저자는 IMR90-hiPSC를 제공 한 Dr. Marc Peschanski (I-Stem, 프랑스, ​​에리)에게 감사 드리고 싶습니다. Dr. Giovanna Lazzari와 Dr. Silvia Colleoni (Avantea srl, Cremona, Italy); Dr. Simone Haupt (독일 본 (Bonn) 대학교); Tiziana Santini 박사 (이탈리아 기술 연구소, 로마), 면역 형광 염색 평가에 대한 조언 제공. RPPA 분석 및 항체 검증에 대한 공헌으로 Benedetta Accordi 박사, Elena Rampazzo 박사 및 Luca Persano 박사 (University of Padua, 이탈리아) 자금 조달 :이 작업은 EU가 지원하는 &quot;SCR &amp; Tox&quot;프로젝트 (보조금 협약 N ° 266753)에 의해 지원되었습니다.

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functions+in+astrocytes:+neuroprotective+implications+from+oxidative+damage+by+rotenone.">Mitochondrial functions in astrocytes: neuroprotective implications from oxidative damage by rotenone.</a> Neurosci Res. 74 (2), 80-90 (2012).</li><li>Swarnkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+activation:+a+key+step+in+rotenone+induced+cytotoxicity+and+DNA+damage.">Astrocyte activation: a key step in rotenone induced cytotoxicity and DNA damage.</a> Neurochem Res. 37 (10), 2178-2189 (2012).</li><li>Canovas-Jorda, D., Louisse, J., Pistollato, F., Zagoura, D., Bremer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+toxicology:+the+role+of+stem+cells+in+the+development+of+chronic+toxicities.">Regenerative toxicology: the role of stem cells in the development of chronic toxicities.</a> Expert Opin Drug Metab Toxicol. 10 (1), 39-55 (2014).</li></ol>

저자는 공개 할 것이 없습니다.

이 연구는 IMR90-hiPSC를 사후 유사 분열 뉴런 및 신경 교세포로 분화시키기위한 견고하고 비교적 빠른 프로토콜을 기술한다. hESCs와 hiPSCs를 기반으로 이전에 발표 된 신경 세포 분화 프로토콜은 일반적으로 높은 비율의 신경 전구체 25 , 26 과 상당한 숫자의 연결 대상 세포 27 , 28 , 29 , 30 , 31 , 32 , 33을 생산 합니다. 유사하게, 여기에 설명 된 분화 프로토콜은 glia와 nestin + 세포의 이산 비율과 함께 GABA 성, glutamatergic 및 dopaminergic 신경 세포의 이종 배양을 생성하는 데 적합합니다. glutamatergic (~ 35-42 %) 및 GABA (~ 15-20 %) 신경 세포의 존재는이 문화는 forebrain, 피질과 같은 기능을 보유하고 dopaminergic 뉴런 (~ 13-20 %)의 이산 번호의 존재는 또한 midbrain 특이성을 나타낼 수 있습니다. 또한, neestrogen + 세포의 겸손한 비율의 영속성은 neurogenesis의 연구와 NSC에 대한 화학 물질의 가능한 영향을 입증 할 수 있으며, NSC는 주로 해마와 뇌의 뇌실 영역 (SVZ) 모두에 국한되어있다. 추가 면역 세포 화학 및 유전자 발현 분석은 분화 된 세포 유도체의 지역 특이성을보다 잘 정의하는 데 도움이됩니다. 이 문서에서 설명하는 분화 프로토콜의 가장 중요한 두 단계는 다음과 같습니다 : (i) 균질 한 조각으로 hiPSC 콜로니를 절단하는 것 (균일 한 크기의 EB 생성에 중요합니다) 및 (ii) 신경 외배엽 구조 절단 ) NSC 차별화에는 상당한 수작업 기술이 필요합니다.분화시 얻은 뉴런 및 신경 교세포의 비율을 감소시킬 수있는 중배엽 및 내배엽 세포를 수집하는 것을 피하기위한 정밀성을 제공한다. 팽창하는 동안 (미분화 된 식민지 또는 NSCs와 같은) 모든 분화 단계에서 세포의 표현형을 특성화하는 것이 중요합니다. 특히, 신경 세포 / 신경 교세포 유도체의 유전자 및 단백질 발현 프로파일은 뉴런 관련 신호 경로의 상향 조절 및 활성화를 보여야하지만, 다 능성 마커의 발현은 감소되어야한다. EBs와 neuroectodermal 유도체 (rosettes)의 생성은 수동으로 도전하고 가변성이 있습니다. 이러한 이유로 우리는 로제트 유도 NSC의 확장 및 신경 세포 / 신경 교세포로의 추가 분화를위한 프로토콜을 개발했습니다. 이 분화 프로토콜의 가능한 한계는 주로 (i) 상대적으로 낮은 비율의 d(ii) 성숙한 신경 네트워크 기능의 결핍 (파열의 부족으로 나타남)과 관련이있다. 또한, 성상 세포의 특정 subpopulations은 기본 progenitors 또는 NSCs 35 로 기능 할 수 있습니다. 이 분화 된 세포 배양에서 nestin / GFAP 이중 양성 세포가 관찰되지 않았지만 (데이터는 표시되지 않음), 이러한 혼합 배양 물에서 GFAP + 세포는 성상 세포 전구 세포 및 성상 세포 인 것으로 가정합니다. 분화 시간을 연장함으로써 성 장군의 숫자가 증가하고 그 형태가보다 성숙해질 가능성이있다 (Zhang의 36 , 37) . 새로운 독성 시험 패러다임에서는 화학적 역경을 평가할 때 생물학적 경로의 화학적 섭동에 대한 지식이 가장 중요합니다. 따라서 체외 시험 시스템은불리한 결과 경로 (AOP)의 개념에 따라 신호 경로의 교란에 부작용을 연관시킨다. 개념 증명으로 로테 논은 Nrf2 경로의 활성화를 평가하는 데 사용할 수 있습니다.이 경로는 산화성 또는 친 전자 성 스트레스에 대한 세포 방어에 관여합니다. 38 산화 스트레스는 다음과 관련된 다양한 AOP에서 중요하고 공통적 인 핵심 이벤트입니다. 발달 및 성충 신경 독성 39 . 로 테논은 Nrf2 단백질의 핵 전좌와 NQO1과 SRXN1을 포함한 Nrf2 표적 효소의 발현 증가에 의해 증명 될 수있는 Nrf2 경로의 활성화를 이끌어 내야한다. 로테 논은 성상 세포 활성화 40 , 41 을 나타내는 GFAP 단백질 수준의 용량 ​​의존적 인 증가를 유도한다는 것이 밝혀졌습니다. Rotenone은 또한 dopaminergic (TH + ) 세포의 수를 감소 시키며, 이는 previ와 일치한다이 유형의 뉴런은 특히 산화 적 스트레스에 민감하기 때문에, 로테 논 의존성 도파민 성 세포 사멸을 보여주는 시험관 내 및 생체 내 연구 21 , 22 , 23 . 결론적으로이 hiPSC 유래 신경 세포 및 신경교 세포 배양 모델은 Nrf2 경로 활성화를 초래하는 산화 스트레스를 유발하는 화학 물질의 신경 독성 효과를 평가하는 데 유용한 도구입니다. 이 분화 프로토콜은 신경 세포 (GABA 성, 도파민 성 및 글루탐산 작용 성 뉴런) 및 성상 교세포의 혼합 배양 물의 생성을 허용하기 때문에 신경 퇴행성 질환과 같은 생리적 및 병리학 적 조건에서 뉴런과 글 리아 사이의 크로스 토크를 연구하는 데 적합 할 수 있습니다 예 : 파킨슨 병). 또한 상당량의 NSC가 존재하면 신경계에 화학 물질이 미치는 영향을 평가하는 데 도움이 될 수 있습니다화학적으로 유도 된 돌연변이 또는 바이러스 감염의 주요 표적이 될 수있는 것으로 알려져있다.

미국 국립 연구위원회 보고서 1 은 규제 독성 시험이 동물에서 관찰 된 표현형 변화에 의존하는 접근법 에서 인간 세포를 이용한 역학 시험 법에 초점을 맞춘 접근법으로 바뀌는 새로운 독성 시험 패러다임을 구상했습니다. 다 능성 줄기 세포 (PSC) 유도체는 얻은 세포가 인간 조직의 생리 조건과 더 유사 할 수 있고 화학 유발 유해 영향을 연구하기 위해보다 관련성이 높은 도구를 제공하기 때문에 암 세포 모델의 대안이 될 수 있습니다. 독성 시험에 가장 유망한 두 가지 유형의 PSC 배양은 인간 배아 줄기 세포 (human embryonic stem cell, hESCs)와 인간 유도 다 능성 줄기 세포 (human induced pluripotent stem cell, hiPSCs)로 현재 기초 연구 및 재생 의학 분야에서 널리 사용되고있다. 이 전문 지식은 이제 새로운 종류의 toxicolo 개발에 활용 될 수 있습니다.생체 내 부작용의 발병과 관련된 섭동 한 생리 학적 경로를 확인하기위한 생체 외 시험. 그러나 hESC에 기초한 규제 안전성 평가 시험 방법은 가능한 모든 윤리적 문제와 배아 유래 세포의 사용을 규제하는 다양한 국가 입법 정책으로 인해 모든 EU 회원국과 전 세계 국가에서 받아 들여지지 않을 것입니다. hiPSCs는 hESCs 4 , 5 와 유사한 특성을 공유하며, 안전성 평가뿐만 아니라 치료 표적 을 확인 하기위한 시험관 내 방법에 대한 큰 가능성을 가지고 있습니다. 또한, hiPSC 기술은 제한된 기증자 집단의 제약과 배아 유래 세포와 관련된 윤리적 우려를 완화합니다. hiPSC에 대한 주요 도전 과제는 이러한 세포가 독성 학적으로 관련된 세포 유도체의 상당 부분을 재현 가능하게 만들 수 있다는 데모이다.인간의 조직에 전형적으로 나타나는 특성과 반응을 보입니다. 선택된 마커의 미리 정의 된 수준은 일반적으로 차별화 프로세스 이후 세포 집단을 특성화하고 차별화 프로세스의 안정성에 대한 통찰력을 제공하는 데 사용됩니다. 이전의 연구는 hiPSC가 신경 및 신경 교세포의 혼합 배양 물을 생성하고 미토콘드리아 호흡 복합체 I의 억제제 인 로테 논이 항산화 제 방어 기전의 핵심 조절 인자 인 Nrf2 경로의 활성화에 미치는 영향을 평가했다. 많은 세포 유형 6 , 7 . 이 작품은 연결 / glial 차별화시 활성화 신호 경로 (유전자 및 단백질 수준)에 대한 자세한 내용을 제공, 혼합 신경 및 glial 문화로 hiPSCs의 차별을 위해 사용되는 프로토콜을 설명합니다. 또한,이 작업을 통해hiPSC에서 유래 한 신경 세포 및 신경 교세포 모델은 로테 논으로 급성 (24 시간) 치료에 의해 유발 된 Nrf2 신호 전달 활성화를 평가하는 데 사용할 수있어 산화 스트레스 유도 평가가 가능합니다. IMR90 섬유 아세포는 pMIG 벡터를 사용하여 2 개의 전사 인자 (Oct4 및 Sox2)의 바이러스 성 전달에 의해 I-Stem (프랑스)에서 hiPSCs로 재 프로그램되었다. 유사한 hiPSC 모델도 적용 할 수 있습니다. 아래에 설명 된 프로토콜은 신경 줄기 세포 (NSCs)로 hiPSCs의 차별화의 모든 단계를 요약하고 사후 mitotic 뉴런과 glial 세포의 혼합 문화 (단계 1과 2, 또한 자세한 내용은 EURL ECVAM DBALM 웹 사이트를 참조하십시오 프로토콜) 8 . NSC의 혼합 뉴런 및 신경 교세포로의 분리, 확장, 저온 보존 및 추가 분화에 대한 추가 프로토콜은 단계 3 및 4에서 상세히 설명된다 (EURL ECVAM DBALM이 프로토콜에 대한 자세한 설명은 bsite를 참조 하십시오.) 9 . 5 단계는 헌신과 분화의 여러 단계 동안 세포의 표현형 정체성을 평가하기 위해 수행 할 수있는 분석을 설명합니다.

protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing

인간의 다 능성 줄기 세포는 인체 시험관 독성 시험 에 적용 할 수있는 다양한 세포 유형으로 분화 할 수 있습니다. 인간의 배아 줄기 세포 (hESCs)의 사용과 관련된 윤리적 및 입법상의 문제를 피하기 위해 인간 유도 다 능성 줄기 세포 (hiPSCs)를 생산하기위한 체세포의 재 프로그램화가 가장 큰 장점이다. HiPSCs는 확대 될 수 있으며 독성 테스트를위한 테스트 시스템으로, 특히 신경 독성과 관련된 다양한 경로의 평가를 위해 여러 유형의 신경 및 신경 교세포로 효율적으로 분화 될 수 있습니다. 이 작품은 연결 및 glial 세포의 혼합 문화에 hiPSCs의 차별을위한 프로토콜을 설명합니다. 신경 세포 분화 (neuronal differentiation)에 의해 조절 및 / 또는 활성화되는 신호 전달 경로가 정의된다. 이 정보는 새로운 독성 테스트 패러다임에 대한 세포 모델의 적용에 중요합니다.이 패러다임에서는 화학 물질이 자신의 능력에 따라 평가됩니다rturb 생물학적 경로. 개념 증명으로 미토콘드리아 호흡 복합체 I의 억제제 인 로테 논 (rotenone)을 사용하여 산화 스트레스에 대한 항산화 - 반응 요소 - (ARE) - 유도 세포 방어 기작의 핵심 조절자인 Nrf2 신호 전달 경로의 활성화를 평가했다 .

Watch this Scientific Journal Video about Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing at JoVE.com

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

인간 유도 다 능성 줄기 세포 (hiPSCs)는 약물 및 화학 스크리닝 및 신경 독성을 포함한 독성 시험을위한 새로운 시험 관내 모델 개발을위한 강력한 도구로 간주됩니다. 여기, hiPSCs의 뉴런과 glia 로의 차별화에 대한 자세한 프로토콜이 설명되어 있습니다.

인간 독성 Pluripotent 줄기 세포의 신경 독성 테스트를위한 뉴런과 Glia의 혼합 문화로의 분화를위한 프로토콜

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

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22.9K 조회수.  Joint Research Centre.  인간 유도 다 능성 줄기 세포 (hiPSCs)는 약물 및 화학 스크리닝 및 신경 독성을 포함한 독성 시험을위한 새로운 시험 관내 모델 개발을위한 강력한 도구로 간주됩니다. 여기, hiPSCs의 뉴런과 glia 로의 차별화에 대한 자세한 프로토콜이 설명되어 있습니다. 

비디오: Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

인간 유도 만능 줄기 세포에서 파생 된 피라미드 뉴런에서 돌기 쪽의 3 차원 정량화

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

마이크로 전극 배열에 네트워크 활동을 측정하기위한 유도 다 능성 줄기 세포의 빠른 신경 세포의 분화

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

인간 다 능성의 체외 분화에서 융모 세포로 줄기 세포

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

인간의 배아 또는 유도 만능 줄기 세포에서 망막 안료 상피 세포의 급속 한, 감독 차별화

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

인간의 기본 섬유 아 세포의 유도 만능 줄기 세포로 재프로그래밍 RNA 기반

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

인간 다능성 줄기 세포를 간 세포로 효율적으로 분화

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

코드 혈액 파생에서 3D 피부 Organoid의 생성 유도 만능 줄기 세포

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...