Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells

Abstract

We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.is a neurodegenerative disorder characterized by progressive and selective loss of dopaminergic (DA) neurons in the midbrain substantia nigra (1). Currently, the prevailing strategy for the treatment of PD is pharmacological. However, pharmacological treatment with L-DOPA works initially, but over time, the effectiveness of L-DOPA wanes and side effects develop (2). An alternative approach may be the transplantation of DA-synthesizing cells. One source of DA-synthesizing cells is embryonic stem cells (ESCs). ESCs are pluripotent and capable of self-renewal (3-5). For the purpose of applying the ESCs to PD, many researchers have tried to develop protocols by which ESCs from some species can differentiate into DA neuronal phenotypes (6-11). Although some progress has been made in the generation of DA neurons from human ESCs (hESCs) (12-22), there are still many technical improvements to be made before the application of hESCs to treat PD. Examples include increasing the purity of DA neurons, supplying a sufficient quantity of DA neurons for clinical applications, decreasing tumor formation after transplantation, and clearly demonstrating the functionality of hESC-derived DA neurons in a parkinsonian animal model.Here, we introduce a method that allows us to differentiate hESCs into functional tyrosine hydroxylase-positive (TH ϩ ) neurons up to near 86% of the total hESC-derived neurons, which is the highest purity ever reported. Achieving high efficiency of DA neuronal derivation is an important issue in cell therapy, because it would not only increase the effica...

Cellular Repair in the Parkinsonian Nonhuman Primate Brain

“…Several methods have been reported, with functional improvements in rodent models, but the results have been less successful than had been hoped. [49][50][51][52][53] Several studies 190 REDMOND ET AL.…”

Cell Therapeutics in Parkinson's Disease

“…So far, the most promising results have been obtained using embryonic stem (ES) cells [52][53][54][55][56][57][58][59][60][61][62] or therapeutically cloned ES cells [63]. The examples shown (Fig.…”

“…Other types of stem cells, such as NSCs and progenitors from the embryonic ventral mesencephalon [64][65][66][67], adult NSCs from the subventricular zone [68], bone marrow stromal cells [69], and fibroblast-derived induced pluripotent stem (iPS) cells [61,[70][71][72] have also been used for this purpose. It has also been shown that human stem cell-derived DA neuroblasts, which will be required for patient application, can survive in animal models of PD and after maturation exert functional effects [54,58,66,69,71,72]. However, some properties that will be fundamental for successful clinical translation have not been demonstrated yet for human stem cell-derived DA neurons, including those that they can substantially reinnervate striatum, restore DA release in vivo, and markedly improve deficits resembling the symptoms experienced by patients with PD.…”

Induced human pluripotent stem cells: promises and open questions

“…Such methods established for human ES-derived cells are lineage-specific directed differentiation and selection techniques to generate pure populations of specialised cells. For example, human ES cells have been used to generate functional neurons essential to cure senso-motoric deficits in an animal model of Parkinson's disease (Cho et al, 2008) or to develop insulin-producing cells that reverted glycaemia in diabetic animals (Kroon et al, 2008). At first, these techniques have to be adapted to reprogrammed iPS cells.…”