Transplanted human neurons derived from a teratocarcinoma cell line (NTera‐2) mature, integrate, and survive for over 1 year in the nude mouse brain

Abstract

Retinoic acid (RA) induces a human teratocarcinoma cell line (NTera-2 or NT2) to give rise exclusively to post-mitotic neuron-like (NT2N) cells, but NT2N cells never acquire a fully mature neuronal phenotype in vitro. To determine whether NT2N cells can mature into adult neuron-like cells in vivo, purified NT2N cells were grafted into different regions of the central nervous system (CNS) of adult and neonatal athymic mice, and the grafts were examined immunohistochemically by light, confocal, and electron microscopy using antibodies to a panel of developmentally regulated neuronal polypeptides. NT2N grafts were distinguished from endogenous mouse neurons with antibodies that recognize human or murine specific epitopes in selected neuronal polypeptides. Viable NT2N cells were identified in > 89% of graft recipients (N = 90), and some grafts survived 14 months. Within 3 weeks of implantation, grafted NT2N cells re-extended their processes, and the location of the grafts (e.g., septum versus neocortex) appeared to determine the extent to which processes were elaborated. Within the early post-transplantation period, grafted NT2N cells expressed the same neuronal polypeptides as their in vitro counterparts. However, between 6 weeks and 4-6 months post-implantation, the grafted NT2N cells progressively acquired the molecular phenotype of fully mature in vivo neurons as evidenced by dramatically increased expression of the most highly phosphorylated isoforms of the heavy neurofilament subunit, and the de novo expression of adult CNS tau. Notably, the time course for the extension of processes and the expression of neuronal polypeptides by NT2N grafts was similar in neonatal and adult mice. Although grafted NT2N cells formed synapse-like structures and elaborated dendrites and axons, these axons remained unmyelinated. Finally, none of the transplanted NT2N cells reverted to a neoplastic state. These studies demonstrate that pure populations of grafted human NT2N cells acquire a fully mature neuronal phenotype in vivo, and that these cells integrate and survive for > 1 year post-implantation in the mouse CNS. These human neuron-like cells are an attractive model system for studies of neuronal development, polarity and transplantation.

Embryonic Stem Cell in the Therapy of Neurodegenerative Diseases

“…Mouse ESCs transplanted into adult mouse brain and kidney capsule developed large numbers of cells exhibiting neuronal morphology including DA neurons. TH positive axons from intracerebral grafts grew into regions of the dopamine-lesioned host striatal gray matter (Kleppner et al, 1995;Morassutti et al, 1994;Wojcik et al, 1993;Miyazono et al, 1995;Isacson & Deacon 1996;Hemmati-Brivanlou & Melton, 1997). Then a further study in which with low numbers of undifferentiated mouse ESCs implanted into the rat DA-depleted striatum, the cells proliferated and differentiated into functional DA neurons that reduced Parkinsonism (Björklund et al, 2002).…”

Stroke repair with cell transplantation: neuronal cells, neuroprogenitor cells, and stem cells

“…When transplanted, these neuronal cells survived, extended processes, expressed neurotransmitters, formed functional synapses, and integrated with the host. 16,33 During the retinoic acid induction process, significant changes were seen in the LBS neuron precursor cells that resulted in the loss of neuroepithelial markers and the appearance of neuronal markers. The final cell product was a Ն 95% pure population of human neuronal cells that appeared virtually indistinguishable from terminally differentiated, postmitotic neurons.…”

“…Animal transplantation studies of LBS neurons reveal graft survival, a mature neuronal phenotype, and integration into the host brain in vivo. 16,33,34 Layton BioScience neurons xenografted into different regions of the CNS of nude mice showed viable cells in 90% of recipients, with some grafts surviving for up to 14 months. Grafted LBS neurons initially remained similar to their in vitro counterparts, but then progressively acquired the phenotype of fully mature neurons in vivo.…”

“…Grafted LBS neurons initially remained similar to their in vitro counterparts, but then progressively acquired the phenotype of fully mature neurons in vivo. 3,16 Transplanted neurons formed synapselike structures and elaborated dendrites and axons, and have been tested electrophysiologically. In our first human trial, fluorodeoxyglucose-18 positron emission tomography showed increased uptake at the target site that correlated with the clinical response, and an autopsy evaluation of a graft 27 months after surgery showed surviving donor cells.…”

Cell therapy for stroke

“…14,19 They fail to acquire a fully mature phenotype in culture and terminally differentiate only when transplanted to the animal brain. 20 Grafted NT2N cells to the brains of adult nude mice show evidence for terminal differentiation by 6-8 weeks and elaborate processes within 3 weeks. The cells survive up to 14 months without reverting to a neoplastic state, 20 a significant concern given that these cells derive from a cancer cell line.…”