Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions  

Abstract

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs. Stem Cells 2010;28:1893–1904

Cell Therapy for Multiple Sclerosis

“…Although the use of ES cells dictates an allogeneic donor cell source, the recent generation of induced pluripotential stem (iPS) cells from somatic mouse [213] cells and from human [214,215] [216][217][218] and oligodendrocytes [219,220]. It will now be necessary to explore the potential for generating populations of iPS-derived oligodendrocytes for autologous grafting in the myelin disorders.…”

Treatment of Parkinson's disease using cell transplantation

“…This is unlikely to occur if the technology is used to create a cell bank with human leukocyte antigen haplotype-matching iPS cells to be used in allografting experiments [51]. Extensive axonal outgrowth from the human iPS cellderived dopaminergic neurons implanted into the denervated rodent striatum has not been convincingly demonstrated [38, [46][47][48][49][50], and their capacity to improve behavioural deficits relevant for the clinical condition is incompletely known. A rich dopaminergic terminal network extending throughout the striatum will definitely be needed for clinical efficacy.…”

Isolation of Human Neural Stem Cells from the Amniotic Fluid with Diagnosed Neural Tube Defects

“…To direct the differentiation of specific cell types, AF-NSCs were seeded on plates coated with 100 mg/mL poly-L-lysine at a density of 5 · 10 4 cells/cm 2 in the presence of the NS-A proliferation medium. After attachment, the medium could be changed to the specific induction medium that would produce astrocytes, oligodendrocytes, and dopaminergic neurons according to previously published protocols [26]. AF-NSCs were cultured in the DMEM/F12 medium (Life Technologies) supplemented with 1 · NEAA (Life Technologies), 2 mM lglutamine (Life Technologies), 1 · N2 (R&D Systems), and 1 · B27 (Life Technologies) for 2 weeks to differentiate into astrocytes.…”