Contributed by Fred H. Gage, June 7, 2012 (sent for review March 10, 2012)
Images related to CB-iNCs derivation and characterization. (A) Timeline of CB CD133+ stem cell conversion into neuronal-like cells. (B) Representative phase-contrast images of passage-0 and passage-2 CB-iNCs generated by overexpression of only Sox2 and relative alkaline phosphatase (AP) staining. (C) Sox2 CB-iNCs expressed TUJ-1, microtubule-associated protein 2 (MAP2), and NF but were negative for OCT4 and NANOG. Blue indicates nuclei stained with DAPI. (Scale bars: Upper, 50 μm; Lower, 100 μm.). (D) Representative phase contrast images of passage-0 and passage-2 CB-iNCs generated with Sox2 and c-Myc and relative AP staining. (E) The colonies contained neuronal cells positive for TUJ-1, MAP2, PAX6, and DCX. Blue indicates nuclei stained with DAPI. (Scale bar, 50 μm.)
Characterization and gene-expression profile of CB-derived neurons. (A) CB-iNCs after 6 wk of differentiation on top of human astrocytes acquired a more mature phenotype highlighted by the expression of the excitatory markers VGLUT1 and the dendritic marker MAP2. (Scale bar, 30 μm.) (B) CB-derived neurons were positive for inhibitory markers such as GABA. (Scale bar, 80 μm.) (C) Synaptic buttons on CB-derived neurons were highlighted by the expression of synapsin puncta on TUJ-1–positive cells. (Scale bar, 10 μm.) (D) Heat map of genes differentially expressed in microarray analysis performed on CB CD133+ cells, CB-iNCs, and CB-derived neurons. Human ES-derived (HUES6) NPCs and HUES6-derived neurons were used as points of reference for neuronal phenotype. (E) Average global gene expression patterns were compared between CB CD133+ (n = 3 replicates) CB-iNCs (n = 3 replicates), CB-derived neurons (n = 3 replicates), and HUES6-NPCs (n = 2 replicates). Some neural-specific genes are highlighted in the plots (Map1, Map2, VGlut1, Pax6, NF, NeuroD1, Ncam, and Synapsin1). We found up-regulation of Fez1, Tead2, and NNAT, typical markers for dorsal neurons. (F) ChIP assay shows SOX2 binding to the regulatory regions of the indicated genes. Levels were determined by quantitative PCR and are expressed as fold change vs. the input. The positions of the amplicons are indicated in kilobases from the transcription start site (TSS). The mean and SD of three independent experiments is shown.
Activity-dependent calcium transients in CB-derived neurons. (A) Representative example of Syn::DsRed cultures of CB-derived mature neurons used for calcium signal traces. (Scale bar, 50 μm.) (B) Red traces correspond to the calcium rise phase detected by the algorithm used (SI Materials and Methods). Example of fluorescence intensity changes reflecting intracellular calcium fluctuations in CB-derived neurons before and after glutamate receptor antagonist (CNQX/APV) treatment. Each number on the left corresponds to the tracing of a different neuron on the plate. (C) Effects of TTX (1 μM) and CNQX/APV (10 μM/20 μM) on intracellular calcium transient frequency of individual neurons analyzed simultaneously by calcium imaging. (D) Analysis of spontaneous intracellular calcium transients in CB-derived neurons after 4 wk of differentiation. CB-derived neurons differentiated from two distinct CB-iNCs showed similar prevalence of calcium signaling as well as similar transient frequency within the neuronal population. Data shown as mean ± SEM.
Electrophysiology and in vivo grafting of CB-derived neurons. (A) Representative fluorescence micrograph of CB-derived neurons in culture expressing Synapsin::DsRed. (Scale bar, 10 μm.) (B and C) Whole-cell recordings obtained in vitro. (B) Transient Na+ currents and sustained K+ currents in response to voltage step (cell voltage-clamped at −70 mV while transient steps at 5-mV increments were applied). The trace highlighted in red was obtained in response to a step of +45 mV from resting −70 mV. (C) Action potentials evoked by somatic current injections (cell current-clamped at approximately −70 mV, currents from 50 to 150 pA at 50-pA steps). (D) Spontaneous action potentials when the cell was current-clamped at −60 mV. (E) CD133+ cells grafted in the hippocampus, as controls, did not integrate into the host tissue and did not express the neuronal markers TUJ-1 (arrow). (F) Representative image of CB-derived neurons 4 wk after transplantation show increase in colocalization with the mature neuronal marker NEUN (arrow). (Scale bars, 50 μm.) (G) Quantification of percentage of CB-derived neurons positive for TUJ-1 and NEUN 4 wk after transplantation. (H) Detail of a GFP+ CB-derived neuron 3 mo after transplantation shows extensive arborization and colocalization with the mature neuronal marker NEUN. (Scale bar, 50 μm.) (I–K) Whole-cell recordings obtained from a GFP+ CB-derived neuron 3 mo after its transplantation in a young mouse hippocampus from cell represented in I demonstrate that CB-derived neural progenitors can develop into functional neurons and survive in the mouse brain. (J) Action potentials evoked by somatic current injections [cell current-clamped at approximately −70 mV (−2 pA) while increments of 2 pA were applied]. (K) Transient Na+ currents and sustained K+ currents in response to voltage step (cell voltage-clamped at −70 mV while transient steps at 5-mV increments were applied). The traces highlighted in red were obtained in response to steps of +20 pA (J) or +45 mV (K).
Files in this Data Supplement:
