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. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

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. For this reason, the demand for a safe and efficient multipotent cell source that can differentiate into various neural cells is increasing. In this study, we evaluated the efficacy and safety of human polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive neural precursor cells (hNPCsPSA-NCAM+) as a treatment for SCI. METHODS: One hundred thousand hNPCsPSA-NCAM+ isolated from human embryonic stem cell-derived NPCs were transplanted into the lesion site by microinjection 7 days after contusive SCI at the thoracic level. We examined the histological characteristics of the graft and behavioral improvement in the SCI rats 10 weeks after transplantation. RESULTS: Locomotor activity improvement was estimated by the Basso-Beattie-Bresnahan locomotor rating scale. Behavioral tests revealed that the transplantation of the hNPCsPSA-NCAM+ into the injured spinal cords of rats significantly improved locomotor function. Histological examination showed that hNPCsPSA-NCAM+ had differentiated into neural cells and successfully integrated into the host tissue with no evidence of tumor formation. We investigated cytokine expressions, which led to the early therapeutic effect of hNPCsPSA-NCAM+, and found that some undifferentiated NPCs still expressed midkine, a well-known neurotrophic factor involved in neural development and inflammatory responses, 10 weeks after transplantation. CONCLUSION: Our results demonstrate that hNPCsPSA-NCAM+ serve as a safe and efficient cell source which has the potential to improve impaired motor function following SCI.

Conditioned Medium of Human Pluripotent Stem Cell-Derived Neural Precursor Cells Exerts Neurorestorative Effects against Ischemic Stroke Model.

Previous studies have shown that early therapeutic events of neural precursor cells (NPCs) transplantation to animals with acute ischemic stroke readily protected neuronal cell damage and improved behavioral recovery through paracrine mechanisms. In this study, we tested the hypothesis that administration of conditioned medium from NPCs (NPC-CMs) could recapitulate the beneficial effects of cell transplantation. Rats with permanent middle cerebral artery occlusion (pMCAO) were randomly assigned to one of the following groups: PBS control, Vehicle (medium) controls, single (NPC-CM(S)) or multiple injections of NPC-CM(NPC-CM(M)) groups. A single intravenous injection of NPC-CM exhibited strong neuroregenerative potential to induce behavioral recovery, and multiple injections enhanced this activity further by suppressing inflammatory damage and inducing endogenous neurogenesis leading to histopathological and functional recovery. Proteome analysis of NPC-CM identified a number of proteins that are known to be associated with nervous system development, neurogenesis, and angiogenesis. In addition, transcriptome analysis revealed the importance of the inflammatory response during stroke recovery and some of the key hub genes in the interaction network were validated. Thus, our findings demonstrated that NPC-CM promoted functional recovery and reduced cerebral infarct and inflammation with enhanced endogenous neurogenesis, and the results highlighted the potency of NPC-CM in stroke therapy.

Trophoblast glycoprotein is a new candidate gene for Parkinson's disease.

Parkinson's disease (PD) is a movement disorder caused by progressive degeneration of the midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNc). Despite intense research efforts over the past decades, the etiology of PD remains largely unknown. Here, we discovered the involvement of trophoblast glycoprotein (Tpbg) in the development of PD-like phenotypes in mice. Tpbg expression was detected in the ventral midbrain during embryonic development and in mDA neurons in adulthood. Genetic ablation of Tpbg resulted in mild degeneration of mDA neurons in aged mice (12-14 months) with behavioral deficits reminiscent of PD symptoms. Through in silico analysis, we predicted potential TPBG-interacting partners whose functions were relevant to PD pathogenesis; this result was substantiated by transcriptomic analysis of the SNc of aged Tpbg knockout mice. These findings suggest that Tpbg is a new candidate gene associated with PD and provide a new insight into PD pathogenesis.

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. TPBG-targeted cell sorting enriched FOXA2+LMX1A+ vmDA precursors and helped attain efficient behavioral recovery of rodent PD models with increased numbers of TH+, NURR1+, and PITX3+ vmDA neurons in the grafts. Additionally, fewer proliferating cells were detected in TPBG+ cell-derived grafts than in TPBG- cell-derived grafts. Our approach is an efficient way to obtain enriched bona fide vmDA precursors, which could open a new avenue for effective PD treatment.

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. After subretinal injection in rat animal models, retinal function was determined with electroretinography and optokinetic response detection, and immunofluorescent staining was performed to assess the survival of the injected cells. Results: Cocultured cells were positive for rhodopsin, red and blue opsin, recoverin, and phosphodiesterase 6 beta on immunofluorescent staining and RT-PCR. Serial detection of stem cell-, neural precursor-, and photoreceptor-specific markers was noted in each stage of differentiation with microarray analysis. Increased cGMP hydrolysis in light-exposed conditions compared to that in dark conditions was observed. After the subretinal injection in the rats, preservation of optokinetic responses was noted up to 20 weeks, while electroretinographic response decreased. Survival of the injected cells was confirmed with positive immunofluorescence staining of human markers at 8 weeks. Conclusions: Cells showed photoreceptor-specific features when stem cell-derived neurogenic precursors were cocultured with RPE cells.

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. In addition, FVIII expression was confirmed in YCMi001-B-1-derived endothelial cells.

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. After differentiation into mDA neurons, most PITX3 immunoreactivity overlapped with the red fluorescence of mCherry. This reporter cell line may be used to study the development of mDA neurons or to enrich mDA populations for transplantation.

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. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIII gene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.

Defined Conditions for Differentiation of Functional Retinal Ganglion Cells From Human Pluripotent Stem Cells.

Purpose: We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. Methods: To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. Results: Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. Conclusions: This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

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. Cell survival, proliferation, apoptosis, and differentiation on days 1, 3, 5, and 7 post treatment were analyzed. RESULTS: Treatment with 6% sevoflurane increased cell viability (P = 0.046) and decreased apoptosis (P = 0.014) on day 5, but the effect did not persist on day 7. Survival and apoptosis were not affected by 3% and 8% sevoflurane; there was no effect of proliferation at any of the tested concentrations. The differentiation of cells exposed to 6% or 8% sevoflurane decreased on day 1 (P = 0.033 and P = 0.036 for 6% and 8% sevoflurane, respectively) but was again normalized on days 3-7. CONCLUSION: Clinically relevant treatment with sevoflurane for 2 h induces no significant changes in the survival, proliferation, apoptosis, and differentiation of human neural precursor cells, although supraclinical doses of sevoflurane do alter human neurogenesis transiently.

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. METHODS: We used the National Health Insurance Service National Sample Cohort. In this study, data finally included 472,250 patients who were newly diagnosed with dental caries during 2003-2013. We performed survival analysis using cox proportional hazard model, negative binomial-regression, and regression analyses using generalized estimating equation models. RESULTS: There were 48.49 % outpatient dental care visit during study period. Individuals with water fluoridation had a lower risk of dental care visits (HR = 0.949, 95 % CI = 0.928-0.971). Among the individuals who experienced a dental care visit, those with water fluoridation program had a lower number of dental care visits (ß = -0.029), and the period of water fluoridation had an inverse association with the dental care expenditures. CONCLUSION: The implementation of water fluoridation programs and these periods are associated with reducing the utilization of dental health care. Considering these positive impacts, healthcare professionals must consider preventive strategies for activating water fluoridation programs, such as changes in public perception and relations, for the effective management of dental care in South Korea.

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. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

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. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells (10(5) cells/5 µL) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. RESULTS: The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. CONCLUSION: Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease.

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. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.

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. In this report, we describe our method step by step, covering not only how to differentiate hESCs into DA neurons at a high yield, but also how to amplify, freeze and thaw the SNMs, which are the key structures that make our protocol unique and advantageous. Although the whole process of generation of DA neurons from hESCs takes about 2 months, only 14 d are needed to derive DA neurons from the SNMs.

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. 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.

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. As an alternative, cell transplantation therapy has been tried. Although transplantation of fetal ventral mesencephalic cells looks promising for the treatment of PD in some cases, ethical and technical problems in obtaining large numbers of human fetal brain tissues also lead to difficulty in its clinical application. Our recent studies showed that a high yield of DA neurons could be derived from embryonic stem (ES) cells and they efficiently induced behavioral recovery in a PD animal model. Here we summarize methods for generation of functional DA neurons from ES cells for application to PD models.