodendrocytes. Ten days after growth factor withdrawal, many of the cells showed a multipolar morphology characteristic of immature oligodendrocytes (Fig. 1F, 1G). Prolonged growth factor withdrawal for more than 20 days promoted further oligodendroglial differentiation (Fig. 1H, 1I), and these cells expressed oligodendrocyte markers including oligodendrocyte surface protein O4 (Fig. 2Ae), O1 (Fig. 2Af), and MBP (Fig. 2Ag). O4 is an antigen on the surface of immature oligodendrocytes to oligodendrocytes and O1 is a protein expressed on the surface of oligodendrocytes . MBP is localized to the major dense lines of myelin and confined to the interior of oligodendrocytes . After differentiation, only a small percentage (less than 5%) of neurons (Fig. 2Ah) and astrocytes (Fig. 2Ai; inset, positive control for astrocytes) were detected.
The expression of several markers was also analyzed by RT-PCR through the differentiation procedure (Fig. 2B). Nestin, a marker of neural precursors, was detected in neural precursor expansion culture (stage III). The reduction in nestin expression was associated with the increased expression of oligodendrocyte precursor markers following differentiation. The expression of PDGF-R and NG2 (oligodendrocyte precursor markers) was strongly upregulated during the induction of oligodendrocyte precursors (stage IV) and was downregulated thereafter. After final differentiation (stage V), the expression of oligodendrocyte-specific genes, such as MBP and PLP, was enhanced. The marker of astrocytes, GFAP, and neuronal marker neurofilament were not nearly detectable after differentiation, showing a good correlation with immunostaining results. This result indicates that the stage-specific marker genes are well expressed following the differentiation procedure and there is enrichment of the oligodendrocyte lineage relative to neurons or astrocytes.
After differentiation, we counted oligodendrocyte precursor cells (stage IV) and oligodendrocytes (stage V) at the cellular level. The majority of the cells were PDGF-R (82%) or A2B5-positive (94%) at stage IV, and O1-positive (89%) at stage V. Flow cytometry analysis (Fig. 3) revealed that most of the cells expressed PDGF-R (81%), A2B5 (90.4%), NG2 (91.3%), and O1 (81%), which is similar to the results of random field counting. These results suggest that our in vitro protocol results in efficient induction of oligodendrocytes and their progenitors from human ES cells.
Figure Figure 3.. Flow cytometry analysis of oligodendrocytes and their progenitors. Antibodies against PDGF-R (A), A2B5 (B), NG2 (C) (oligodendrocyte precursor markers), and O1 (D) (oligodendrocyte marker) were used for the detection of oligodendrocytes and their progenitors. Stained cells were analyzed using a FACScan. This analysis revealed that 81%–91% of total cells were oligodendrocyte progenitors and ∼81% of total cells were oligodendrocytes. Abbreviation: PDGF-R, platelet-derived growth factor receptor.
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To determine whether these cells were biologically functional in terms of myelination activity, the oligodendrocyte precursor cells were cocultured with hippocampal neurons (Fig. 4). The hippocampal neurons were dissected from the brains of 18-day-old rat fetuses, treated with trypsin, and dissociated by pipetting. After the neurons had attached on culture dishes, oligodendrocyte precursors at a concentration of 2 × 104 per cm2 were added and incubated for 5 weeks to induce myelination of the axons. The electron micrograph in Figutre 4A–4C shows a transverse section of a representative myelinated axon by coculture. As shown in Figure 4B and 4C, the myelin sheets compactly enclosed the axon of a neuron. Myelination of neurons (red: arrow) were also confirmed by immunostaining using anti-MBP (red) and β-III-tubulin (green) antibodies (Fig. 4D). MBP was hardly detected in cultures of hippocampal neurons alone (Fig. 4E), indicating that myelination of neurons was due to oligodendrocytes differentiated from human ES cells, but not endogenous oligodendrocytes. This fact was confirmed using antibody against human nuclei (Fig. 4F–4H). MBP-positive oligodendrocytes (Fig. 4F, 4G, green) were human nuclei-positive (red), yielding yellow color (big arrows). In contrast, hippocampal neurons (Fig. 4H, big arrow; Fig. 4F and 4G, small arrows) were human nuclei-negative (blue). Fgiure 4J and 4K showed that ∼25.8% of the hippocampal neurons in the coculture were myelinated MBP-positive cells, whereas ∼1.6% of the neurons were MBP-positive in the culture of hippocampal neurons alone.
Figure Figure 4.. Human embryonic stem (ES) cell-derived cells myelinate axons in cocultures with fetus hippocampal neurons. Oligodendrocyte precursor cells were cocultured with hippocampal neurons from the brains of 18-day-old rat fetuses for 5 weeks. (A–C): The transmission electron micrograph illustrates that the axons of hippocampal neurons are surrounded by multilayered compact myelin sheets ([A], ×70,000; [B], ×140,000; [C], ×720,000). (D): Double immunostaining of MBP (red) and β-III-tubulin (green) after coculturing for 5 weeks. Myelination of axons (arrow, red) is shown. (E): Hippocampal neurons cultured without human ES cell-derived oligodendrocytes were stained with anti-β-III-tubulin (green) and anti-MBP antibodies (red), but MBP-positive cells (red) as shown in (D), are hardly detectable. (F, G): Double immunostaining of MBP (green) and human nuclei (red) after coculture shows that the myelinating MBP-positive cells are human cells (big arrows, yellow). Hippocampal neurons are shown as small arrows. [H]: Costaining of β-III-tubulin (red) and human nuclei (green) after coculture. β-III-tubulin-positive cells (hippocampal neurons) were not labeled with anti-human nuclei antibody (big arrow). Human cells are shown as small arrows (green). 4′,6-Diamidino-2-phenylindole staining (blue) is also shown in (D–H). Scale bar, 50 μm. (I–K): Flow cytometry analysis using antibodies against β-III-tubulin and MBP. Of total cells, ∼21% (∼25.8% of the neurons) were MBP-positive cells in the coculture (J), whereas ∼1.2% of total cells (∼1.6% of the neurons) were MBP-positive cells in the culture of hippocampal neurons alone (K). Cells stained only with secondary antibodies were used as the control (I). Abbreviation: MBP, myelin basic protein.
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