• BMB Reports
• /
• 제46권5호
• /
• pp.276-281
• /
• 2013

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures.

• Nutrition Research and Practice
• /
• 제4권4호
• /
• pp.276-282
• /
• 2010

The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2004년도 제4회 발생공학 국제심포지움 및 학술대회
• /
• pp.59-59
• /
• 2004

• BMB Reports
• /
• 제56권3호
• /
• pp.153-159
• /
• 2023

Neuronal differentiation is highly coordinated through a cascade of gene expression, mediated via interactions between trans-acting transcription factors and cis-regulatory elements of their target genes. However, the mechanisms of transcriptional regulation that determine neuronal cell-fate are not fully understood. Here, we show that the nuclear transcription factor Y (NF-Y) subunit, NFYA-1, is necessary and sufficient to express the flp-3 neuropeptide gene in the IL1 neurons of C. elegans. flp-3 expression is decreased in dorsal and lateral, but not ventral IL1s of nfya-1 mutants. The expression of another terminally differentiated gene, eat-4 vesicular glutamate transporter, is abolished, whereas the unc-8 DEG/ENaC gene and pan-neuronal genes are expressed normally in IL1s of nfya-1 mutants. nfya-1 is expressed in and acts in IL1s to regulate flp-3 and eat-4 expression. Ectopic expression of NFYA-1 drives the expression of flp-3 gene in other cell-types. Promoter analysis of IL1-expressed genes results in the identification of several cis-regulatory motifs which are necessary for IL1 expression, including a putative CCAAT-box located in the flp-3 promoter that NFYA-1 directly interacts with. NFYA-1 and NFYA-2, together with NFYB-1 and NFYC-1, exhibit partly or fully redundant roles in the regulation of flp-3 or unc-8 expression, respectively. Taken together, our data indicate that the NF-Y complex regulates neuronal subtype-specification via regulating a set of terminal-differentiation genes.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2004년도 제4회 발생공학 국제심포지움 및 학술대회
• /
• pp.57-58
• /
• 2004

• The Korean Journal of Physiology and Pharmacology
• /
• 제19권2호
• /
• pp.105-109
• /
• 2015

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

• 한국안광학회지
• /
• 제18권4호
• /
• pp.541-548
• /
• 2013

목적: 골수기질세포는 생체 내 외에서 신경세포와 신경교세포로 교차분화 할 수 있는 능력을 가지고 있는 것으로 밝혀져 있다. 발생 중인 숙주 환경에 따라 이식된 골수기질세포의 생존여부, 형태학적 그리고 분자적 분화영향을 조사하기 위해 브라질산 주머니쥐 안구에 마우스 골수기질세포를 이식하였다. 방법: GFP를 발현하는 골수기질세포를 발생 중인 브라질산 주머니쥐의 각 시기별로 이식하여, 이식 후 최대 4주까지 생존시킨 후 각 시기별로 면역조직화학법을 시행하였다. 결과: 이식한 골수기질세포의 일부는 숙주동물의 유리체 내에서 생존하며 일부 돌기를 내는 신경세포로 형태학적 분화가 됨을 관찰할 수 있었다. 또한 유리체에 존재하는 일부 세포는 신경세포 표지인자인 TuJ1(class III ${\beta}$-tubulin), 신경교세포 표지인자인 GFAP(glial fibrillary acidic protein), 또는 신경줄기세포 표지인자인 Nestin 단백질을 발현하였다. 게다가, 일부 골수기질세포는 신경절세포층으로 이동함을 관찰했으나, 이동한 세포들은 형태학적 또는 분자적 분화를 나타내지는 않았다. 결론: 이번 연구에서 가장 효율적인 이식시기는 생후 16일째의 포유류 망막으로, 이는 망막세포의 분화양상과 층분화 패턴으로 미뤄볼 때 생후 4~5일 정도의 마우스 망막과 발생학적으로 상동함을 알 수 있었다. 또한 이식 받은 숙주 망막의 미세환경이 이식된 세포운명에 영향을 미치는 것을 확인할 수 있었다.