Name: reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program
Uploaded: 2016-12-16T12:30:00
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.

윤리 문 : 마우스 세포주가 시내 산에서 의학의 아이칸 학교의 동물 관리 지침에 따라 유도하고, 호스트 기관 준수 수행해야합니다. C57BL / 6 마우스 1. 마우스 배아 섬유 아세포 (MEF) 분리 <ol><li> 시간 제한 짝짓기 (13)을 설정합니다. 질 플러그가 가시화되면,이 날 0.5을 고려하십시오. </li><li> 플러그 날짜에 연결 여성을 분리하고 임신을 확인하기 위해 하루 10 ~ 11에이를 확인. </li><li> ?...

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쉽게 달성 체세포에서 HSPCs 새로이 생성하는 것은 잠재적으로 시험관 내에서 조혈 및 기회를 연구 인간 시스템에이 기술을 적용하는 독특한 방법을 제공한다. 이 번역은 접시에 인간의 혈액 질환을 연구뿐만 아니라 새로운 치료제 또는 HSC 이식 수많은 장애를 치료 할 수있는 기회를 대상으로 약물 테스트 플랫폼과 유전자를 제공하기 위해 새로운 도구를 생성합니다. 필드에서, 최근?...

조혈는 조혈 줄기 세포 (조혈 모세포)가 같은 대동맥 - 생식소 - Mesonephros로 배아 조혈 사이트와 태반 1,2의 다양한 존재 hemogenic 내피 세포를 싹 복잡한 발달 과정이다. 체외에서 배양 조혈 모세포에 대한 무능력이 과정의 깊이 분석뿐만 아니라 이러한 연구의 임상 응용 프로그램을 방지 할 수 있습니다. 이 제한을 회피하기 위해, 기존의 연구는 유도하기 위해 시도 조혈 모세포 드 노보 중 재 프로그래밍 미디어 4,5를 사용하는 다 능성 줄기 세포 (PSC를) 3, 또는 체세포에서 유도 소성 감독 분화의 차별화를 통해. 이 연구는, 그러나, 임상 적으로 안전 engraftable 세포를 생성하거나 결정적인 발달 조혈의 연구를 허용하지 않습니다 &quot;접시를.&quot; 체세포 섬유 아세포의 페이지에서 야마나카 연구팀에 의해 설립 된 신규 작업을 유도 생성하는 다 능성 줄기 세포 (iPSCs)전사 인자 (TF) 프로그래밍 세포 운명 6,7에 기반 과발현 전략을위한 프레임 워크를 rovides. 이 작품은 쉽게 얻을 체세포의 TF의 재 프로그래밍을 통해 선택의 세포 유형을 생성하기 위해 여러 분야의 연구자를 묻는 메시지가있다. 여기에 설명 된 프로그래밍 전략 목표는 마우스 체세포 시험 관내에서 인간 조혈을 연구하기 위해 환자 별 섬유 아세포를 재 프로그램 할 수있는 인간의 시스템에 이들 결과를 변환하는 목표와 TF 기반 프로그래밍 방법을 사용하여 세포로부터 hemogenic 처리를 유도하는 것이다 질병 모델 약물 테스트에 대한 환자 - 특정 혈액 제제를 생성하고, 줄기 세포 이식. 이러한 마우스 시스템의 적절한 프로그래밍을 위해 첫 번째 단계는 CD34 발현 내피 전구 세포 및 조혈의 공지 된 마커에 대한 판독 역임 리포터 라인을 개발 하였다. 이렇게하려면 huCD34-TTA와 TETO-H2BGFP 유전자 변형 마우스 라인 g을 사용 하였다enerate 이중 형질 전환 마우스 배아 섬유 아 세포 (MEFs에)는, 이제 CD34 발기인 (8)의 활성화에 따라 녹색 형광 34 / H2BGFP를 나타낸다. 이는 조혈 명세서 및 개발하는 동안 다른 지점에서 요구되는 공지의 TF의 다양한 스크리닝시켰다. PMX retrovial 벡터 18과 TF를 시작으로 34 / H2BGFP MEFs에는 AFT024 기질 세포를 HSC는지지의 모든 요소 배양과 형질 도입 된, (34 / H2BGFP 마우스를 설명 문학 마이닝 및 이전에서 조혈 모세포를 유지 GFP 라벨의 프로파일 링을 통해 결정). 기자의 활성화를위한 TF가 최적의 설정이 확인 될 때까지 34 / H2BGFP 활성화의 검출 후, TF가이 연속적으로 재 프로그래밍 칵테일에서 제거되었습니다. 이 초기 화면 후에 인자하여 TFS의 발현을 제어 할 수 있도록하는 유도 DOX pFUW 벡터 시스템으로 옮겼다. 이 두 DOX 제어 시스템 (34 / H2BGFP 세포와 pFUW 유도 벡터)에서 MEFs에 호환되지 않는 때문에야생형 C57BL / 6 마우스가 필요했다. hemogenesis 진행하고 multilineage 클론 원성 전구 세포를 만들 수 있도록 적절한 미세 환경을 제공하는 것도 필요했다. 조혈 줄기 및 전구 세포로 (HSPCs)에 체세포를 재 프로그래밍을 시도하는 현재의 연구는 성공 9-11의 다양한 수준을 만났다. 현재까지, 마우스 및 장기 자기 갱신 다시 채우기 능력이 인간의 이식 가능한 HSPCs 모두의 발생과 TF들의 동일한 세트를 사용하여 달성되어 있지 않다. 이 프로토콜에서는 재현 된 MEFs에서 hemogenesis을 유도하는 미리 설정된 전략에 대한 상세한 설명을 제공한다. 우리는 TF가 (Gata2, Gfi1b, cFos를, 그리고 Etv6)의 최소한의 도입을 보여 발달 조혈 및 조혈 프로그래밍의 임상 응용 프로그램이 더 (12)를 연구 할 수있는 플랫폼을 제공 체외에서 복잡한 개발 프로그램을 선동 할 수있다.

<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.

조혈 다양한 배아 사이트에서 시작 복잡한 발달 과정이다. Hemogenic 내피 세포는이 사이트에있는 세포는 23 신진를 통해 조혈 모세포에 상승을 제공합니다. 이 과정은 현재 HSPC 확장 생체 또는 새로이 생성하여 역시 든 시험관 내에서 이들 세포를 수득하는 방법을 필요로 배양에서 조혈 모세포 또는 조혈 전구체를 배치하여 재생할 수 없다. 이 프...

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.

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

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

인간 만능 줄기 세포에서 전사 인자의 과발현에 의해 직접 Hemogenic 내피 세포의 유도 및 혈액

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

연구

발생학

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

생성 CRISPR은 / Cas9 중재 Monoallelic 삭제할 경우는 마우스 배아 줄기 세포의 증강 기능을 연구하는

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

소설<em&gt; 예 비켜</em&gt; 밴딩 기법 닭 배아 시스템에서 심장 내 혈류를 변경할

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

쥐 배아 Hemogenic 내피 세포의 분리

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

영양막 줄기 세포로 쥐 배아 섬유 아세포의 직접적인 변환을위한 프로토콜

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

무 혈청의 수집 및 공급 장치없이 마우스 배아는 세포 자유로운 접근을위한 세포 조절 중간 줄기

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

마우스 배아 섬유 아 세포의 전사 인자 매개 재 프로그래밍에 따라 심근 서브 타입을 평가

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

프로 거리 신호 억제 유도 Cardiomyocytes에 마우스 미 발달 섬유 아 세포의 프로그래밍 요소 중재 Potentiates

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

리니지 Reprogramming 성인 마우스 구와 Erythroid 창시자에의 직접

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

인간의 기본 섬유 아 세포의 유도 만능 줄기 세포로 재프로그래밍 RNA 기반

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