JQW, SZ, SD and KC are supported by grant from the National Institutes of Health and the Staman Ogilvie Fund&#8212;Memorial Hermann Foundation.

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The authors have nothing to disclose.

哺乳動物のトランスクリプトームは、34〜38は非常に複雑です。 RNA-配列技術はまた、遺伝子発現解析のための他の方法に勝る多くの利点を有する新規な転写産物の検出および一塩基変化の検出など 、トランスクリプトーム解析の研究においてますます重要な役割を果たしている。冒頭で述べたように、マイクロアレイのハイブリダイゼーションのアーチファクトを克服す...

造血幹細胞は、成体骨髄ニッチに主に存在する稀な血液細胞である。これらは、血液および免疫系1を補充するために必要な細胞の産生を担う。幹細胞の種類としては、HSCは、自己再生および分化の両方が可能である。 HSCの運命決定を制御するメカニズムの解明、自己複製または分化のどちらかに向かって、血液疾患の研究や臨床使用量2のためのHSCの操作に貴重なガイダンスを提供します。研究者が直面する問題の一つは、HSCが維持され、非常に限られた範囲でインビトロで増殖することができることである。その子孫の大部分は、部分的に文化2で分化されている。 ゲノムワイドスケールでの自己再生および分化の過程を制御する重要な調節因子を同定するために、我々は、モデル系としてマウス原始造血前駆細胞株EMLを使用した。目細胞株は、マウス骨髄3,4から導出された。異なる成長因子を供給すると、EML細胞は、in vitroで 5 において赤血球、骨髄、およびリンパ系細胞に分化することができる。重要なことに、この細胞株は、幹細胞因子（SCF）を含む培地中で大量に増殖させることができ、それでもそれらの多能性を維持する。 EML細胞は、自己再生リン-SCA + CD34 +表面マーカーCD34およびSCA 6に基づいて、部分的に分化したリン-SCA-CD34-細胞の亜集団に分けることができる。短期HSCのと同様に、SCA + CD34 +細胞は、自己複製が可能である。 SCFで処理した場合、LIN-SCA + CD34 +細胞は急速に林-SCA + CD34 +およびLIN-SCA-CD34-細胞の混合集団を再生成し、6を増殖し続けることができます。つの集団は、形態学的に類似しており、c-キットmRNAおよびタンパク質6の同様のレベルを有する。リン-SCA-CD34-細胞はIL-3の代わりに、SCF 3を含有する培地中で増殖させることが可能である。 UnveilingのEML細胞の運命決定に重要な調節因子は、造血時の初期発生の移行における細胞および分子メカニズムのより良い理解を提供します。 自己複製リン-SCA + CD34 +および部分的に分化リン-SCA-CD34-細胞との間の根本的な分子の違いを調べるために、我々は、差次的に発現される遺伝子を同定するために、RNA配列を使用した。転写因子は、細胞の運命を決定する上で重要であるように、特に、我々は、転写因子に焦点を当てる。 RNA-seqがゲノム7,8から転写されたRNAをプロファイリングし、定量化するために、次世代シーケンシング（NGS）技術の能力を利用して最近開発されたアプローチです。簡単に述べると、トータルRNAは、ポリ選択と初期template.The RNAテンプレートは次いで逆転写酵素を用いてcDNAに変換されるように、断片化されている。 cDNAライブラリーを構築するために、無傷の、非分解RNAを用いて、完全長のRNA転写物をマッピングするために重要である。 PURのためシークエンシングの姿勢は、特定のアダプター配列は、cDNAの両端に付加される。そして、ほとんどの場合、cDNA分子は、PCRによって増幅され、ハイスループット様式で配列決定した。 シーケンシング後、得られたは参照ゲノムとトランスクリプトームデータベースに整列させることができる読み取ります。数は、参照遺伝子のマップがカウントされ、この情報は、遺伝子発現のレベルを推定するために使用できることを読み取る。読み込みは、非モデル生物9にトランスクリプトームの研究を可能にする、参照ゲノムなしデノボ組み立てることができる。 RNA-seqの技術は、スプライスアイソフォーム10-12、新規な転写物13および14の遺伝子融合を検出するために使用されてきた。タンパク質をコードする遺伝子の検出に加えて、RNA-配列はまた、新規に検出し、そのような18の非コードRNA 15,16、マイクロRNA 17は、siRNA などの長い非コードRNAの転写レベルを分析するために使用され得る。なぜなら、tの彼は、この方法の精度は、単一ヌクレオチド変異19,20の検出に利用されている。 RNA-配列技術の出現の前に、マイクロアレイは、遺伝子発現プロファイルを分析するために使用される主要な方法であった。予め設計されたプローブは、マイクロアレイスライド21を形成するように合成し、続いて固体表面に付着される。 mRNAを抽出し、cDNAに変換される。逆転写プロセスの間に、蛍光標識されたヌクレオチドは、cDNAに組み込まれ、cDNAはマイクロアレイスライドにハイブリダイズさせることができる。特定の場所から収集された信号の強度は、そのスポット21上の特異的プローブに結合したcDNAの量に依存する。 RNA-配列技術と比較して、マイクロアレイはいくつかの限界がある。 RNA-配列技術は、その使用が制限されるとき、比較的高いバックグラウンドレベルで新規な転写物を検出することが可能であるが、まず、マイクロアレイは、遺伝子アノテーションの既存の知識に依存している葛ね発現レベルが低い。による背景信号が飽和し、マイクロアレイの精度は両方の高度に発現される遺伝子と低い7,22のため制限され、一方でまた、RNA-配列技術は、検出（8000倍）7の非常に高いダイナミックレンジを有する。最後に、マイクロアレイプローブは、一つのサンプル23内の異なる転写物の相対発現レベルを比較した場合、結果はあまり信頼性を高めるそれらのハイブリダイゼーションの効率、が異なる。 RNA-配列がマイクロアレイに比べて多くの利点を有するが、そのデータ分析は複雑である。これは、多くの研究者がまだRNA-配列の代わりに、マイクロアレイを使用する理由の一つです。様々なバイオインフォマティクスツールは、RNA配列のデータ処理および分析24のために必要とされる。 いくつかの次世代シーケンサー（NGS）のプラットフォームのうち、454、イルミナ、SOLIDとイオントレントは、最も広く使用されているものである。 454は最初の商用NGSプラットフォームだった。他のシーケンシングプラットフォームとは対照的にそのようなイルミナ固体として、454プラットフォームは、より長い読みの長さ（平均700ベースの読み取り）25を生成します。長くは、その高い効率25を組み立てるためにtranscriptiomeの初期特性評価のための優れている読み取ります。 454プラットフォームの主な欠点は、シーケンスのメガベース当たりのコストが高いことである。イルミナとSOLIDプラットフォームは増大した数と短い長さで読み込み、生成する。シーケンスのメガベース当たりのコストは、454プラットフォームよりもはるかに低い。が短いため、大量のデータ分析がはるかに計算集約され、イルミナとSOLIDプラットフォーム用に読み取ります。イオントレントプラットフォーム用のシーケンシングするための機器および試薬の価格は安いですし、シーケンシング時間は25短い。しかし、エラーレートとシーケンスのメガベース当たりのコストは、イルミナとSOLIDプラットフォームに比べて高くなっている。異なるプラットフォームは、独自の長所と短所を持っており、データ解析のために異なる方法を必要とする。ナンプラーTFORMは、配列決定の目的や資金の利用可能性に基づいて選択する必要があります。 本稿では、例として、イルミナRNA-のSeqプラットフォームを取る。我々は、EMLの細胞の自己再生および分化において重要な調節因子を調査するためのモデル系としてEML細胞を使用し、発現量の計算および新規な転写産物を検出するためのRNA-配列ライブラリーの構築およびデータ分析の詳細な方法を提供した。我々は、機能テスト（ 例えば shRNAのノックダウン）と結合EMLモデルシステム2のRNA-seqの研究は、造血分化の初期段階の分子機構を理解する上での強力なアプローチを提供し、として機能することができるという本発明者らの以前の刊行物に示されている一般に、細胞の自己再生および分化の分析のためのモデル。

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			<td>15240-062</td>
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			<td>Anti-Mouse Ly-6A/E (Sca-1) PE</td>
			<td>eBioscience</td>
			<td>12-5981-81</td>
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			<td>BD FACSAria Cell Sorter</td>
			<td>BD Biosciences</td>
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			<td>FACS sorting</td>
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			<td>Corning&#8482; Cell Culture Treated Flasks 75cm2</td>
			<td>Corning incorporated</td>
			<td>430641</td>
			<td>Cell culture</td>
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		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 25cm2</td>
			<td>Corning incorporated</td>
			<td>430639</td>
			<td>Cell culture</td>
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			<td>Deoxyribonuclease I, Amplification Grade</td>
			<td>Invitrogen</td>
			<td>18068-015</td>
			<td>Library preparation</td>
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			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
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			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>Cell culture</td>
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			<td>HI FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16140071</td>
			<td>BHK cell culture</td>
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			<td>Horse Serum</td>
			<td>Invitrogen</td>
			<td>16050-122</td>
			<td>EML cell culture</td>
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			<td>IMDM</td>
			<td>HyClone</td>
			<td>SH30228.02</td>
			<td>EML cell culture</td>
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			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>Cell culture</td>
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			<td>Lineage Cell Depletion Kit, mouse&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Isolation of lineage negative cells</td>
		</tr>
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			<td>NanoVue Plus spectrophotometer&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9569-62</td>
			<td>Quality control</td>
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			<td>Thermo Scientific&#8482; Napco&#8482; 8000 Water-Jacketed CO2 Incubators</td>
			<td>Thermo Scientific</td>
			<td>15-497-002&#160;</td>
			<td>Cell culture</td>
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			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>EML cell culture</td>
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			<td>TRIzol&#174; Reagent</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
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			<td>TruSeq&#8482; RNA Sample Prep Kit v2 -Set B (48rxn)</td>
			<td>Illumina</td>
			<td>RS-122-2002</td>
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			<td>2100 Electrophoresis Bioanalyzer Instrument&#160;</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td>Quality control</td>
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			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200</td>
			<td>Cell culture</td>
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			<td>0.45 &#181;m Syringe Filters</td>
			<td>Nalgene</td>
			<td>190-2545</td>
			<td>Cell culture</td>
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identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis

造血幹細胞（HSC）は、白血病やリンパ腫などの多くの疾患において、患者の造血系を再構築するために、移植治療のために臨床的に使用されている。 HSCの自己再生および分化を制御するメカニズムを解明することは研究および臨床用途のためのHSCのアプリケーションのために重要である。しかし、によりインビトロで増殖することができないことにHSCを大量に得ることができない。このハードルを克服するために、我々は、この研究のためのモデル系として、マウス骨髄由来の細胞株、EML（赤血球、骨髄、およびリンパ球）細胞株を使用した。 RNAシークエンシング（RNA-配列）がますます遺伝子発現研究のためのマイクロアレイを置き換えるために使用されてきた。ここではEML細胞の自己再生および分化の調節において潜在的な主要因を調査するためにRNA-配列技術を使用する具体的な方法を報告する。このホワイトペーパーで提供されるプロトコルは、3つの部分に分かれています。最初のパーtはどのように文化のEML細胞と独立したLIN-CD34 +およびLIN-CD34-細胞に説明しています。プロトコルの第2の部分は、トータルRNA調製およびハイスループット配列決定のための後続のライブラリー構築のための詳細な手順を提供しています。最後の部分は、RNA配列データ解析のための方法を説明し、リン、CD34 +およびリン-CD34-細胞との間の差次的に発現する転写因子を同定するためにデータを使用する方法について説明します。最も有意に差次的に発現される転写因子はEML細胞の自己再生および分化を制御する潜在的な重要な調節因子であると同定された。本稿の議論のセクションでは、この実験の成功パフォーマンスのための主要なステップを強調表示します。 要約すると、本論文では、EML細胞中で自己複製と分化の潜在的な調節因子を同定するために、RNA-配列技術を使用する方法を提供しています。特定された主要な要因は、in vitroおよびiの下流の機能解析に供されているn個の生体内。

リン- CD34 +およびCD34-リン-EML細胞において差次的に発現される遺伝子を分析するために、我々は、RNA配列の技術を使用した。 図1は、手順のワークフローを示している。磁気細胞選別による系統陰性細胞の単離後、我々は、LIN-SCA + CD34 +およびFACSアリアを使用してLIN-SCA-CD34-細胞を分離した。リン富化EML細胞を、抗CD34、抗のSca1および系統カクテル抗体で染色した。唯一のリニア細?...

Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

RNAシークエンシングおよびバイオインフォマティクス解析は、マウスEMLcellsのリン、CD34 +およびリン-CD34-亜集団において有意にかつ差次的に発現する転写因子を同定した。これらの転写因子は、自己再生リン-CD34 +を、部分的に分化したリン-CD34-細胞との間の切り替えを決定する際に重要な役割を果たしている可能性がある。

RNAシーケンシング解析によりEML造血前駆細胞における自己再生および分化を調節する重要な要因の同定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

identification-key-factors-regulating-self-renewal-differentiation

Research

JoVE Journal

Biology

12.2K ビュー.  The University of Texas Graduate School of Biomedical Sciences at Houston.  RNAシークエンシングおよびバイオインフォマティクス解析は、マウスEMLcellsのリン、CD34 +およびリン-CD34-亜集団において有意にかつ差次的に発現する転写因子を同定した。これらの転写因子は、自己再生リン-CD34 +を、部分的に分化したリン-CD34-細胞との間の切り替えを決定する際に重要な役割を果たしている可能性がある。 

ビデオ: Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

LIFの不在下で自己再生MESCをサポートするためのSC1（Pluripotin）の使用

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

マウス胚性幹細胞の維持と自己複製を制御する遺伝子を研究するためにOct4GiPレポーターアッセイ

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

山中因子の過剰発現によって多能性の成体体細胞のin vivoでの再プログラミング中

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

急成長腫瘍生成細胞によって分泌される因子によって募集のモニタリング機能と血管系の形態

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

マウス胚のレトロウイルス感染は、造血分化の解析のための細胞由来の胚様体幹細胞

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

スペクトラルフローサイトメトリーにより固形組織から単離された細胞懸濁液の分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...