作者想感谢淳哲红（医学博士，副教授，妇产科学系，医学院高丽大学）提供胎盘组织。这项工作是由来自韩国的韩国国家研究基金会（NRF），补助（R1211902）的部分资助。

伦理声明:人类胎盘的研究进行前瞻性，与机构审查委员会对人类的高丽大学（AN09085-001）研究批准。所有的实验都是在无尘无菌室设施，在韩国大学医学中心进行。实验设计，并使用hPSCs程序批准了韩国大学医学中心的机构审查委员会（AN12277-003）。 1.准备仪器，文化传媒，和菜名<ol><li>涂层的所有培养皿用0.1％明胶。高压釜的磷酸盐缓冲盐水（PBS）和钳子。下15磅psi的消毒手术剪，微量离心管，并纱布在121℃下为15分钟以上。 </li><li>制备的预热0.25％胰蛋白酶 - 乙烯胺四乙酸（胰蛋白酶-EDTA），并含有20％胎牛血清（FBS），100U / ml青霉素预热过滤培养培养基（DMEM），和100微克/毫升streptomyc在开始操作前。 </li></ol>从人胎盘2.细胞分离<ol><li>获得胎盘组织，后剖腹产分娩，从健康的孕妇获得书面知情同意后进行正常或治疗性流产在7-32孕周。 </li><li>有从胎盘产科专科手术分离人胎盘绒毛膜板（高性能计算机）;在37℃下在0.25％胰蛋白酶-EDTA孵育这些20分钟，然后冲洗，用5ml PBS中。 </li><li>隔离用剪刀高性能计算机绒毛;剁碎的绒毛和用1ml PBS冲洗三次。切碎后，将绒毛成用枪头一个离心管中;洗用500μl的PBS将样品通过上下抽吸，并离心管120×g离心3分钟。用500μl预热的培养基洗两次组织。 </li><li>培养的细胞在明胶包被的平板培养基在37℃下，在5％CO 2 </子&gt;和下95％的湿度。预涂层在37℃下所有平板用0.1％明胶1小时。成纤维细胞样细胞的集落将形成并在相同的培养基中开始培养后约5-7天贴壁生长。 </li><li>交换介质每2-3天，直到第三通道。在培养期间，除去细胞碎片漂浮在培养基中;不断增长的胎盘成纤维细胞会附着到板上。培养物从HPC到第 10位的通道，然后在成纤维细胞样细胞后胰蛋白酶消化和丝裂霉素-C（10微克/毫升）处理收获它们用于人类胚胎干细胞培养物。 </li><li>之前冻结高性能计算机作为股票样本，使用逆转录聚合酶链反应（RT-PCR），以确认高性能计算机不沾染病原体通常感染胎盘，包括支原体（M. fermentans，M猪鼻，M精氨酸，男。ORALE，M. salivarium，人型支原体，M pulmonis，M. arthritidis，M. neurolyticum，猪肺炎支原体，和M.山羊 ）和细菌种类， 解脲脲原体 ，通过使用支原体检测试剂盒。 <ol><li>按照制造商的说明来进行RT-PCR，请使用套件中提供的所有引物和混合物。使用已培养了36天的支原体检测细胞的上清。加入3微升这些样品在PCR混合物并执行第1次的PCR程序为35个循环，每个包括94℃，30秒，55℃2分钟，72℃1分钟。 </li><li>小心加入0.5微升第1次PCR产物的PCR反应混合物，并执行第2 次的PCR程序，使用相同的条件前30个周期。分析扩增产物以1％琼脂糖凝胶电泳;申请10微升每个第1 和第 2 次 PCR产物向凝胶。 </li></ol></li><li>如果发现高性能计算机与病原体污染，消除这些使用抗生素的组合（BM-细胞周期蛋白），按照制造商的说明进行操作。添加含BM-周期蛋白1的10微克/毫升新培养基3天，然后处理细胞用5微克/毫升的BM-周期蛋白2另外4天。重复这个循环两次，然后检查是否有支原体污染了。 </li></ol> 3.收获来自人胎盘的细胞条件培养基（hPCCM） <ol><li>不含污染后，处理的贴壁细胞（85-90％汇合;（1.5×10 7个细胞/瓶）用丝裂霉素C（10微克/毫升）处理2小时和30分钟，并洗涤所述细胞三次，用10ml的PBS孵育细胞用预热的介质而不丝裂霉素-C 24小时。 </li><li>处理后一天，孵育细胞在10ml培养基（DMEM-F12补充20％敲除血清替代品[KOSR]，0.1毫β巯基乙醇，1％NEAA，和1％青霉素 - 链霉素）。经过24小时培养的，收获的CM在一个15毫升锥形管中，并使用0.22微米的注射器过滤器过滤的CM。 </li><li>添加另一个10毫升新媒体和孵化。每天收集所有上清液从培养1周和冻结所收获的培养基中于-80℃。避免反复冻融。 </li></ol> 4.文化人类胚胎干细胞（胚胎干细胞）和多能干细胞诱导（iPS细胞） <ol><li>获得人胚胎干细胞系，H1和H9细胞（分别在名义WA01和WA09美国国立卫生研究院的hESC注册表中列出），并诱导多能干细胞iPSC的1:的iPS（包皮）-1和iPSC的2（IISH1i- BM）从WiCell研究所（麦迪逊，威斯康星州，美国）。细胞应解冻和培养按照制造商的说明进行操作。 </li><li>为对照组，在广泛使用的无饲养和无血清定义培养基，在37℃基底膜基质被覆菜肴和在5％CO 2培养细胞。最初，种子80-100团块hPSCs的我ñ35毫米的菜肴。生长细胞的这些菜，直到70-80％汇合，并定期通过细胞传代培养每5-6天一次，使用机械或酶方法（中性蛋白酶）。与介质洗涤细胞两次和板其中以1:4的比例。每天更换新鲜培养基介质。 </li><li>对于实验组，培养细胞中hPCCM上碗碟预涂覆有0.1％明胶和执行例行传代，每5天一次，通过机械或酶。板的细胞在1的范围内的比率:6-1:10。 hPSCs转移后附着到明胶内2天。每天更换新鲜培养基介质。 </li></ol>人多能干细胞的5.表征注意:表征使用几种方法，诸如碱性磷酸酶染色，免疫细胞化学，定量实时PCR（QRT-PCR）和免疫印迹hPSCs。 <ol><li>免疫荧光染色<ol><li>对于immunofluorescencE染色，培养hPSCs和固定细胞在8孔载玻片室用4％（重量/体积）低聚甲醛10分钟;然后，透化的细胞用0.1％（体积/体积）的Triton-X100 15分钟，并阻断含有0.1％1小时的细胞用3％（体积/体积）正常马血清的PBS（体积/体积）吐温20。两次每一步之间的5分钟洗涤PBS。 </li><li>孵育细胞与第一抗体（OCT-4，SSEA4，TRA-1-81，TRA-1-60，稀释1:1000）O / N在4℃下，并与二级抗体。 </li><li>在安装前，孵育细胞4&#39;，6-二脒基-2-苯基吲哚（DAPI）中5分钟，在黑暗中。洗涤细胞严格三次，每次5分钟，并干细胞完全，在黑暗中。保存细胞荧光封固剂，观察他们在荧光显微镜下。 </li></ol></li><li>定量RT-PCR <ol><li>分离总RNA，从使用根据制造商的规格的RNeasy小型试剂盒的细胞和量化使用纳米掉落分光光度计提取的RNA。 </LI&gt; <li>合成cDNA通过添加2微克的总RNA，以含寡20微升的反应混合物（dT）的引物和标II逆转录酶;按照此过程中的制造商的说明。 </li><li>扩增合成的cDNA用Bio-Rad公司的iCycler iQ的系统中，采用的iQ SYBR绿定量PCR主混合物和补充表S1中所描述的引物序列。正常化的目的基因的循环阈值的那些的甘油醛-3-磷酸脱氢酶（GAPDH）。 </li></ol></li><li>碱性磷酸酶（AP）染色<ol><li>检测使用的ES细胞表征试剂盒AP活性，根据制造商的协议。 </li><li>在第5天，吸出介质并固定在PBS中的4％多聚甲醛的hPSCs 12分钟。不要overfix;定影单元的时间超过2分钟，将导致在碱性磷酸酶的失活。吸出固定液，并用清水冲洗冲洗1X缓冲。别让井步骤之间干燥。 </li><li>加入足够的染色溶液来覆盖每个孔（每35毫米培养皿的孔1ml）。孵育板在黑暗中在RT下15分钟。 </li><li>孵育后，清洗餐具用1X冲洗缓冲。盖细胞用1×PBS，以防止干燥和使用Olympus显微镜检查和图像的染色细胞。 </li></ol></li><li> Western印迹<ol><li>执行如先前在9中所述免疫印迹分析。简单地说，确定蛋白质浓度在细胞裂解物。解决蛋白上的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶等量的蛋白质转印到聚偏二氟乙烯（PVDF）膜。 </li><li>块印迹和不同的初级抗体探查，O / N在4℃;随后孵育用HRP缀合的二级抗体的印迹1小时。对于保温之间，每次10分钟洗膜三次。检测用ECL试剂的信号。 </li&gt; </ol></li><li>短串联重复序列（STR）分析<ol><li> hPSCs从对照组和实验组在同一通道收获。使用DNA微试剂盒提取基因组DNA，根据生产商的说明。 </li><li>放大用于使用AmpF / STR PCR扩增试剂盒16不同基因位点的基因组DNA，根据制造商说明和毛细管电泳是使用遗传分析仪进行的。 </li></ol></li></ol>

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作者指出无利益冲突。

该模型的开发是为了成功传播hPSCs，同时保持它们的特性，在人源化无饲养培养条件不加入外源重组生长因子，例如bFGF的或胰岛素，使hPSCs的操纵在人源化的微环境。外源性bFGF补充是常见和基质如基质胶或层粘连蛋白的应用是用于无饲养培养hPSCs 14是必不可少的。 此协议是简单，易于实现。然而，有些步骤是其成功的关键。最重要的步骤是从胎盘绒毛膜细胞的无菌分?...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table><tbody><tr><td>Mitomycin-C</td><td>Sigma-Aldrich Corporation</td><td>M4287</td><td>10 &amp;mu;g/ml</td></tr><tr><td>Mycoplasma detection kit</td><td>TaKaRa Bio Inc.</td><td>#6601</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>#354277</td><td></td></tr><tr><td>mTeSR1</td><td>STEMCELL Technologies Inc.</td><td>#05850</td><td></td></tr><tr><td>Dispase</td><td>Worthington Biochemical Corporation</td><td>LS02100</td><td>1 mg/ml</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich Corporation</td><td>G2500</td><td>0.10%</td></tr><tr><td>BM-Cyclin</td><td>Roche</td><td>799 050</td><td>10 &amp;mu;g/ml</td></tr><tr><td>RNeasy mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Nano Drop Spectrophotometer</td><td>Thermo Fisher Scientific Inc</td><td></td><td></td></tr><tr><td>iQ SYBR Green qPCR Master Mix&amp;nbsp;</td><td>Bio-Rad Laboratories</td><td>#170-8882AP</td><td></td></tr><tr><td>ES Cell Characterization kit</td><td>Chemicon International, Inc.,</td><td>SCR001</td><td></td></tr><tr><td>Power cDNA Synthesis Kit&amp;nbsp;</td><td>iNtRON Biotechnology</td><td>25011</td><td></td></tr><tr><td>QIAamp&amp;reg; DNA Micro kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>AmpF/STR&amp;reg; Identifiler&amp;reg; PCR Amplification kit</td><td>Applied Biosystems Inc.</td><td>4322288</td><td></td></tr><tr><td>Applied Biosystems&amp;reg; 3130xl Genetic Analyzer&amp;nbsp;</td><td>Applied Biosystems Inc.</td><td></td><td></td></tr></tbody></table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

此HPSC传播方法的一个优点是，它使用了由人细胞分泌的成分。在协议A最ciritical步骤是人胎盘来源的细胞的分离和培养。这需要从HPC板绒毛的精确部分准确清扫。 图1示出了用于来自人胎盘的细胞分离的步骤。这个过程是简单的和方便的细胞的分离，并且可以很容易地在1小时内进行。如在图2中描述的，组织附着到发芽鲁棒明胶涂层盘和成纤维细胞样细胞。在传代通过胰蛋...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

a-novel-culture-model-for-human-pluripotent-stem-cell-propagation-on

Research

JoVE Journal

Developmental Biology

7.8K 次观看.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

视频: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

可扩展的96孔板基于IPSC的文化和生产用机器人液体处理系统

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

科研

发育生物学

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

生物工程

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

免疫与感染

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

使用自动化培养系统诱导多能干细胞分化

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

作者聚焦:自动化 iPSC 培养以提高可重复性 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

用于维持和分化人诱导的多能干细胞的自动化培养系统

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

从 iPSC 高效生成人肠道类器官的基质比较

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

作者聚焦：使用 iPSC 推进用于药物开发的类器官生成

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

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此视频中的章节

可扩展的96孔板基于IPSC的文化和生产用机器人液体处理系统

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

人胎盘及蜕膜器官培养研究在母胎界面感染

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究