Human embryonic stem cells for brain repair?

Abstract

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

Figure 1

Stereotypic neuronal specification from human ES cells. (a) Schematic procedures for inducing dopamine neuron (DA) and motor neuron (MN) differentiation. ES cells were differentiated to primitive neuroepithelial cells (NE) and then neuroepithelial cells that exhibit neural tube-like rosettes in two weeks of differentiation in a chemically defined neural medium (Zhang et al. 2001) without the presence of morphogens. (b) Treatment with FGF8 and SHH after neuroepithelial cells have formed resulted in differentiation of dopamine neurons that possess forebrain features (upper row), whereas the same set of morphogens at the primitive NE stage caused differentiation of dopamine neurons with midbrain characters including the expression of En-1 (lower row; Yan et al. 2005). (c) Similarly, neuroepithelial cells, after treatment with RA and SHH, differentiated to neurons that expressed Isl1, but not the motor neuron-specific transcription factor HB9+ (upper row). In contrast, treatment of primitive neuroepithelial cells with the same set of morphogens resulted in the production of motor neurons that express HB9+ (Li et al. 2005). Blue indicates Hoechst stained nuclei. Bar=50 μm. (b) and (c) are reproduced from Yan et al. (2005) and Li et al. (2005), respectively, with permission.

Figure 2

Functional properties of human ES cell-derived midbrain DA neuron and spinal motor neurons. (a) Electrophysiological recording indicated that action potentials were evoked by depolarization current steps in TH+ dopamine neurons. Immunostaining showed that the recorded neuron (positive for biocytin, from the recording electrode) was TH+. (b) High performance liquid chromatography measurement indicated that dopamine was released from dopamine neuronal cultures spontaneously and the release was increased by depolarization (56 mM KCl in HBSS). (c) Electrophysiological analyses indicated that hES cell-derived motor neurons received both inhibitory and excitatory synaptic currents from neighbouring neurons (i). The outward inhibitory currents were blocked by bicuculline (20 μM) and strychnine (5 μM) (ii). Subsequent application of d(−)-2-amino-5-phosphonopentanoic acid (AP-5; 40 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 μM) blocked the remaining the inward excitatory currents (iii). (d) Co-culture of human ES-derived motor neurons with C2C12 myoblasts resulted in aggregation of acetylcholine receptors on myotubes as shown by α-bungarotoxin (BTX) staining in areas where synapsin+ or ChAT+ fibres landed. Bar (a)=50 μm and (d)=30 μm. (a+b) and (c+d) are reproduced from Yan et al. (2005) and Li et al. (2005), respectively, with permission.