• International Journal of Stem Cells
• /
• 제16권2호
• /
• pp.156-167
• /
• 2023

Background and Objectives: Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency. Methods and Results: Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome "recalcitrant" nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells. Conclusions: Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

• Molecules and Cells
• /
• 제23권1호
• /
• pp.49-56
• /
• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

• International Journal of Stem Cells
• /
• 제17권2호
• /
• pp.158-181
• /
• 2024

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.75-75
• /
• 2003

Since the establishment of embryonic stem cell, pluripotency of the cells was known to allow differentiation of the cells into various cell types consisting whole body. Several protocols have been developed to induce expression of specific genes.. However, no precise protocol that will generate a single type of the cells from stem cells has been reported. In order to produce cells suitable for transplantion into brain of PD animal model, which arouse due to a progressive degeneration of dopaminergic neurons in midbrain, human embryonic stem cell (hESC, MB03) was transfected with cDNAs cording for tyrosine hydroxylase (TH). Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by the two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA/ascorbic acid (AA), embryoid bodies (EB, for 4days) derived from hES cells were exposed to RA (10$^{-6}$ M)/AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. By indirect immunocytochemical studies, proportion of cells expressing NF200 increased rapidly from 20% at 7 days to 70 % at 28 days in RA/AA-treated group, while those cells expressing NF160 decreased from 80% at 7 days to 10% at 28 days upon differentiation in N2 medium. However, in differentiation by RA/AA treatment system, there was a significant increase in proportion of neuron maturity (73%) at day 14 after N2 medium. TH#2/MB03 cells expressing TH are >90% when matured at the absence of either bDNF or TGF-$\alpha$. These results suggested that TH#2/MB03 cells could be differentiated in vitro into mature neurons by RA/AA.

• Biomolecules & Therapeutics
• /
• 제28권1호
• /
• pp.34-44
• /
• 2020

Mesenchymal stem cells (MSCs) have been proposed as an alternative therapy to be applied into several pathologies of the nervous system. These cells can be obtained from adipose tissue, umbilical cord blood and bone marrow, among other tissues, and have remarkable therapeutic properties. MSCs can be isolated with high yield, which adds to their ability to differentiate into non-mesodermal cell types including neuronal lineage both in vivo and in vitro. They are able to restore damaged neural tissue, thus being suitable for the treatment of neural injuries, and possess immunosuppressive activity, which may be useful for the treatment of neurological disorders of inflammatory etiology. Although the long-term safety of MSC-based therapies remains unclear, a large amount of both pre-clinical and clinical trials have shown functional improvements in animal models of nervous system diseases following transplantation of MSCs. In fact, there are several ongoing clinical trials evaluating the possible benefits this cell-based therapy could provide to patients with neurological damage, as well as their clinical limitations. In this review we focus on the potential of MSCs as a therapeutic tool to treat neurological disorders, summarizing the state of the art of this topic and the most recent clinical studies.

• Biomolecules & Therapeutics
• /
• 제31권3호
• /
• pp.264-275
• /
• 2023

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by tremors, bradykinesia, and rigidity. PD is caused by loss of dopaminergic (DA) neurons in the midbrain substantia nigra (SN) and therefore, replenishment of DA neurons via stem cell-based therapy is a potential treatment option. Astrocytes are the most abundant non-neuronal cells in the central nervous system and are promising candidates for reprogramming into neuronal cells because they share a common origin with neurons. The ability of neural progenitor cells (NPCs) to proliferate and differentiate may overcome the limitations of the reduced viability and function of transplanted cells after cell replacement therapy. Achaete-scute complex homolog-like 1 (Ascl1) is a well-known neuronal-specific factor that induces various cell types such as human and mouse astrocytes and fibroblasts to differentiate into neurons. Nurr1 is involved in the differentiation and maintenance of DA neurons, and decreased Nurr1 expression is known to be a major risk factor for PD. Previous studies have shown that direct conversion of astrocytes into DA neurons and NPCs can be induced by overexpression of Ascl1 and Nurr1 and additional transcription factors genes such as superoxide dismutase 1 and SRY-box 2. Here, we demonstrate that astrocytes isolated from the ventral midbrain, the origin of SN DA neurons, can be effectively converted into DA neurons and NPCs with enhanced viability. In addition, when these NPCs are inducted to differentiate, they exhibit key characteristics of DA neurons. Thus, direct conversion of midbrain astrocytes is a possible cell therapy strategy to treat neurodegenerative diseases.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.79-79
• /
• 2003

Embryonic stem cells are capable of differentiating into a variety of cell lineages. However, the ultimate results of differentiation in vitro greatly depend on the duration of treatment and kinds of differentiating inducers added. In order to investigate the efficiencies of various differentiation inducers and the methods of treatment, we examined differentiation patterns of human embryonic stem cell (hESC, MB03) according to several different protocols. Exp. I) Upon differentiation using retinoic acid and ascorbic acid (RA/AA), embryoid bodies (EB, for 4days) derived from hESC was exposed to Rh (10$^{-6}$ M) and AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. Exp. III) In addition, to examine the effects of neurotrophic factors in the production of mature neurons, groups of cells were exposed to either BDNF (5 ng/ml) or TGF-$\alpha$(10 ng/ml) during the 28 days of final differentiation. Differentiation patterns of RA/AA or bFGF treated groups were very similar; approximately 82% and 83% of the cells, respectively, were positive for anti-NF200 antibody, while it was about 10% and 11%, respectively, for anti-NF160 antibody in 28 days in N2 medium. Alsor, cells expressing TH were as low as 5%, while the cells doubled when matured at the presence of either BDNF or TGF-$\alpha$. Cells immunoreactive to anti-GAD antibody were approximately 20%. These results suggest that a maturation step rather than differentiation induction step, which is formation of EB, effects more decisively to the ultimate differentiation pattern.

• 한국약용작물학회지
• /
• 제17권2호
• /
• pp.145-150
• /
• 2009

This study investigated the protective roles and mechanism of magnolol, from the stem bark of Magnolia officinalis against potential neurotoxin 3-hydroxykynurenine (3-HK)-induced neuronal cell death. For the evaluation of protective role of magnolol, we examined cell viability, apoptotic nuclei, change of mitochondrial membrane potential and caspase activity in human neuroblastoma SH-SY5Y cells. It was found that 3-HK induces neuronal cell death in the human neuroblastoma SH-SY5Y cell line. The reduced cell viability produced characteristic features such as cell shrinkages, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation. The cells treated with 3-HK showed an increase in the concentration of reactive oxygen species (ROS) as well as in caspase activity. In addition, both are involved in the 3-HK-induced apoptosis. Magnolol attenuated the cell viability reduction by 3-HK in both a dose- and time-dependent manner. Optical microscopy showed that magnolol inhibited the cell morphological features in the 3-HK-treated cells. Furthermore, the increase in the ROS concentration and the caspase activities by 3-HK were also attenuated by magnolol. These results showed that magnolol has a protective effect on the 3-HK induced cell death by inhibiting ROS production and caspase activity.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.19-19
• /
• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.

• IMMUNE NETWORK
• /
• 제11권3호
• /
• pp.135-154
• /
• 2011

The great discovery of microRNAs (miRNAs) has revolutionized current cell biology and medical science. miRNAs are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3' untranslated region of specific messenger RNAs for degradation or translational repression. New members of the miRNA family are being discovered on a daily basis and emerging evidence has demonstrated that miRNAs play a major role in a wide range of developmental process including cell proliferation, cell cycle, cell differentiation, metabolism, apoptosis, developmental timing, neuronal cell fate, neuronal gene expression, brain morphogenesis, muscle differentiation and stem cell division. Moreover, a large number of studies have reported links between alterations of miRNA homeostasis and pathological conditions such as cancer, psychiatric and neurological diseases, cardiovascular disease, and autoimmune disease. Interestingly, in addition, miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from cancer to myocardial infarction. miRNAs can repress the gene translation of hundreds of their targets and are therefore well-positioned to target a multitude of cellular mechanisms. As a consequence of extensive participation in normal functions, it is quite logical to ask the question if abnormalities in miRNAs should have importance in human diseases. Great discoveries and rapid progress in the past few years on miRNAs provide the hope that miRNAs will in the near future have a great potential in the diagnosis and treatment of many diseases. Currently, an explosive literature has focussed on the role of miRNA in human cancer and cardiovascular disease. In this review, I briefly summarize the explosive current studies about involvement of miRNA in various human cancers and cardiovascular disease.