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.

declaração de ética: linhas de células do rato são derivados seguindo as orientações de cuidados de animais da Icahn Escola de Medicina Monte Sinai, e deve ser feito em conformidade com qualquer instituição de acolhimento. 1. rato embrionário de fibroblastos (MEF) Isolamento de ratinhos C57BL / 6 <ol><li> Configurar o acasalamento cronometrado 13. Uma vez que um plug vaginal é visualizado, considerar este dia 0,5. </li><li> Separar as fêmeas ligados na data plugue e consultá-los no dia ...

<ol>
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Gerando hspCs de novo a partir de células somáticas facilmente atingíveis oferece um método único para estudar a hematopoiese in vitro, e a oportunidade para potencialmente aplicar esta tecnologia para o sistema humano. Esta tradução iria gerar uma nova ferramenta para o estudo da doença hematológica humano em um prato, bem como fornecer plataformas de teste de drogas e direccionamento de genes oportunidades para o tratamento de numerosas desordens com novos agentes terapêuticos ou d...

A hematopoiese é um processo complexo de desenvolvimento em que as células estaminais hematopoiéticas (HSCs) brotam endotélio hegemônicos presente numa variedade de sítios hematopoiéticas embrionárias, tais como a aorta-gônadas-Mesonefros e da placenta 1,2. A incapacidade de cultura hsc in vitro evita a análise em profundidade deste processo, bem como a aplicação clínica destes estudos. Para ultrapassar esta limitação, estudos anteriores têm tentado derivar HSCs de novo, quer por meio de diferenciação das células estaminais pluripotentes (PSCs) 3, ou plasticidade induzida em células somáticas e diferenciação dirigida utilizando meios de reprogramação 4,5. Estes estudos, no entanto, não geram células engraftable clinicamente seguras ou permitir o estudo da hematopoiese desenvolvimento definitivo &quot;em um prato.&quot; O romance de trabalho estabelecido por Yamanaka e seus colegas para gerar células-tronco pluripotentes induzidas (iPSCs) a partir de fibroblastos somática pferece um quadro de fator de transcrição (TF) estratégias superexpressão baseados em célula destino reprogramação 6,7. Este trabalho levou os pesquisadores em vários campos para gerar tipos de células de escolha através de TF reprogramação de células somáticas de fácil obtenção. O objetivo da estratégia de reprogramação descrito aqui é para induzir um processo hegemônicos a partir de células somáticas de rato usando uma abordagem de reprogramação TF base com o objetivo de traduzir esses achados para o sistema humano para reprogramar fibroblastos específicos do paciente, a fim de estudar hematopoiese humana in vitro e gerar produtos derivados de sangue específicos do paciente para a modelagem de doenças, testes de drogas e transplante de células estaminais. O primeiro passo para assegurar reprogramação adequada neste sistema de ratinho foi desenvolver uma linha repórter que serviu como uma leitura para a expressão de CD34, um marcador conhecido nas células progenitoras endoteliais e HSCs. Para fazer isso, as linhagens de camundongos transgênicos huCD34-tTA e TetO-H2BGFP foram usadas para generate rato transgénico fibroblastos embrionários duplos (MEFs), agora denotado 34 / H2BGFP, que apresentam fluorescência verde após a ativação do promotor CD34 8. Isto permitiu o rastreio de uma variedade de TFs conhecidos que sejam necessários em diferentes pontos durante a especificação e desenvolvimento hematopoiético. Começando com 18 TFs em PMX vetores retrovial (determinado através da mineração literatura e profiling de etiqueta GFP manter HSCs do descrito anteriormente 34 / H2BGFP ratos), 34 / H2BGFP MEFs foram transduzidas com todos os factores e cultivadas em AFT024 HSC-apoiando células estromais. Após a detecção de 34 / H2BGFP ativação, TFs foram posteriormente removido do cocktail reprogramação até o melhor conjunto de TFs para ativação repórter foi identificado. Após esta primeira tela, os factores foram transferidos para um sistema de vector indutível pFUW DOX para permitir a expressão controlável de TFS. Uma vez que estes dois sistemas controláveis ​​DOX são incompatíveis (34 células / H2BGFP e os vectores indutíveis pFUW), MEFs deDe tipo selvagem C57BL / 6 murganhos foram necessários. Foi também necessário para proporcionar um microambiente adequado permitir hemogenesis para prosseguir e criar progenitores clonogï¿½icas multilinhagem. Os estudos atuais que tentam reprogramar células somáticas em células-tronco e progenitoras hematopoiéticas (hspCs) reuniram-se níveis variados de sucesso 9-11. Até à data, a geração de ambos rato transplantáveis ​​e hspCs humanos com a longo prazo e capacidade de repovoamento auto-renovação não foi alcançado utilizando o mesmo conjunto de FT. Neste protocolo, nós fornecemos uma descrição detalhada da estratégia previamente estabelecido para induzir reproducibly hemogenesis em MEFs. Nós demonstramos que a introdução de um conjunto mínimo de TFs (GATA2, Gfi1b, CFOs e ETV6) é capaz de instigar um programa complexo de desenvolvimento in vitro que fornece uma plataforma através da qual hematopoiese desenvolvimento e aplicação clínica de reprogramação hematopoiéticas podem ser mais bem estudadas 12.

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			<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>
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		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12605-010</td>
			<td></td>
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		<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>
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		<tr>
			<td>Flt3L</td>
			<td>R &#38; D Systems</td>
			<td>427-FL</td>
			<td></td>
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		<tr>
			<td>IL-3</td>
			<td>R &#38; D Systems</td>
			<td>403-ML</td>
			<td></td>
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		<tr>
			<td>IL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML</td>
			<td></td>
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		<tr>
			<td>TPO</td>
			<td>R &#38; D Systems</td>
			<td>488-TO</td>
			<td></td>
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		<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>
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		<tr>
			<td>Methylcellulose media</td>
			<td>Stem Cell Technologies</td>
			<td>Methocult M3434</td>
			<td></td>
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		<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.

A hematopoiese é um processo de desenvolvimento complexo que começa em vários locais embrionárias. Células endoteliais hegemônicos residem nestes locais e dar origem a HSCs via celular brotamento 23. Este processo actualmente não pode ser reproduzido pela colocação de HSCs ou precursores hematopoiéticos em cultura, que necessitam de uma metodologia para a obtenção de alguma forma destas células in vitro, quer por HSPC expansão ex vivo ou geraç?...

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.

Reprogramação do rato embrionárias fibroblastos com Fatores de transcrição para induzir um Programa hegemônicos

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

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