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Dual-stimuli responsive silica-based nanoparticles for co-delivery of small molecules to promote neural differentiation of human iPSCs

  • 유정현 (Jeong Hyun You, 서강대학교 일반대학원)

초록 (요약문)

The differentiation of human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs) is a promising approach for the treatment of neurodegenerative diseases and regenerative medicine. Dual SMAD inhibition using small molecules has been identified as a key strategy for directing the differentiation of hiPSCs into NPCs by regulating specific cell signaling pathways. However, the conventional culture methods are time-consuming and exhibit the low differentiation efficiency in neural differentiation. The nanocarriers can address these obstacles as an efficient method for the controlled release and accurate delivery of small molecules. In this paper, I developed the calcium phosphate-coated mesoporous silica nanoparticles capable of delivering multiple small molecules including LDN193189 as a BMP inhibitor and SB431542 as a TGF-beta inhibitor, for the directed differentiation of hiPSCs- towards NPCs. Our results demonstrated that this nanocarrier-mediated small molecule release system not only enhanced the in vitro formation of neural rosettes but also modulated the expression levels of key markers. Specifically, it downregulated OCT4, a marker of pluripotency, while upregulating PAX6, a critical marker for neuroectoderm. These finding suggest that our controlled small molecules release system holds significant potential for therapeutic applications in neural development and neurodegenerative diseases.

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초록 (요약문)

인간 유도만능 줄기세포(hiPSCs)를 신경 전구세포(NPCs)로 분화하는 기술은 신경 퇴행성 질환 및 재생의학 치료를 위한 유망한 접근법이다. 저분자 화합물을 이용한 이중 SMAD 억제는 두 종류의 특정 세포 신호 전달 경로를 조절하여 hiPSCs 를 NPCs 로 분화하시키는 핵심 전략이다. 그러나 기존 배양 방법은 여전히 시간이 많이 걸리고 신경 분화 효율이 낮다는 문제가 존재한다. 이러한 문제점들을 극복하기 위해서 나노 캐리어가 저분자 화합물의 제어 방출 및 정확한 전달을 위한 효율적인 방법으로 제시되었다. 본 논문에서는 hiPSCs 로부터 NPCs 로의 분화를 유도하기 위해 BMP 저해제로 LDN193189, TGF-beta 저해제로 SB431542 를 이용하였고, 여러 저분자 화합물을 전달할 수 있는 인산 칼슘이 코팅된 메조 다공성 실리카 나노 입자를 개발하였다. 연구 결과, 이 나노 캐리어 매개 저분자 화합물 방출 시스템은 시험관 내 신경 로젯 구조의 형성을 촉진할 뿐만 아니라 신경 발달과 관련된 주요 마커의 발현 수준을 조절한다는 것을 보여주었다. 구체적으로 전분화능 마커인 OCT4 를 하향 조절하고 신경 외배엽 마커인 PAX6 를 상향 조절하였다. 이러한 연구 결과는 제어된 저분자 화합물 방출 시스템이 신경 발달 및 신경 퇴행성 질환 치료 응용에 중요한 가능성을 지니고 있음을 시사한다.

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목차

I. Introduction 6
II. Materials and Methods 10
i. Chemicals and Reagents 10
ii. MSN synthesis and modification 11
iii. Synthesis of MSN-CaP 11
iv. Small molecule loading into MSN-CaP 12
v. Characterization of MSN-CaP 13
vi. Small molecule release test 14
vii. Observation of dual stimuli(pH/GSH) responsive degradation of MSN-CaP 14
viii. hiPSCs culture 15
ix. Cytotoxicity test 15
x. Cellular uptake analysis of MSN-CaP 16
xi. Neural induction of hiPSCs treated with small molecule encapsulated MSN-CaP 16
xii. Immunofluorescence analysis 18
xiii. Quantitative pcr analysis 19
xiv. Western blotting analysis 21
III. Results and discussions 22
i. Morphological observation and elemental analysis of MSN-CaP 22
ii. Physicochemical characterization of MSN-CaP 22
iii. Analyze small molecule content and release characteristics of nanoparticles 27
iv. Evaluation of pH-sensitive small molecule release behavior 28
v. pH and GSH dual-sensitized nanoparticle degradation morphology analysis 29
vi. Evaluation of the biocompatibility of the nanoparticles 30
vii. Evaluation of endocytosis of nanocarriers 31
viii. Immunofluorescence analysis 35
ix. Quantitative PCR analysis 36
x. Western blot analysis 39
IV. Conclusion 40
V. Supporting Information 41
Reference 42

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