Important precautions when deriving patient-specific neural elements from pluripotent cells

Abstract

Multipotent human neural stem cells (hNSC) have traditionally been isolated directly from the central nervous system (CNS). To date, as a therapeutic tool in the treatment of neurologic disorders, the most promising results have been obtained using hNSC isolated directly from the human fetal neuroectoderm. The propagation ability of such tissue-derived hNSC is often limited, however, making it difficult to establish a large-scale culture. Following engraftment, these hNSC often show low efficiency in generating the desired neuronal cells necessary for reconstruction of the damaged host milieu and, as a result, have failed to give satisfactory results in clinical trials so far. Alternatively, human embryonic stem cells (hESC) offer a pluripotent reservoir for in vitro derivation of a rich spectrum of well-characterized neural-lineage committed stem/progenitor/precursor cells that can, theoretically, be picked at precisely their safest and most efficacious state of plasticity to meet a given clinical challenge. However, the need for 'foreign' biologic additives and multilineage differentiation inclination may make direct use of such cell-derived hNSC in patients problematic. The hNSC, when derived from pluripotent cells under protocols presently employed in the field, tend to display not only a low efficiency in neuronal differentiation, but also an inclination for phenotypic heterogeneity and instability and, hence, increased risk of tumorigenesis following engraftment. For hNSC derived in vitro to be used safely in therapeutic paradigms, it requires conversion of human pluripotent cells uniformly to cells that are restricted to the neural lineage in need of repair. Developing strategies for direct induction of human pluripotent cells exclusively into neural-committed progenies at a broad range of developmental stages will allow a large supply of optimal therapeutic hNSC tailor-made for safe and effective treatment of particular neurologic diseases and injuries in patients.

Figure 1

Primary hNSC isolated directly from the human fetal neuroectoderm. (A) A phase image of the CNS-derived hNSC in culture. (B) The hNSC (>95%) express neural stem/progenitor marker nestin (green) and vimentin (green). All cells are revealed by DAPI staining of their nuclei (blue). (C) When provided with appropriate cues, the hNSC (c. 5–10%) differentiate into neurons [labeled for β-III-tubulin (red)] that express Nurr-1 (green), a marker associated with the midbrain dopaminergic phenotype. Scale bars: (A) 50 µm; (B, C) 5 µm.

Figure 2

Conversion of pluripotent hESC exclusively to a neuronal lineage. (A) A phase image of neuronal cells with extensive processes and networks directly induced from pluripotent hESC. (B) The neuronal cells induced from hESC express neuronal marker Map-2 (green); DAPI staining is blue. (C, D) The hESC-derived cells pursue a neuronal fate, as indicated by co-labeling with β-III-tubulin (red) and Map-2 (green). All cells are indicated by DAPI nuclear staining (blue). (E, F) A large subpopulation of the hESC-derived neuronal cells progress to expression of TH (red, c. 60%) and Nurr-1 (green, >95%), markers associated with the midbrain dopaminergic phenotype. Scale bars: (A) 100 µm; (B, C, E) 25 µm; (D, F) 5 µm.