Studies on the Differentiation of Dopaminergic Traits in Human Neural Progenitor Cells in Vitro and in Vivo

Abstract

The development of cell replacement therapies for the treatment of neurodegenerative disorders such as Parkinson's disease (PD) may depend upon the successful differentiation of human neural stem/progenitor cells into dopamine (DA) neurons. We show here that primary human neural progenitors (HNPs) can be expanded and maintained in culture both as neurospheres (NSPs) and attached monolayers where they develop into neurons and glia. When transplanted into the 6-hydroxydopamine-lesioned rat striatum, undifferentiated NSPs survive longer (60% graft survival at 8-16 weeks vs. 30% graft survival at 8-13 weeks) and migrate farther than their attached counterparts. While both NSP and attached cells continue to express neuronal traits after transplantation, the spontaneous expression of differentiated transmitter-related traits is not observed in either cell type. However, following predifferentiation in culture using a previously described cocktail of reagents, approximately 25% of HNPs can permanently express the DA enzyme tyrosine hydroxylase (TH), even following replating and removal of the DA differentiation cocktail. When these predifferentiated HNPs are transplanted into the brain, however, TH staining is not observed, either because expression is lost or TH-expressing cells preferentially die. Consistent with the latter view is a decrease in total cell survival and migration, and an enhanced glial response in these grafts. In contrast, we found that the overall survival of HNPs is improved when cells engraft near blood vessels or CSF compartments or when they are placed into an intact unlesioned brain, suggesting that there are factors, as yet unidentified, that can better support the development of engrafted HNPs.

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

“…Further differentiation of DA traits in Stage V was achieved by incubating cells seeded onto poly-ornithine coated slides for an additional 1-7 days in DM supplemented with 1mM dibutyryl cAMP (dbcAMP) as described in earlier studies (20,23,24). Following this treatment, no cells expressed stem cell markers like Oct-4 ( Fig.…”

“…Therefore, to learn whether the DA differentiation process could be initiated in culture with the current protocol but continued in situ in the brain, hES cells from H9 (N=3), HUES7 (N=2) and HUES8 (N=5) lines were harvested at the neural progenitor stage (Stage IV) for transplant. Approximately 2-5 × 10 5 cells were stereotaxically implanted into the striata rats with unilateral 6-OHDA lesions as described previously (24,25). Upon sacrifice 2-3 weeks later, robust cell survival was seen in 7 of the 10 engrafted animals (no viable hES cells were found in one H9 and two HUES8 grafts).…”

“…During this period, whether cells are attached to a substrate or differentiated in suspension culture, they can elicit an extensive highly branched network of processes that make harvest untenable without irreparable mechanical damage to cells. Ultimately, few, if any, of these cells survive transplantation into the brain (19,24).…”

Transplantation of Human Neural Stem Cells Exerts Neuroprotection in a Rat Model of Parkinson's Disease

“…Even with daily immunosuppression, graft survival of v-myc oncogeneprimed HB1.F3 cells was ϳ1% at 1 month after transplantation, which is relatively low compared with at least 5% graft survival with other neural stem/progenitor cells (Borlongan et al, 1998;Yang et al, 2004;Harrower et al, 2006). In addition, almost all transplanted cells resided at the original implantation site.…”

Providing healthy diets for young children: the experience of parents in a UK inner city

“…The significant and abiding consequences of obesity are widely evidenced and include physical co-morbidities such as diabetes and heart disease (Huang et al, 2015; Martinez, Milagro, Claycombe, & Schalinske, 2014). In addition, associations between obesity, particularly in childhood, and psychological disorders such as depression and emotional and behavioural disorders have been found (Rankin et al, 2016; Yang, Donaldson, Marshall, Shen, & Iacovitti, 2004). Obesity has remained a significant and costly public health issue in the UK for a number of years (Wang et al, 2015) and there is now a considerable and consistent body of evidence to demonstrate that obesity in childhood tends to continue into adolescence and through to adulthood resulting in adverse consequences for physical morbidity and possibly premature mortality (Reilly & Kelly, 2011).…”