Neuroectodermal differentiation from mouse multipotent adult progenitor cells
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Fig. 1.mMAPCs were cultured sequentially for 7 days with 100 ng/ml bFGF, 10 ng/ml FGF8 and 100 ng/ml SHH, and 10 ng/ml BDNF on fibronectin-coated chamberslides. After 7, 10, and 21 days, cells were fixed and stained with antibodies against nestin and Nurr1 followed by secondary Cy5- and Cy3-coupled antibodies, respectively (d7); NF-200 and GFAP followed by secondary Cy3- and Cy5-coupled antibodies, respectively (1) and NF-200 and MBP followed by secondary Cy3- and Cy5-coupled antibodies, respectively (2) (d10); and GABA and DDC followed by secondary Cy5- and Cy3-coupled antibodies, respectively (1), TrH and TH followed by secondary Cy5- and Cy3-coupled antibodies, respectively (2), and microtubule-associated protein and Tau followed by secondary Cy3- and Cy5-coupled antibodies, respectively (3) (d21).
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Fig. 2.eGFP-transduced mMAPCs (28% transduction efficiency) were cultured on fibronectin-coated chamberslides sequentially for 7 days with 100 ng/ml bFGF, 10 ng/ml FGF8 and 100 ng/ml SHH, 10 ng/ml BDNF, and finally with E16 fetal mouse brain astrocytes plated on coverslips that were placed upside down in the chamberslides. After a total of 28 days, cells were fixed and stained. Slides were analyzed for the presence of GFP-positive cells and cells costaining with Cy3- or Cy5-labeled antibodies. (A) Cells labeled with antibodies against GABA and DDC. (A1-A3) Single fluorescence color analysis of cells stained with antibodies against GABA followed by secondary Cy3-coupled antibody, eGFP-labeled cells, and cells stained with antibodies against DDC followed by secondary Cy5-coupled antibody, respectively. (A4-A6) Overlay pictures of GFP/anti-GABA-Cy3, anti-GABA Cy3/anti-DDC-Cy5, and GFP/anti-DDC-Cy5, respectively. Shown is that GFP-positive cells acquired morphological and phenotypic features of GABA-ergic and dopaminergic neurons, whereas a fraction of cells with morphological and phenotypic features of GABA-ergic and dopaminergic neurons was GFP-negative. (B) Cells labeled with antibodies against TrH and dopamine. (B1-B3) Single fluorescence color analysis of cells stained with antibodies against TrH followed by secondary Cy3-coupled antibody, eGFP-labeled cells, and cells stained with antibodies against dopamine followed by secondary Cy5-coupled antibody, respectively. (B4-B6) Overlay pictures of GFP/anti-TrH-Cy3, anti-TrH-Cy3/anti-dopamine-Cy5, and GFP/anti-dopamine-Cy5, respectively. Shown is that GFP-positive cells acquired morphological and phenotypic features of serotonergic and dopaminergic neurons, whereas a fraction of cells with morphological and phenotypic features of serotonergic and dopaminergic neurons was GFP-negative.
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Fig. 3.Spiking behavior and voltage-gated currents from MAPCs in coculture with fetal mouse brain astrocytes. (A) Current-clamp recordings from a MAPC that had been cocultured with astrocytes for 8 days. Illustrated in the bottom three panels are the voltage responses elicited by the current-injection protocol shown (a 17-pA current-injection step, Top). The repetitive spiking recorded in this cell was blocked reversibly by TTX. The current-injection protocol reports the current injected relative to a negative DC current that was injected into the cell to “hold” it near -100 to -130 mV. (B) Voltage-clamp recordings of leak-subtracted currents from the same cell shown in A. (Top) The voltage-clamp protocol used to elicit the families of currents shown in the bottom three panels. A large transient inward current was evident that could be blocked reversibly by TTX. (C) Current-clamp records obtained from a MAPC that had been in culture with astrocytes for 8 days. In this example, the cell produced only one spike in response to depolarizing current injections (Δ pA = 7). The arrows point to possible synaptic potentials.
Footnotes
- Copyright © 2003, The National Academy of Sciences
