Name: directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells
Uploaded: 2017-11-01T12:30:00.000+00:00
Duration: 14 min 37 s
Description: Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

この作品は、血液部門内科学、ワシントン州大学医科大学院によってサポートされています。CD は、国民の中心、肺および血の協会からの T32HL007088 によって支えられました。CMS は、血液学者賞のアメリカの社会によって支えられました。

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著者は、彼らは競合する金銭的な利益があることを宣言します。

このプロトコルでは、プリミティブ型または決定的な造血前駆細胞の分化の急速、血清無料、実質無料のメソッドについて説明します。プリミティブ型または決定的な造血前駆細胞の中胚葉性仕様は、一意にカノニカル WNT シグナルの小分子阻害剤を悪用当社のプロトコルを使用して確実に実現できます。CHIR9902133 GSK3β 阻害剤によるステージ固有 wnt シグナルの活性化を生?...

ひと多能性幹細胞 (hPSCs) ヒト胚性幹細胞 (hESCs)、ひと誘導多能性幹細胞 (hiPSCs) を包含として定義され、適切な成長条件下で自己更新を受けていない唯一のユニークな能力を持っています。また、あらゆる細胞に分化する能力が 3 つの胚葉から派生: 外胚葉、中胚葉、内胚葉の1。これらのユニークな能力のためは、hPSCs は、再生医療、疾患モデル作製、細胞ベースの治療2に偉大な約束を保持します。種類の細胞は、hPSCs から正常に分化されている、1 つの重要な課題は大人っぽい hPSC 由来造血幹細胞 (造血)、決定的な造血前駆細胞の in vitro仕様。
HPSCs から人間の HSCs の発展に 1 つの可能性があります障壁ヒト胚3で複数の造血プログラムの存在であります。現れる、「原始的な造血、」と呼ばれる最初のプログラムは胚の卵黄嚢の組織内で起きると巨核球、マクロファージ、赤芽球前駆細胞 (EryP フロン) の一時的な生産が最大の特徴です。特に、このプログラムは、造血に上昇を与えないもそれは T および B リンパ球前駆細胞に上昇を与えるが。ただし、卵黄嚢では、エリスロ骨髄前駆 (EMP4,5,6,7,8) など、制限された決定的な造血前駆細胞に上昇を与えるは一過性赤血球欠乏リンパ プライミング多能性前駆 (LMPP9)。ただし、EMPs でも LMPPs でもない、完全に多能性または成人レシピエントにおける HSC のような生着が可能です。対照的に、開発で後で古典的な定義の「決定的な」造血プログラムは、HSC を含むすべての成人造血系統に上昇を与える、適切な胚大動脈性腺発生地域で指定されます。これら胎児内決定的な造血細胞の仕様は、hemogenic (彼) 内皮3,10,11 から内皮-造血移行を介しての Notch 依存的ファッション ,12,13,14。溶解能力は別として潜在的な multilineage とこれらの細胞の依存性のノッチを EMP と (文献参照3,13 LMPP からこれらの決定的な造血前駆細胞を区別するために使用できます。).
HPSCs からプリミティブと決定的な造血仕様を支配する機構を理解することは可能性が高いさまざまな hPSC ラインの決定的な造血前駆細胞の再現性のある生産に不可欠。最近まで、プリミティブと決定的なの多能性造血前駆細胞を分離できれば hPSC 微分のプロトコルには、15,16,17,18がありませんでした。 19,20,21,22,23,24,25。最初に牛胎児血清 (FBS) および/または間質共培養を使用多くのアプローチ説明プリミティブと決定的な造血潜在的な15,16、混合 hPSC 分化の造血の可能性 17,19,22,23,25。さらに、造血無血清の多くのプロトコルは、港湾の造血潜在的な18,20,21, hPSCs から中胚葉の仕様は信号の要件を説明しています。24します。 ただし、これらのメソッドはまだ両方のプログラムの異種混合物に上昇を与えた、と臨床応用と理解の発達メカニズムの使用は限定される場合があります。
我々 は最近概説アクチビン/節点と hPSC から派生した中胚葉18,26 からプリミティブと決定的な造血仕様における WNT シグナルのステージ固有の信号要件を持つこれらの研究で構築して.ステージ固有 WNT 信号操作の使用は、専らプリミティブまたは決定的な造血前駆細胞26排他的のいずれかの仕様に、後者は特にユニークです。中胚葉仕様、中に PORCN 阻害剤 IWP2 とカノニカル WNT シグナル伝達の阻害の結果 CD43 の仕様で+ EryP フロン ・骨髄前駆細胞、リンパ球検出可能性のないです。対照的に、カノニカル WNT シグナル GSK3β 阻害剤、CHIR99021 と分化の段階で同じ刺激検出 CD43 の完全な不在で起因した+ EryP フロン、同時にリードしながらに、CD34 の仕様+CD43−彼。この人口が骨髄性、所有していたHBG-赤芽球と T リンパ球の可能性を表現します。以降の解析には、CD7327,28 ・ CD18428、その造血可能性の表現としてはノッチ依存28だったこれが識別されます。さらに、単一細胞のクローン性解析は、これらの決定的な造血系統の多能性の単一細胞28が由来することを示した。一緒に取られて、これらの研究は、純粋な原始造血前駆細胞や多能性 NOTCH 依存的決定的な造血前駆細胞、ステージ固有 WNT 操作ができますを示します。
ここでは、我々 は WNT シグナリング中胚葉のパターン形成とその下流の造血系統の試金の操作を介して排他的プリミティブまたは決定的な造血系細胞を生成する我々 の差別化戦略を概説します。このプロトコルは、再生医療用 hPSCs からプリミティブ型または決定的な造血前駆細胞の生産に興味を持っている研究者に大きな価値です。

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMD)</td><td>Corning</td><td>10-016</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), ES cell rated</td><td>Gemini Bioproducts</td><td>100-500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30396.03</td><td></td></tr><tr><td>L-glutamine, 200 mM solution</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Gelatin, porcine skin, Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Alpha-MEM</td><td>Life Technologies</td><td>12000-022</td><td></td></tr><tr><td>DMEM-F12</td><td>Corning</td><td>10-092-CV</td><td></td></tr><tr><td>Knock-out serum replacement</td><td>Life Technologies</td><td>10828028</td><td>&quot;KOSR&quot;</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>b-mercaptoethanol, 55 mM solution</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Fraction V, Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP1605</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Corning</td><td>10-080</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td></td></tr><tr><td>B27 supplement, no vitamin A</td><td>Life Technologies</td><td>12587010</td><td></td></tr><tr><td>Stempro-34 (SP34)</td><td>Life Technologies</td><td>10639011</td><td>&quot;SP34&quot;</td></tr><tr><td>Growth factor reduced Matrigel</td><td>Corning</td><td>354230</td><td>&quot;MAT&quot;</td></tr><tr><td>L-absorbic acid</td><td>Sigma-Aldrich</td><td>A4403</td><td></td></tr><tr><td>Human serum transferrin</td><td>Sigma-Aldrich</td><td>10652202001</td><td></td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Collagenase B</td><td>Roche</td><td>11088831001</td><td></td></tr><tr><td>Collagenase II</td><td>Life Technologies</td><td>17101015</td><td></td></tr><tr><td>DNaseI</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Life Technologies</td><td>14190144</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td></td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D Systems</td><td>338-AC</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td></td></tr><tr><td>SCF</td><td>R&amp;amp;D Systems</td><td>255-SC</td><td></td></tr><tr><td>IGF-1</td><td>R&amp;amp;D Systems</td><td>291-G1</td><td></td></tr><tr><td>IL-3</td><td>R&amp;amp;D Systems</td><td>203-IL</td><td></td></tr><tr><td>IL-6</td><td>R&amp;amp;D Systems</td><td>206-IL</td><td></td></tr><tr><td>IL-7</td><td>R&amp;amp;D Systems</td><td>207-IL</td><td></td></tr><tr><td>IL-11</td><td>R&amp;amp;D Systems</td><td>218-1L</td><td></td></tr><tr><td>TPO</td><td>R&amp;amp;D Systems</td><td>288-TP</td><td></td></tr><tr><td>EPO</td><td>Peprotech</td><td>100-64</td><td></td></tr><tr><td>Flt-3 ligand (FLT3-L)</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>IWP2</td><td>Tocris</td><td>3533</td><td></td></tr><tr><td>Angiotensin II</td><td>Sigma-Aldrich</td><td>A9525</td><td></td></tr><tr><td>Losartan Potassium</td><td>Tocris</td><td>3798</td><td></td></tr><tr><td>CD4 PerCP Cy5.5 Clone RPA-T4</td><td>BD Biosciences</td><td>560650</td><td>Dilution 1:100; T cell assay</td></tr><tr><td>CD8 PE Clone RPA-T8</td><td>BD Biosciences</td><td>561950</td><td>Dilution 1:10; T cell assay</td></tr><tr><td>CD34 APC Clone 8G12</td><td>BD Biosciences</td><td>340441</td><td>Dilution 1:100; EHT assay</td></tr><tr><td>CD34 PE-Cy7 Clone 8G12</td><td>BD Biosciences</td><td>348801</td><td>Dilution 1:100; Hemogenic endothelium</td></tr><tr><td>CD43 FITC Clone 1G10</td><td>BD Biosciences</td><td>555475</td><td>Dilution 1:10; Hemogenic endothelium</td></tr><tr><td>CD45 APC-Cy7 Clone 2D1</td><td>BD Biosciences</td><td>557833</td><td>Dilution 1:50; T cell assay</td></tr><tr><td>CD45 eFluor450 Clone 2D1</td><td>BD Biosciences</td><td>642284</td><td>Dilution 1:50; EHT assay</td></tr><tr><td>CD56 APC Clone B159</td><td>BD Biosciences</td><td>555518</td><td>Dilution 1:20; T cell assay</td></tr><tr><td>CD73 PE Clone AD2</td><td>BD Biosciences</td><td>550257</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>CD184 APC Clone 12G5</td><td>BD Biosciences</td><td>555976</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>BD Biosciences</td><td>564907</td><td>Dilution 1:10,000; T cell assay</td></tr><tr><td>OP9 DL4 cells</td><td></td><td></td><td>Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156</td></tr><tr><td>MethoCult H4034</td><td>Stemcell Technologies</td><td>4034</td><td>&quot;MeC&quot;</td></tr><tr><td>Milli-Q water purification system</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>5% CO2 incubator</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>Multigas incubator</td><td></td><td></td><td>Set at 37 C, 5% CO2, 5% O2</td></tr><tr><td>6 well tissue culture plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>24 well tissue culture plate</td><td>Corning</td><td>353226</td><td></td></tr><tr><td>6 well low-adherence tissue culture plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>24 well low-adherence tissue culture plate</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>35 mm tissue culture dishes</td><td>Corning</td><td>353001</td><td></td></tr><tr><td>Blunt-end needle, 16 gauge</td><td>Corning</td><td>305198</td><td></td></tr><tr><td>3 cc syringes</td><td>Corning</td><td>309657</td><td></td></tr><tr><td>5 mL polypropylene test tube</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>5 mL polystyrene test tube</td><td>Corning</td><td>352058</td><td></td></tr><tr><td>15 mL polypropylene conical</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50 mL polypropylene conical</td><td>Corning</td><td>430921</td><td></td></tr><tr><td>2 mL serological pipette</td><td>Corning</td><td>357507</td><td></td></tr><tr><td>5 mL serological pipette</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Corning</td><td>4489</td><td></td></tr><tr><td>Cell scrapers</td><td>Corning</td><td>353085</td><td></td></tr><tr><td>2.0 mL cryovials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>5 mL test tube with 40 &amp;micro;M cell strainer</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>40 &amp;micro;M cell strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Cell culture centrifuge</td><td></td><td></td><td></td></tr><tr><td>Biosafety hood</td><td></td><td></td><td></td></tr><tr><td>FACS AriaII or equivalent</td><td></td><td></td><td></td></tr><tr><td>LSRii or equivalent</td><td></td><td></td><td></td></tr><tr><td>FlowJo software</td><td>TreeStar</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>0.22 &amp;micro;M filtration system</td><td>Corning</td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>4 C refrigerator</td><td></td><td></td><td></td></tr><tr><td>-20 C Freezer</td><td></td><td></td><td></td></tr><tr><td>-80 C Freezer</td><td></td><td></td><td></td></tr></tbody></table>

directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells

再生医療の主な目的の 1 つは、生成、造血幹細胞 (造血) ひと多能性幹細胞 (hPSCs) 由来のメンテナンスです。最近まで、造血に hPSCs を区別するための努力は主に HSC 可能性を欠いて、代わりに卵黄嚢造血のような造血前駆細胞を生成しています。これらの結果の造血前駆細胞は、様々 な成人造血器疾患、リンパ系統の特にそれらの in vitro疾患モデル作製のためのユーティリティ限られている可能性があります。しかし、最近ここの概要ステージ固有分化プロトコルを使用して hPSCs からエリスロ骨髄リンパ多能決定的な造血前駆細胞を生成する方法を説明しました。基底膜マトリックス コーティング容器に hPSCs の酵素分解による胚様体 (EBs) が形成されています。EBs は、中胚葉に GSK3β 阻害剤の CHIR99021 でその後決定的な造血プログラムに指定されている組換え BMP4 によって区別されます。また、原始造血は PORCN 阻害剤の IWP2 によって指定されます。造血はさらに VEGF 組換え添加および支持造血サイトカインを介して駆動されます。このメソッドを使用して生成された結果の造血前駆細胞疾患と発達モデル、体外に使用される可能性があります。

HPSCs からプリミティブと決定的な造血前駆細胞の誘導を描いた回路図は図 1に示します。中胚葉分化、造血前駆仕様に続いての 2 ~ 3 日中に発生するカノニカル WNT によるパターニングします。
代表フローサイトメトリーによる解析とコロニー形成 hPSC 由来造血分化文化のセルロースの試金は<strong clas...

Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

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

ここで、提示ひと多能性幹細胞 (hPSC) 培養のプロトコル、CD34 に hPSCs を区別するために使用される+造血前駆細胞。このメソッドは、排他的に決定的なまたは原始的な造血プログラムのいずれかのセルを指定するカノニカル WNT のステージ固有操作を使用します。

ひと多能性幹細胞からプリミティブかつ決定的な造血前駆細胞の分化を監督

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

directed-differentiation-primitive-definitive-hematopoietic

Research

JoVE Journal

Developmental Biology

10.9K ビュー.  Washington University in St. Louis. ここで、提示ひと多能性幹細胞 (hPSC) 培養のプロトコル、CD34 に hPSCs を区別するために使用される+造血前駆細胞。このメソッドは、排他的に決定的なまたは原始的な造血プログラムのいずれかのセルを指定するカノニカル WNT のステージ固有操作を使用します。

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

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

ヒト多能性幹細胞における転写因子の過剰発現によって、造血内皮と血液の直接誘導

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

発生生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

転写因子との再プログラミングマウス胚線維芽細胞造血プログラムを誘導するために

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Tリンパ球に向かって誘導多能性幹細胞の分化の指示

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

免疫・感染学

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

ヒト臍帯血由来CD34+造血幹細胞および前駆細胞のパンミエロイド分化

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

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.

転写因子の強制発現によるヒト線維芽細胞のヘモジェニックリプログラミング

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

ヒト多能性幹細胞からドーパミン作動性ニューロンの分化を監督

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

神経科学

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

ヒト人工多能性幹細胞からの破骨細胞の分化と特性評価

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

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.

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

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