PMC

In vitro differentiation pattern of iPS-dNSCs(+DLL4) 1 week following plating with 1% fetal bovine serum shows greater neural differentiation compared to iPS-dNSCs(−DLL4). (A) Immunofluorescence images of neural markers (GFAP; astrocytes. Olig2; oligodendrocytes and precursors. PDGFrα; immature oligodendrocytes. βIII-tublin; neurons. Nestin; NSCs). (B) Cell counting for immunofluorescent-positive cell shows the differentiation pattern for iPS-dNSCs cultured±DLL4. iPS-dNSC(+DLL4) yielded predominantly terminally differentiated neural cells, specifically astrocytes, while the majority of iPS-dNSC differentiated cells remained Nestin-positive or were undefined (C5-4A iPSC line) mean±SEM; *P<0.05 scale bar represents 50 μm.

iPS-dNSCs cultured in definitive media conditions with DLL4 exhibit a more neural-specific gene expression profile compared to iPS-dNSCs cultured in definitive media alone. Selected neural (A), pluripotency (B), and nonectoderm (C) gene markers were examined in the iPS-dNSCs (C5-4A; left and B1-1G; right) cultured with and without DLL4. The addition of DLL4 significantly reduced residual pluripotency and endodermal markers, while maintaining the neural character of the cells. Cyropreserved passage 4 definitive neurospheres for C5-4A cell line cultured in definitive media without DLL4 (upper panels) or with continuous DLL4 treatment (lower panels) were sectioned and stained for NSC markers [(A) nestin, (B) Olig2], a pluripotency marker [(C) Oct4], and a pan-endoderm [(D) Afp] (C5-4A iPSC line). ND denotes not detected; mean±SEM; n=4–5, *P<0.05 compared to iPS-dNSC grown without DLL4; scale bar represents 100 μm.

Noggin increases primary iPSC-derived neurosphere generation. (A) The number of primary primitive neurospheres generated following 7 days of culture in SFM + LIF with and without Noggin was evaluated from iPSCs grown on MEFs with and without Noggin pretreatment. The presence of Noggin in the SFM + LIF evoked a significant increase in neurosphere number regardless of Noggin pretreatment. Selected neural (B), pluripotency (C), and endodermal (D) gene markers were examined in the passage 3 pNSCs cultured without Noggin (No Noggin), with a single passage with Noggin (1×Noggin), or with continuous Noggin treatment (3×Noggin). No significant differences were detected. C5-4A iPSC line. ND denotes not detected; mean±SEM; n=3, *P<0.05. SFM, serum-free media; pNSC, primitive NSC.

ESCs and iPSCs have variable neural propensity using the default pathway conditions. (A) Phase-contrast microscopy images of ESCs (upper, left) and iPSCs (lower, left) on a feeder layer, LIF-dependent primitive neurospheres (pNS; center), FGF-dependent definitive neurospheres (dNS; right). The ES-derived dNS (upper, right) show a free-floating phenotype, while the iPS-derived dNS (lower, right) show a considerable amount of adhesion and differentiation. (B–D) iPS-dNSCs show an increase in neural gene transcription. However, iPS-dNSCs retained pluripotency and endodermal gene expression compared to ES-dNSC controls. (B) Neural-specific markers are significantly increased in the ESC- and iPSC-derived dNSCs. (C) Pluripotency genes are altered as iPSCs follow the default pathway, however, to a lesser extent than the ESCs. (D) Endodermal lineage gene markers remain present in the iPS-dNSCs. ND denotes not detected; mean±SEM; n=3; scale represents 75 μm (R1 and G4 ES lines. C5, C5-4A, and B1-1G iPSC lines). ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; LIF, leukemia inhibitory factor; FGF, fibroblast growth factor; NSCs, neural stem cells; dNSCs, definitive NSCs; ES, embryonic stem; ESC, embryonic stem cells; SEM, standard error of the mean.

The addition of DLL4 significantly increased NOTCH pathway genes in iPS-dNSCs. (A) The number of neurospheres per 1,000 cells in definitive media conditions alone, with DLL4 or with DAPT was evaluated. The addition of the NOTCH pathway inhibitor DAPT resulted in significantly fewer neurospheres and failure to form spheres beyond the second passage. Significantly more definitive neurospheres were formed in the presence of DLL4. (B) Phase-contrast microscopy images of passage 4 iPS-derived definitive neurospheres (left) and iPS-derived definitive neurospheres with DLL4 (right). The iPS-derived definitive neurospheres show a considerable amount of adhesion and differentiation compared to iPS-derived definitive neurospheres cultured with DLL4. [(C, E) left] Notch1 gene expression is significantly increased in iPS-dNSCs by the fourth passage in definitive conditions with DLL4 in the media compared to the definitive media alone. [(D, E) right] HES3, a downstream mediator of NOTCH, increases in iPS-dNSCs with a single passage in definitive media regardless of DLL4 treatment, however, without exogenous DLL4 treatment, HES3 expression returns to baseline levels by passage 4. (F) Immunolabeling for receptor Notch1 on cryopreserved definitive neurospheres with and without DLL4 agonism. C5-4A iPSC line. mean±SEM; n=4–5, *P<0.05 scale bar represents 75 μm. DLL4, Delta-like ligand 4; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.

Electrophysiological profile of differentiated cells derived from iPSCs. Using the nystatin configuration of the patch-clamp electrophysiology method, both inward and outward currents were recorded in differentiated cells. In some cells, a distinct inward current starting at −30 mV and terminating around −10 mV (A) was observed. Inset depicts a typical current profile of voltage steps from −100 mV to +80 mV. During current-clamp studies, neuronal-like cells were observed to have reversible depolarizing transients that were sensitive to bath application of the voltage sensitive Na+ channel blocker tetrodotoxin (TTX 0.5 mM) (B). After electrophysiological experiments, the cells were backfilled with Lucifer yellow [(C) green) and cells were found to be immunopositive for the neuronal marker β-3 tubulin (red). In contrast, glial cells did not exhibit any inward current profile, although they did show current/voltage relationships typical for oligodendroglial precursor cells [OPC; (D)], mature oligodendrocytes (E), and astrocytes (F). After electrophysiological recordings, cells were backfilled with Lucifer yellow (LY; green). Cells were identified as being OPCs when stained positive for PDGFα-R [(D); red), as mature oligodendrocytes when positive for myelin basic protein [(E); MBP; red], or astrocytes when positive for GFAP [(E); red]. Table (G) summarizes the electrophysiological profile of the above cells indicating resting membrane potential (Em in mV), input resistance (MΩ), membrane capacitance (pF), and number of cells recorded (N).