Name: reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program
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Description: Watch this Scientific Journal Video about Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program at JoVE.com

This work was supported by an NHLBI grant to K.M. and I.R.L. (1RO1HL119404). Carlos-Filipe Pereira was the recipient of a Revson Senior Biomedical Fellowship. We gratefully acknowledge the Mount Sinai hESC/iPSC Shared Resource Facility and S. D'Souza for assistance with materials and protocols. We also thank the Mount Sinai Flow Cytometry, Genomics, and Mouse facilities.

伦理学声明：小鼠细胞系衍生下列医学学院伊坎在西奈山的动物护理指导原则，并应遵守任何主办机构来完成。 C57BL / 6小鼠1.鼠标胚胎成纤维细胞（MEF）隔离<ol><li>设置定时交配13。一旦阴道塞的可视化，可以考虑0.5本节。 </li><li>分离插头上的日期插入女性和检查他们在10-11天，以确认妊娠。 </li><li>在13.5-14.5日通过CO安乐死怀孕的雌性小鼠吸入2颈椎脱位。 </li><li>浸泡怀孕?...

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从容易实现的体细胞产生骨髓干细胞从头提供了一个独特的方法来研究体外造血，并有机会可能这个技术应用到人的制度。这个翻译将产生一个新的工具来研究人类血液病在培养皿中，以及提供药物测试平台和基因定位机会以治疗与新疗法或HSC移植众多障碍。在该领域，最近的研究上的能力扩展到产生骨髓干细胞从头 ，展示的重编程过程的几个特征的重要性。这些措施包括起...

造血是一个复杂的发育过程，其中的造血干细胞（HSCs）出芽存在于各种胚胎造血网站，如主动脉-性腺-中肾和胎盘1,2 hemogenic内皮。无法培养造血干细胞在体外阻止此过程的深入分析，以及这些研究的临床应用。规避此限制，先前的研究试图获得造血干细胞从头或者通过使用重新编程媒体4,5-多能干细胞（的PSCs）3，或在体细胞诱导的可塑性和定向分化的分化。这些研究，但是，不产生临床安全engraftable细胞或允许最终造血发育研究“的菜。” 从体细胞的成纤维细胞p由山及其同事建立的新颖工作以产生诱导性多能干细胞（iPS细胞）rovides转录因子（TF）在重编程的细胞命运6,7-基于过表达策略的框架。这项工作已经促使调查人员在若干领域通过容易获得体细胞重编程TF产生细胞类型的选择。这里所描述的重编程策略的目标是诱导从使用具有转换这些发现对人体系统重新编程特定病人的成纤维细胞，以研究人类造血体外的目标基于TF重新编程的方法和小鼠体细胞hemogenic过程生成患者特异性血制品用于疾病建模，药物检验，和干细胞移植。 第一步，以确保在该小鼠系统适当重新编程是开发担任的读出为CD34的表达，在内皮祖细胞和造血干细胞的已知标记的报道一致。要做到这一点，huCD34-和tTA的TETO-H2BGFP转基因小鼠系被用于与Generate双转基因小鼠胚胎成纤维细胞（MEF），现在表示34 / H2BGFP，发荧光绿于CD34启动子8的激活。这允许各种已知被要求在不同的点造血说明书和发育过程中的TF的筛选。在PMX retrovial载体18开始的TF（通过文献挖掘和GFP标签保留从之前造血干细胞的分析确定描述34 / H2BGFP只），34 / H2BGFP MEF中与AFT024 HSC-支持基质细胞的所有因素和培养转。检测34 / H2BGFP激活后，转录因子随后从重新编程的鸡尾酒删除，直到转录因子的活化记者一组最佳被确定。此初始屏幕之后，将因素转移到DOX诱导pFUW载体系统，以允许转录因子的控制的表达。由于这两个DOX可控系统是不兼容的（34 / H2BGFP细胞和pFUW诱导矢量），由MEF野生型C57BL / 6小鼠被要求。此外，还必须提供适当的微环境，使hemogenesis继续创造多向祖细胞克隆。 试图体细胞重编程到造血干细胞和祖细胞（骨髓干细胞）目前的研究已达到成功的9-11不同层次。迄今为止，小鼠和具有长期和自我更新的重新填充能力的人类移植骨髓干细胞的产生尚未使用同一组转录因子实现。在这个协议中，我们提供先前建立的策略的详细描述可重复地诱导hemogenesis在MEF中。我们证明，引进了全套转录因子（GATA2，Gfi1b，首席财务官和ETV6）的最小的能够煽动体外复杂的发展计划，规定了可发展的造血和造血重新编程的临床应用，可进一步研究12的平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45uM filters</td>
			<td>Corning</td>
			<td>430625</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini&#39;s Benchmark</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needles</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>20G needles</td>
			<td>BD</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>25G needles</td>
			<td>BD</td>
			<td>305125</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Sigma</td>
			<td>C0130-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Myelocult media</td>
			<td>Stem Cell Technologies</td>
			<td>M5300</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>R &#38; D Systems</td>
			<td>455-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Flt3L</td>
			<td>R &#38; D Systems</td>
			<td>427-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>R &#38; D Systems</td>
			<td>403-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>R &#38; D Systems</td>
			<td>488-TO</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>AL-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore 0.65uM membrane filters</td>
			<td>Millipore</td>
			<td>DVPP14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose media</td>
			<td>Stem Cell Technologies</td>
			<td>Methocult M3434</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Stem Cell Technologies</td>
			<td>07904</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G-1890 100g</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUW-tetO</td>
			<td>Addgene</td>
			<td>Plasmid #20321</td>
			<td></td>
		</tr>
		<tr>
			<td>Gata2</td>
			<td>Origene</td>
			<td>MR226728</td>
			<td></td>
		</tr>
		<tr>
			<td>Gfi1b</td>
			<td>Origene</td>
			<td>MR204861</td>
			<td></td>
		</tr>
		<tr>
			<td>cFos&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #19259</td>
			<td></td>
		</tr>
		<tr>
			<td>Etv6</td>
			<td>Origene</td>
			<td>MR207053</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>Plasmid #12260</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>Plasmid #12259</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>FUW-M2rtTA</td>
			<td>Addgene</td>
			<td>Plasmid #20342</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm culture dishes</td>
			<td>Thermo Scientific</td>
			<td>171099</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

造血是一个复杂的发展过程，在不同的胚胎网站开始。 Hemogenic内皮细胞存在于这些网站，并给通过手机初露头角的23引起造血干细胞。这个过程目前还无法通过将造血干细胞或造血前体文化，因此需要一种方法在体外以某种方式获得这些细胞进行复制，或者通过HSPC 体外扩增或产生从头 。该协议表明了我们试图通过获得TF表达，这些细胞在MEF中...

Watch this Scientific Journal Video about Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program at JoVE.com

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

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.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

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

The overall goal of this hemogenic induction protocol is to instigate a developmental process in mouse fibroblasts through transcription factor overexpression to generate hematopoietic precursor cells in vitro that product hematopoietic cells upon prolonged culture. This method can help answer key questions in the hematopoietic programming field such as whether or not a protocol can be developed to obtain bonafide HSCs de novo that are capable of multi-lineage and graftment. The main advantage of this technique is that through overexpression of a minimal set of transcription factors, the developmental process can be initiated in reprogrammed cells that does not require travel through a pluripotent intermediate.After culturing mouse embryonic fibroblasts or MEFS, and producing virus according to the text protocol, use 0.1 percent gelatin in PBS to pre-coat 6-well plates. Ensuring that the gelatin covers the entire area of the well. Place the plates in the 37 degrees celsius incubator for at least 20 minutes.Then, remove the plates and aspirate the gelatin. Plate MEFs with standard DMEM at a density of 25, 000 cells per well and allow the cells to settle at 37 degrees celsius overnight. Prepare a 100 microliter virus cocktail by mixing 50 microliters of M2RTTA and 12.5 microliters each of GATA2, GFI1B, CFOS and ETV6.Aspirate the medium from the MEFS and add two milliliters of standard DMEM with 8 micrograms per milliliters of hexadimethrine bromide. Then, transduce each well of MEFs with 15 microliters of the virus cocktail and incubate at 37 degrees celsius overnight. Following 16 to 20 hours of incubation, replace the medium in each well with two milliliters of fresh standard DMEM, supplemented with one microgram per milliliter of DOX.Incubate at 37 degrees Celsius for three days. On day four, prepare hematopoietic culture medium supplemented with hydrocortisone, stem cell factor, MFS-related tyrosine kinase three ligand, interleukin three, interleukin six and DOX. Next, aspirate the medium from the MEFs and use one milliliter of PBS to wash the cells.Then, after aspirating the PBS, add one milliliter of 0.05 percent trypsin per well to dissociate and collect the cells. Using the hemocytometer, count the cells, then spin at 300 times g for five minutes and use 12 milliliters of the hematopoietic medium just prepared to resuspend the cells. Then, plate 10, 000 cells per well on gelatin-coated six-well plates.Every six days, replace the medium with fresh supplemented hematopoietic culture medium for the duration of the culture. Analyze the cells according to the text protocol. After dissecting placentas from pregnant mice according to the text protocol, use two to five milliliters of 0.2 percent collagenase type one in PBS with 20 percent FBS to wash the tissue and using an 18 gauge needle fitted on a five milliliter syringe, mechanically dissociate the tissue in the PBS solution.Incubate the placental cells in two to five milliliters of collagenase solution at 37 degrees Celsius for 1.5 hours. Then, passage the cells several times through 20 to 25 gauge needles fitted on 5 milliliter syringes to further mechanically dissociate them. Next, filter single cell suspensions through 70 micron cell strainers.Then, using a hemocytometer, count the cells and irradiate at 2, 000 centigray volts for mitotic inactivation. Add ten milliliters of hematopoietic culture medium supplemented with the following to a ten centimeter dish. Place the 0.65 micron filter directly on the culture medium in the dish, allowing it to float to create a liquid-gas interface and set the dish aside.Dissociate day 25 transduced MEFs as demonstrated earlier in this video and mix 33, 000 of the transduced MEFs with 167, 000 cells of the mitotically inactivated placental aggregates. To generate an aggregate, spin down the MEF and placental cell mix at 300 times g for five minutes. Aspirate the medium and use 30 to 50 microliters of standard DMEM to resuspend the pelleted cells, drawing the single cell suspension into a 200 microliter non-beveled pipette tip.Next, with paraffin film, occlude the pipette tip. Then, place the blocked tip into a 15 milliliter centrifuge tube and spin it down at 300 times g for five minutes so that a pellet forms at the covered end of the tip. Carefully remove the tip from the 15 milliliter tube and place it onto the end of a P200 pipette.Discard the paraffin film and extrude the cell pellet onto the filter floating in the dish of culture medium. Culture the aggregates on the floating filters at 37 degrees Celsius and five percent carbon dioxide for four to five days. Using a cell scraper, collect the aggregates then combine them and use 0.5 percent collagenase to digest them.After dissociation, use two to five milliliters of PBS with five percent FBS to wash the aggregates and spin down at 300 times g for five minutes. Resuspend the aggregates in 500 microliters of DMEM without FBS, Pen-Strep or L-glutamine. Add the entire suspension to three milliliters of one percent of methylcellulose medium supplemented with ten nanograms per milliliter of Tepo, carefully avoiding the formation of bubbles.Plate equal volumes of this mixture in three, 35 by ten millimeter non-treated culture dishes for CFU assays. As a control, culture irradiated placental aggregates. Finally, count hematopoietic colonies manually under a microscope after ten to 14 days of culture at 37 degrees Celsius with 5 percent carbon dioxide.In the process of reprogramming MEFs to HSCs, cells are transduced with transcription factors as shown here. After three days of expansion and exposure to DOX to begin transgene activation, cells are dissociated, split and grown on gelatin-coated plates and supplemented hematopoietic medium. At day 20 in culture, post-transduction with reprogramming medium, 34/H2BGFP MEFs adopt clear morphological changes including endothelial morphology distinct from MEFs.Further culture to day 35 results in several round hematopoietic-like cells emerging from the endothelial-like intermediates that are GFP positive and stain positive for the hematopoietic markers Sca1 and CD45. Further culture results in cells expressing CD45 while maintaining expression of the human CD34 reporter. Upon placental aggregation, culture cells adopt clonogenic potential and generate colonies in methylcellulose containing various hematopoietic morphologies and blast-like cells.While attempting this procedure, it's important to remember to perform each section slowly so as to not get mixed up. Any mistakes will be hard to notice until weeks after you start the reprogramming. Following this procedure, other methods such as cobblestone-forming cell assays and long-term culture initiating cell assays can be performed in order to answer additional questions such as if the derived cells truly possess hematopoietic function as demonstrated in the functional assays.After its development, this technique paved the way for researchers in the field of hematopoietic stem cell programming to explore other reprogramming strategies and optimization protocols in blood, endothelial and other cell types. After watching this video, you should have a good understanding of how to induce a hemogenic program in mouse fibroblasts, the overexpression of transcription factors and how to functionally assay the derived cell. Thank you for watching and good luck with your experiments.

Research

JoVE Journal

Developmental Biology

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

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

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

科研

发育生物学

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

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

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

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

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

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

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

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

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