Neuroectodermal differentiation from mouse multipotent adult progenitor cells

Abstract

We recently showed that a rare cell from murine bone marrow, which we termed multipotent adult progenitor cells (MAPCs), can be expanded for >120 population doublings. Mouse (m)MAPCs differentiate into mesenchymal lineage cells as well as endothelium and endoderm, and, when injected in the blastocyst, mMAPCs contribute to most if not all somatic cell lineages including the different cell types of the brain. Our results, reported herein, demonstrate that mMAPCs can also be induced to differentiate into cells having anatomical and electrophysiological characteristics similar to those of midbrain neurons. Differentiation to a neuronal phenotype was achieved by coculturing mMAPCs with astrocytes, suggesting that neuronal differentiation may require astrocyte-derived factors similar to what is required for the differentiation of embryonic stem cells and neural stem cells to neurons. Differentiation of mMAPCs to neuron-like cells follows similar developmental steps as described for embryonic stem cells and neural stem cells. MAPCs therefore may constitute a source of cells for treatment of central nervous system disorders.

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).

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.

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.