Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny

Neurological diseases today afflict about a billion people, accounting for 12% of human mortalities worldwide, and the incidence is expected to rise with an aging population1. The existence of stem cells in the adult mammalian2, particularly adult human3 central nervous system (CNS) makes it feasible for neurological injuries to undergo repair by endogenous mechanisms. Unfortunately, adult neurogenesis is likely not robust enough to address the severity of many injuries4,5. As another option, neural stem cells (NSCs) and precursor cells (NPCs) can be harvested from a donor, and then expanded in tissue culture for the purpose of later transplantation. Indeed, neural cell replacement therapy is a promising method to help regenerate the afflicted CNS, and the promise of this approach has inspired enormous amounts of global research. In light of the numerous types of neurodegenerative diseases and neurological insults diagnosed increasingly on an annual basis, it would seem that these research efforts are well placed. NSCs and NPCs have been transplanted as heterogeneous, undifferentiated material by many research groups, in animal models as well as clinically1,4,6,7. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate deleteriously within the host8,9. (2) There is little control over the donor cells’ terminal differentiation, e.g., a graft intended to replace lost neurons might choose a predominantly glial fate10,11,12,13,14. (3) There is insufficient ability of researchers to manage and modulate the specific combination of terminal cell types in pursuit of a precise injury treatment (i.e., there is limited investigative power). Controlling the terminal phenotypic fate of grafted cells has long been a challenge in the field. NSCs and NPCs implanted into the CNS have primarily become astrocytes10,11,12,13,14, which are inadequate by themselves to constitute neural networks and can even have adverse effects such as allodynia10,15. Shortcomings such as these have inspired many groups to innovate ways of manipulating donor cells in vitro, prior to transplant, with the aim of enhancing transplant precision and functional outcome16,17,18. Here we demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in tissue culture, followed by isolation of the neuronal progeny from the glia. We reasoned that by providing heterogeneous hNPCs with neuronal-inducing factors in vitro, and then subjecting the differentiated cells to a phenotype-based separation technique before grafting, a high yield of donor astrocytes would be avoided. We hypothesized that transplanting a high concentration of immature neurons into the host CNS would result in fewer surviving donor astrocytes, as compared to transplanting a heterogeneous population of undifferentiated hNPCs. The phenotypic predictability of such a pre-defined, neuronally-enriched human donor cell graft after a prolonged period in vivo has not been directly investigated. In multiple experiments in this study, either undifferentiated hNPCs, or a defined concentration of hNPC-derived immature neurons were transplanted into immune-compromised mice. The two graft types were compared with regard to their in vivo survival, proliferative capacity and phenotypic fate. We present evidence suggesting that pre-differentiated, purified grafted cells survive as well in vivo as their heterogeneous, undifferentiated progenitors, and undergo less proliferation and less astrocytic differentiation. We also demonstrate accompanying procedures for improved hNPC low-temperature preservation and portability, vitally necessary components in “off-the-shelf” cell-based strategies of replacing tissue lost to injury or disease.