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Quantitation and functional characterization of neural cells derived from ES cells using nestin enhancer-mediated targeting in vitro

Nibedita Lenka*,{ddagger}, Zhong J. Lu, Philipp Sasse, Jürgen Hescheler and Bernd K. Fleischmann

Institute of Neurophysiology, University of Cologne, Cologne, Germany
* Present address: National Centre For Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, Maharashtra, India



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  		Fig. 5. Time-lapse experiments showed live the neural progenitor proliferation and differentiation into neurons as well as glia. (A) Transmitted light and fluorescence monitoring of isolated EB-derived cells. Starting from a single progenitor (arrow), characterized by its typical morphology and prominent EGFP expression, cell division (2.5 h, 6 h, 36.5 h) and differentiation into neuronal (unipolar, 11.5 h; bipolar, 12 h) cells (arrowhead) was observed in a representative experiment. Upon migration and further differentiation the EGFP fluorescence declined. (B) In line with the retained capacity of proliferation of glia (arrow), prior to division rounding-up of the glial cell was noticed (54.5 h). After division, migration, cellular interaction as well as flattening of the cells occurred. The cell labelled with arrowhead is likely of other origin. Bars, 30 µM (A); 50 µM (B).

 


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  		Fig. 1. Human nestin intron-II-driven EGFP expression in undifferentiated ES cells and corresponding immunocytochemical expression pattern. (A) A single NeoR clone after electroporation of h-Nestin-EGFP construct and G418 selection for 12 days shows heterogeneous EGFP expression. (B,C) The EGFP expression in ES cells (arrow) remains patchy during propagation on mitotically inactive feeders (arrow head) in clone-1. (D,E) Weak EGFP expression in one of the few (<15%) EGFP-positive ES-cell clones transfected with tkEGFP. (B,D) Combined transmission and fluorescent light; (C,E) fluorescence light alone. (G,H) A complete overlap between EGFP expression (G) and (H) nestin-immunoreactivity was observed in undifferentiated ES cells during feeder-free propagation. Bars, 100 µM (A), 75 µM (D,E), 50 µM (B,C,G,H).

 


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  		Fig. 2. RT-PCR revealed the presence of nestin transcripts in undifferentiated ES cells as well as murine blastocysts. The upper panel shows nestin transcripts from D3 undifferentiated ES cells (lane 2), transgenic h-Nestin-EGFP ES cells (n-ES cells; lane 5) grown on feeders and from D3 undifferentiated ES cells grown without feeders (lane 8). The lower panel shows nestin transcripts in the transgenic ES cells without feeders (lane 2) and in the murine blastocysts (lanes 8, 14). The length of the nestin PCR product corresponds to the expected size of 478 bp. The corresponding products for housekeeping HPRT (upper panel, lanes 4,7,10; lower panel, lanes 5,10) and ß-actin (lower panel, lanes 6,12) are shown as controls. Lanes 3,6,9 (upper panel) and 3,4,7,9,11,13 (lower panel) represent the respective negative controls where the reverse transcriptase was omitted from the reaction.

 


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  		Fig. 3. EGFP expression profile during differentiation and neural specification. The expression was prominent in progenitors and downregulated in differentiating neurons. Localized EGFP expression in the EB (A,B; d7+2) and in outgrowths (arrow) of the EB (C; d7+4) post-plating. (C) Arrowhead, central part of EB. (D) EBs (d7+7) show differentiating neurons (arrow) at the periphery with EGFP expression (see inset); arrowhead shows the central part of the EB. (E) d7+14; three clusters of neurons (1,2,3) with short and long processes. (F) (d7+20); with further plating time cluster 2 shows an extension of neurite outgrowths from the central cell mass. (G,H) Magnified view of the same cluster, where some neurites are still EGFP positive. The arrowhead indicates the presence of EGFP-positive neural progenitors in the vicinity of mature neurons, and the arrow indicates the lengthening of neurite processes with longer plating time from d7+14 to d7+20 (E-H). (J,K) Almost all of the EGFP-expressing cells (J) in EBs (d7+7) are nestin positive (K); the yellow colour indicates the superimposed pattern between EGFP and Cy3. (L,M) In the differentiated neurons labelled with MAP2 (M, double filter), a weak or no EGFP signal (L, d7+7) was observed. Combined transmission and fluorescent light: A,C,D-F,G. Fluorescent light alone: B,H,J,L,inset D. Bars, 50 µM (A,B,inset D); 100 µM (C,D,G,H,J-M); 250 µM (E,F).

 


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  		Fig. 4. Isolated cells mimic neurogenesis observed in whole EBs (d7+7). (A,B) Differentiating neurons with bipolar-(arrow) and multipolar (arrowhead) morphology were seen after plating of isolated cells A: combined transmission and fluorescence; B: fluorescence. (C,D) A high overlap (D, yellow) between EGFP expression (C) and nestin labelling was observed. Besides the generation of neurons, EGFP-positive and -negative glial cells (E) were identified based on GFAP staining (F, superimposed). Flat glial cells were weak/negative for EGFP (arrowhead), while elongated fibre-like glial cells were EGFP positive (arrow). (G,H) The formation of synaptic connections between isolated neurons was confirmed by synaptophysin staining (H). As expected for differentiated neurons, low or no EGFP signal (G) was detected in synaptophysin-positive cells. Little overlap (K, superimposed) between MAP-2 staining (K) and EGFP expression (J) was detected. The flat glial cells (green) and differentiating neurons (red) were found to closely associate. Bars, 25 µM.

 


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  		Fig. 6. The quantitative evaluation of in vitro neurogenesis using flow-cytometry revealed a development-dependent distribution pattern. A clear delay in the generation of strong EGFP-positive progenitors was observed in the retinoic acid untreated (right panels) versus the treated (left panels) EBs. The maximal generation of progenitors occurred between d4 and d10 after plating in the RA-treated cells. Notice the decline in the number of EGFP-positive cells accompanied by a compensatory increase in EGFP-negative cells, suggesting a transition to the differentiated neuronal population. While the population of undifferentiated D3 ES cells (A) was used for calibration of the system (estimate of the range of auto-fluorescence), a relatively homogenous EGFP expression was noticed in the undifferentiated h-nestin-EGFP cells (B). The EGFP fluorescence intensity on the horizontal axis is displayed as log scale; the vertical axis represents the percentage of gated cells. Numbers in the top-left corner indicate the differentiation stage. (B) A quantitative evaluation of the in vitro neurogenesis was obtained by averaging two to three experiments per time point of differentiation. This analysis clearly showed that the decline in the strong EGFP-positive (EGFP+++ and EGFP++) and weak EGFP-positive (EGFP+) cell population was accompanied by a parallel increase in EGFP-negative (EGFP-) cells.

 


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  		Fig. 7. The functional characterization of EGFP-positive cells demonstrated a development-dependent pattern of ion channel expression. Ramp depolarizations (150 ms from -100 mV to 50 mV, Hp -80 mV) showed that at the apolar stage (A) no voltage-dependent ion channels were detected, whereas unipolar stages (B) expressed voltage-activated outward currents. Starting from the bipolar stage, TTX hypersensitive INa was detected (C,D). In the early developmental stage (EDS), bipolar neurons INa were detected first, whereas IBa appeared at later stages (E). While INa density in bipolar neurons increased (P=0.01) during further development (F), the mean current density in multipolar neurons was less (P=0.05) than that of bipolar neurons (G).

 


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  		Fig. 8. Functional expression of IBa during early stages of neuronal development. (A) The IBa current-voltage relationship (voltage steps lasting for 150 milliseconds, from -60 to +50 mV in 10 mV increments, HP -80 mV) in a representative bipolar LDS neuronal cell. The threshold of activation was between -60 and -50 mV and peak currents were measured at -10 mV. (B) The fraction of different IBa subtypes composing the whole cell current was evaluated using selective antagonists. (C,D) The percentage of the different current components did not significantly differ between bipolar- and multipolar neurons.

 


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  		Fig. 9. Functional expression of receptor-operated channels during early neuronal development. GABA (A) as well as glycine (B) evoked fast-activating and inactivating inward currents (HP -80 mV) in bipolar strong EGFP-positive cells. Co-application of the selective antagonists Bicuculline (A) and Strychnine (B) almost completely inhibited the activation of GABA- and glycine-evoked inward currents, respectively. The ionic nature was determined by performing ramp depolarizations (from -100 mV to 50 mV in 150 milliseconds) in the presence and absence of the agonist yielding a reversal potential of -27 mV, as expected for a Cl- current (see inset in A).

 

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© The Company of Biologists Ltd 2002