우리는 박사 마이클 워드 (NIH)에 대 한 기술적인 조언 iNeuron 차별화, 그리고 사바 Barlas 예비 이미지 분석에 대 한 이러한 절차의 디자인에 지적 입력을 위한 박사 에릭 Ullian (UCSF)를 감사 하 고 싶습니다.

저자는 공개 없다.

이 프로토콜에서 우리는 신경 cocultures의 3D 분야의 생산을 위한 체계적인 방법을 설명합니다. 분야 이다와는 독립적으로 파생 hPSCs에서 뉴런으로 구성 됩니다. 비록 하지이 프로토콜의 초점, 이다 hPSCs28 에서 순수한 인구의 세대는 중요 한 단계 이며 사전 경험 없이 수행 하는 경우에 기술적으로 도전적 일 수 있다. 이러한 시 냅 스 칩의 세대에서이 첫 번째 단계는 세심 한 타이밍 ...

이다는 구조 지원 이상 기능적인 책임의 다양 한 중앙 신경 시스템 (CNS) 내에서 매우 풍부한 glial 세포 유형입니다. 수용 성 synaptogenic 요인의 세포 외 기질 (ECM) 분 비를 통해 이다 설립 및 개발1동안 성숙한 시 냅 스의 클러스터링에 도움이. 그들은 또한 건강과 extracellular 신호2,3,,45, 시 냅 스의가 소성 유지에 중요 한 역할을 하 고 항상성의 장기 안정성을 세포 외 칼륨과 조미료, 뿐만 아니라 에너지 기질과 ATP6,,78의 분 비를 조절 하 여 환경. 마지막으로, 그들은9, extrasynaptic 전류 영향을 미치는 의해 neurotransmission에 기여할 수 있는 고 수 직접 다른 세포 유형 myelination10홍보 등을 통해 활동 영향. 중요 한 것은, 비정상적인 또는 이다의 부전 이어질 수 많은 neurodevelopmental 증후군 성인 neuropathology, 이므로 개선에 대 한 순서로 함께 설계 된 신경망에서 뉴런 이다를 포함 하는 명백한 필요가 내 생 두뇌 환경 모델입니다. 이다의 필수적인 특성 신경 시 냅 스1,,1112양식 동적 상호 작용을 그들의 능력입니다. 명과의 부재에서 신경 시 냅 스, 일반적으로 또한 기능 성숙13부족의 제한 된 수를 형성 한다.
인간의 이다 표시, transcriptional, 형태학 및 기능적 특성-분기, 뿐만 아니라 종의 유전자의 증가 크기와 복잡성 등-는 설치류12,14에 지 하지는 15. 그 결과, 인간의 만능 줄기 세포 (hPSC)를 활용 하 여 연구-파생된 신경 세포 소설 치료, 부상, 모델과 문화 패러다임16 개발 하는 동안 생체 외에서 CNS 관련 질병 검사의 수단으로 널리 수용 되는 ,17. 또한, hPSCs 인간 시 냅 스 형성 및 기본 조직18,19에 대 한 필요 없이 기능 연구를 허용합니다.
시 냅 스 회로 기능에 기여 하는 다양 한 세포 유형 및 신호에 대 한 우리의 이해에 방 벽은 인간 두뇌의 관련 모델의 부족 이다. 높은 정확도와 재현성의 시 냅 스 네트워크를 정리 하는 적절 한 플랫폼에 대 한 필요가 있다. 최근, 관심 3D 문화 시스템의 생산에서 나왔다 ('organoids,'로 널리 알려진 'spheroids,' 또는 '미니 두뇌') 휴대 및 매크로 수준에서 구조 하는 복잡 한 3 차원 (3D)를20 . 3D 문화 시스템은 일반적으로 결 석 하 또는 제한 된 일반적인 2D coculture 패러다임21,22동안 ECM 및 셀 상호 작용을 유지 합니다. 기술의 풍부한 3D 신경 spheroids23,,2425; 경작에 대 한 존재 그러나, 많은 긴 문화 기간 필요 (년 개월) 자연 개발 및 레이어 보존, 사용자 아주 작은 제어할 출력 전시.
여기, 우리가 설명 하는 체계적인 방법을 빠르게 하 고 지속적으로 여러 세포 유형 (사전 차별화 된 뉴런을 이다) 간의 bioengineer 신경 상호 작용 영역 cocultures ('소행성')26으로 셀을 조립 하 여 hPSCs에서 파생 된 는 정리 차원에서 인간의 특정 형태학 복잡. 이 고밀도 신경 시스템 균등 하 게 분산 된 신경 하위 시간이 지남에 성숙한 속성에 걸릴 하 고 상영 하거나 높은 처리량 방식에서 분석을 생성 합니다. 처음으로 인간의 이다 이러한 3D cocultures에서 시 냅 스 네트워크 버스트 활동 유도 대 한 설명 합니다. 또한,이 프로토콜은 다른 크기, CNS의 다른 지역 id에 지정 된 셀을 사용 하 고 여러 다른 종류의 세포로 원하는 상호 작용을 공부 하는 분야를 생성에 쉽게 적응.

<table>
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>Circular glass coverslips</td>
			<td>Neuvitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium with 10% DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332100</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>22-037-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332500</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>DLAB</td>
			<td>8030170200</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Tissue embedding solution for cryosectioning</td>
		</tr>
		<tr>
			<td>Pap Pen (Aqua Hold)</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Acros Organics</td>
			<td>169650025</td>
			<td>HAZARD: Toxic if inhaled. Working concentration: 4% in PBS</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Rectangular glass cover slips</td>
			<td>Fisherfinest Premium Superslip</td>
			<td>12-545-88</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Chemical</td>
			<td>S5-3</td>
			<td>Working concentration: 20% or 30% in PBS</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>Terg-A-zyme</td>
			<td>Sigma</td>
			<td>Z273287-1EA</td>
			<td>Detergent. Working concentration: 1%</td>
		</tr>
		<tr>
			<td>TeSR-E8 basal medium</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Human pluripotent stem cell (hPSC) medium</td>
		</tr>
		<tr>
			<td>TeSR-E8 supplements</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Supplements for human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td>Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Thermofisher</td>
			<td>A-11029</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluor 594</td>
			<td>Thermofisher</td>
			<td>A-11037</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ezrin</td>
			<td>Thermofisher</td>
			<td>MA5-13862</td>
			<td>Primary antibody; astrocytes perisynaptic</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Chemicon</td>
			<td>MAB360</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Glt1</td>
			<td>Gift from Dr. Jeffrey Rothstein</td>
			<td>n/a</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Homer</td>
			<td>Synaptic Systems</td>
			<td>160 011</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>MAP2</td>
			<td>Synaptic Systems</td>
			<td>188 004</td>
			<td>Primary antibody; neurons</td>
		</tr>
		<tr>
			<td>PSD95</td>
			<td>Abcam</td>
			<td>ab2723</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>Abcam</td>
			<td>ab868</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Synapsin 1</td>
			<td>Synaptic Systems</td>
			<td>106 103</td>
			<td>Primary antibody; neurons, pre-synaptic</td>
		</tr>
		<tr>
			<td>TuJ1/&#946;3-tubulin (TUBB3)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>Primary antibody; neurons</td>
		</tr>
	</tbody>
</table>

synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells

시 냅 스 회로 기능에 기여 하는 다양 한 세포 유형 및 신호에 대 한 우리의 이해에 방 벽은 인간의 뇌 연구 관련 모델의 부족 이다. 이 문제를 해결 하려면 한 신흥 기술을 3 차원 (3D) 신경 세포 배양, 'organoids' 또는 'spheroids', extracellular 접착 분자를 포함 하 여 세포 상호 작용의 장기 보존에 대 한 나의 사용 이다. 그러나, 이러한 문화 시스템은 시간 소모 하 고 체계적으로 생성 된. 여기, 우리 신속 하 고 지속적으로 신경의 3D cocultures을 생산 하는 방법에 자세히 설명 하 고 인간 만능에서 이다 줄기 세포. 첫째, 미리 차별화 이다 신경 창시자는 해리와 계산. 다음으로, 셀 영역을 형성 결합 되어 요리로 키 니 아 제 억제제와 재현 크기의 구체를 생산 하는 특정 비율에. 몇 주 후 문화의 부동 구체로, cocultures ('소행성') 마지막으로 immunostaining에 대 한 구분 또는 시 냅 스 밀도 강도 측정 하는 multielectrode 배열에 따라 도금. 일반적으로,이 프로토콜 표시 성숙한 세포 유형 제한 표식, 기능 시 냅 스를 형성 하 고는 자발적 시 냅 스 네트워크 버스트 활동 전시 3D 신경 분야를 얻을 것입니다 예상 된다. 함께,이 시스템 단층 문화에 비해 더 적합 한 모델에서 약물 검사와 질병의 메커니즘에 대 한 조사를 허용 합니다.

제대로 수행 하는 경우이 프로토콜으로 이다28,33,34 와 뉴런35 (그림 1A-1C), hPSCs에서 생성 된 기능 cocultures의 정의 된 인구를 생산할 예정 이다 26 이전 상세 하 고 여기에 설명 된 단계 2.1-2.2. 이 단계적 절차, microwell 접시를 사용 하 여...

Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

이 프로토콜에서 결합 해리 인간의 pluripotent 줄기 세포 파생 뉴런에 대 한 재현 방법에 설명 하 고 이다 3D에 범위 cocultures, 무료 부동 조건, 그리고 이후 이러한 분야를 유지 하 immunoanalysis와 multielectrode 배열 녹음 분야의 측정 시 냅 스 회로 활동.

3D Cocultures 이다 인간 만능 줄기 세포에서 신경 세포의 시 냅 스 마이크로 회로 모델링

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

synaptic-microcircuit-modeling-with-3d-cocultures-astrocytes-neurons

Research

JoVE Journal

Developmental Biology

12.1K 조회수.  Houston Methodist Research Institute. 이 프로토콜에서 결합 해리 인간의 pluripotent 줄기 세포 파생 뉴런에 대 한 재현 방법에 설명 하 고 이다 3D에 범위 cocultures, 무료 부동 조건, 그리고 이후 이러한 분야를 유지 하 immunoanalysis와 multielectrode 배열 녹음 분야의 측정 시 냅 스 회로 활동.

비디오: Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

정의 및 간세포와 같은 세포의 확장 성 생성 인간 다 능성 줄기 세포에서

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

말초 혈액 유래 인간 유래 다 능성 줄기 세포에서 연골 세포 분화

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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 ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

분화를 촉진하기 위해 DMSO를 사용하여 인간 다능성 줄기 세포의 일시적인 치료

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...