Figure 1.
Cell surface marker screen of cell cultures at distinct phases of SFEB neural induction of H9 hESC.
(A) Diagrammatic representation of the SFEB neural induction and isolation of NSC (see Methods for details). EB = embryoid body(ies); EB-rosette(+) = EB at a stage of differentiation when columnar rosettes are present in many EB; NSC = neuronal stem cells expanded from rosettes that were handpicked from EB-rosette(+); NSC contaminant = culture of intermittent contaminant of handpicked and expanded NSC cultures. (B) Heat map showing percent positive (% +ve) of representative cell surface markers in a FACS-based screen. EB-rosette(-) = EB depleted of rosettes. Markers identified from the screen to be potential positive (pos) or negative (neg) selection candidates (Hits) are noted. (C) Examples of intensity distributions of EB-rosette(+) cultures stained with candidate cell surface markers. The percentage reflects the population in the “positive” population for each marker. (D) Examples of an image screen of cell surface markers on neuron enriched cultures induced to differentiate from NSC for 3 weeks. CD24 clearly stains neurons whereas CD44, and CD15 do not. CD184 appears to stain some neurons and other cell types. (E) Same as c but with neuron-enriched cultures induced to differentiate from NSC. Scale bar is 50 µm.
Figure 2.
Sorting NSC from differentiating hESC and hiPSC from SFEB and SDIA neural induction cultures.
(A) Cell sorting strategy of NSC derived from H9 SFEB cultures at the EB-rosette(+) stage. Cells were first selected based on CD184 staining. The CD184+ population was then depleted of cells expressing CD271 and CD44. This cell population was nearly 100% positive for CD24 and contained cells expressing CD15. 10% of the total cells (orange) were CD184+/CD271−/CD44−/CD24+. (B and C) Staining with anti-Sox1, anti-Pax6 and DAPI of CD184+/CD271−/CD44−/CD24+ H9 NSC from SFEB neural induction cultures at the third passage post-FACS. (D) Same as B but stained with anti-Sox2, anti-Nestin, and DAPI. (E and F) Four color intracellular FACS analysis with anti-Sox1, anti-Nestin, anti-Sox2 and anti-Oct3/4 of CD184+/CD271−/CD44−/CD24+ H9 NSC from SFEB at the third passage post-FACS. (G) CD184+/CD271−/CD44−/CD24+ H9 NSC expanded to passage 7 and induced to differentiate for 3 weeks and stained with anti-Nestin, anti-β-III tubulin and DAPI. (H-J) The CD184+/CD271−/CD44−/CD24+ H9 NSC were differentiated for 3 weeks and stained with anti-Map2b, anti-Ki-67 and DAPI; anti-GFAP, anti-synaptophysin (SYP) and DAPI. (K and L) Staining with anti-Sox1, anti-Pax6 and DAPI of CD184+/CD271−/CD44−/CD24+ HUES-9 NSC from SDIA PA6 neural induction cultures at the fourth passage post-FACS. (m) Same as k but stained with anti-Sox2, anti-Nestin and DAPI. (N-P) Same as K-M but with hiPSC, NDC3.1 NSC from SDIA PA6 neural induction cultures. Scale bar is 50 µm.
Figure 3.
Sorting neurons and glia from cultures of sorted and subsequently expanded and differentiated NSC.
NSC were differentiated for 3 weeks in neuron differentiation medium prior to FACS. (A) Cell sorting strategy of differentiated H9 NSC using CD184−/CD44−/CD15LOW/CD24+ and CD184+/CD44+. Similar populations were isolated from differentiated cultures of HUES-9 NSC and NDC3.1 NSC both derived from SDIA PA6 co-culture. (B) Sorted CD184−/CD44−/CD15LOW/CD24+ cells were cultured in neuron differentiation medium for 2 days post-FACS and subsequently stained with anti-Map2b, anti-Nestin, anti-Ki-67 and DAPI. White arrow indicates the presence of one Ki-67+, Nestin+ cell in this field. (C) Same as B except cells were stained with anti-Sox2 instead of Ki-67. One Nestin+ cell is evident in this field. (D) CD184−/CD44−/CD15LOW/CD24+ sorted cells were cultured in neuron differentiation medium for 7 days post FACS and subsequently stained with anti-β-III tubulin, anti-Map2b, anti-Ki-67 and DAPI. No Ki-67+ cells are observed in this field. (E) Same as D but stained with anti-β-III tubulin, anti-Nestin, anti-GFAP and DAPI. No GFAP+ cells are evident and one Nestin+ cell is evident in this field. (F) Same as E but from HUES-9. (G) Same as E but from hiPSC, NDC3.1. (H) Electrical recordings of patched neurons from a 3-week differentiated HUES-9 NSC culture. All cells exhibited Na+ current (right panel). 7 of 8 cells tested fired action potentials when depolarized with current (left panel). (I) Electrical recordings of sorted neurons from a 3-week differentiated HUES-9 NSC culture. Sorted neurons were cultured for an additional 3 weeks prior to recording. All cells exhibited Na+ current (right panel). 6 of 8 cells fired action potentials when depolarized with current (left panel). (J) CD184+/CD44+ sorted cells derived from H9 were cultured in neuron differentiation medium for 7 days post-FACS and stained with anti-β-III tubulin, anti-Nestin, anti-GFAP and DAPI. (K) Same as H except cells were cultured in astrocyte culture medium for 7 days prior to imaging. (L) Same as K, but from HUES-9. (M) CD184+/CD44+ cells from NDC3.1 were cultured for 6 passages in astrocytes medium and stained for GFAP and DAPI. Scale bar is 50 µm. (N) 2-color intra-cellular FACS analysis of NSC sorted from H9 and then induced to differentiate for 3 weeks prior to sorting cell populations based on different combinations of CD184 and CD44 immunoreactivity. The unsorted population is composed of a Nestin+ population that is highly enriched for mitotic Ki-67+ cells whereas the Nestin−/LOW population is quiescent. Sorting of a CD184−/CD44− population enriches for Nestin−/LOW quiescent cells. Isolation of CD184+/CD44+ and CD184+/CD44− cells enriches for the Nestin+ cycling population. Percentages for these 2 populations are indicated.
Figure 4.
Spinal transplantation of differentiated NSC cultured cells in rats with spinal ischemic injury.
(A) In animals with previous ischemic injury hNUMA+ grafted cells (blue) were identified in the intermediate zone or in the ventral horn (VH) 2 weeks after grafting. Scale bar is 100 µm. (B, C) Numerous hNUMA+ cells were DCX immunoreactive and showed extensive projection of DCX+ processes towards the ventral horn (yellow arrows). Scale bar is 40 µm. Yellow dotted box represents expanded view in C. (D, E) In control animals injected with medium only, no hNUMA or DCX immunoreactivity was identified. (F, G) A subpopulation of grafted hNUMA-positive cells showed colocalization with GFAP but were DCX-negative (yellow arrows). Scale bar is 10 µm. (H) At 2 weeks after grafting hNestin-positive cells were seen in the core of the graft and were DCX negative. Scale bar is 20 µm. (I–K) Proliferating cells were identified by colocalization of Ki-67 and hNUMA immunoreactivity and were primarily seen 2 weeks after grafting. Scale bar is 20 µm.
Figure 5.
Spinal transplantation of proliferating NSC cultured cells in rats with spinal ischemic injury.
(A) At 10 weeks after grafting a high density of DCX+ grafted neurons (red) in the center of intermediate zone were identified. Numerous solitary DCX+ neurons which migrated into ventral horns (yellow arrows) were also seen. Scale bar is 100 µm. (B, C) High density of hSYN punctata-like immunoreactivity in the regions containing grafted DCX+ neurons was seen. (D) hSYN immunoreactivity showed spatial co-localization with DCX+ processes (yellow arrows). Scale bar is 20 µm. (C-insert, E) Numerous hNSE immuoreactive neurons in the core of grafted regions were identified (yellow arrows). Scale bar is 20 µm. (F–I) A subpopulation of grafted cells acquired an oligodendrocyte phenotype and were NG2 or Olig2 immunoreactive. Scale bar is 20 µm. (J, K) Transgenic SOD1 mutant rats were grafted with CD184+/CD44+ sorted glial cells derived from differentiated HUES-9 NSC cultures. Numerous hNUMA/GFAP-positive astrocytes were identified in the core of the graft 2 weeks after grafting but no DCX-positive neurons were seen. Scale bar is 20 µm. (L) 2 weeks after grafting numerous hNestin-positive cells typically localized in the core of the graft. Scale bar is 20 µm. (M–O) Costaining with hNUMA and Ki-67 antibody revealed occasional proliferating cells (yellow arrow). Scale bar is 20 µm.
Figure 6.
Diagram of stages and defined markers for isolation of NSC, neurons and glia from neural induction cultures starting with pluripotent stem cells.