Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

这手稿是由来自多伦多总与西方基金会和班廷和最佳糖尿病中心，大学健康网络研究生奖资金支持。

1.解决方案和媒体的制备注:准备细胞培养的所有媒体在无菌环境中。媒体已作出，并立即使用。在材料表中提供的试剂的信息。 <ol><li> 媒体分化 <ol><li>制备0天分化培养基:RPMI培养基中以1％谷氨酰胺，2微米CHIR 99021，100纳克/ ml激活素A，10 4M的 MTG。 </li><li>准备1-2天分化培养基:RPMI培养基用1％谷氨酰胺，100毫微克/毫升激活素A，10 4M的 MTG，5毫微克/毫升的bFGF，50μg/ ml的抗坏血酸。 </li><li>制备天3-5分化培养基:RPMI培养基中以1％谷氨酰胺，1％B27，10 4M的 MTG，0.75μMDorsomorphin，50纳克/毫升FGF10。 </li><li>用1％谷氨酰胺，1％B27，50微克/毫升抗坏血酸，50纳克/毫升FGF10，0.25μMSANT-1，2μM维甲酸，50纳克/ ml成头蛋白DMEM:准备6-7天分化培养基。 注:维甲酸酸应最后添加到媒体和媒体应加以保护，以防止氧化降解15。 </li><li>制备天8-12分化培养基:DMEM，用1％谷氨酰胺，1％B27，50微克/毫升抗坏血酸，50纳克/ ml成头蛋白，100ng / ml的人表皮生长因子，10毫烟酰胺。 </li></ol></li><li>制备FACS缓冲液:在PBS中的10％FBS，减去钙和镁。 </li></ol> 2. HESC分化注:HESC解冻，传代，并在补充有bFGF的16基于KOSR介质的存在下对照射的小鼠胚胎成纤维展开。胚胎干细胞是准备分化时细胞达到80-95％汇合。此时的菌落大有确定的边界和一个&#39;domelike&#39;结构（ 图1A）。所有细胞培养在平底，涂有0.1％明胶组织培养处理板中进行。通常情况下，将细胞在6生长或12孔板，在分别2毫升或1毫升，介质卷。 <ol><li>在第0天，通过与第0天分化培养基替换基于KOSR媒体开始分化。 </li><li>第1天和第2，轻轻摇动板吸取之前删除从单层的死细胞。用1-2天分化培养基更换。 </li><li>在第3天，收获的细胞进行流式细胞术。如果细胞表达90％以上的CXCR4 + / CD117 +，继续执行步骤2.4。 </li><li>在天3和5，轻轻摇动板去除单层任何死细胞吸取之前。用3-5天分化培养基更换。 </li><li>天6和7，更换6-7天分化培养基。 </li><li>在天8，10和12，更换8-12天分化媒体。 </li><li>第13天，收获细胞流式细胞仪测定PDX1和NKX6-1的表达。 </li></ol> 3.收获细胞的流式细胞仪分析<ol><li>分离与细胞根据制造商的协议，并在37°C孵育3分钟1×商业胰蛋白酶溶液。 </li><li>在1000微升FACS缓冲液与30微升的DNA酶I.删除商业胰蛋白酶溶液和悬浮单个细胞</li><li>使用35微米的尼龙网细胞滤网，并转移到微量离心管过滤器的细胞。 </li><li>离心机的细胞在455 XG 5分钟。制备用于任一活的或固定染色细胞。 </li></ol> 4.染色的流式细胞仪<ol><li> 流准备活染第3天 <ol><li>悬浮细胞在1000微升FACS缓冲。转移200微升（约1-3×10 5个细胞）到96孔板的两口井（用于未染色和染色的样本）。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li> APC和CD117:原发性标记的抗体，CXCR4染色PE（参见主材料板表 ）30分钟，在室温下避光保存。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li>重悬的样品在100μlFACS缓冲液。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li>悬浮每个样品和传送在300-500微升FACS缓冲液的总体积至1ml微试管或5毫升的圆底管中。 </li><li>在流式细胞仪17的流量运行样品。如果样品不能立即运行，存储在4℃避光保存。 </li></ol></li><li> 流准备固定染色第13天 <ol><li>重悬在商业固定/透化溶液中的细胞沉淀，在4℃下24小时。 </li><li>离心样品以455×g离心5分钟。悬浮于400μl彼尔姆/洗涤溶液。 </li><li>转移样品（约0.5-1×10 6细胞），以总重量为200微升Ø96孔板的孔（IgG的控制和PDX1 / NKX6-1染色）。离心机931 XG为2分钟。 </li><li>在重悬含有抗PDX1和抗NKX6-1小学或同型对照抗体100微升渗透/洗涤溶液中的细胞团（见材料表 ）。在4℃孵育过夜。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。悬浮样品100微升彼尔姆/洗涤液。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li>重悬在100μl彼尔姆/洗涤含有在室温下的二抗的样品1小时避光。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li>悬浮样品100微升彼尔姆/洗涤溶液。 </li><li>离心板在931×g离心2分钟。通过倒转板除去上清液。 </li><li>众议员吃一步4.2.9和4.2.10。 </li><li>悬浮每个样品和传送在300-500微升FACS缓冲液的总体积至1ml微试管或5毫升的圆底管中。 </li><li>在流式细胞仪17的流量运行样品。如果样品不能立即运行，存储在4℃避光保存。 </li></ol></li></ol>

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成功地从hPSCs 在体外产生NKX6-1 +胰腺祖依赖于使用hPSCs高品质的文化和定向分化涉及胰腺发育过程中的关键管理发展阶段的特定信号通路的精确调节的。虽然该协议可以用于诱导在各种HPSC线NKX6-1稳健表达如先前3所示，以确保有效的NKX6-1代以下考虑应。重要的是，所有的细胞培养和介质制剂在无菌环境中，以防止污染进行。重要的是，在胚胎干细胞集落已达到适当?...

糖尿病的发病率正在增加，根据加拿大糖尿病协会，据估计超过11亿人在加拿大是糖尿病或糖尿病前期，与具有1型糖尿病（T1D）1这些个体的5-10％。 T1D]是由位于胰岛内产生胰岛素的β细胞的破坏引起的自身免疫性疾病。目前，生活在1型糖尿病人需要胰岛素2的外源性来源。尽管在胰岛素治疗的进展，患者1型糖尿病继续有困难的时候，他们的调节血糖水平，并继续遭受既低血糖症和高血糖。治疗的一个有前途的形式恢复正常血糖在T1D是利用人胚胎干细胞（胚胎干细胞），其可以用来产生胰岛素生产在体内和体外 3β细胞的无限供给的， <s向上类="外部参照"&gt; 4，5，6，7。分化的胚胎干细胞对β细胞样细胞可以使它可能研究糖尿病体外 ，允许新的治疗靶点的鉴定2型糖尿病，并提供用于移植细胞分化为T1D患者。 在从体外人类胚胎干细胞产生胰岛素生产细胞的最成功的尝试是概括胰腺发育4,5期间发生的胚胎的事件。这涉及到不同的信号传导途径的操纵到显影胰腺的关键阶段准确建模。胰腺发育开始于定形内胚层，其特点是CXCR4和CD117（C-KIT）8，9的表达的诱导。 definit的精确调节IVE内胚层组织是所必需的肠道管，其然后经历前 - 至 - 后和腹侧背侧图案的形成。背侧和腹侧胰芽来自表达胰腺十二指肠同源框基因（Pdx1的 ），这是必要的胰腺发育10的后前肠的区域出现。背，腹芽融合形成胰腺，然后经历了广泛的上皮细胞重建和扩建11。承诺内分泌和外分泌谱系伴随表达多能祖细胞（多用途储值卡）的产生，除其他外，转录因子Pdx1的，NKX6.1和Ptf1a 12,13。多用途储值卡，将成为内分泌和导管细胞继续表达Nkx6-1同时降低Ptf1a表达。与此相反，外分泌谱系细胞会失去expressioNkx6-1的n和维护Ptf1a表达12。 转录因子Nkx6-1在胰腺发育关键作用，特别是在内分泌祖细胞对β细胞的分化。如前所述胰腺发育14中，缺失Nkx6-1导致形成β细胞受损。因此，产生在体外和体内产生胰岛素的β细胞，需要Nkx6-1的有效诱导。 我们最近开发的协议，有效地生成从hPSCs PDX1 + / NKX6-1 +胰腺祖细胞。这些HPSC衍生胰腺祖细胞生成移植后成熟β细胞免疫缺陷小鼠3。该分化方案可以分为4个阶段的特点:1）定形内胚层诱导，2）后前肠图案，3）胰说明书和4）NKX6-1诱导。在这里，我们提供的定向分化的每个步骤的详细描述。

<table><tbody><tr><td>&lt;strong&gt;Media and cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Thioglycerol (MTG)</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC/CF&amp;nbsp;</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Buffer</td><td>BD Bioscience</td><td>554722</td><td></td></tr><tr><td>BD Perm/Wash buffer, 1x</td><td>BD Bioscience</td><td>554723</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>CHIR990210</td><td>Tocris</td><td>4423a</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DNase I</td><td>VWR</td><td>80510-412&amp;nbsp;</td><td></td></tr><tr><td>Dorsomorphin</td><td>Sigma</td><td>P5499</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D</td><td>236-EG</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>88150</td><td></td></tr><tr><td>FGF10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>NO636</td><td></td></tr><tr><td>NOGGIN</td><td>R&amp;amp;D</td><td>3344-NG</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875</td><td></td></tr><tr><td>SANT-1</td><td>Tocris</td><td>1974</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), phenol red</td><td>Life Technologies</td><td>12605-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies for flow cytometry (working dilutions)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD117 PE (1:100)</td><td>Life Technologies</td><td>CD11705</td><td></td></tr><tr><td>CXCR4 APC (1:50)</td><td>BD&amp;nbsp; Bioscience</td><td>551966</td><td></td></tr><tr><td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td><td>Life Technologies</td><td>A-31571</td><td></td></tr><tr><td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.</td><td>705-546-147</td><td></td></tr><tr><td>Isotype Control Mouse IgG&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.&amp;nbsp;</td><td>015-000-003</td><td></td></tr><tr><td>Isotype Control Goat IgG</td><td>R&amp;amp;D&amp;nbsp;&amp;nbsp;</td><td>AB-108-C</td><td></td></tr><tr><td>NKX6-1 (1:2,000)</td><td>DSHB</td><td>F55A10</td><td></td></tr><tr><td>PDX1 (1:100)</td><td>R&amp;amp;D</td><td>AF2419</td><td></td></tr></tbody></table>

efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors

多能干细胞具有自我更新和分化为多种谱系，使它们有吸引力的来源可用于研究和糖尿病的未来治疗胰腺祖细胞的产生的能力。本文概述旨在产生自人胚胎干细胞（胚胎干细胞）的胰腺祖细胞的四阶段分化方案。这个协议可以被应用到许多的人多能干细胞（HPSC）线。采取产生胰腺祖细胞的方法是区分人类胚胎干细胞胰腺发展的关键阶段精确建模。这开始于定形内胚层，这是由在活化素A，碱性成纤维细胞生长因子（bFGF）的和CHIR990210的存在下培养细胞来实现的诱导。进一步的分化和图案与成纤维细胞生长因子10（FGF10）和Dorsomorphin生成类似细胞后前肠。添加的雷廷伊斯兰会议组织酸，头蛋白，圣-1和FGF10区分后前肠细胞进入胰腺内胚层细胞的特性。最后，表皮生长因子（EGF）的结合，烟和头蛋白导致有效地产生PDX1 + / NKX6-1 +细胞。流式细胞仪进行，以确认特定的标志物的表达在胰腺发育的关键阶段。的PDX1 + / NKX6-1 +胰在阶段4的端祖细胞能够在移植后产生的成熟β细胞免疫缺陷小鼠的，并且可以进一步分化以产生在体外产生胰岛素的细胞。因此，有效地产生PDX1的+ / NKX6-1 +胰祖细胞，如在该协议证明的，是非常重要的，因为它提供了一个平台，以研究人类胰腺发育的体外和提供细胞的来源与分化为潜在可能EVβ细胞entually用于糖尿病的治疗。

有效地产生胰祖细胞的依赖于未分化的细胞，随后通过该分化方案中的精确加入特定信号分子的适当生长和维持，如在图1A中示意示出。在第0天，未分化的细胞应该是80-95％汇合和菌落具有限定边缘（ 图1A）。第一阶段期间，媒体将有可能出现混浊，因为细胞死亡是相当普遍的在这个阶段。由第1阶段的末尾，将细胞应共表达定形内胚层标志物，CXC...

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

这里，我们描述一个4级协议分化人胚胎干细胞在体外 NKX6-1 +胰祖细胞。这个协议可以应用于各种人多能干细胞系的。

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

efficient-differentiation-pluripotent-stem-cells-to-nkx6-1-pancreatic

Research

JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

7.9K 次观看.  University Health Network. 这里，我们描述一个4级协议分化人胚胎干细胞在体外 NKX6-1 +胰祖细胞。这个协议可以应用于各种人多能干细胞系的。 

视频: Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

科研

遗传学

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

发育生物学

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

生物学

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

生物工程

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

多能高效分化干细胞NKX6-1

摘要

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此视频中的章节

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1⁺后后福格内胰腺祖细胞

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

人类多能干细胞分化为产生胰岛素的胰岛簇

一个定量测定胰岛素表达细胞集落形成先觉

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

体外人体牙浆干细胞向胰腺血统的诱导

使用定义的分化系统从多能干细胞的肝祖原体规范

多能高效分化干细胞NKX6-1

摘要

探索更多视频

此视频中的章节

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

人类多能干细胞分化为产生胰岛素的胰岛簇

一个定量测定胰岛素表达细胞集落形成先觉

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

体外 人体牙浆干细胞向胰腺血统的诱导

使用定义的分化系统从多能干细胞的肝祖原体规范

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1⁺后后福格内胰腺祖细胞

体外人体牙浆干细胞向胰腺血统的诱导