We thank Evelyn Wang and Gregory Hyde (Whitehead Institute, Cambridge, MA) for cloning and Harvey Lodish (Whitehead Institute) for providing many of the plasmids used for generating the retroviral library. We thank Kavitha Siva and Sofie Singbrant (Department of Molecular Medicine and Gene Therapy, Lund University), G&#246;ran Karlsson and Shamit Soneji (Department of Molecular Hematology, Lund University) for their roles in description of iEP production. We would also like to acknowledge and thank Julian Pulecio (Centre of Regenerative Medicine, Barcelona Biomedical Research Park), Violeta Rayon-Estrada (The Rockefeller University, New York), Carl Walkley (St. Vincent's Institute of Medical Research and Department of Medicine, St Vincent's Hospital, University of Melbourne), &#193;ngel Raya (Catalan Institution for Research and Advanced Studies, Barcelona), and Vijay G. Sankaran (Broad Institute of the Massachusetts Institute of Technology and Harvard, Cambridge) for their previous contributions to this work. This work was supported by the Ragnar S&#246;derberg Foundation (to J.F.); the Swedish Research Council (to J.F.); Stiftelsen Olle Engkvist Byggm&#228;stare (to J.F.); the Swedish Foundation for Strategic Research (to J.F.); &#197;ke Wiberg's Foundation (to J.F.); a Marie Curie integration grant (to J.F.).

1. Establishment and Maintenance of Primary Mouse Tail Tip Fibroblast Cultures
<ol>
	<li>Prepare gelatin-coated dishes (recommend a 10 cm dish for one tail) by covering the surface with 0.1% gelatin and incubating the dishes for approximately 20 min at 37 &#176;C. Aspirate the gelatin solution from the dish and allow it to dry for at least 2 h.</li>
	<li>Euthanize the mice by cervical dislocation. Remove the tail with scissors, cutting at the base of the tail. Put the tail in Dulbecco&#39;s phosphate-buffered saline (DPBS) with 2% fetal bovine serum (FBS) until ready to use. 
	NOTE: For the best results, tails should be taken from mice around 6 to 8 weeks of age. Tails can be taken from mice older than 8 weeks of age, however, as the mice age, the proliferation capability of the fibroblasts and the efficiency of reprogramming decrease11.</li>
	<li>Perform all subsequent steps of this protocol in a tissue culture hood under sterile conditions. Prepare a diluted trypsin solution of 0.02% trypsin-EDTA in DPBS and add 5 mL into an uncoated 10 cm dish.</li>
	<li>Wash the tail, first in 70% ethanol, then in DPBS. In a dish, place the tail flat and use forceps to hold it in place. Make an incision on the tail along its longitudinal axis from the base to the tip.</li>
	<li>Grasp the tail with one pair of forceps and hold it vertically. Using a second pair of forceps, grip the skin beside the incision at the base of the tail and peel it back. Do this at both sides of the incision until the skin can be peeled off by pulling downwards toward the tip of the tail.</li>
	<li>Hold the peeled tail with forceps over the dish containing the trypsin solution and cut the tail into approximately 1 cm long pieces. With the tail pieces in the trypsin solution, use scissors to fragment the pieces into smaller pieces. Incubate the tail pieces in the trypsin solution at 37 &#176;C for 10 min. 
	NOTE: The smaller the pieces are preferred so as to provide each piece a high surface area to volume ratio.</li>
	<li>Quench the trypsin using 2 volumes of fibroblast expansion (FEX) medium (high-glucose Dulbecco Modified Eagle Medium (DMEM) with 15% FBS, 2 mM L-glutamine, Non-essential amino acids (NEAA) and 100 U/mL Penicillin/Streptomycin).</li>
	<li>Collect entire contents of the dish into a 50 mL tube and centrifuge at 350 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend the tail fragments in 10 mL of fresh FEX medium.</li>
	<li>Transfer tail fragments in medium to a gelatin-coated dish and incubate at 37 &#176;C in 5% CO2 and 4% O2, adding fresh FEX medium every 2 days. 
	NOTE: After five to seven days, tail fragments have attached at the bottom of the dish and fibroblasts can be seen moving away from them.</li>
	<li>Once clusters of fibroblasts are spotted, gently shake the dish to dislodge tail pieces and aspirate the medium and all the bone fragments leaving the fibroblasts attached to the plate. Add new FEX medium and culture the fibroblasts until confluent.</li>
	<li>To ensure no contamination of fibroblasts by hematopoietic progenitors, dissociate the cells from the plate using 1x trypsin-EDTA for 5 mins and collect cells. Deplete for cells expressing hematopoietic markers (CD117, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4, and Ter-119) using a magnetic separation system10.</li>
</ol>
2. Retrovirus Production
<ol>
	<li>Seed retroviral packaging cells at approximately 2.5 &#215; 104 cells/cm2 (2.0 &#215; 106 cells for a 10 cm dish) on a tissue culture-treated (by vacuum-gas plasma) dish and culture overnight in high-glucose DMEM with 10% FBS, 10 mM Sodium Pyruvate, and 100 U/mL Penicillin/Streptomycin at 37 &#176;C and 5% CO2.</li>
	<li>On the following morning, change the medium to DMEM with no additives using half the volume used to culture overnight (5 mL for a 10 cm dish). In the afternoon, check that the cells are 70-80% confluent and begin the transfection.</li>
	<li>For each reprogramming factor, Gata1, Tal1, Lmo2, and c-Myc, prepare a 2:1 mixture of the expression vector (pMX) and helper vector (containing gag and pol genes). For a 10 cm dish of fibroblasts, use 6 &#181;g of expression vector and 3 &#181;g of helper vector in a final volume of 100 &#181;L in DMEM medium.</li>
	<li>For each reprogramming factor, prepare 300 &#181;L of room temperature (RT) DMEM in a sterile polystyrene tube and carefully add 27 &#181;L of commercial transfection reagent. 
	NOTE: The transfection reagent should be brought to RT before use and must be added directly into the medium as the electrostatic properties of the compound can make it stick to the plastic wall of the tube.</li>
	<li>Add the plasmid mix into the transfection reagent-containing tube, vortex briefly and incubate the transfection reagent-DNA mixture for 15 min at RT. Briefly vortex the mixture and add it dropwise to the retroviral packaging cells so that the transfection reagent-DNA mix is evenly spread over the culture and incubate at 37 &#176;C overnight.</li>
	<li>24 h after transfection, change the medium to DMEM with 20% FBS and 100 U/mL Penicillin/Streptomycin. 48 h after transfection, collect the supernatant and filter it through a 0.22 &#181;m pore-size syringe filter. 
	NOTE: Viral supernatants can be frozen to -80 &#176;C and kept until required, although transduction is more efficient if fresh viral supernatant is used.</li>
</ol>
3. GTLM transduction and iEP harvest
<ol>
	<li>Seed the tail tip fibroblasts at 1 &#215; 104 cells/cm2 on 0.1% gelatin pre-coated dishes in FEX medium and incubate at 37 &#176;C for 24 h.</li>
	<li>The following day, prepare a transduction mixture as follows.
	<ol>
		<li>Add 1 volume of viral supernatant for each reprogramming factor supplemented with 4 &#181;g/mL of retroviral infection reagent (40%) to 6 volumes of FEX medium (60%).</li>
		<li>For a 10 cm dish of fibroblasts, add 1 mL of each viral supernatant (4 &#215; viruses = 4 mL) supplemented with 4 &#181;g/mL of retroviral infection reagent to 6 mL of FEX medium, giving a total of 10 mL transduction mixture.</li>
	</ol>
	</li>
	<li>Aspirate FEX medium from the fibroblast culture and replace it with the transduction mixture. Incubate the transduction for 4 h at 37 &#176;C in hypoxic conditions (5% CO2 and 4% O2).</li>
	<li>Aspirate the transduction mixture and replace it with fresh reprogramming medium (Serum-free Expansion medium (SFEM), 100 U/mLPenicillin/Streptomycin, 100 ng/mL murine Stem Cell Factor (mSCF), 10 ng/mL murine interleukin-3 (IL3), 2 U/mL human recombinant Erythropoietin (hrEPO), and 100 nM Dexamethasone).</li>
	<li>Incubate for 8 daysat 37 &#176;C in hypoxic conditions, adding fresh reprogramming medium every 2 days. After five to eight days, successful reprogramming will yield clusters of cells that have detached from the plate.</li>
	<li>To collect reprogrammed cells for analysis, gently pipette up and down to harvest them directly from the dish. To harvest untransduced fibroblasts for comparison, dissociate the cells from the plate using 1x trypsin-EDTA and collect.</li>
</ol>

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F., 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=Induction+of+a+hemogenic+program+in+mouse+fibroblasts.">Induction of a hemogenic program in mouse fibroblasts.</a> Cell Stem Cell. 13 (2), 205-218 (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 (5), 1871-1884 (2014).</li><li>Yu, B., 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+fibroblasts+into+bipotential+hepatic+stem+cells+by+defined+factors.">Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.</a> Cell Stem Cell. 13 (3), 328-340 (2013).</li><li>Hendry, C. 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The authors have no conflicts of interest to report.

Overexpression of a four-factor cocktail, GATA1,&#160;TAL1,&#160;LMO2,&#160;and c-MYC (GTLM), is sufficient to reprogram murine and human fibroblasts directly to iEPs10. The reprogrammed erythroid cells greatly resembled bona fide erythroid progenitors in terms of morphology, phenotype, gene expression, and colony-forming ability. This finding corroborates the rationale of using direct ...

Red blood cells (RBCs) are essential in all vertebrates and make up 84% of all cells of human bodies1. From embryonic to adult life, our health is highly dependent on exact regulation of RBC homeostasis. The ongoing production of mature RBCs throughout development into adulthood is known as erythropoiesis. A major challenge in erythropoiesis research is to define the master regulators that orchestrate RBC development and the switch between primitive and definitive erythropoiesis. Direct lineage reprogramming of erythroid progenitors presents an opportunity to further understand erythroid development in vivo.
Direct lineage reprogramming (DLR), also known as transdifferentiation, is the process of reprogramming one cell type directly into another, bypassing pluripotent and multipotent progenitor stages. DLR has thus far been used to produce numerous cell types including neural2, hematopoietic3,4,5, hepatic6 and nephrotic7, progenitor cells. For developmental biologists, DLR has become an important tool for interrogating aspects of lineage commitment and terminal differentiation processes8,9. DLR can complement and partially replace in vivo studies for understanding mechanisms of cell fate determining factors during development. The DLR protocol for reprogramming to erythroid progenitors described in this paper provides the field a complimentary method for developmental studies of erythropoiesis.
We have previously demonstrated that overexpression of a four-factor cocktail, GATA1, TAL1, LMO2, and c-MYC (GTLM), is sufficient to reprogram both murine and human fibroblasts directly to induced erythroid progenitors (iEPs)10. The GTLM-reprogrammed erythroid cells greatly resemble bona fide primitive erythroid progenitors in terms of morphology, phenotype, and gene expression10. Thus iEPs have limited proliferation capacity and mature to nucleated erythrocytes similar to those transiently produced in the early embryo before onset of definitive erythropoiesis. By making changes in the reprogramming conditions (e.g., point mutations in reprogramming factors or addition of other factors), one can understand how this leads to changes in erythroid development and differentiation. We have for example shown that addition of Klf1 or Myb to the GTLM cocktail changes the globin expression pattern from predominantly embryonic (primitive) to mainly adult (definitive). This finding corroborates the validity of using DLR as a tool for defining developmental factors in erythropoiesis.
Here, we outline the process of generating iEPs from mouse tail tip fibroblasts (TTF). In our representative results, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eYFP) from the Rosa26 locus in all cells that have expressed the erythropoietin receptor, allowing easy visualization of commitment to the erythroid lineage. Using this method, YFP positive (EpoR+) cells are present as early as five days after transduction. This protocol, therefore, offers a quick and robust technique for the generation of erythroid progenitors in vitro.

<table border="0" cellpadding="0" cellspacing="0" style="width:1141px;" width="1142">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM without sodium pyruvate</td>
			<td style="width:167px;">GE Life Sciences</td>
			<td style="width:123px;">SH30022.01</td>
			<td style="width:505px;">Culturing media for PhGP cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM with sodium pyruvate</td>
			<td style="width:167px;">GE Life Sciences</td>
			<td style="width:123px;">SH30243.01</td>
			<td>Culturing media for tail tip fibroblasts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">StemSpan Serum Free Expansion Media (SFEM)</td>
			<td style="width:167px;">Stem Cell Technologies</td>
			<td style="width:123px;">9650</td>
			<td>Reprogramming media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal Bovine Serum HyClone</td>
			<td style="width:167px;">GE Life Sciences</td>
			<td style="width:123px;">SH30071.03HI</td>
			<td>Growth factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin/Streptomycin HyClone (100x)</td>
			<td style="width:167px;">Ge Life Sciences</td>
			<td style="width:123px;">SV30010</td>
			<td>Antiboiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Non-Essential Amino Acids (100x)</td>
			<td style="width:167px;">Thermo Fisher</td>
			<td style="width:123px;">11140050</td>
			<td>SNL media is suppplemented with this</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Trypsin HyClone (1x)</td>
			<td style="width:167px;">GE Life Sciences</td>
			<td style="width:123px;">SH30042.01</td>
			<td>Cell dissociation agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Murine Stem Cell Factor (mSCF)</td>
			<td style="width:167px;">Peprotech</td>
			<td style="width:123px;">250-03</td>
			<td>Added to reprogramming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Recombinant Murine Il-3</td>
			<td style="width:167px;">Peprotech</td>
			<td style="width:123px;">213-13</td>
			<td>Added to reprogramming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">human recombinant erythropoietin (hrEPO)</td>
			<td style="width:167px;">Peprotech</td>
			<td style="width:123px;">100-64</td>
			<td>Added to reprogramming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Dexamethasone</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:123px;">50-02-2&#160;&#160;</td>
			<td>Added to reprogramming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Gelatin from porcine skin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:123px;">9000-70-8&#160;</td>
			<td>Dissovled in dH2O and used for coating plates. Ensure sterility before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Blasticidin S hydrochloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:123px;">3/9/3513</td>
			<td>Selection antibiotic. Affects both pro- and eukaryotic cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:167px;">Ge Life Sciences</td>
			<td style="width:123px;">SH30850.03</td>
			<td>Used for washing steps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Polybrene</td>
			<td style="width:167px;">Merck</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>Infection / Transfection&#160; Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">FuGENE HD Transfection Reagent</td>
			<td style="width:167px;">Promega</td>
			<td style="width:123px;">E2311</td>
			<td>Transfection Reagent for PhGP cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Millex-GP Syringe Filter Unit 0.22 &#181;m</td>
			<td style="width:167px;">Merck</td>
			<td style="width:123px;">SLGP033RS</td>
			<td>Used for filtering virus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">BD Emerald Hypodermic Syringe</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:123px;">SKU: 307736</td>
			<td>Used for filtering virus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">100 mm Culture Dish</td>
			<td style="width:167px;">Corning</td>
			<td style="width:123px;">430167</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">6-well plate</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:123px;">10799541</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Jeweler Forceps #5</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:123px;">66-7642</td>
			<td>Used for handling small tail fragments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sklarlite Iris Scissors</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:123px;">23-1149</td>
			<td>Used for cutting the tail into small pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Lineage Cell Depletion Kit, mouse</td>
			<td style="width:167px;">Miltenyi Biotec</td>
			<td style="width:123px;">130-090-858</td>
			<td>For depletion of hematopoietic cells in fibroblast cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">CD117 MicroBeads, mouse</td>
			<td style="width:167px;">Miltenyi Biotec</td>
			<td>130-091-224</td>
			<td>For depletion of hematopoietic cells in fibroblast cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PheonixGP cells</td>
			<td style="width:167px;">ATCC</td>
			<td>CRL-3215</td>
			<td>retroviral packaging cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EcoPAC vector (pCL-Eco)&#160;</td>
			<td>Novus Biologicals</td>
			<td>NBP2-29540</td>
			<td>retroviral helper vector containing gag and pol genes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">pMX-Gata1</td>
			<td style="width:167px;">Cloned in-lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">pMX-Tal1</td>
			<td style="width:167px;">Cloned in-lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">pMX-Lmo2</td>
			<td style="width:167px;">Cloned in-lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">pMX-cMyc</td>
			<td style="width:167px;">Cloned in-lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellSens Standard 1.6 software&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:123px;"></td>
			<td>Cytospin analysis software</td>
		</tr>
	</tbody>
</table>

direct lineage reprogramming of adult mouse fibroblast to erythroid progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present a reproducible protocol for the production of iEPs from adult fibroblasts using transcription factor-driven DLR. We evaluate the cell reprogramming using flow cytometry, colony-forming assays, and gene expression analysis. In order to assist in the visualization of the conversion to erythroid cell fate, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eY...

Watch this Scientific Journal Video about Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors at JoVE.com

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

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

direct-lineage-reprogramming-adult-mouse-fibroblast-to-erythroid

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JoVE Journal

Developmental Biology

6.3K Views.  Lund University. Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Video: Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

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 ...

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 ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...