• International Journal of Stem Cells
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• v.17 no.1
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• pp.59-69
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• 2024

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.

• Molecules and Cells
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• v.42 no.3
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• pp.200-209
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• 2019

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been used as promising tools for regenerative medicine, disease modeling, and drug screening. Traditional and common strategies for pluripotent stem cell (PSC) differentiation toward disease-relevant cell types depend on sequential treatment of signaling molecules identified based on knowledge of developmental biology. However, these strategies suffer from low purity, inefficiency, and time-consuming culture conditions. A growing body of recent research has shown efficient cell fate reprogramming by forced expression of single or multiple transcription factors. Here, we review transcription factor-directed differentiation methods of PSCs toward neural, muscle, liver, and pancreatic endocrine cells. Potential applications and limitations are also discussed in order to establish future directions of this technique for therapeutic purposes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.10-10
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• 2006

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.67-74
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• 2004

Objective: This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). Materials and Methods: Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA, embryoid bodies (EB, for 4 days) derived from TH#2/MB03 cells were exposed to RA ($10^{-6}M$)/AA ($5{\times}10^{-2}mM$) for 4 days, and were allowed to differentiate in N2 medium for 7, 14 or 21 days. Exp. II) When b-FGF was used, neuronal precursor cells were expanded at the presence of b-FGF (10 ng/ml) for 6 days followed by a final differentiation in N2 medium for 7, 14 or 21 days. Neuron differentiation was examined by indirect immunocytochemistry using neuron markers (NF160 & NF200). Results: After 7 days in N2 medium, approximately 80% and 20% of the RA or b-FGF induced Th#2/MB03 cells were immunoreactive to anti-NF160 and anti-NF200 antibodies, respectively. As differentiation continued, NF200 in RA treated cells significantly increased to 73.0% on 14 days compared to that in b-FGF treated cells (53.0%, p<0.05), while the proportion of cells expressing NF160 was similarly decreased between two groups. However, throughout the differentiation, expression of TH was maintained ($\sim$90%). HPLC analyses indicated the increased levels of L-DOPA in RA treated genetically modified hES cells with longer differentiation time. Conclusion: These results suggested that a genetically modified hES cells (TH#2/MB03) could be efficiently differentiated in vitro into mature neurons by RA induction method.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2004.07a
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• pp.21-21
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• 2004

• IMMUNE NETWORK
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• v.14 no.1
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• pp.54-65
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• 2014

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

• International Journal of Oral Biology
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• v.33 no.2
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• pp.59-64
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• 2008

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.

• Journal of Life Science
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• v.28 no.12
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• pp.1397-1405
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• 2018

Mitochondria have multiple functions in cells: providing chemical energy, storing cellular $Ca^{2+}$, generating reactive oxygen species, and regulating apoptosis. Through these functions, mitochondria are also involved in the maintenance, proliferation, and differentiation of stem/progenitor cells. In the brain, the subventricular zone (SVZ) is one of the neurogenic regions that contains neural stem cells (NSCs) throughout a lifetime. However, reports on the role of mitochondria in SVZ NSCs are scarce. Here, we show that rotenone, a complex I inhibitor of mitochondria, inhibits the proliferation and differentiation of SVZ NSCs in different ways. In proliferating NSCs, rotenone decreases mitosis as measured through phosphorylated histone H3 detection; moreover, apoptosis is not induced by rotenone at 50 nM. In differentiating NSCs, rotenone blocks neurogenesis and oligodendrogenesis while glial fibrillary acidic protein-positive astrocytes are not affected. Interestingly, in this study there were more cells in the differentiating NSCs treated with rotenone for 4-6 days than in the vehicle control group which was a different effect from the reduced number of cells in the proliferating NSCs. We examined both apoptosis and mitosis and found that rotenone decreased apoptosis as detected by staining cleaved caspase-3 but did not affect mitosis. Our results suggest that functional mitochondria are necessary in both the proliferation and differentiation of SVZ NSCs. Furthermore, mitochondria might be involved in the mitosis and apoptosis that occur during those processes.

• Journal of Korean Ophthalmic Optics Society
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• v.18 no.4
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• pp.541-548
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• 2013

Purpose: Marrow stromal cells (MSCs) have been known for their potential to trans-differentiate into neural and glial cells in vitro and in vivo. To investigate the influence of the developing host environment on the survival and morphological and molecular differentiation, murine MSCs transplanted into the eye of Brazilian opossum (Monodelphis domestica). Methods: Enhanced green fluorescent protein (GFP) - expressing MSCs were transplanted into developing Brazilian opossums. Animals were allowed to survive for up to 4 weeks after transplantation, at which time the eyes were prepared for immunohistochemical analysis. Results: Some transplanted MSCs survived and showed morphological differentiation into neural cells with some processes within the host vitreous chamber. Some transplanted cells expressed class III ${\beta}$-tubulin (TuJ1, a marker for neuronal cells) or glial fibrillary acid protein (GFAP, a marker for glial cells) or Nestin (a marker for neural stem cells). In addition, some transplanted cells were located in ganglion cell layer but did not show morphological and molecular differentiation. Conclusions: Our result show that the most effective stage of development for transplantation into the retina was postnatal day 16, which retinas developmentally corresponded to postnatal day 4-5 days mouse retina based on cell differentiation and lamination patterns. The present findings suggest that the age of the host appears to play a key role in determining cell fate in vivo.

• Journal of Korean Neurosurgical Society
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• v.38 no.2
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• pp.121-125
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• 2005

Objective : Many recent reports have shown that the mature mammalian brain harbors multipotent stem cells, rendering the brain capable of generating new neurons and glia throughout life. Harvested stem cells from an adult rat are transplanted in order to evaluate the cell survival and differentiation. Methods : Using a percoll gradient with a high speed centrifugation method, we isolate neural stem/progenitor cells were isolated from the subventricular zone[SVZ] of a syngeneic adult Fisher 344 rats brain. For 14days expansion, the cultured cells comprised of a heterogeneous population with the majority of cells expressing nestin and/or GFAP. After expanding the SVZ cells in the presence of basic fibroblast growth factor-2, and transplanting then into the hippocampus of normal rats, the survival and differentiation of those cells were examined. For transplantation, the cultured cells were labeled with BrdU two days prior to use. In order to test their survival, the cells were transplanted into the dorsal hippocampus of normal adult Fisher 344 rats. Results : The preliminary data showed that at 7days after transplantation, BrdU+ transplanted cells were observed around the injection deposition sites. Immuno-fluorescent microscopy revealed that the cells co-expressed BrdU+ and neuronal marker ${\beta}$-tubulin III. Conclusion : The data demonstrate that the in vitro expanded SVZ cells can survive in a heterotypic environment and develop a neuronal phenotype in the neurogenic region. However more research will be needed to examine the longer survival time points and quantifying the differentiation in the transplanted cells in an injured brain environment.