Human embryonic stem cell‐derived neural precursors as a continuous, stable, and on‐demand source for human dopamine neurons

Abstract

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 · 10 4 -folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.

Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture

“…However, the expression levels of three germ layer markers were significantly reduced in late passage. Ko et al (2007) also reported that the degree of hESC differentiation varied depending on the passage number of undifferentiated hESCs in HSF-6 hES cell line. These results indicated that although long-term cultured hESCs retained somewhat their pluripotency, they also possessed a tendency to maintain an undifferentiated state rather than to differentiate.…”

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease

“…The hiPSCs and hESCs used in this study are listed in Tables 1 and 2. Undifferentiated hiPSCs and hESCs were propagated on MEF feeder and differentiated toward midbrain-type NPCs on feeder layers of MS5 and subsequently on MS5-SHH as previously described (17,45). bFGF (20 ng/ml) was supplemented during the first week of the coculture period.…”

“…Since individual ES cell (ESC) and iPSC lines are known to have different propensities to differentiate into specific cell lineages (11)(12)(13), we first optimized in vitro differentiation methods to generate neuronal precursor cells (NPCs) and DA neurons from these diverse hiPSC lines. Based on previous studies showing efficient neural induction and/or proliferation of NPCs on different feeder cells (14)(15)(16)(17)(18)(19), we first optimized the stromal coculture method to ensure efficient neural induction from hiPSC and hESC lines. As schematized in Figure 1A, undifferentiated hiPSCs maintained on mouse embryonic fibroblasts (MEFs) were sequentially cocultured onto MS5 feeder cells and MS5 stably expressing sonic hedgehog (MS5-SHH).…”

Dopaminergic Neurons Derived from Human Embryonic Stem Cell Derived Neural Progenitors: Biological Relevance and Application

“…These neurons were transplanted into the nigral-stratial pathway with negative consequences including proliferation following transplantation and terotoma formation (Brederlau et al, 2006). Differentiation of H9 hESCs on a SHH secreting M5S stromal feeder layer with bFGF lead to no teratoma formation when transplanted into the SN, but few TH+ cells survived (Ko et al, 2007). Attempts at differentiation with a bone marrow stromal cell feeder layer and FGF8/SHH lead to 40% TH+ cells but no cells survived the graft.…”