Little is known to date about neural development of pig and directed differentiation of porcine pluripotent stem cells (PSCs) to neuronal cells remains elusive. To determine whether soluble factors from glioblastoma multiforme (GBM) promoted the neural differentiation from porcine induced PSCs (iPSCs), cells were treated cultured media of GBM cells. First of all, we isolated and established primary GBM cell line (WHO grade IV). The cellular morphology of GBM cancer cell line are dendritic-like with positive expression in NESTIN, SOX2, VIMENTIN and GFAP using immunofluorescence analysis. G-banded karyotype from primary GBM cell line revealed severe numerical chromosomal aberrations. GBM-cultured medium (CM) treated iPSC-NPCs survive well in vitro when supplemented with a combination of growth factors, including EGF and bFGF. The GBM-CM treated differentiated cells showed an increased mRNA expression level of astrocyte marker, GFAP and the dopaminergic neuron marker, tyrosine hydroxylase (TH). However, there was no significant difference in mRNA expression level of oligodendrocyte marker, MBP. The protocol developed in the present study for large animal models might provide an exciting tool to bridge the present gaps in neuroscience studies between rodents and humans.

Introducing lineage-specific transcription factors (TFs) into somatic cells enables the induction of distinct cellular identities without the need to first pass through a pluripotent stem cell (PSC) state. We and others have demonstrated the direct conversion of somatic cells into adult stem cells or progenitor cells, such as angioblast-like progenitor cells, hematopoietic stem cells, and neural stem cells. The process underlying direct conversion is known to be relatively simpler and faster than that of induced pluripotent stem cell (iPSC) generation. Furthermore, directly converted cells have been shown to exhibit therapeutic potential following transplantation into respective disease models without obvious evidence for tumor formation. Thus, TF-mediated direct conversion technology has been considered as an alternative to iPSC technology for patient-specific cell- and tissue-replacement therapies. Here we show our recent findings describing the robust direct conversion of differentiated somatic cells into distinct cellular identities. Furthermore, we also show the recent 3D organoid technology for generating brain tissues from human pluripotent stem cells.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Neural stem cells (NSCs) are self-renewing tripotent cell populations and have capacity of neuronal (neurons) and glial (astrocytes and oligodendrocytes) differentiation. Many researchers have reported that NSCs have therapeutic effects in neurological disease by transplantation. However, it is not easy to obtain NSCs in vitro. Recently, Yamanaka and colleagues showed that somatic cells could be reprogrammed into pluripotent state by enforcing reprogramming factors. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. Direct differentiation into a specialized cell types from iPS cells hold considerable promise for regenerative medicine as well as basic research. Here, we induced differentiation of iPS cells into NSCs in vitro and in vivo, which were compared with embryonic stem (ES) cell-derived NSCs and brain derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from brain derived NSCs and stained positive for NSCs markers Nestin and Sox2. ES cells derived NSCs were transcriptionally distinguishable from brain derived NSCs. However, global gene expression pattern were similar but distinct between iPS derived NSCs and brain derived NSCs. Moreover, iPS derived NSCs were spontaneously aggregated upon passaging, formed ES cell like colonies, and finally reactivated Oct4-GFP. The spontaneously reverted GFP-positive cells (iPS-NSC-iPS) expressed similar levels of pluripotency markers (Oct4,Nanog) to ES and iPS cells, and could form germ line chimera. One possible explanation for this phenomenon is that spontaneously re-reprogramming was associated with transgene re-activation when iPS cells were differentiated into NSCs. However, NSCs from dox-inducible iPScells could not be reprogrammed into pluripotent state without doxycycline. Taken together, iPS derived NSCs were morphologically and similar to brain derived NSCs, but differ in gene expression pattern and maintenance. * This work was supported by the Next Generation Bio-Green21 Program funded by the Rural Development Administration (Grant PJ008009).

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of Tuj1 increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous Igf2 may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

Cell transplantation therapy using adult stem cells has recently been identified as a potential treatment for spinal cord injury (SCI). But, recovery after traumatic SCI is very limited. As dogs are physiologically much more similar to human compared with other traditional mammalian models in disease presentation and clinical responses, a number of researches demonstrated canis familiaris is a suitable model for human diseases. This study investigated the effect of transplantation of canine Mesenchymal Stem Cells (cMSC) and neural-induced cMSC (nMSC) to understand how these cells improve neurological function in canine SCI model. The differentiation of cMSC into neural precursor cells was induced in dulbecco’s modified eagle’s medium supplemented with N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. SCI was induced between T1 and T2 by surgical hemi-section in adult dogs, and then assigned to two groups according to the applied cell types (cMSC vs nMSC). Pelleted cMSC or nMSC were transplanted directly into the injured site after SCI, respectively. Analysis of motor function after transplantation was evaluated by modified Olby score. Magnetic resonance imaging (MRI), histological and immunohistichemical analysis were also performed. Functional recovery in group of cMSC was increasing gradually after transplantation and was higher than nMSC. In MRI, we could not confirm any difference between the cMSC and nMSC experimental groups. Immunohistochemically, beta3-tubuline and nestin were observed in injury site of two experimental groups with the expression level close to non-injured groups. Transplantation of mesenchymal stem cells could promote neuronal reconstruction and repair motor function in SCI. These showed mesenchymal stem cells could be a great candidate as a therapeutic tools in degeneration disease, and dogs could be used to explore human regenerative medicine as a promising animal model. This research was supported by iPET (Grants 110056032CG000), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

This study was designed to investigate the changes in the properties of the neuronal setm cells or progenitor cells associated with age-related decline in neurogenesis of the hippocampal dentate gyrus (DG). Active whole cells cycle marker Ki67 (a marker of whole cell cycle)-positive and S phase marker bromodeoxyuridine (BrdU)-positive. Neural stem cells gradually were reduced in the hippocampal subgranular zone (SGZ) in an age-dependant manner after birth (from P1 month to P1 year). The ratio of BrdUpositivecells/Ki67-positive cells was gradually enhanced in an age-dependent manner. The ratio of Ki67-positive cells/accu-mulating BrdU-positive cells at 3 hrs after BrdU injection was injected once a day for consecutive 5 days gradually decreased during ageing. TUNEL- and caspase 3 (apoptotic terminal caspase)-positive cells gradually decreased in the dentate SGZ during ageing and immunohistochemical findings of glial fibrillary acid protein (GFAP) were not changed during ageing. NeuN, a marker of mature neural cells, and BrdU-double positive cells gradually decreased in an age-dependent manner but differentiating ratio and survival rate of cells were not changed at 4 wks after BrdU injection once a day for consecutive 5 days. The number of BrdU-positive cells migrated from the hippocampal SGZ into granular layer and its migration speed was gradually declined during ageing. These results suggest that the adult neurogenesis in the mouse hippocampal DG gradually decrease through reducing proliferation of neural stem cells accompanying with cells cycle change and reduced cells migration rather than changes of differentiation.

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III β-tubulin and MAP2ab were observed. Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo. Human ES cells have the capacity to differentiate into various types of cells in the body. Human ES cells are indefinite source of cells for cell therapy in various degenerative disorders including neuronal disorders. Directed differentiation of human ES cells is a prerequisite for their clinical application. The objective of this study is to develop the culture condition for the derivation of neural precursor cells from human ES cells. Neural precursor cells were derived from human ES cells in a stepwise culture condition. Neural precursor cells in the form of neural rosette structures developed into neurospheres when cultured in suspension. Suspension culture of neurospheres has been maintained over 4 months. Expressions of nestin, soxl, sox2, pax3 and pax6 transcripts were upregulated during differentiation into neural precursor cells by RT-PCR analysis. In contrast, expression of oct4 was dramatically downregulated in neural precursor cells. Immunocytochemical analyses of neural precursor cells demonstrated expression of nestin and SOX1. When induced to differentiate on an adhesive substrate, neuro-spheres were able to differentiate into three lineages of neural systems, including neurons, astrocytes and oligo-dendrocytes. Transcripts of sox1 and pax6 were downregulated during differentiation of neural precursor cells into neurons. In contrast, expression of map2ab was elevated in the differentiated cells, relative to those in neural precursor cells. Neurons derived from neural precursor cells expressed NCAM, Tuj1, MAP2ab, NeuN and NF200 in immunocytochemical analyses. Presence of astrocytes was confirmed by expression of GFAP immuno-cytochemically. Oligodendrocytes were also observed by positive immuno-reactivities against oligodendrocyte marker O1. Results of this study demonstrate that a stepwise culture condition is developed for the derivation of neural precursor cells from human ES cells.