We thank Mrs. Matsumoto for the technical assistance.

1.细胞培养<ol><li>获得的细胞以诱导四倍体。迄今为止，已经证实，该技术可以应用到人类的成纤维细胞系的TIG-1，BJ，IMR-90和端粒酶永生TIG-1（TIG-HT）。 </li><li>生长在基本培养基细胞用α修改或适合于细胞类型进行研究补充有10％（V / V）热灭活的胎牛血清（FBS）的通过，在5％孵育（体积/任何其他细胞培养基ⅴ） 的 CO 2气氛中于37℃。传代细胞每3天或4天不超过汇合密度。 注:群体倍增水平（PDL）应在传代每次评估细胞生长和细胞年龄计算。的PDL可以从接种到培养皿（N1），并在使用下述式（X是初始PDL）的下传代（N 2）收获细胞数量的细胞数来计算。 PDL = X +（日志N1 - 日志N2）/ 2日志</li></ol> 2.诱导和埃斯塔从二倍体成纤维细胞四倍体细胞blishment <ol><li>诱导四倍体没有动摇过。 注意:某些细胞株（ 如 TIG-1）可与demecolcine（DC）的如下成为连续处理几乎完全四倍体。 <ol><li>传代细胞插入每天一或两个100毫米培养皿治疗前以诱导四倍体。一般，准备每盘5×10 5到1×10 6个细胞用于四倍体细胞的诱导。 </li><li>治疗指数生长的细胞含有0.1微克/毫升DC 4天网上平台。 </li><li>替换包含直流介质与不含药物的培养基，并在37℃孵育，在5％（体积/体积） 的 CO 2气氛中的细胞。细胞通常在1周内恢复增殖。 </li></ol></li><li>诱导四倍体与摇断。 注意:大多数细胞成为上述（2.1）中描述的，与直流的简单处理的结果是二倍体和四倍体群体的混合物。因此，p的一些修改rocedure消除二倍体人口是必要的如下（ 图1）。 <ol><li>传代细胞分为几个T75烧瓶一天治疗前诱导四倍体。通常，至少准备5×10 6个细胞（每烧瓶1×10 6个细胞）为四倍体细胞的诱导。 </li><li>治疗指数生长的细胞含有0.1微克/毫升DC 16中 - 18小时逮捕细胞的有丝分裂。逮捕在有丝分裂细胞中所需要的时间可能取决于靶细胞，并应通过初步实验来确定，以获得尽可能多的有丝分裂细胞成为可能。例如，速度较慢的生长的细胞应与直流待处理的速度比生长细胞较长的时间。 注:如果细胞可用于本步骤太长治疗，他们可能会经历有丝分裂滑脱并附着在餐具表面，使得不可能以收集通过摇断丝分裂细胞中的下一个步骤。 </li><li>收集DC-逮捕有丝分裂细胞通过摇动关方法，WHICH松散地粘附的有丝分裂的细胞由烧瓶中并用培养基洗涤后离心的轻轻摇动（300 XG，5分钟）收集。通过在这个步骤中的适当的方法计数细胞数目。 </li><li>补种收集有丝分裂细胞成60个100mm培养菜肴和治疗与0.1微克/毫升的DC细胞额外3天。种子每盘大于1×10 6个有丝分裂的细胞，因为细胞的小于10％的可存活这种治疗。 </li><li>含直流用不含药物的培养基介质更换和等待细胞恢复增殖，通常在1周内出现。 </li></ol></li><li>传代细胞原二倍体细胞相类似。 </li></ol> 3. DNA倍体的检查注意:这是一个行之有效的方法和技术手册应提交详细的程序和技术技巧。 <ol><li>收获的细胞（5×10 5 - 1 10 6）通过用溶液CON培养细胞泰宁0.05％胰蛋白酶和0.02％EDTA的10分钟，在37℃，并用含10％FBS的培养基中和胰蛋白酶。 </li><li>离心细胞介质和通过在5ml磷酸盐缓冲盐水的（PBS）然后离心（300 XG，5分钟）再悬浮清洗。 </li><li>通过以下两种方法之一制备用于DNA分析的细胞<ol><li>一种分析非固定细胞10方法 注意:可以使用用于细胞周期分析市售的试剂盒来进行此方法。用这种方法制备的样品应当立即进行分析，因为细胞不固定在过程和染色的核将随着时间的推移损坏。 <ol><li>洗用含有250mM的蔗糖的40mM柠檬酸盐缓冲液的细胞。随后处理细胞用0.1％（体积/体积）的Nonidet P40和30微克/毫升胰蛋白酶250微升在含有250mM的蔗糖在室温下10分钟200 40微升mM柠檬酸盐缓冲液中。 </li><li>加入0.5微克/毫升胰蛋白酶INH 200微升ibitor和0.1微克/毫升RNA酶A在含有250mM的蔗糖的40mM柠檬酸盐缓冲液，并温育细胞，在室温下10分钟。 </li><li>加入200微升的0.4毫克/在含有250mM的蔗糖的40mM柠檬酸盐缓冲溶液的碘化丙啶（PI），并在室温下孵育10分钟的细胞进行染色细胞核。 </li></ol></li><li>一种分析固定的细胞的方法<ol><li>修复用80％乙醇的细胞通过在1毫升的PBS悬浮细胞，加入4毫升100％乙醇的溶液，同时混合该细胞悬浮液，并离开它在室温下30分钟。在此固定剂的细胞可以储存在-20℃下几个星期。 </li><li>通过在5mL PBS中重悬它们洗涤细胞，然后离心（300 XG，5分钟）。治疗细胞用0.5毫克/毫升RNA酶A并用50微克/毫升的碘化丙啶（PI）（0.5毫克/毫升RNA酶A 475微升和1毫克25微升/毫升，PI），在室温下30分钟染色它们。 </li></ol></li></ol></li><li>分析的T DNA含量他用流式细胞仪用488 nm激光和红色通道排放过滤器网格（长传&gt; 610纳米）。分析在同一时间从真正二倍体细胞制备来评估靶细胞的倍性的参比样品。 <ol><li>备选地，添加荧光标准珠以评估二倍体细胞对珠的荧光强度比。使用"脉冲高度与脉冲宽度"流选项仪单四倍体细胞和二倍体细胞11团块区别对待。 </li></ol></li></ol> 4.检查染色体计数及核型分析注意:这些都是行之有效的程序和技术手册或制造商的协议应提交详细的程序和技术技巧。 <ol><li>治疗指数生长的细胞用0.1微克/毫升DC 4小时，逮捕细胞分裂中期。 </li><li>准备染色体核型。 <ol><lI&gt;收获细胞（至少5×10 5个细胞）通过胰蛋白酶消化，在含有FBS的培养基中止。 </li><li>离心细胞在培养基（300 XG，5分钟），并用在37℃的低渗溶液（5mL，0.075M的KCl）10分钟治疗。 </li><li>修复和暂停在卡诺固定液细胞（甲醇:乙酸= 3:1）。 </li><li>细胞悬液 - （35微升25）通过删除一小体积散布细胞到载玻片。如暴露于热蒸汽附加步骤可能是必要的，以改善染色体蔓延。 </li></ol></li><li>染色细胞原位杂交染色体计数或核型分析或多色荧光（渔业部）。 <ol><li>染色体计数。 <ol><li>染色细胞用5％吉姆萨溶液（或含有荧光显微镜0.5毫克/毫升RNA酶A 5微克/毫升，PI）15 - 20分钟。 </li><li>数字拍摄至少50中期细胞。 </li><li>使用图像分析softwa计数每个细胞的染色体数目再接触并使用照片编辑软件重叠染色体手动分离后。虽然染色体数目可以手动进球，推荐电脑计分。 </li></ol></li><li>核型分析 注:本分析应该由经过培训的技术人员或专业的公司来进行。 <ol><li>根据一个标准的G显带技术12染色的细胞。 </li><li>分析染色体，使标准karyograms 12。 </li></ol></li><li>渔业部 注:该步骤是可选的，并且如果必要应进行。 <ol><li>使用用于根据制造商的方案13的人染色体的多色FISH探针染色的细胞。 </li><li>分析使用带有适当的滤波器荧光显微镜和渔业部分析13软件的染色体。 </li></ol></li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

从二倍体细胞通过化学试剂四倍体诱导的主要问题，无论是由胞质抑制剂或由主轴抑制剂，是细胞常成为二倍体和四倍体群体的混合物，和四倍体细胞必须从二倍体细胞中分离出来。免费二倍体细胞的四倍体种群隔离最常用的方法通过有限稀释使用FACS或克隆。然而，这些程序是费力和不容易执行。在这份报告中，我们提出了一个新的协议建立无正常人成纤维细胞二倍体人口增殖细胞四倍体。该协...

多倍体已经观察到，不仅在哺乳动物物种的专门的组织，而且在各种病理状况，如癌症和变性疾病。多倍体（主要是四倍体）细胞通常在人体组织中，诸如Barrett食管1,2或鳞状子宫颈3,4上皮内病变的癌前病变观察到的，并已经被认为是恶性非整倍体细胞的来源在那些组织5 6。虽然有人建议，四倍体转化为异倍体细胞可以是在肿瘤发生的早期阶段的关键事件，参与这一过程的机制尚未完全了解。这部分是因为没有体外模型已经可以在那里未转化多倍体人体细胞可以传播。 一些研究人员已经通过代双核细胞通过异烟肼诱导四倍体在非转化人类上皮细胞ibiting胞质7-9。在该方法中，然而，不必要的二倍体细胞，必须通过荧光激活细胞分选（FACS）消除7,8- 或通过有限稀释9克隆。因为这些程序是艰苦和不易执行，简单的方法来建立非转化四倍体细胞被期望在这一领域的研究。 在本报告中，我们描述了一个协议，通过相对简单的程序，以建立正常的人类成纤维细胞或端粒酶永生化人成纤维细胞增殖四倍体细胞。该过程使用的主轴毒药demecolcine（DC）逮捕由摆脱与DC进一步处理收集在有丝分裂二倍体细胞，并有丝分裂细胞。与DC治疗时间延长二倍体有丝分裂细胞转化为四倍体G1期细胞，而这些细胞增殖生长停滞了以下药物清除几天后的四倍体细胞。该协议提供用于创建一个有用的模型来研究染色体的不稳定性和多倍体的人类细胞的致瘤潜力之间的关系的有效方法。

<table border="0" cellpadding="0" cellspacing="0" width="687">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">MEM-&#945;</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">M8042-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Trypsin-EDTA</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">T4174</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FBS</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">172012-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Demecolcine solution (10 &#956;g/mL in HBSS)</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">D1925-10ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">BD CycleTES Plus DNA Reagent Kits</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:136px;">#340242</td>
			<td style="width:245px;">For examination of DNA ploidy by flow cytometry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:205px;">Human chromosome multicolor FISH probe 24XCyte</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;">#D-0125-060-DI</td>
			<td style="width:245px;">Specialized filter set and software for mFISH analysis are necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Isis imaging system with mFISH&#160; software&#160;</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;"></td>
			<td style="width:245px;">Specialized probe kit is necessary</td>
		</tr>
	</tbody>
</table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

根据我们的经验，TIG-1细胞可以几乎完全四倍体用0.1微克/毫升直流4天（ 图2A）制成由简单的连续治疗。与此相反，其他成纤维细胞株，如BJ或IMR-90，和TIG-HT细胞，成为由摇离方法二倍体和按照相同的治疗四倍体群体，和有丝分裂细胞的分离的混合物中的过程中是必要的的直流处理（通常为16 -治疗开始后18小时）（ 图2B，C）。与DC的额外治疗3天后?...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

establishment-proliferative-tetraploid-cells-from-nontransformed

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  Saitama Medical University. Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

视频: Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

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 ...

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.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

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

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

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.

从人多能干细胞的黑素细胞的无饲养推导

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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

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

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.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

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

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 ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...