• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제40권1호
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• pp.3-10
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• 2014

Objectives: Although nerve growth factor (NGF) could promote the functional regeneration of an injured peripheral nerve, it is very difficult for NGF to sustain the therapeutic dose in the defect due to its short half-life. In this study, we loaded the NGF-bound heparin-conjugated fibrin (HCF) gel in the NGF-delivering implants and analyzed the time-dependent release of NGF and its bioactivity to evaluate the clinical effectiveness. Materials and Methods: NGF solution was made of 1.0 mg of NGF and 1.0 mL of phosphate buffered saline (PBS). Experimental group A consisted of three implants, in which $0.25{\mu}L$ of NGF solution, $0.75{\mu}L$ of HCF, $1.0{\mu}L$ of fibrinogen and $2.0{\mu}L$ of thrombin was injected via apex hole with micropipette and gelated, were put into the centrifuge tube. Three implants of experimental group B were prepared with the mixture of $0.5{\mu}L$ of NGF solution, $0.5{\mu}L$ HCF, $1.0{\mu}L$ of fibrinogen and $2.0{\mu}L$ of thrombin. These six centrifuge tubes were filled with 1.0 mL of PBS and stirred in the water-filled beaker at 50 rpm. At 1, 3, 5, 7, 10, and 14 days, 1.0 mL of solution in each tubes was collected and preserved at $-20^{\circ}C$ with adding same amount of fresh PBS. Enzyme-linked immunosorbent assay (ELISA) was done to determine in vitro release profile of NGF and its bioactivity was evaluated with neural differentiation of pheochromocytoma (PC12) cells. Results: The average concentration of released NGF in the group A and B increased for the first 5 days and then gradually decreased. Almost all of NGF was released during 10 days. Released NGF from two groups could promote neural differentiation and neurite outgrowth of PC12 cells and these bioactivity was maintained over 14 days. Conclusion: Controlled release system using NGF-HCF gel via NGF-delivering implant could be an another vehicle of delivering NGF to promote the nerve regeneration of dental implant related nerve damage.

• 한국발생생물학회지:발생과생식
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• 제10권3호
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• pp.169-175
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• 2006

전세계적으로 널리 복용되고 있음에도 불구하고 인삼이 중추신경계에 미치는 효과에 대한 세포수준에서의 증거는 별로 없다. 따라서 본 연구자들은 지금까지 보고된 30여종 이상의 ginsenosides 중에서 인삼 효과의 주된 활성성분으로 알려져 있는 Rb1과 Rg1을 이용해 해마신경전구세포의 분화와 증식에 미치는 효과를 연구하였다. 증식실험결과 Rb1과 Rg1을 3일 동안 해마신경전구세포에 처리하면 대조군에 비해 BrdU(+)세포수가 상당히 감소한 반면에 분화조건에서 Rb1과 Rg1을 처리했을 때는 신경세포특이적인 MAP2 단백질을 발현하는 세포수가 증가하였다. 전구세포의 신경세포로의 분화 결정에 관여한다고 잘 알려져 있는 proneural 전사인자인 Ngn1과 Hes1 유전자의 발현양상도 대조군에 비해 증가하였다. 따라서 본 연구 결과, ginsenoside Rb1과 Rg1은 해마신경전구세포의 증식을 감소시키고 대신 신경세포로의 분화를 촉진시킴으로써 해마신경발생에 관여함을 보여준다.

• Journal of Ginseng Research
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• 제45권2호
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• pp.325-333
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• 2021

Background: Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders. Enhancing hippocampal neurogenesis by promoting proliferation and differentiation of neural stem cells (NSCs) is a promising therapeutic strategy for AD. 20(S)-protopanaxadiol (PPD) and oleanolic acid (OA) are small, bioactive compounds found in ginseng that can promote NSC proliferation and neural differentiation in vitro. However, it is currently unknown whether PPD or OA can attenuate cognitive deficits by enhancing hippocampal neurogenesis in vivo in a transgenic APP/PS1 AD mouse model. Here, we administered PPD or OA to APP/PS1 mice and monitored the effects on cognition and hippocampal neurogenesis. Methods: We used the Morris water maze, Y maze, and open field tests to compare the cognitive capacities of treated and untreated APP/PS1 mice. We investigated hippocampal neurogenesis using Nissl staining and BrdU/NeuN double labeling. NSC proliferation was quantified by Sox2 labeling of the hippocampal dentate gyrus. We used western blotting to determine the effects of PPD and OA on Wnt/GSK3β/β-catenin pathway activation in the hippocampus. Results: Both PPD and OA significantly ameliorated the cognitive impairments observed in untreated APP/PS1 mice. Furthermore, PPD and OA significantly promoted hippocampal neurogenesis and NSC proliferation. At the mechanistic level, PPD and OA treatments resulted in Wnt/GSK-3β/β-catenin pathway activation in the hippocampus. Conclusion: PPD and OA ameliorate cognitive deficits in APP/PS1 mice by enhancing hippocampal neurogenesis, achieved by stimulating the Wnt/GSK-3β/β-catenin pathway. As such, PPD and OA are promising novel therapeutic agents for the treatment of AD and other neurodegenerative diseases.

• The Journal of Korean Physical Therapy
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• 제13권2호
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• pp.433-444
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• 2001

This review highlights the ontogenesis and the differentiation of motor neuron in spinal cord, and introduce the survival motor neuron(SMN) which is associated with growth and survival of motor neurons. The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. This signal is Sonic hedgehog(Shh). The early development of motor neurons involves the inductive action of Shh. The SMN gene is essential for embryonic viability. SMN mRNA is also expressed in virtually all cell types in spinal cord, including large motor neurons. The SMN protein is involved in RNA processing and during early embryonic development is necessary fer cell survival. Two SMN genes are present in 5q 13 in humans: the telomeric gene(SMNt), which is the SMA-determining gene, and the centromeric analog gene(SMNc). The majority of transcripts from the SMNt gene are full length but, major transcripts of the SMNc gene have a high degrees of alternative splicing and tend to have little or no exon 7. The SMN is involved in the RNA processing(the biogenesis of snRNPs and pre-mRNA splicing), the anti-apoptotic effects, and regulating gene expression.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.229-233
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• 2010

Pluripotency and self-renewal capacity of human embryonic stem cells (hESCs) are retained by hESCs related genes as OCT4, SOX2 and NANOG. These genes are shown high expression level in diverse cancer cells and have potential role in the carcinogenesis. On the contrary to this, several genes which are up-regulated in the differentiated hESCs are involved to suppress the carcinogenesis or proliferation of cells. We discovered several genes in immortalized lung fibroblast (WI-38 VA13) by suppression subtractive hybridization. Among them, we focused chromosome 6 open reading frame 62 (C6orf62) which is uncharacterized, mapped to 6p22.3 and generated to Hepatitis B virus X-transactivated proteins (HBVx-transactivated proteins, XTP). Aim of this study was to characterize C6orf62 through analyzing of expression pattern in various cell lines. Expression of C6orf62 was significantly upregulated in diverse normal cell lines than cancer cell lines. And C6orf62 was up-regulated in differentiated hESCs (endothelial cells, neural cells) compared to those of undifferentiated hESCs. Also, C6orf62 in WI-38 cells was highly up-regulated during G1/S transition of the cell cycle. Taken together, C6orf62 is shown expression pattern similar to differentiated hESCs-associated genes which down-regulated in cancer cells. Therefore, we assume that C6orf62 may participate to suppress the proliferation and to induce differentiation through regulating the cell cycle.

• Journal of Gastric Cancer
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• 제3권4호
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• pp.201-205
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• 2003

Purpose: Telomerase activity is generally absent in primary cell cultures and normal tissues. Telomerase is known to be induced upon immortalization or malignant transformation of human cells. Telomerase activity can be increased in immature lymphocytes and activated lymphocytes, but it is not detected in the peripheral blood of normal persons. The authors analyzed peripheral blood telomerase from patients of gastric cancer to evaluate the possibility of using it for diagnosis and as a prognostic factor. Materials and Methods: We obtained blood samples from 11 inflammatory patients and 64 gastric cancer patients. The telomerase activity was measured using the [PCR-ELISA] method. The results were correlated with the T, N, M stage, cell differentiation, vascular, neural, and lymphatic invasion, tumor size, and tumor location. Results: In the 11 inflammatory patients, telomerase activity was not detected while in the gastric cancer patients, a positive rate of $28.1\%$ was noted. The peripheral telomerase activity was not related with tumor size, tumor site, lymphatic and vascular invasion, stage, or histologic differentiation. Conclusion: The peripheral blood telomerase activity for patients of gastric cancer can be utilized as a marker for the diagnosis of not only advanced gastric cancer, but also relatively early stage gastric cancer, but not as a prognostic factor.

• Molecules and Cells
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• 제43권6호
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• pp.581-589
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• 2020

Neurons have multiple dendrites and single axon. This neuronal polarity is gradually established during early processes of neuronal differentiation: generation of multiple neurites (stages 1-2); differentiation (stage 3) and maturation (stages 4-5) of an axon and dendrites. In this study, we demonstrated that the neuron-specific n-glycosylated protein NELL2 is important for neuronal polarization and axon growth using cultured rat embryonic hippocampal neurons. Endogenous NELL2 expression was gradually increased in parallel with the progression of developmental stages of hippocampal neurons, and overexpression of NELL2 stimulated neuronal polarization and axon growth. In line with these results, knockdown of NELL2 expression resulted in deterioration of neuronal development, including inhibition of neuronal development progression, decreased axon growth and increased axon branching. Inhibitor against extracellular signal-regulated kinase (ERK) dramatically inhibited NELL2-induced progression of neuronal development and axon growth. These results suggest that NELL2 is an important regulator for the morphological development for neuronal polarization and axon growth.

• 한국동물생명공학회지
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• 제35권1호
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• pp.42-49
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• 2020

As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

• Journal of Korean Neurosurgical Society
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• 제61권2호
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• pp.258-266
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• 2018

Objective : The diagnosis of insufficiency fractures of the sacrum in an elder population increases annually. Fractures show very different morphology. We aimed to classify sacral insufficiency fractures according to the position of cortical break and possible need for intervention. Methods : Between January 1, 2008 and December 31, 2014, all patients with a proven fracture of the sacrum following a low-energy or an even unnoticed trauma were prospectively registered : 117 females and 13 males. All patients had a computer tomography of the pelvic ring, two patients had a magnetic resonance imaging additionally : localization and involvement of the fracture lines into the sacroiliac joint, neural foramina or the spinal canal were identified. Results : Patients were aged between 46 and 98 years (mean, 79.8 years). Seventy-seven patients had an unilateral fracture of the sacral ala, 41 bilateral ala fractures and 12 patients showed a fracture of the sacral corpus : a total of 171 fractures were analyzed. The first group A included fractures of the sacral ala which were assessed to have no or less mechanical importance (n=53) : fractures with no cortical disruption ("bone bruise") (A1; n=2), cortical deformation of the anterior cortical bone (A2; n=4), and fracture of the anterolateral rim of ala (A3; n=47). Complete fractures of the sacral ala (B; n=106) : parallel to the sacroiliac joint (B1; n=63), into the sacroiliac joint (B2; n=19), and involvement of the sacral foramina respectively the spinal canal (B3; n=24). Central fractures involving the sacral corpus (C; n=12) : fracture limited to the corpus or finishing into one ala (C1; n=3), unidirectional including the neural foramina or the spinal canal or both (C2; n=2), and horizontal fractures of the corpus with bilateral sagittal completion (C3; n=8). Sixty-eight fractures proceeded into the sacroiliac joint, 34 fractures showed an injury of foramina or canal. Conclusion : The new classification allowes the differentiation of fractures of less mechanical importance and a risk assessment for possible polymethyl methacrylate leaks during sacroplasty in the direction of the neurological structures. In addition, identification of instable fractures in need for laminectomy and surgical stabilization is possible.