The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Sk&#229;ne are acknowledged for generous financial support. This work was supported by a grant from Olle Engkvists Stiftelse (194-0694 to Filipe Pereira) and PhD scholarships from Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (PTDC/BIM-MED/0075/2014 to Filipe Pereira, and SFRH/BD/135725/2018 and SFRH/BD/51968/2012 to Rita Alves and Andreia Gomes).&#160;This study was also supported by funds from NIH and NYSTEM (1R01HL119404 and C32597GG to Kateri A. Moore).

This protocol was performed according to Lund University&#39;s human research ethics committee guidelines and should be done in accordance with individual institutional guidelines.
1. Reagent Preparation
<ol>
	<li>For Dulbecco&#39;s modified Eagle's medium (DMEM)/20% fetal bovine serum (FBS), mix high glucose DMEM containing sodium pyruvate with 20% FBS, 1% penicillin-streptomycin (pen/strep), 1% L-glutamine, 1% non-essential amino acids and 10-4-M 2-mercaptoethanol.</li>
	<li>For complete DMEM, mix high glucose DMEM containing sodium pyruvate with 10% FBS, 1% Pen/strep and 1% L-glutamine.</li>
	<li>For hematopoietic medium, mix hematopoietic medium (Table of Materials) with 10-6 M hydrocortisone and 1% pen/strep.</li>
	<li>Use phosphate-buffered saline (PBS) without calcium or magnesium.</li>
</ol>
2. Human Dermal Fibroblast Isolation
NOTE: HDFs can be purchased from certified suppliers (Table of Materials). In that case, expand fibroblasts and use them directly in reprogramming experiments (section 4). Alternatively, HDFs can be isolated from donors. If fibroblasts are isolated from different donors, keep the samples separated from each other at all steps of the protocol. Label plates/wells and collection tubes with the identification number of each donor.
<ol>
	<li>Obtain HDFs from 3 mm round skin punch biopsies performed by qualified physicians.</li>
	<li>Coat three wells of a tissue culture-treated 6-well plate with 500 &#956;L of 0.1% gelatin and incubate for 20 min at 37 &#176;C.</li>
	<li>Aspirate the remaining gelatin solution and add 750 &#956;L of DMEM/20% FBS to each well. The entire surface of the well should be covered with medium.</li>
	<li>Add 1.5 mL of DMEM/20% FBS to the inside surface of a sterile 100 mm petri dish lid and spread the drop with the aid of a 5 mL serological pipette.</li>
	<li>Place the skin biopsy in the medium on the lid with sterilized forceps.</li>
	<li>Dissect the skin biopsy into nine identical sections, using one sterilized scalpel to hold the biopsy in place and a second scalpel to cut.</li>
	<li>Place three biopsy pieces per well using pointed forceps. Make sure the pieces attach to the bottom of the well.</li>
	<li>Lay a 22 mm coverslip on top of the pieces and apply some pressure.</li>
	<li>Incubate the plate at 37 &#176;C, 5% CO2, for a week. Check cells daily and add 200 &#956;L of DMEM/20% FBS every 2 days to replace evaporated medium.</li>
	<li>After one week, add up to 2 mL of DMEM/20% FBS and replace medium every 2&#8722;3 days.</li>
	<li>Passage cells at 1:4 ratio when wells are confluent (about 4&#8722;8 weeks).
	<ol>
		<li>Prepare 0.1% gelatin coated tissue culture-treated 6-well plates.</li>
		<li>Aspirate medium from wells at 80% confluency and wash once with 1 mL of PBS.</li>
		<li>Remove coverslip with sterile forceps and place the coverslip into a new well of a 6-well plate, with the tissue side up. 
		NOTE: Cells that remained attached to the coverslip will also be harvested.</li>
		<li>Add 500 &#956;L of dissociation solution (Table of Materials) per well (including wells with the coverslips) and incubate at 37 &#176;C, 5% CO2 for 5&#8722;10 min. Check when cells start to rise from the bottom of the well and inactivate the dissociation solution by adding 500 &#956;L of DMEM/20% FBS to each well.</li>
		<li>Collect fibroblasts from all the wells into a 15 mL conical tube. Add extra medium to the wells to collect the remaining cells. Centrifuge the tube at 350 x g for 5 min.</li>
		<li>In the meantime, add 500 &#956;L of DMEM/20% FBS to each well of previously gelatin coated plates.</li>
		<li>Aspirate medium and resuspend fibroblasts in 6 mL of DMEM/20% FBS.</li>
		<li>Add 500 &#956;L of fibroblast suspension to each well (two 6-well plates per sample/donor in total). Incubate cells overnight at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>On the next day, add 1 mL of DMEM/20% FBS to each well. Replace medium with 2 mL of DMEM/20% FBS every 2&#8722;3 days until wells are 80% confluent.</li>
	<li>Repeat section 2.11 for three confluent wells until third passage is reached.</li>
	<li>Freeze fibroblasts from confluent wells (passages 1 and 3).
	<ol>
		<li>Aspirate medium from the wells and wash once with 1 mL of PBS.</li>
		<li>Dissociate and collect fibroblasts as described in steps 2.11.4 and 2.11.5.</li>
		<li>Count cells with a hemocytometer and centrifuge the tube at 350 x g for 5 min.</li>
		<li>After centrifugation, aspirate medium and resuspend fibroblasts in FBS with 10% DMSO at a density of 5 x 105 cells/mL.</li>
		<li>Add 1 mL of the cell suspension per cryovial and freeze cells overnight at -80 &#176;C using a freezing container. Move vials to -150 &#176;C (liquid nitrogen) for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Lentiviral Production
<ol>
	<li>Grow HEK293T cells in a 100 mm tissue culture-treated dish with 10 mL of complete DMEM, at 37 &#176;C, 5% CO2, until confluency is reached.</li>
	<li>On the day prior to transfection, aspirate medium and wash the dish carefully with 5 mL of PBS.</li>
	<li>After removing PBS, add 1.5 mL of dissociation solution and incubate at 37 &#176;C, 5% CO2 for 5&#8722;10 min to dissociate cells from the dish. 
	NOTE: It is recommended to warm both PBS and dissociation solution before using, so that cells do not suffer a thermal shock.</li>
	<li>Inactivate dissociation solution with 3 mL of complete DMEM and transfer the cell suspension to a 15 mL conical tube. Wash the dish with 5 mL of complete DMEM to remove remaining attached cells and transfer this volume to the 15 mL conical tube.</li>
	<li>Centrifuge cell suspension at 350 x g for 5 min.</li>
	<li>Aspirate supernatant and split the cell pellet evenly between six 100 mm tissue culture-treated dishes in a final volume of 10 mL of complete DMEM per dish. Cells should be approximately 60% confluent by the time of transfection.</li>
	<li>On the next day, transfect cells with the plasmid mixes as follows: 
	NOTE: This part of the protocol describes the production of lentiviruses in one 100 mm tissue culture-treated dish per plasmid mix. To obtain higher volumes of lentiviral supernatant for concentration, use at least four 100 mm HEK293T cell culture dishes per mix.
	<ol>
		<li>In a 15 mL conical tube, add 10 &#956;g of the three transfer plasmids together: 3.33 &#956;g of pFUW-tetO-GATA2 (Addgene plasmid #125028)14, 3.33 &#956;g of pFUW-tetO-GFI1B (Addgene #125597)14 and 3.33 &#956;g of pFUW-tetO-FOS (Addgene #125598)14, plus 10 &#956;g of the 2nd generation psPAX2 packaging vector encoding the Gag, Pol, Tat and Rev genes (Addgene #12260) and 5 &#956;g of pMD2.G envelope vector encoding the VSV-G gene (Addgene #12259). Add water up to 500 &#956;L.</li>
		<li>In two new 15 mL conical tubes add 10 &#956;g of FUW-M2rtTA plasmid (Addgene #20342)17, 10 &#956;g of psPAX2 packaging vector and 5 &#956;g of pMD2.G envelope vector to each tube. Add water up to 500 &#956;L. One tube is going to be used as a control.</li>
		<li>To each tube add 62.5 &#956;L of 2 M CaCl2. Next, release bubbles into each mixture using a Pasteur pipet inserted into a pipet controller. While bubbles are forming, pipette 500 &#956;L of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffered saline (pH 7.1, 25 &#176;C), with a P1000 pipette, drop-wise against the Pasteur pipet and onto the mixture.</li>
		<li>Incubate tubes at room temperature for at least 15 min. The mixtures will appear slightly cloudy after some time.</li>
	</ol>
	</li>
	<li>Meanwhile, aspirate medium from HEK293T cell dishes (passaged the day before) and add 10 mL of complete DMEM without antibiotics. Be careful and add medium slowly as HEK293T cells are semi-adherent.</li>
	<li>Distribute each individual mixture (approximately 1 mL) evenly and drop-wise into separate dishes and incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>Replace medium with 4 mL of complete DMEM, 24 h after incubation. Incubate overnight at 37 &#176;C, 5% CO2. If available, incubate instead at 32 &#176;C, 5% CO2, as the reduced temperature will increase the half-life of the lentiviral particles.</li>
	<li>Collect supernatant with lentiviral particles three times to a 50 mL conical tube. Do not mix different lentiviral particles at this point. Each dish will result in 12 mL of lentiviral supernatant. Four dishes of the same viral preparation fit into one 50 mL conical tube. 
	CAUTION: Perform lentiviral collection in a biosafety level-2 laboratory in a laminar flow hood dedicated for lentiviral work and place viral contaminated waste (tubes, tips, dishes) in an appropriate container for biohazardous materials.
	<ol>
		<li>Do the first collection 16 h after the last incubation and add 4 mL of complete DMEM. Incubate at 37 &#176;C, 5% CO2.</li>
		<li>Do the second collection 8 h after the first to the same tube, add 4 mL of complete DMEM and incubate at 37 &#176;C, 5% CO2.</li>
		<li>Do the third collection 16 h after the second to the same tube and discard the dishes. 
		NOTE: Store lentiviral supernatants at 4 &#176;C after each collection.</li>
	</ol>
	</li>
	<li>Filter each lentiviral supernatant using a 0.45 &#956;m low-protein binding filter with a cellulose acetate membrane (Table of Materials) to a clean tube.</li>
	<li>Add a maximum of 15 mL of filtered supernatant to a centrifugal filter unit with a regenerated cellulose membrane (Table of Materials) and spin at 4,000 x g for 25 min, at 4 &#176;C. Discard flow-through. A viscous liquid containing lentiviruses will remain in the filter unit.</li>
	<li>Repeat step 3.13 by adding 15 mL of supernatant on top of the filter unit, until there is no more lentiviral supernatant left. 
	NOTE: When there are only a few milliliters of supernatant to concentrate, decrease the spinning time to 10 min. If there is still extra liquid (non-viscous) on the filter, centrifuge for an additional 10 min.</li>
	<li>Make aliquots (50&#8722;200 &#956;L depending on the initial supernatant volume) of each type of concentrated lentiviruses and store at -80 &#176;C for long-term storage (1&#8722;2 years) or at 4 &#176;C for short-term storage (1&#8722;2 weeks). 
	NOTE: Concentrated or non-concentrated lentiviruses can also be used fresh. Do not re-freeze and thaw as this results in reduced titer.</li>
</ol>
4. Hemogenic Reprogramming
NOTE: Use HDFs with a passage number of three (P3) or higher (until P10) to perform reprogramming experiments.
<ol>
	<li>Coat a 100 mm tissue culture-treated dish with 5 mL of 0.1% gelatin and incubate at 37 &#176;C for 20 min. Aspirate the remaining gelatin solution.</li>
	<li>Thaw a fibroblast vial and plate cells in the 0.1% gelatin-coated dish. Incubate overnight at 37 &#176;C, 5% CO2. If necessary, expand fibroblasts for a longer period of time until the desired passage and confluency are reached.</li>
	<li>Coat a 6-well tissue culture-treated plate with 500 &#956;L of 0.1% gelatin solution and incubate at 37 &#176;C for 20 min. Remove extra gelatin.</li>
	<li>Plate HDFs at a density of 150,000 cells per plate (25,000 cells per well) in 2 mL of complete DMEM per well. Incubate overnight at 37 &#176;C, 5% CO2, to allow cell attachment.</li>
	<li>Replace medium with 2 mL of complete DMEM plus 8 &#956;g/mL polybrene. Prepare a 1:1 ratio mix of pool-produced TF lentiviruses and M2rtTA in a new microcentrifuge tube. 
	NOTE: In this protocol, pool-production of lentiviruses for the three TFs is performed, which, in authors&#39; hands, results in higher reprogramming efficiency. Alternatively, it is suggested to perform a titration of the individual lentiviral particles by qPCR18, on a standard cell line. This will be used to define the volume of individual viruses necessary to meet a multiplicity of infection (MOI) optimal for co-transduction and hemogenic reprogramming.</li>
	<li>Distribute 10 to 100 &#956;L of lentiviral mixture per well, to transduce HDFs. This is day -2 of reprogramming. 
	NOTE: Defining the optimal volume of lentiviral mix for efficient reprogramming, without compromising cell viability, requires optimization (see Supplementary Figure 1 for more details). HDFs with more than 7 passages may require higher volumes of viruses than cells with lower passages.</li>
	<li>After 16 h of incubation, remove viruses and add complete DMEM. Allow cells to recover for 6&#8722;8 h.</li>
	<li>After recovery, aspirate medium and add 2 mL of complete DMEM with 8 &#956;g/mL polybrene.</li>
	<li>Do a second transduction as described in step 4.6 and incubate at 37 &#176;C, 5% CO2 for 16 h. This is day -1 of reprogramming. The lentiviral mix can be prepared on day -2 for both transductions and kept at 4 &#176;C.</li>
	<li>On the next day, remove the viruses and add complete DMEM supplemented with 1 &#956;g/mL DOX. This is day 0 of reprogramming. Incubate at 37 &#176;C, 5% CO2 for 48 h.</li>
	<li>At day 2 of reprogramming, split each well at 1:2 ratio.
	<ol>
		<li>Aspirate medium and wash cells with 1 mL of PBS.</li>
		<li>Aspirate PBS and dissociate cells with 500 &#956;L of dissociation solution. Incubate 5&#8722;10 min at 37 &#176;C, 5% CO2.</li>
		<li>Inactivate the dissociation solution with 1 mL of complete DMEM and collect cells into a conical tube. Centrifuge at 350 x g for 5 min.</li>
		<li>Resuspend the pellet in hematopoietic medium (see step 1.3), supplemented with 1 &#956;g/mL DOX, and plate cells into new tissue culture-treated 6-well plates coated with 0.1% gelatin to a final volume of 2 mL per well.</li>
	</ol>
	</li>
	<li>Change medium (hematopoietic medium plus DOX) twice a week for the duration of the reprogramming cultures (25 days).</li>
	<li>Analyze resulting reprogrammed cells at different time points by brightfield or fluorescence microscopy (see Supplementary Figure 2), flow cytometry, bulk and single-cell RNA sequencing, and transplantation assays for the acquisition of hematopoietic morphology, presence of endothelial and hematopoietic markers, acquisition of endothelial/hematopoietic gene expression profile and regeneration capacity14.</li>
</ol>
5. Optimization of Fibroblast Expansion for ChIP-seq Analysis at&#160;the Onset of Hemogenic Reprogramming
<ol>
	<li>Plate 300,000 HDFs (&#60;P8) in 0.1% gelatin coated tissue culture-treated 6-well plates with complete DMEM to a final volume of 2 mL per well. Incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>On the following day, replace medium with complete DMEM supplemented with 8 &#956;g/mL polybrene.</li>
	<li>Transduce cells with individual factors: pFUW-tetO-FOS14, pLV-tetO-HA-GFI1B (Addgene #125599)14 and pFUW-tetO-3xFLAG-GATA2 (Addgene #125600)14 or with a pool of the three factors, plus FUW-M2rtTA at 1:1 ratio. Use 10&#8722;20 &#956;L total virus (individual TF + M2rtTA or three TFs + M2rtTA). Incubate cells overnight at 37 &#176;C, 5% CO2. 
	NOTE: It is recommended to use twelve 6-well plates per condition (for each individual TF and the three TFs combined).</li>
	<li>Remove lentiviruses and add complete DMEM 16 h after the first transduction. Let cells recover for 6&#8722;8 h.</li>
	<li>Transduce cells a second time with the same amount of virus per condition and incubate at 37 &#176;C, 5% CO2.</li>
	<li>On the next day remove viruses and add complete DMEM. Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	<li>Re-plate each well into a 0.1% gelatin coated tissue culture-treated 100 mm dish with complete DMEM to a final volume of 10 mL per dish. This represents approximately a 1:6 passage.</li>
	<li>Allow cells to grow for 6 days at 37 &#176;C, 5% CO2.</li>
	<li>On day 6 after re-plating, aspirate medium and add complete DMEM with 1 &#956;g/mL DOX. Incubate cells at 37 &#176;C, 5% CO2 for 2 days.</li>
	<li>Collect fibroblasts and analyze genomic binding sites of the three TFs transduced individually or in combination, by ChIP-seq 2 days after DOX supplementation14. 
	NOTE: The final seventy-two 100 mm dishes will contain between 20&#8722;50 x 106 cells, sufficient to perform ChIP-seq experiments and replicates.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Signatures+of+Tissue-Specific+Microvascular+Endothelial+Cell+Heterogeneity+in+Organ+Maintenance+and+Regeneration.">Molecular Signatures of Tissue-Specific Microvascular Endothelial Cell Heterogeneity in Organ Maintenance and Regeneration.</a> Developmental Cell. 26, 204-219 (2013).</li><li>Batta, K., Florkowska, M., Kouskoff, V., Lacaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Reprogramming+of+Murine+Fibroblasts+to+Hematopoietic+Progenitor+Cells.">Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells.</a> Cell Reports. 9, 1871 (2014).</li><li>Cheng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+mouse+fibroblasts+into+engraftable+myeloerythroid+and+lymphoid+progenitors.">Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors.</a> Nature Communications. 7, 1-15 (2016).</li></ol>

The authors have nothing to disclose.

In this article, a method is described to generate hematopoietic progenitor cells directly from human fibroblasts, which go through an HP cell intermediate, similarly to definitive HSCs14.
Pool-production of lentiviral particles encoding GATA2, GFI1B and FOS was preferred over individual production, since in our hands it results in higher reprogramming efficiencies (unpublished data). Lentiviruses, as members of the Retroviridae&#160;family, normally contain two copies of positive single-stranded RNA19. The increased reprogramming efficiency may be due to packaging of two different transgenes in the same lentiviral particle, resulting in increased number of cells co-transduced with the three transcription factors. To ensure the success of this protocol, it is necessary to transduce HDFs with adequate amount of virus depending on the cell passage to obtain an optimal balance between reprogramming efficiency and cell viability, as recommended in step 4.6. Moreover, fresh non-concentrated viruses can be used. It is recommended to transduce cells with 0.5-3 mL of 3TFs pool and M2rtTA. Also, cell density should be adjusted according to the application. 150&#160;000 HDFs per 6-well plate (step 4.4) provided the optimal density to perform FACS, transplantation and flow cytometry analysis of reprogrammed cells. For ChIP-seq experiments, more cells were required from the beginning (step 5.1). It is important to check cells regularly for morphological changes and replace hematopoietic medium twice a week to support the emergence of induced hematopoietic cells. Addition of hematopoietic cytokines or co-culture in feeder layers may increase reprogramming efficiency.
With this method, we can identify new hematopoietic markers that are dynamically expressed during hemogenic reprogramming. CD9, which was shown to be up-regulated in reprogrammed cells at the transcriptional level14, is rapidly expressed at the cell surface in the initial phases of reprogramming together with CD49f and CD143, serving as a novel marker of human HSC precursors. We also show that ITGA6 and ACE are direct targets of GATA2 during the initial stages of hemogenic reprogramming, in addition to CD9 and CD3414, providing a direct mechanistic link between human hemogenic precursor phenotype and GATA2.
One advantage of this system resides in the use of relatively homogeneous fibroblast cultures. While PSCs are easily expanded and maintained in vitro, differentiation protocols generate heterogeneous populations that include hematopoietic progenitors, which engraft poorly5,6,7. Moreover, there is a risk of tumorigenesis when transplanting PSC-derived HSPCs, since undifferentiated PSCs may still remain in culture even after employing differentiation protocols. Alternatively to fibroblasts, direct reprogramming to HSCs has been applied to blood-committed progenitors20 and endothelial cells21. However, starting with blood-restricted progenitor cells hinders therapeutic application of the resulting HSCs if the patient carries mutations that affect the stem/progenitor hematopoietic population22. In the case of endothelial cells, these are more difficult to obtain compared to fibroblasts, and constitute a very heterogeneous cell population in terms of phenotype, function and structure, which are organ-dependent23. Other studies have succeeded in reprogramming mouse fibroblasts into engraftable hematopoietic progenitors24,25 yet, so far, no other protocol describes the generation of HSPC-like cells from human fibroblasts.
This approach, coupled with pharmacological inhibition, gene knock-out, or knock-down permits to define individual or combination of factors that are required to directly induce human HSCs. Employing high efficiency screening methodologies based on recent CRISPR-Cas9 technologies in HDFs prior to reprogramming, represents an exciting endeavor for defining novel regulators of human definitive hematopoiesis. In the future, reprogramming non-blood related human cell types such as fibroblasts will serve as a platform to generate healthy patient-tailored hematopoietic progenitor cells for clinical applications.

An erratum was issued for: <a href="https://www.jove.com/video/60112/hemogenic-reprogramming-human-fibroblasts-enforced-expression">Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors</a>. The author affiliations were updated.
The second author affiliation was&#160;updated from:
2Wallenberg Center for Molecular, Lund University
to:
2Wallenberg Center for Molecular Medicine, Lund University

An erratum was issued for: <a href="https://www.jove.com/video/60112/hemogenic-reprogramming-human-fibroblasts-enforced-expression">Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors</a>. The author affiliations were updated.

Erratum: Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Definitive hematopoietic stem and progenitor cells (HSPCs) emerge in the aorta-gonad-mesonephros (AGM) region and placenta from endothelial precursors with hemogenic capacity, through an endothelial-to-hematopoietic transition (EHT)1,2. Hemogenic precursors (HPs) express both endothelial and hematopoietic markers, but their precise identification remains elusive, particularly in the human system. Despite being a relatively conserved process in mammals, hematopoietic stem cell (HSC) development still differs significantly between humans and mouse models3,4. Therefore, in vitro approaches to recapitulate human HSC development are needed.
Differentiation of pluripotent stem cells (PSCs) to HSCs, although promising, has met limited success over the past 20 years, mostly due to the available differentiation protocols, which result in primitive hematopoietic progenitors with poor engraftment ability5,6,7. Alternatively, direct cell reprogramming methodologies have been applied to generate HSPC-like cells from multiple cell types, using transcription factors (TFs)8,9. In particular, the overexpression of three TFs, Gata2, Gfi1b and cFos, converted mouse embryonic fibroblasts into HSPCs through an HP intermediate with a defined phenotype (Prom1+Sca-1+CD34+CD45-)10. This process resembled the EHT that occurs in the embryo and placenta, during specification of definitive hematopoiesis. This phenotype enabled the identification and isolation of a population of HPs in the mouse placenta that after short-term culture and Notch activation generated serially transplantable HSCs11.
So far, no phenotype has been established that distinguishes human HSCs from their precursors, but some molecules are known to be expressed in emerging HSCs. Integrin alpha 6 (ITGA6 or CD49f) is highly expressed in long-term repopulating HSCs, the most immature cells in the HSC compartment12, and angiotensin-converting enzyme (ACE or CD143) is present in CD34 negative hematopoietic precursors in embryonic blood-forming tissues13.
Recently, we have demonstrated that human versions of the three TFs, GATA2, FOS and GFI1B reprogram human dermal fibroblasts (HDFs) into HPs with short-term engraftment capacity14. In the initial phases of reprogramming, GATA2 engages open chromatin and recruits GFI1B and FOS to repress fibroblast genes and activate endothelial and hematopoietic genes. Induced cells highly expressed CD49f and ACE, and contained a small percentage of cells expressing the HSPC marker CD34. The CD9 gene, which is expressed in HSCs15 and is important for HSC homing16, was shown to be a direct target of GATA2 and among the most up-regulated genes in reprogrammed cells14. CD9 may therefore constitute an additional marker for HPs of human definitive hematopoiesis.
In this protocol, we describe the generation of HSPC-like cells from human fibroblasts through enforced expression of GATA2, GFI1B and FOS, as well as an adapted method for chromatin immunoprecipitation (ChIP)-sequencing (seq) analysis at the onset of reprogramming. TFs were encoded in a doxycycline (DOX)-inducible lentiviral vector (pFUW-tetO) that contains a tetracycline response element (TRE) and a minimal CMV promoter, and were transduced together with a constitutive vector containing the reverse tetracycline transactivator protein (pFUW-M2rtTA). When DOX (analog of tetracycline) is added after transduction, it binds to the rtTA protein which interacts with the TRE allowing TF transcription (Tet-On system). The procedure requires 25 days to complete. For ChIP-seq experiments, HDFs were transduced with tagged versions of GATA2 (pFUW-tetO-3xFLAG-GATA2) and GFI1B (pLV-tetO-HA-GFI1B), plus pFUW-tetO-FOS and TF binding sites were analyzed two days after DOX supplementation.
Ultimately, hemogenic reprogramming of human fibroblasts provides an in vitro tractable system to study the mechanisms underlying human developmental hematopoiesis and a potential source of patient-specific HSPCs for future clinical application.

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			<td>Human Dermal Fibroblasts (HDFs)</td>
			<td>ScienCell</td>
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			<td>Sigma-Aldrich</td>
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			<td>PE anti-human CD9 Antibody clone HI9a</td>
			<td>BioLegend</td>
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			<td>Addgene</td>
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hemogenic reprogramming of human fibroblasts by enforced expression of transcription factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

A schematic representation of the reprogramming approach using HDFs is illustrated in Figure 1A. Fibroblasts are acquired from commercial sources or collected from human donors and expanded in vitro previous to reprogramming. After plating, cells are transduced twice with GATA2, GFI1B and FOS (and M2rtTA) lentiviruses, and doxycycline is added at day 0 of reprogramming. On day 2, cells are split and plated in hematopoietic medium until day 25 of culture. Reprogrammed cells may be generated at different time points for multiple applications including transplantation in immunocompromised mice, single-cell RNA-sequencing (scRNA-seq) of purified cell populations (day 2 unsorted, day 15 CD49f+ CD34 and day 25 CD49f+CD34+ cells), as well as microscopy and flow cytometry analysis for the cell surface markers CD49f, CD34, CD9 and CD143. Representative cytometry plots show ~17% of reprogrammed cells expressing both CD49f and CD9 (Figure 1B, left panel), after 25 days of reprogramming. The majority of double positive cells express CD143 (~86%), and a small population express CD34 (0.9%), suggesting a dynamic hemogenic fate induction. These markers are not activated in M2rtTA transduced HDFs cultured for 25 days (Figure 1B, right panel). Immunofluorescence images confirm expression of CD9 and CD143 in adherent and round cells, morphologically distinct from fibroblasts that are negative for these markers (Figure 1C). Human hemogenic colonies also express CD49f and CD3414. ScRNA-seq analysis of HDFs, day 2 unsorted cells, and purified reprogrammed cells at day 15 (CD49f+CD34-) and day 25 (CD49f+CD34+) show a stepwise increase in CD49f, CD9 and CD143 expression from day 2 to day 25. CD49f and CD9 positive cells appear first during the reprogramming process, between day 2 and 15, indicating that these molecules may represent markers of early human hemogenesis. CD143 expression starts to be detected at day 15 and CD34 expressing cells are detected only at later time points (day 25). CD34+ umbilical cord blood (UCB) cells were used as reference (Figure 1D).
Figure 2A describes a modified protocol to generate sufficient number of cells for ChIP-seq analysis at the initial stages of hemogenic reprogramming (day 2). First, HDFs are plated at a density two times higher than in the standard protocol (300,000 cells versus 150,000 cells per plate). After transduction, each well is re-plated into a 100 mm dish allowing cells to expand for 6 days before supplementing medium with DOX. Cells are analyzed 2 days after adding DOX and consequent TF expression. Figure 2B shows genome browser profiles of GATA2 binding to genomic regulatory regions of ITGA6 and ACE when cells are co-transduced with the three factors (3TFs) or GATA2 individually. GATA2 also binds to open chromatin regions of CD9 and CD34 genes14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60112/60112fig1a.jpg" /> 
Figure 1: Induction of hemogenic fate in human dermal fibroblasts. (A) Experimental strategy for hemogenic reprogramming of human dermal fibroblasts (HDFs). Fibroblasts from skin punch biopsies are collected from donors, expanded and transduced with GATA2, GFI1B, FOS and M2rtTA lentiviruses. Doxycycline (DOX) is added to the culture at day 0 of reprogramming and cells are analyzed at several time points until day 25. scRNA-seq, single cell RNA-sequencing. FACS, Fluorescence-Activated Cell Sorting. (B) Gating strategy used to evaluate the expression of hemogenic/hematopoietic markers by flow cytometry at day 25 after transduction with the three transcription factors (3TFs). Cytometry plots show percentage of double positive cells for CD49f and CD9, gated in the live-cell population (DAPI-negative). Within the double positive population, the expression of CD143 and CD34 is shown. HDFs transduced only with M2rtTA virus under the same culture conditions are used as control. (C) Immunofluorescence images of day 25 reprogrammed colonies confirming the expression of CD9 (upper panel) and CD143 (lower panel). Cells were stained with antibodies (Table of Materials) diluted 1:100 in PBS/2% FBS with mouse serum, incubated 20 min at 37 &#176;C, 5% CO2, washed three times and imaged in PBS/2% FBS. Phase, phase-gradient contrast. Scale bars = 50 &#181;m. (D) ScRNA-seq analysis of 253 cells at different time points. Expression of ITGA6, CD9, ACE and CD34 is activated during reprogramming. Cells are collected at day 2 (unsorted), day 15 (CD49f+CD34-) and day 25 (CD49f+CD34+). HDFs and CD34+ umbilical cord blood (34+UCB) cells are used as references. <a href="https://www.jove.com/files/ftp_upload/60112/60112fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60112/60112fig02.jpg" /> 
Figure 2: Expansion of human dermal fibroblasts for ChIP-seq analysis. (A) Experimental strategy depicting a modified protocol to generate high numbers of transduced human dermal fibroblasts (HDFs) for ChIP-seq at day 2 of reprogramming. 300,000 cells are plated in 6-well plates and transduced twice with individual factors (pFUW-tetO-FOS, pLV-tetO-HA-GFI1B or pFUW-tetO-3xFLAG-GATA2) or a combination of the three factors (plus M2rtTA). After removing viruses, fibroblasts are expanded for six days in 100 mm dishes. Doxycycline (DOX) is added at day 0 and cells are collected two days after DOX addition. (B) Genome browser profiles highlighting GATA2-binding sites (grey boxes) at ITGA6 and ACE loci two days after transduction with the three transcription factors (3TFs) or with GATA2 alone. The total number of mapped reads is represented on the y-axis. <a href="https://www.jove.com/files/ftp_upload/60112/60112fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60112/60112suppfig1.jpg" /> 
Supplementary Figure 1: Defining an optimized lentiviral volume for efficient hemogenic reprogramming. Increasing volumes of concentrated (10 to 100 &#956;L) pool-produced lentiviral particles (3TFs: GATA2, GFI1B and FOS) are used to transduce human dermal fibroblasts (HDFs), together with M2rtTA at a ratio of 1:1, following steps 4.5&#8722;4.12 of the protocol. Reprogrammed cells are analyzed at day 25 to define an optimal volume of transduction for hemogenic reprogramming, given by the percentage of CD49f+CD9+ cells gated in live-cells (DAPI-negative). Cell viability can be assessed by quantifying the absolute number of live cells at day 25. HDFs transduced with M2rtTA (100 &#956;L) are used as negative control. <a href="https://www.jove.com/files/ftp_upload/60112/60112suppfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/60112/60112suppfig2.jpg" /> 
Supplementary Figure 2: Morphology changes during hemogenic reprogramming of human dermal fibroblasts. Human Dermal Fibroblast (HDF) cultures are imaged at the day of the first transduction (day -2), when DOX is added to the cultures (day 0), two days (day 2) and fifteen days (day 15) after DOX supplementation, and at the end-point of the experiment (day 25). Hemogenic colonies at days 15 and 25 are highlighted. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60112/60112suppfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors at JoVE.com

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

hemogenic-reprogramming-human-fibroblasts-enforced-expression

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5.8K Views.  Lund University. This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Video: Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

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

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

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

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

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

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.

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

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

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.

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.

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

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

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.

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