这项工作得到了新泽西州州长自闭症医学研究和治疗委员会 (CAUT13APS010) 的支持;CAUT14APL031;CAUT15APL041), 南茜. Mindworks 标志着家庭基金会, 慈善领导信任, 以及更大 MetroWest 新泽西州的犹太社区基金会。

作者声明他们没有竞争的财政利益。

这里提出的协议说明了快速和简单的方法来研究基本神经发育过程和测试生长因子和药物使用 hiPSC 衍生神经前体细胞。hiPSC 技术已经革命性的研究神经发育疾病的发病机制, 为我们提供了前所未有的机会, 从受影响的人的活体神经细胞细胞。事实上, 已经有许多 hiPSC 研究的神经发育紊乱, 包括 Rett 综合征, 蒂莫西综合征, 脆性 x 综合征, 精神分裂症, 发现了疾病特异的畸变在树突, 突触和神经元函数<s...

使用简单的有机体和老鼠模型阐明了基本的大脑发展和疾病发病机制。尽管有这些进展, 许多精神疾病的病因仍然难以捉摸, 因为并非所有的研究结果都与人类疾病的复杂方面直接相关。此外, 人脑的更大复杂性往往使人的发育和动物疾病的建模变得困难。随着人类诱导多潜能干细胞 (hiPSCs) 技术的发展和发展, 体细胞可以重新编程成干细胞, 然后分化为神经细胞来研究人类疾病。hiPSCs 和 "涨价可能" 技术的进步 (基因组学、转录组学、蛋白质组学、新陈代谢) 有望彻底改变人类大脑发育的认识。这些技术现在可以在个案的基础上对神经精神疾病的特征进行 "精确医学" 处理。
目前在 hiPSC 疾病建模领域的主要内容是将细胞分化成单层的特定神经元亚型, 或者使用一个叫做 organoid 的3D 培养系统来重述大脑发育的各个方面1,2, 3。这些系统在研究和揭露人类发展和疾病的独特方面非常有价值4,5,6,7。然而, 在他们准备学习之前, 神经元文化和 organoids 通常需要从几周到几个月的文化。这些协议的耗时性和维护这些文化系统所需的资源量常常限制可以进行的实验数量和可测试的变量 (如生长因子或药物) 的数量。此外, 许多利用有丝分裂后神经元和 organoids 的研究都集中在后来的发展过程, 如枝晶突起或突触形成。虽然这些过程牵涉到发育紊乱的病理学, 如自闭症和精神分裂症, 早期的发展事件发生之前, 明确的神经元分化也重要的疾病发病机制8 ,9,10,11,12,13。事实上, 最近的基因组研究表明, 由增殖、过程和迁移组成的中胎儿期在自闭症发病机制中尤为重要11,14。因此, 研究神经干细胞和祖细胞群, 以更好地了解这些早期的过程是很重要的。Organoid 系统由于其3D 性质和组织结构而被认为可以更好地概括人脑发育, 但它包含了一个前体池, 用于研究这些早期事件。然而, organoids 的祖群往往稀疏, 更像是径向胶质细胞, 而不是神经干细胞或祖细胞5,15。因此, 有一个高吞吐量的方法来研究神经发育在积极增殖细胞的早期阶段是有益的。
在实验室中, 我们创建了一个使用 hiPSC 神经前体细胞 (npc) 的协议, 一个神经干细胞和祖细胞的混合种群, 高度增生, 以研究神经发育过程, 如增殖、细胞迁移和初始过程 (突起) 扩展。这些化验是从我们实验室使用的技术中开发的, 数十年来成功地研究了老鼠和老鼠皮层培养的神经发育 16, 17, 18, 19, 20, 21,22,23。重要的是, 它还表明, 在鼠和小鼠培养系统中定义的表型和调控信号是高度预测的机制, 是活跃的在体内, 表明这些技术的价值16, 17,18,19,24。在 hiPSCs 与 npc 的初步分化之后, 这些方法使我们能够在几天内研究重要的发育过程。这些方法有许多优点: (1) 它们需要很少的复杂设备, 易于实现, (2) 可以在短时间内进行大量的实验复制, 从而快速确认结果的重现性, (3)培养变量, 如涂层基质, 生长因子的影响, 药物的活性, 可以快速和经济高效地测试。此外, 我们利用细胞外生长因子作为不同发育过程的关键调控者的良好作用。npc 被暴露在选择发展信号, 直接刺激诸如扩散, 突起和细胞迁移等事件, 并发现它们增强了识别在控制条件下不明显的缺陷的能力19,25,26,27,28. 同样, 评估药物的容易性为采用精确的医学技术来测试各种治疗干预措施的功效提供了有力的途径。因此, 本议定书促进了高通量、重现性和直截了当的方法, 研究早期大脑发育、疾病发病机制以及生长因子和药物对神经发育表型的潜在有益影响。

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:323px;">PSC Neural Induction Medium: 
			Protocol Link: https://goo.gl/euub7a&#160;</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">A1647801</td>
			<td style="width:472px;">This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Advanced DMEM/F12 Medium</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">12634-010</td>
			<td style="width:472px;">Component of 100% Expansion Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Neurobasal Medium</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">21103049</td>
			<td style="width:472px;">Component of both NIM and 100% Expansion Medium&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:323px;">hESC-qualified Matrigel</td>
			<td style="width:185px;">Corning</td>
			<td style="width:111px;">354277</td>
			<td style="width:472px;">hESC-qualified extracellular matrix-mimic gel (ECM-mimic gel)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Y-27632 (2HCl), 1 mg</td>
			<td style="width:185px;">Stem Cell Technologies</td>
			<td style="width:111px;">72302</td>
			<td style="width:472px;">ROCK inhibitor</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:323px;">6 well plates</td>
			<td style="width:185px;">Corning</td>
			<td style="width:111px;">COR-3506</td>
			<td style="width:472px;">Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24 well plates</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">2021-05</td>
			<td style="width:472px;">Polystyrene plates: Used for NPC DNA Synthesis Assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">35 mm dishes</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">2021-01</td>
			<td style="width:472px;">Polystyrene plates: Used for NPC S-Phase Entry and Neurite Assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Natural Mouse Laminin</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:111px;">23017-015</td>
			<td style="width:472px;">Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fibronectin</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:111px;">F1141</td>
			<td style="width:472px;">Substrate for coating plates: Used for Neurite Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Poly-D-Lysine</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:111px;">P0899</td>
			<td style="width:472px;">Substrate for coating plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Penicillin/Streptomycin</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">15140122</td>
			<td style="width:472px;">Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">StemPro Accutase</td>
			<td style="width:185px;">Gibco</td>
			<td style="width:111px;">A11105-01</td>
			<td style="width:472px;">1X Cell Detachment Solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2.5% Trypsin (10X)</td>
			<td style="width:185px;">Gibco</td>
			<td style="width:111px;">15090-046</td>
			<td style="width:472px;">10X enzymatic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5 M EDTA</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td>AM9261</td>
			<td style="width:472px;">used in trypsin solution for lifting cells for DNA synthesis assay</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">tritiated [3H]-thymidine</td>
			<td style="width:185px;">PerkinElmer</td>
			<td style="width:111px;">NET027E001</td>
			<td style="width:472px;">Radioactive tritium, thymidine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fisherbrand 7 mL HDPE Scintillation Vials</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:111px;">03-337-1</td>
			<td style="width:472px;">Vials for liquid scintillation counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EcoLite(+)</td>
			<td style="width:185px;">MP Biomedicals</td>
			<td style="width:111px;">0188247501&#160;</td>
			<td style="width:472px;">Liquid scintillation cocktail</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">LS 6500 multi-purpose liquid scintillation counter</td>
			<td style="width:185px;">Beckman Coulter</td>
			<td style="width:111px;">8043-30-1194</td>
			<td style="width:472px;">Liquid Scintillation Counter</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:323px;">Skatron Semi-automactic Cell Harvester Type 11019</td>
			<td style="width:185px;">Molecular Devices &#38; Skatron Instruments, Inc.</td>
			<td style="width:111px;"></td>
			<td style="width:472px;">Semi-automatic cell harvester</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Click-iT EdU Alexa Fluor&#174; 488 Imaging Kit</td>
			<td style="width:185px;">ThermoFisher Scientific</td>
			<td style="width:111px;">C10337</td>
			<td style="width:472px;">EdU and staining kit for S-Phase Entry Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:185px;">ThermoFisher Scientific</td>
			<td style="width:111px;">15250061</td>
			<td style="width:472px;">Assessing viability of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Grade GF/C filter paper</td>
			<td style="width:185px;">GE Healthcare Life Sciences, Whatman</td>
			<td style="width:111px;">1822-849</td>
			<td style="width:472px;">Glass fiber filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Human Basic FGF-2</td>
			<td style="width:185px;">Peprotech</td>
			<td style="width:111px;">100-18B</td>
			<td style="width:472px;">growth factor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38)</td>
			<td>BACHEM</td>
			<td>H-8430</td>
			<td>neuropeptide</td>
		</tr>
	</tbody>
</table>

rapid detection of neurodevelopmental phenotypes in human neural precursor cells (npcs)

人类大脑发育通过一系列精确编排的过程进行, 早期的阶段以增殖、迁移和突起为特色;后阶段的特征是轴突/枝晶生长和突触形成。在神经发育紊乱中, 通常有一个或多个过程被打乱, 导致大脑形成和功能异常。随着人类诱导的多潜能干细胞 (hiPSC) 技术的出现, 研究人员现在有大量的人类细胞供应, 可以被区分成几乎任何细胞类型, 包括神经元。这些细胞可以用来研究正常的大脑发育和疾病发病机制。一些使用 hiPSCs 模型神经精神疾病的协议使用晚期分化的神经元或使用3D 的文化系统称为 organoids。这些方法在研究人类疾病发病机制方面已证明是宝贵的, 但也存在一些弊端。hiPSCs 分化为神经元和 organoids 的产生是冗长而昂贵的过程, 可能会影响可以评估的实验和变量的数量。此外, 在有丝分裂后的神经元和 organoids 允许研究与疾病有关的过程, 包括枝晶生长和 synaptogenesis, 它们排除了对早期的过程, 如扩散和迁移的研究。在神经发育紊乱, 如自闭症, 丰富的遗传和验尸证据表明早期发育过程中的缺陷。神经前体细胞 (npc), 一个高度增生的细胞群体, 可能是一个合适的模型, 其中提出问题的发育过程和疾病的启动。我们现在扩展了从研究老鼠和大鼠皮质文化的发展到人类 npc 的方法学。使用 npc 可以让我们调查与疾病有关的表型, 并定义不同的变量 (例如, 生长因子, 药物) 如何影响发展过程, 包括增殖, 迁移, 并在短短几天的分化。最终, 此工具集可用于重现性和高通量的方式, 以确定神经发育紊乱疾病的特定机制和表型。

这些研究的目的之一是确定 npc 的增殖活动, 即细胞数量的增加。这是通过评估 DNA 合成的总细胞总数, 一个高通量的方法, 测量将放射性示踪剂氚胸苷纳入细胞提取物, 并反映所有从事 s-阶段的细胞, 无论是合成5分钟或整整两个小时。此外, 这些化验可以确定进入 S 相和总细胞数的细胞比例, 一种更劳动密集型的单细胞检测。细胞在 s-阶段合成 DNA, 这是有丝分裂和细胞分裂之...

Watch this Scientific Journal Video about Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs at JoVE.com

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

神经发育过程, 如增殖, 迁移和突起的生长, 往往是不安的精神疾病。因此, 我们提出的协议, 快速和重现性评估这些神经发育过程中的人类 iPSC 派生的 npc。这些协议还允许评估相关生长因子和疗法对 NPC 发展的影响。

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

9.9K 次观看.  Rutgers Robert Wood Johnson Medical School. 神经发育过程, 如增殖, 迁移和突起的生长, 往往是不安的精神疾病。因此, 我们提出的协议, 快速和重现性评估这些神经发育过程中的人类 iPSC 派生的 npc。这些协议还允许评估相关生长因子和疗法对 NPC 发展的影响。

视频: Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

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.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

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.

人诱导多能干细胞体外生成体细胞衍生物的研究

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

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...