[보고서]IL-6에 의한 인체 조직-유도 만능 신경 줄기 세포(h-iPSCs)로부터 유도된 신경 줄기 세포(NSCs)의 신경분화에 관한 연구

Klaus Heese

[국가R&D연구보고서] IL-6에 의한 인체 조직-유도 만능 신경 줄기 세포(h-iPSCs)로부터 유도된 신경 줄기 세포(NSCs)의 신경분화에 관한 연구
Investigation of IL-6-mediated neuronal differentiation of human fibroblast / induced pluripotent stem cells (h-iPSCs)-derived neuronal stem cells (NSCs) 원문보기

보고서 정보
주관연구기관	한양대학교 HanYang University
연구책임자	Klaus Heese
보고서유형	최종보고서
발행국가	대한민국
언어	대한민국
발행년월	2016-06
과제시작연도	2015
주관부처	교육부 Ministry of Education
연구관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO202100002359
과제고유번호	1345237912
사업명	이공학개인기초연구지원
DB 구축일자	2021-06-26

초록

Abstract ▼

iPSC has opened the possibility for personalized regenerative treatment via autologous transplantation from patient’s own tissue that can open avenues to treat neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and other related degenerative diseases. However, the practical application for neural cell replacement is limited by heterogeneity of neural stem cell (NSC) progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. Previous studies have shown that Hyper interleukin-6 (HIL-6), a fusion protein of interleukin (IL-6) and its soluble receptor sIL-6R, has neurogenic activities via the activation of a mitogen activated protein kinase (MAPK) pathway. Therefore, in this study we investigated the application of IL-6 and sIL-6R for inducing neurogenesis on human iPSC-derived NSCs. We found, unlike the previous findings that reported the activation of IL-6/STAT3 axis to be gliogenic, that IL-6 activation is neurogenic to the level comparable with BDNF. Moreover, the addition of a STAT3-specific inhibitor abolished the neurogenicity of IL-6. Patch clamp recordings also showed that the IL-6-dependent neurons were functional and able to generate multiple-bursts of action potentials. Thus activating IL-6 signaling can be used to improve neuron yield of iPSC-derived NSCs and the obtained neurons can be fluorescence-activated cell sorting (FACS)-purified to generate a pure and functional neural population that can be used for further IL-6 signaling studies in a iPSC development model or as a source for regenerative therapy to tackle the challenge of incurable neurodegenerative diseases, such as AD or PD.

(출처 : ABSTRACT 2p)

목차 Contents

COVER ... 1
ABSTRACT ... 2
INTRODUCTION ... 3
MATERIAL AND METHODS ... 5
RESULTS ... 14
Integration-free episomal plasmid-mediated reprogramming of human normal dermal fibroblast ... 14
Dual SMAD inhibition for NSC differentiation ... 17
IL-6 signaling mediates neurogenesis of iPSC-derived NSC ... 19
BDNF but not IL-6 improves neuron maturation of iPSC-derived NSC ... 21
iPSC-derived NSC differentiated into functional neuron ... 28
DISCUSSION ... 31
References ... 35
End of Page ... 37

표/그림 (14)

표 Episomal plasmid used for integration-free reprogramming. Episomal plasmid containing reprogramming factors Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 cloned inside replicating EBNA-1 vector were used to reprogram fibroblast into iPSC
표 iPSC colony formation from human normal dermal fibroblast. (A) An iPSC colony formed among the fibroblast during the second week of reprogramming. (B) The resulting iPSC colonies showing characteristic morphology with distinct colony border and uniform monolayer cells with large nuclei. (C) AP staining of iPSC colonies stained strong positive staining. These images are taken under phase-contrast microscopy in 5x, 10x, and 20x magnification, left to right, respectively. Scale bar indicates 175, 75, and 37.5 μM, respectively
표 Expression of pluripotency marker of generated iPSC. The fibroblast-derived iPSCs showed positive result when stained by pluripotency marker SSEA3, SSEA4, TRA-1-60, TRA-1-81, OCT4, top to bottom. Scale bars indicate from left to right 175 μm, 75 μm, 37,5 μm, respectively
표 Western blot of Oct4 of the iPSC. Same line of iPSC showed a strong expression of 46 kDa band of Oct4. GAPDH was used as internal standard
표 NSC induction by using SMAD/BMP chemical inhibition. iPSCs were differentiated into NSC by inhibiting the activity of SMAD and BMP signaling by using chemical Dorsomorphin and SB431542 for 12 days. (A) The morphology of iPSC colonies after 7 days of the dual DM and SB induction in 5x, 10x, and 20x, left to right, respectively. Scale bar indicates 175, 75, and 37.5 μM, respectively (B) The appearance of neural rossetes during iPSC differentiation (left panel 10x magnification, middle and right 20x magnification) (C) FACS gating strategy of cells after 12 days NSC induction using dual chemical inhibition by using surface marker CD184, CD44, CD271, CD24, and CD15. NSCs were sorted based on the criteria of CD184+, CD 44-, CD271-, and CD24+. Gating strategy used as follows: P1 was live cells gating, P2 was doublet discrimination, P3 was CD184-APC population, P4 is CD271-PerCP-Cy5.5 and CD44-PerCP-Cy5.5 double negative population, and P5 is CD24-PE population
표 iPSC-derived NSC expresses strong NSC and proliferation marker in a pure culture. iPSC cells were induced by Dorsomorphin and SB431542 for 12 days and cells were dissociated for FACS analysis. After FACS cells were cultured for additional 2 passages and stained with NSC marker Nestin and Sox2 (left) and proliferation marker Ki67 (right). Scale bar indicates 62.5 μM
표 7. iPSC-derived NSCs are able to differentiate into neuron and astrocyte. Immunofluorescence MAP2 and GFAP staining of differentiated NSCs. NSCs were isolated using FACS based on the selection marker in figure 7 after two round of FACS with 2 passages of culture in between. During differentiation, NSCs were introduced to nothing (control), IL-6 + sIL-6R at 100 ng/ml each, IL-6 + sIL-6R + 2.5 μM of Stattic, or BDNF at 80 ng/ml for 40 days. Images were taken at 20x magnification, with scale bar indicates 62.5 μM
표 iPSC-derived NSCs are able to differentiate into mature neuron. Immunostaining of differentiated NSC using axonal marker Tau and mature neuron marker NeuN. NSCs were isolated using FACS based on the selection marker in figure 7 after two round of FACS with 2 passages of culture in between. During differentiation, NSCs were introduced to nothing (control), IL-6 + sIL-6R at 100 ng/ml each, IL-6 + sIL-6R + 2.5 μM of Stattic, or BDNF at 80 ng/ml for 40 days. Images were taken at 20x magnification, with scale bar indicates 62.5 μM
표 Extracellular FACS analysis reveals that IL-6 and BDNF increase neurogenesis and gliogenesis. After 40 days of differentiation, cells from all of the experimental groups were dissociated and stained using surface marker antibodies. The neurons are gated by CD44- , CD84-, and CD24 + selection criteria, while glia was gated by CD44+, CD84+. (A)The neuron and glia population identified by the surface markers. The population was normalized to the population of single live cells, i.e. after doublet discrimination. Error bar indicates the standard error of means from three experiments (n=3). (B) Representative result from each experimental group of FACS analysis for neuron and glia markers. Percentage of the population are indicated in the inset. The percentage of population normalized to single live cells are indicated in the table. Live cell gating and doublet discrimination gating are not shown
표 IL-6 and BDNF up-regulate Tuj1 expression. Western blot analysis showed that Tuj1 expression was upregulated in IL-6 + sIL-6R and BDNF group when normalized to control group after 40 days of differentiation of iPSC-derived NSC. The upregulation of Tuj1 is reversed by the addition of Stattic. (A) Western blot of Tuj1 protein from each experimental group. GAPDH was used for internal standard. (B) Fold-change quantification of Tuj1 expression (normalized to GAPDH) when compared to control group (n=2)
표 Immunofluorescence staining of sorted neuron showed pure culture with neural morphology. NSCs were differentiated for 40 days according to the experimental group and collected for FACS (see Fig 8). Sorted neurons were then replated in the presence of 20 ng/ml BDNF for all experimental group to improve survival after plating. Neurons were maintained for seven days before fixed and stained using Tuj1 antibody. Top row of each group are images taken using 20x magnification and the bottom row are taken using 40x magnification. Neurons derived from IL-6+sIL-6R+Stattic group exhibited simpler neural processes when compared to control, while BDNF-derived neurons exhibited more extensive neural processes. Scale bar indicates 62.5 μm (20x) or 30 μm (40x)
표 Flow cytometry analysis for intracellular NeuN staining. NSCs were differentiated for 42 days and intracellularly stained using anti-Neun antibody and coupled with Alexa488-labeled secondary antibody. NeuN protein is detected using FITC filter (X-axis) with cell count as the Y-axis. Secondary antibody only control (top row) was used for determining gating
표 Integration-free iPSC-derived NSCs can develop into functional neuron. NSCs were differentiated for 40 days and sorted as in fig 12 and were grown for additional 14 days before patchclamp analysis. Left, representative sodium currents elicited from cell by step depolarizations (holding - 60 mV, step pulse from - 70 mV to 70 mV during 1 sec) for each experimental group. These currents were activated in a graded and voltage-dependent manner. Right, Action potential from the same cell (holding current 0 A, pulse 200 pA, during 500 ms)
표 Neurogenic-to-gliogeneic switch does not occur in iPSC-derived NSC as opposed to its SVZderived counterpart. Present study raises hypothesis that STAT3 activation in iPSC-derived NSC does not give rise to glia because it escaped the neurogenic-to-gliogenic transition that is mediated by Notch signaling and its partners/downstream regulators. Therefore, IL-6 activation leads to increased neurogenesis in iPSC-derived NSC, possibly by rapidly induce neurogenic-commitment through activation of pro-neural genes

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