A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

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The authors have nothing to disclose.

本研究は、心臓転写のレトロウイルス媒介発現を介して、心臓のサブタイプの多様なセットにしたMEFの変換のためのダイレクトリプログラミング戦略を提供することはGATA4、MEF2C、TBX5、およびHand2（GHMT）要因。 PM固有レポーターマウスと組み合わせて多重免疫染色法を用いて、我々は、単一細胞の解像度でIAM、IVMS、およびIPMSを識別することができます。このようなアッセイは、サブタイプの...

心臓は胚1、2に開発した最初の機能的器官です。循環器系に関連して、開発中に、酸素、栄養素、および廃棄物処理機構に供給する。三週間受精後、人間の心は初めて打つとその適切な規制は、心筋細胞（のCM）によって維持されています。これらの特殊化した細胞の不可逆的損失は、したがって、進行性の心不全の根底にある基本的な問題です。このようなゼブラフィッシュおよびアフリカツメガエルのようないくつかの生物は、心臓の再生の可能性を有しているが、大人の哺乳類の心臓は3、5、6、より限定されています。このように、心の重要な機能を考えると、心臓病は米国だけで60万人が死亡7を占め、世界における死亡の主な原因であることは驚くべきではありません。ザrefore、効率的に負傷した心筋を修理または交換するために、細胞ベースの治療は臨床上非常に興味深いものです。 山中ら8の精液の研究は、4つの転写因子の強制発現は、多能性幹細胞に完全に分化した線維芽細胞に変換するのに十分であることを示しました。しかし、すべての多能性幹細胞戦略の腫瘍形成能力は、治療目的のためのそれらの使用における重大な関心事となっています。これは、多能性段階を回避しながら細胞を分化転換するための代替方法を検索するための科学的なフィールドをやる気。最近、いくつかのグループは、転写の異所性発現は、GATA4、MEF2C、TBX5、および後に、Hand2（GMTとGHMT因子を用いて誘導心筋細胞様細胞（iCLMs）にマウス線維芽細胞の直接変換を表示することにより、この戦略の実現可能性を示していますそれぞれ）9,10。 Furthermore、同じ戦略は、 インビボおよびヒト由来の組織9、11、12で行うことができます。最近の研究では、追加の因子またはさらなる心臓再プログラミング効率13、14、15を改善するために調節することができるシグナル伝達経路を強調しています。まとめると、これらの研究は、再生治療のために向かう分化転換の可能性を示します。しかし、方法論的差異16にCMの再プログラミング、未知の分子機構、一貫性のない再現性の低効率、およびiCLMsの不均一な性質は、アドレス指定されないままです。 直接iCLM不均一性を評価するために、我々は、サルコメアの開発および心臓系統specificatioの同定のために離散的で堅牢な単一細胞アッセイを設計しましたn-2の機能的心筋細胞の必要な特性。それらの場所でユニークな電気的特性によって定義されたCMの少なくとも三つの主要なタイプは、心臓である：心房（AM）は、心室（VM）及びペースメーカー（PM）17、18、19、20。オーケストレーションの組み合わせで、それらは、血液の適切なポンピングを可能にします。心臓損傷の間、一つまたはすべてのサブタイプが影響を受ける可能性があり、および細胞治療のタイプは、ケースバイケースで対処する必要があります。少し作業は、サブタイプの仕様を調節する分子メカニズムを研究するために行われている間、現在、ほとんどの戦略は、心筋細胞の全体的な世代に焦点を当てています。 以下の研究は、適切によく組織サルコメアを定量化し、心筋細胞のサブタイプの多様なセットを識別する方法について詳しく説明します。ペースメーカー（PM）特異的レポーターマウスを用いて、Iを適用することができます誘導された心房のような筋細胞（IAM）、誘導された心室のような筋細胞（IVM）、および誘導されたPM-様筋細胞（IPMS）21を区別するためにmmunocytochemicalアプローチ。我々の観察に基づいて、サルコメア組織を示す細胞のみが自発的な拍動することが可能です。このユニークな再プログラミングプラットフォームは役割を評価することができます サルコメア組織、サブタイプの仕様、および単一セルの解像度でCMの再プログラミングの効率の特定のパラメータの。

<table><tbody><tr><td>DMEM</td><td>Sigma</td><td>D5796</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Medium 199</td><td>Thermo Fisher Scientific</td><td>11150059</td><td>Component of iCLM media</td></tr><tr><td>Fetal bovine serrum (FBS)</td><td>Sigma</td><td>F2442</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Insulin-Transferrin-Selenium G</td><td>Thermo Fisher Scientific</td><td>41400-045</td><td>Component of iCLM media</td></tr><tr><td>MEM vitamin solution</td><td>Thermo Fisher Scientific</td><td>11120-052</td><td>Component of iCLM media</td></tr><tr><td>MEM amino acids</td><td>Thermo Fisher Scientific</td><td>1601149</td><td>Component of iCLM media</td></tr><tr><td>Non-Essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td>Component of iCLM media</td></tr><tr><td>Antibiotic-Antimycotics</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Component of iCLM media</td></tr><tr><td>B-27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>Component of iCLM media</td></tr><tr><td>Heat-Inactivated Horse Serum</td><td>Thermo Fisher Scientific</td><td>26050-088</td><td>Component of iCLM media</td></tr><tr><td>NaPyruvate</td><td>Thermo Fisher Scientific</td><td>11360-70</td><td>Component of iCLM media</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>1514022</td><td>Component of Plat-E media and fibroblast media</td></tr><tr><td>Puromycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11139-03</td><td>Component of Plat-E media</td></tr><tr><td>Blasticidin&amp;nbsp;&amp;nbsp;</td><td>Gemini Bio-Products</td><td>400-128P</td><td>Component of Plat-E media</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>Component of Fibroblast media</td></tr><tr><td>Confocal laser scanning LSM700</td><td>Zeiss</td><td></td><td>For confocal analysis</td></tr><tr><td>FuGENE 6 transfection Reagent</td><td>Promega</td><td>E2692</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985-070</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td>Induction reagent. Use at a final concentration of 8 &amp;mu;M/mL</td></tr><tr><td>Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td><td>CellBiolabs</td><td>RV-101</td><td>Retroviral pacaking cell line</td></tr><tr><td>Trypsin 0.25% EDTA</td><td>Thermo Fisher Scientific</td><td></td><td>For MEFs and Plat-E dissociation</td></tr><tr><td>Mouse anti &amp;alpha;-Actinin (Clone EA-53)</td><td>Sigma</td><td>A7811</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Chicken anti-GFP IgY</td><td>Thermo Fisher Scientific</td><td>A10262</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Rabbit Pab anti-NPPA&amp;nbsp;</td><td>Abgent</td><td>AP8534A</td><td>Antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Rabbit Pab anti Myl2 IgG&amp;nbsp;</td><td>ProteinTech</td><td>10906-1-AP</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Vectashield solution with DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Vector Labs</td><td>H-1500</td><td>Dye for confocal analysis</td></tr><tr><td>Superfrost Plus Microscope slides</td><td>Thermo Fisher Scientific</td><td>12-550-15</td><td>25 x 75 x 1.0 mm</td></tr><tr><td>BioCoat Fibronectin 12 mm coverslips</td><td>NeuVitro Corp</td><td>GG-12-1.5</td><td>Coverslips for confocal analysis</td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Thermo Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>0.45 &amp;mu;m Syringes filters SFCA 25MM</td><td>Thermo Fisher Scientific</td><td>09-740-106</td><td>For virus filtration</td></tr><tr><td>6 mL Syringes</td><td>Covidien</td><td>8881516937</td><td>For virus filtration</td></tr><tr><td>Goat anti-Chicken IgY (H&amp;amp;L) A488</td><td>Abcam</td><td>AB150169</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Donkey anti-rabbit A647 IgG(H+L)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A31573</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Goat anti-mouse IgG(H+L) A555</td><td>Thermo Fisher Scientific</td><td>A21422</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443-100ml</td><td>For cell permeabilization&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s PBS without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt; (D-PBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Power Block 10x Universal Blocking reagent</td><td>Thermo Fisher Scientific</td><td>NC9495720</td><td>Dilute to 1x in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>16% Paraformaldehyde aqueous solution (PFA)</td><td>Electro Microscopy Sciences&amp;nbsp;</td><td>15710</td><td>Use at 4% diluted in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>6 cm&amp;nbsp; plates</td><td>Olympus</td><td>25-260</td><td></td></tr><tr><td>6-well plates</td><td>Genesee Scientific</td><td>25-105</td><td></td></tr><tr><td>24-well plates</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>10 cm Tissue culture dishes</td><td>Corning</td><td>4239</td><td></td></tr><tr><td>15 cm&amp;nbsp; Tissue culture dishes</td><td>Thermo Fisher Scientific</td><td>5442</td><td></td></tr><tr><td>15 mL Conical tubes</td><td>Corning</td><td>4308</td><td></td></tr><tr><td>50 mL Conical tubes</td><td>Corning</td><td>4249</td><td></td></tr><tr><td>0.4% Trypan blue solution</td><td>Sigma</td><td>T8154</td><td>For viability</td></tr><tr><td>Ethyl Alcohol 200 proof</td><td>Thermo Fisher Scientific</td><td>7005</td><td></td></tr><tr><td>Bleach</td><td>Thermo Fisher Scientific</td><td>6009</td><td></td></tr></tbody></table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

PM固有のレポーターマウスを利用して、我々は、 図1に示されるように、多様な内因性の筋細胞を同定するための多重免疫戦略を開発しました。 図2に示す書き換え手順に続いて、サブタイプ特異的のCMの誘導は、低レートではあるが、早ければ4日目21として検出することができます。 14日目では、実験は、サルコメア組織（...

Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

assessing-cardiomyocyte-subtypes-following-transcription-factor

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.4K ビュー.  UT Southwestern Medical Center. This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5. 

ビデオ: Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

MYH6プロモーター - 選択と組み合わせたES細胞のプログラミングに基づいて、マウス心臓ペースメーカー細胞凝集体の生成

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

発生生物学

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

GATA4、MEF2C及びTBX5の最適な比率を表現ポリシストロン構築物を用いて誘導心筋細胞の改善された世代

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

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 ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

体外ヒト間葉系幹細胞の心筋細胞のような細胞への分化

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

プロ線維性シグナル伝達の抑制を促進する誘導心筋細胞マウス胚性線維芽細胞のリプログラミング因子を介した

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

GCaMP3レポーターによる心臓特異的カルシウムフラックス測定による心臓リプログラミングのインビトロ評価

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...