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.

Etikk uttalelse: Musecellelinjer er avledet følge retningslinjene fra Icahn School of Medicine ved Mount Sinai dyr omsorg, og bør gjøres i samsvar med enhver vertsinstitusjonen. 1. Mouse Embryonic fibroblast (MEF) Isolering av C57BL / 6 Mus <ol><li> Sett opp tidsbestemt parring 13. Når en vaginal plugg er visualisert, tenk på dette Dag 0,5. </li><li> Skill plugget kvinner på pluggen dato og se på dem på dag 10-11 for å bekrefte graviditeten. </li><li> På dag 13,5 til 14,5 avlive gravide hu...

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Generering HSPCs de novo fra lett oppnåelige somatiske celler tilbyr en unik metode for å studere hematopoiesis in vitro, og muligheten til å potensielt bruke denne teknologien til det menneskelige systemet. Denne oversettelsen vil generere et nytt verktøy for å studere menneskelig hematologisk sykdom i en skål, samt gi narkotika testing plattformer og genet målretting muligheter til å behandle en rekke lidelser med nye terapeutiske eller HSC transplantasjoner. I feltet, har nyere studier utvid...

Hematopoiesis er en kompleks utviklingsprosess der blodkreft stamceller (HSCs) knopp av hemogenic endotelet til stede i en rekke embryonale blodkreft steder som Aorta-Gonade-Mesonephros og morkaken 1,2. Den manglende evne til kulturen HSCs in vitro hindrer grundig analyse av denne fremgangsmåten, så vel som den kliniske anvendelsen av disse studiene. For å omgå denne begrensningen, har tidligere studier forsøkt å utlede HSCs de novo enten via differensiering av pluripotente stamceller (PSC avtaler) 3, eller indusert plastisitet i somatiske celler og rettet differensiering ved hjelp omprogrammering medier 4,5. Disse studiene har imidlertid ikke generere klinisk trygge engraftable celler eller tillate studie av definitive utviklings hematopoiesis &quot;i en skål.&quot; Romanen arbeid etablert av Yamanaka og kolleger til å generere induserte pluripotente stamceller (iPSCs) fra somatisk fibroblaster provides et rammeverk for transkripsjon faktor (TF) basert overekspresjon strategier i omprogrammering celle skjebne 6,7. Dette arbeidet har bedt etterforskerne på flere områder for å generere celletyper av valg via TF omprogrammering av lett oppnåelig somatiske celler. Målet med reprogrammerings strategien som er beskrevet her er for å indusere en hemogenic prosess fra musesomatiske celler ved hjelp av et TF basert reprogrammerings tilnærming med det mål å oversette disse funnene til det menneskelige system for å omprogrammere pasientspesifikke fibroblaster for å undersøke human hematopoese in vitro og generere pasientspesifikke blodprodukter til sykdom modellering, narkotika testing, og stamcelletransplantasjon. Det første skrittet for å sikre riktig omprogrammering i denne musen systemet var å utvikle en reporter linje som fungerte som en lese-out for CD34 uttrykk, et kjent merke i endoteliale progenitorceller og HSCs. For å gjøre dette, ble de huCD34-TTA og Teto-H2BGFP transgene mus linjer som brukes til å generate doble transgene mus embryonale fibroblaster (MEFs), nå betegnet 34 / H2BGFP, som fluoresce grønt ved aktivering av CD34 arrangøren 8. Dette tillot screening av et utvalg av TFS som er kjent for å være nødvendig ved forskjellige punkter i løpet av hematopoetiske spesifikasjon og utvikling. Fra og med 18 TFS i PMX retrovial vektorer (bestemt gjennom litteratur gruvedrift og profilering av GFP etikett beholde HSCs fra de tidligere omtalte 34 / H2BGFP mus), 34 / H2BGFP MEFs ble omformet med alle faktorer og kultivert på AFT024 HSC-støtte stromale celler. Etter påvisning av 34 / H2BGFP aktivering, ble TF&#39;er deretter fjernet fra reprogrammerings-cocktail inntil den optimale settet med TFS for reporter-aktivering ble identifisert. Etter denne startbildet, ble faktorer overført til en DOX induserbar pFUW vektorsystem for å tillate kontrollerbar ekspresjon av TFS. Siden disse to DOX kontrollerbare systemer er uforenlig (de 34 / H2BGFP celler og pFUW induserbare vektorer), MEFs fravilltype C57BL / 6 mus ble nødvendig. Det var også nødvendig å gi en passende mikromiljøet for å tillate hemogenesis å fortsette og lage multilineage klonogene stamfedre. Aktuelle studier som forsøker å omprogrammere somatiske celler i hematopoietiske stamceller og stamceller (HSPCs) har møtt ulike nivåer av suksess 9-11. Til dags dato har generering av både mus og mennesketrans HSPCs med langsiktig og selvfornyende repopulating evne ikke er oppnådd ved hjelp av det samme settet av TFS. I denne protokollen, gir vi en detaljert beskrivelse av tidligere etablert strategi for å reproduserbart indusere hemogenesis i MEFs. Vi viser at innføring av et minimalt sett med TFS (Gata2, Gfi1b, cFos, og Etv6) er i stand til å sette i gang en kompleks utviklings program in vitro som gir en plattform der utviklings hematopoiesis og klinisk anvendelse av blodkreft omprogrammering kan bli ytterligere undersøkt 12.

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			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
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			<td>0.45uM filters</td>
			<td>Corning</td>
			<td>430625</td>
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			<td>Amicon Ultra centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC900324</td>
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			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
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			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td></td>
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		<tr>
			<td>FBS</td>
			<td>Gemini&#39;s Benchmark</td>
			<td>100-106</td>
			<td></td>
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			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
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			<td>18G needles</td>
			<td>BD</td>
			<td>305195</td>
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			<td>20G needles</td>
			<td>BD</td>
			<td>305175</td>
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			<td>25G needles</td>
			<td>BD</td>
			<td>305125</td>
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			<td>Collagenase Type I</td>
			<td>Sigma</td>
			<td>C0130-100MG</td>
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			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12605-010</td>
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			<td>Myelocult media</td>
			<td>Stem Cell Technologies</td>
			<td>M5300</td>
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			<td>SCF</td>
			<td>R &#38; D Systems</td>
			<td>455-MC</td>
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			<td>Flt3L</td>
			<td>R &#38; D Systems</td>
			<td>427-FL</td>
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			<td>IL-3</td>
			<td>R &#38; D Systems</td>
			<td>403-ML</td>
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			<td>IL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML</td>
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			<td>TPO</td>
			<td>R &#38; D Systems</td>
			<td>488-TO</td>
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			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D9891-1G</td>
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			<td>Polybrene (hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>AL-118</td>
			<td></td>
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			<td>Durapore 0.65uM membrane filters</td>
			<td>Millipore</td>
			<td>DVPP14250</td>
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			<td>Methylcellulose media</td>
			<td>Stem Cell Technologies</td>
			<td>Methocult M3434</td>
			<td></td>
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			<td>Hydrocortisone</td>
			<td>Stem Cell Technologies</td>
			<td>07904</td>
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			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
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		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G-1890 100g</td>
			<td></td>
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		<tr>
			<td>pFUW-tetO</td>
			<td>Addgene</td>
			<td>Plasmid #20321</td>
			<td></td>
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		<tr>
			<td>Gata2</td>
			<td>Origene</td>
			<td>MR226728</td>
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			<td>Gfi1b</td>
			<td>Origene</td>
			<td>MR204861</td>
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			<td>cFos&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #19259</td>
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		<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.

Hematopoiesis er en kompleks utviklingsprosess som begynner i ulike embryonale nettsteder. Hemogenic endotelceller bor i disse områdene og gi opphav til HSCs via celle spirende 23. Denne prosessen foreløpig ikke kan reproduseres ved å plassere HSCs eller blodkreft forløpere i kultur, nødvendiggjør en metode for å liksom få disse cellene in vitro, enten ved HSPC utvidelse ex vivo eller generasjon de novo. Denne protokollen viser vår nye tekno...

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.

Omprogrammering mus embryonale Fibroblaster med transkripsjonsfaktorer for å indusere en Hemogenic Program

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

Direkte Induksjon av Hemogenic endotelet og Blood av Overekspresjon av transkripsjonsfaktorer i Human Pluripotent stamceller

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

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

Generering CRISPR / Cas9 mediert Monoallelic Slett å studere Enhancer funksjon i mus embryonale stamceller

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

En novelle<em&gt; Ex Ovo</em&gt; Banding Technique Alter Intrakardial hemodynamikken i en Embryonic Chicken System

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

Isolering av Murine Embryonic Hemogenic endotelceller

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

Protokoll for direkte konvertering av Murine embryonale Fibroblaster i trophoblast stamceller

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

Innsamling av serum-og mater-fri Mouse Embryonic Stem Cell kondisjonerte Medium for en Cell-fri tilnærming

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

Vurdere kardiomyocytt Subtyper Etter transkripsjonsfaktor-mediert omprogrammering av mus embryonale Fibroblaster

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

Undertrykkelse av Pro-fibrotiske signalering Potentiates faktor-mediert omprogrammering av mus embryonale fibroblaster til indusert Cardiomyocytes

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

Direkte avstamning omprogrammering av voksen mus Fibroblast å Erythroid Progenitors

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

RNA-baserte omprogrammering av menneskelig primære fibroblaster til indusert Pluripotent stamceller

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