Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease.

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities.

Transplantation of PSA-NCAM-Positive Neural Precursors from Human Embryonic Stem Cells Promotes Functional Recovery in an Animal Model of Spinal Cord Injury.

Background: Spinal cord injury (SCI) results in permanent impairment of motor and sensory functions at and below the lesion site. There is no therapeutic option to the functional recovery of SCI involving diverse injury responses of different cell types in the lesion that limit endogenous nerve regeneration. In this regard, cell replacement therapy utilizing stem cells or their derivatives has become a highly promising approach to promote locomotor recovery.

Trophoblast glycoprotein is a marker for efficient sorting of ventral mesencephalic dopaminergic precursors derived from human pluripotent stem cells.

Successful cell therapy for Parkinson's disease (PD) requires large numbers of homogeneous ventral mesencephalic dopaminergic (vmDA) precursors. Enrichment of vmDA precursors via cell sorting is required to ensure high safety and efficacy of the cell therapy. Here, using LMX1A-eGFP knock-in reporter human embryonic stem cells, we discovered a novel surface antigen, trophoblast glycoprotein (TPBG), which was preferentially expressed in vmDA precursors.

Embryonic stem cell-derived photoreceptor precursor cells differentiated by coculture with RPE cells.

Purpose: To describe the derivation of photoreceptor precursor cells from human embryonic stem cells by coculture with RPE cells.

Methods: Human embryonic stem cells were induced to differentiate into neural precursor cells and then cocultured with RPE cells to obtain cells showing retinal photoreceptor features. Immunofluorescent staining, reverse transcription-PCR (RT-PCR), and microarray analysis were performed to identify photoreceptor markers, and a cGMP assay was used for in vitro functional analysis.

Generation of a gene edited hemophilia A patient-derived iPSC cell line, YCMi001-B-1, by targeted insertion of coagulation factor FVIII using CRISPR/Cas9.

Hemophilia A is an ideal target for cell or gene therapy because a mild increase in coagulation factor VIII (FVIII) improves symptoms in patients with severe hemophilia A. In this study, we used CRISPR/Cas9 to insert FVIII cDNA into exon 1 of the mutant FVIII locus in induced pluripotent stem cells (iPSCs) from a hemophilia A patient. This gene-modified YCMi001-B-1 line maintained its pluripotency, formed all three germ layers, and had a normal karyotype.

Establishment of PITX3-mCherry knock-in reporter human embryonic stem cell line (WAe009-A-23).

Pituitary homeobox 3 (Pitx3) is a key transcription factor that plays an important role in the development and maintenance of midbrain dopaminergic (mDA) neurons. Here, we established a PITX3-mCherry knock-in reporter human embryonic stem cell (hESC) line using the CRISPR/Cas9 system. PITX3-mCherry hESCs maintained pluripotency marker expression and exhibited the capacity to generate all 3 germ layers and a normal karyotype.

Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs.

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient.

Effects of short-term exposure to sevoflurane on the survival, proliferation, apoptosis, and differentiation of neural precursor cells derived from human embryonic stem cells.

Purpose: Data from animal experiments suggest that exposure to general anesthetics in early life inhibits neurogenesis and causes long-term memory deficit. Considering short operating times and the popularity of sevoflurane in pediatric anesthesia, it is important to verify the effects of short-period exposure to sevoflurane on the developing brain.

Methods: We measured the effects of short-term exposure (2 h) to 3%, 6%, or 8% sevoflurane, the most commonly used anesthetic, on neural precursor cells derived from human embryonic stem cells, SNUhES32.

The differences in healthcare utilization for dental caries based on the implementation of water fluoridation in South Korea.

Background: There were some debates about the water fluoridation program in South Korea, even if the program had generally substantial effectiveness. Because the out-of-pocket expenditures for dental care were higher in South Korea than in other countries, an efficient solution was needed. Therefore, we examined the relationship between the implementation of water fluoridation and the utilization of dental care.

Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors.

Subretinal transplantation of putative retinal pigment epithelial cells derived from human embryonic stem cells in rat retinal degeneration model.

Objective: To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases.

Methods: The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage.

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways.

Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale.

Cell-replacement therapy using human embryonic stem cells (hESCs) holds great promise in treating Parkinson's disease. We have recently reported a highly efficient method to generate functional dopaminergic (DA) neurons from hESCs. Our method includes a unique step, the formation of spherical neural masses (SNMs), and offers the highest yield of DA neurons ever achieved so far.

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

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.

Derivation of functional dopamine neurons from embryonic stem cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra of the midbrain. Pharmacological treatment of PD has been a prevailing strategy. However, it has some limitations because its effectiveness gradually decreases and side effects develop.

Efficient induction of oligodendrocytes from human embryonic stem cells.

Oligodendrocytes form myelin sheaths around axons to support rapid nerve conduction in the central nervous system (CNS). Damage to myelin can cause severe CNS disorders. In this study, we attempted to devise a protocol for the induction of oligodendrocytes from human embryonic stem (ES) cells to treat demyelinated axons.

Dopamine neurons derived from embryonic stem cells efficiently induce behavioral recovery in a Parkinsonian rat model.

To test the in vivo effect of a high yield of dopaminergic (DA) neurons (90% of total neurons) which had been generated from a genetically modified mouse embryonic stem cell line, N2, the cells were transplanted into a rat model of Parkinson's disease (PD). The PD animals grafted with N2-derived cells showed significant behavior improvements compared with sham controls from 2 weeks posttransplantation, whereas animals with naïve D3-derived cells ( approximately 28% DA neurons of total neurons) showed only a modest recovery. Furthermore, hyperactivity observed in the subthalamic nucleus, pedunculopontine nucleus, and substantia nigra pars reticulata of PD rat models was dramatically reduced by the grafting of N2-derived cells.

Methods for expansion of human embryonic stem cells.

The manipulation of human embryonic stem cells (hESCs) requires refined skills. Here we introduce both mechanical and enzymatic transfer methods for hESCs depending on experimental purpose. We use the mechanical transfer method for maintenance of hESC lines.