Name: mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis
Uploaded: 2014-09-09T15:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

This study was supported by NIH R00HL102154, and an American Society of Hematology scholar award to P Ji.

The experiments described in this protocol were performed in accordance with the guidelines and regulations set forth by Northwestern University Institutional Animal Care and Use Committee.
1. Preparation of Culture Medium
<ol>
	<li>Prepare fibronectin solution. Add 1 ml of water to one vial of human fibronectin (1 mg). Leave solution in the tissue culture hood for 30 min without agitation. Transfer the total liquid to 50 ml of PBS to make a final concentration of 20 &#181;g/ml and gently mix well. Make the fibronectin solution fresh before coating the plate (see below).</li>
	<li>Prepare 50 ml of Epo free medium (SCF medium). Combine the listed ingredients in Table 1. The medium can be stored at 4 &#176;C for 1 month when made fresh.</li>
	<li>Prepare 50 ml of Epo containing medium. Combine the listed ingredients in Table 2. The medium can be stored at 4 &#176;C for 1 month when made fresh.</li>
</ol>
2. Fibronectin Coated Plate
<ol>
	<li>Add 1 ml fibronectin solution to each well of a 6-well plate (or 0.5 ml to each well of a 12 well plate). Leave the plate in the tissue culture hood for 1 hr.</li>
	<li>Rinse wells with sterile water twice and air dry the plate.</li>
	<li>Wrap the plate and save in the cold room at 4 &#176;C.</li>
</ol>
3. Purification of Fetal Liver Erythroblasts
NOTE: Use fetal livers&#160;from E13 to E15 timed pregnant mice (C57BL/6). E13.5 livers are preferred to maximize the yield of CFU-E progenitors. To obtain the timed pregnant mice, 6 to 8 weeks old male and female mice were placed in one cage (usually one male with one or two females). Female vaginal plugs were checked the next morning to determine if the mating was successful. The first day of gestation was the day after the plug was found.
<ol>
	<li>Preparation of the Total Fetal Liver Cells
	<ol>
		<li>Sacrifice the mouse by CO2 inhalation and cervical dislocation. Spray the abdomen with 70% ethanol for disinfection. Open the abdomen with dissecting scissors to remove the uterus. Wash the uterus in 1x PBS.</li>
		<li>Dissect the fetuses under sterile conditions using a tissue culture hood and autoclaved tools and wash in 1x PBS.</li>
		<li>Dissect the fetal livers from the fetuses. Hold the body of the fetus with one forceps, gently pull the fetal liver (pink in color) away from the body with another forceps. Clean the fetal liver with the forceps to remove the associated fibrotic tissues. Place all fetal livers into fresh 1x PBS containing 10% fetal bovine serum.</li>
		<li>Mechanically disrupt fetal livers by pipetting up and down.</li>
		<li>Filter the cell suspension through a 40 &#181;m cell strainer into a 50 ml conical tube. Pellet the cells at 800 x g for 5 min.</li>
		<li>Aspirate the supernatant and resuspend the cells in 1 ml PBS with 10% FBS (approximately 8 E13.5 fetal livers per ml).</li>
	</ol>
	</li>
	<li>Purification of TER119 Negative Erythroblasts
	<ol>
		<li>Block the cells with rat IgG (0.2 mg/ml) on ice for 15 min.</li>
		<li>Without washing or centrifuging, add biotinylated anti-TER119 antibody to the cell suspension (1 &#181;g/ml). Incubate for 15 min on ice.</li>
		<li>Wash in 1x PBS with 10% FBS (1:10) and centrifuge at 800 x g for 5 min. Resuspend the cells in 1 ml of PBS with 10% FBS.</li>
		<li>Add 75 &#181;l streptavidin conjugated with magnetic particles and incubate for 10 mins on ice.</li>
		<li>Add the volume to 2.5 ml with PBS containing 10% FBS. Transfer the cell suspension into a 5 ml polypropylene round bottom tube.</li>
		<li>Insert the tube into a magnetic cell-sorting apparatus and incubate for 10 min. TER119 positive cells will attach to the side of the tube. The unattached TER119 negative erythroblasts will remain in the suspension.</li>
		<li>Pour the cell suspension into a new 5 ml polypropylene round bottom tube.</li>
		<li>Repeat steps 3.2.6 and 3.2.7 to remove the residual TER119 positive cells.</li>
		<li>Pellet the cells at 800 x g for 5 min. Aspirate off the supernatant. Resuspend the cells in 1 ml PBS containing 10% FBS and count live cells with Trypan Blue.</li>
	</ol>
	</li>
</ol>
4. Transduction with Viruses Encoding cDNA or shRNA
<ol>
	<li>Plate the purified TER119 negative fetal liver cells at 3-5 x 105/well in a fibronectin coated 12-well plate (adjust the cell number if using a different plate).</li>
	<li>Add polybrene to a final concentration of 10 &#181;g/ml to facilitate viral transduction. Spin infect the cells with lentiviruses or retroviruses, encoding cDNA or shRNA, at 800 x g for 1-1.5 hr&#160;at 37 &#176;C.</li>
</ol>
5. Culture of the Transduced TER119 Negative Mouse Fetal Liver Erythroblasts
<ol>
	<li>To test gene functions in the early stage of terminal erythropoiesis (Figure 1 dashed lines). <ol. li=""></ol.>
	<ol>
		<li><ol. li="">Culture the transduced cells in Epo free medium (SCF medium) for 12 hr to allow the expression of the transduced genes and maintenance of the progenitor state.</ol.></li>
		<li>Centrifuge the cells at 800 x g for 5 min. Aspirate the supernatant. Resuspend the cells in 1 ml Epo containing medium in each well of a 12-well plate.</li>
		<li>After culture for 24 or 48 hr, harvest the cells for further assays.</li>
	</ol>
	</li>
	<li>Test gene functions in the late stage of terminal erythropoiesis (Figure 1 solid lines).
	<ol>
		<li>Culture the transduced cells immediately in Epo containing medium.</li>
		<li>After culture for 24 or 48 hr, harvest the cells for the further assays.</li>
	</ol>
	</li>
</ol>
6. Staining of the Cultured Fetal Liver Cells for Flow Cytometric Analysis
<ol>
	<li>Wash and resuspend the cells in 1x PBS. Harvest 2 x 105 cultured cells for flow cytometric analysis.</li>
	<li>Incubate the cells with fluorophore-conjugated antibodies for 20 min at room temperature in the dark. To test differentiation, use APC conjugated anti-CD71 and PE conjugated anti-TER119. To test enucleation, use Hoechst 33342 stain in addition to the other antibodies.</li>
	<li>Wash cells with 1x PBS. Resuspend the cells in 0.5 ml PBS containing 1% FBS and propidium iodide (PI) (1 &#181;g/ml) to stain the dead cells and exclude them from analysis.</li>
	<li>Conduct flow cytrometric analysis. Analyze single color stained and unstained control cells first for adjustment and compensation of the flow cytometer.</li>
</ol>

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The authors declare that they have no competing financial interests.

Here we present a unique system to chronologically analyze mouse fetal liver terminal erythropoiesis. Through the application of different culture conditions, we successfully dissected terminal erythropoiesis in early and late stages. This is particularly important to determine the mechanisms of genes with multiple functions. For example, Rac GTPases play important roles in different stages of terminal erythropoiesis. Inhibition of Rac GTPases in the early stage of terminal erythropoiesis influences cell differentiation and proliferation. On the other hand, inhibition the activity of Rac GTPases in the late stage of terminal erythropoiesis blocks enucleation without affecting cell differentiation, proliferation, or survival13.
Several important steps in this protocol should be kept in mind. First, E13.5 mouse fetal livers are preferred for the purification of TER119 negative cells. Older embryos can be used but the proportion of the progenitors out of the total fetal liver cells will be low. Second, it is important to use low endotoxin BSA in Epo medium since it is critical for enucleation, although the mechanism is not clear. Third, although fibronectin was reported to be important for erythropoiesis15, we found that the use of fibronectin coated plate was optional. It does not affect cell proliferation, differentiation, or enucleation in our system. However, fibronectin can be useful for the cells to attach to the plate that might be important for certain applications such as immunostaining. Fourth, a small percentage of reticulocytes or late stage erythroblasts may lose GFP signal that was carried with the transduced cells. Alternatively we use human CD4 as a marker to track the transduced cells. Finally, previous reports recommended removal of erythropoietin from the culture on day 111. We found that it was not necessary to do so since there was no significant difference on cell differentiation, proliferation, or enucleation with or without removal of erythropoietin. On the contrary, change of medium may cause cell loss.
Using the same strategy, we recently discovered more than 30 genes that play novel functions in different stages of terminal erythropoiesis (Zhao et al., unpublished results). Like Rac GTPases, many of these genes play dual functions in both early and late stages of terminal erythropoiesis. This indicates that signaling pathways in terminal erythropoiesis are closely interconnected. Genes involved in actin cytoskeleton remodeling and nuclear condensation are those that tend to play dual functions due to the critical roles of these signaling pathways throughout terminal erythropoiesis. On the other hand, genes involved in pathways such as heme synthesis and generation of erythroid specific plasma membrane tend to be more restricted in the early stage of terminal erythropoiesis16,17. Although there are certain limitations of this experimental strategy, such as relatively low infection efficiency compared to transduction of cell lines, and difficulty of knocking down proteins with long half-life, we recommend a routine practice to use our system given the importance of dissecting gene functions in different stages of terminal erythropoiesis. In addition, this experimental strategy can also be used for other applications. For example, using the SCF medium culture condition, genes that are involved in the maintenance of stem cell properties such as self-renewal or differentiation could be analyzed. Using the method for analyzing late stage erythropoiesis, one could also determine the function of genes that could be involved in autophagy for the removal of organelles.
Broadly, this experimental strategy can be applied to the functional investigation of other hematopoietic systems such as megakaryopoiesis and myelopoiesis. Successful application of this strategy would also reveal the hidden side of the molecular mechanisms of specific genes in different developmental stages of blood cells.

Erythropoiesis is the process of differentiation of multipotent hematopoietic stem cells to mature erythrocytes. This stepwise process includes the formation of committed erythroid burst forming units (BFU-Es), the rapidly dividing erythroid colony forming units (CFU-Es), and morphologically recognizable erythroblasts1,2. Terminal erythropoiesis from CFU-E progenitor cells involves sequential erythropoietin-dependent and independent stages2,3. In the early stage of terminal erythropoiesis, Erythropoietin (Epo) binds to its receptor on the cell surface and induces a series of downstream signaling pathways that prevent cell apoptosis and promote rapid cell divisions and gene expression1,4. In the late stage of terminal erythropoiesis, erythroblasts undergo terminal cell cycle exit, chromatin and nucleus condensation, and extrusion of the highly condensed nuclei5.
Our understanding of terminal erythropoiesis has greatly improved in the last few decades, which is largely due to the successful use of several in vitro and in vivo mouse models6-9. Among these models, in vitro culture of mouse fetal liver erythroblasts provides many advantages including the ease of cell purification, fast proliferation and differentiation, and a closer mimic to human erythropoiesis10,11. In this system, large numbers of erythroid progenitor cells from mouse fetal livers can be easily isolated by the single step purification of TER119 (a marker for the mature erythroid cells) negative erythroblasts. During the two-day culture of the erythroblasts, the differentiation of these cells can be monitored by a flow cytometric analysis based on surface expression of the transferrin receptor (CD71) and the TER119 antigen12. In addition, enucleation of the terminally differentiation erythroblasts can be detected by a DNA maker (Hoechst 33342)13. Furthermore, the purified progenitors can be genetically modified by exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest, which facilitates the mechanistic studies of the functions of gene expression on erythropoiesis11,13,14.
On the other hand, the fast cell growth rate can be a double-edged sword since it is difficult to characterize gene functions in different stages of terminal erythropoiesis. In most cases, it is difficult to determine whether a specific gene functions in the early stage of terminal erythropoiesis since by the time the cDNAs or shRNAs expressed, the cells already passed the early stage. To solve this problem, we developed a unique system to dissect the early and late stages of terminal erythropoiesis. For the early stage of terminal erythropoiesis, genetically modified TER119 negative erythroblasts were cultured in Epo-free medium but containing stem cell factor (SCF), IL-6 and FLT3 ligand to maintain their progenitor status and allow the transduced cDNAs or shRNA to expression13. The cells were changed to Epo containing medium after 12 hr to induce cell proliferation and differentiation. In this way, when the cells started to differentiate, the transduced cDNAs or shRNAs were already expressed. For the late stage of terminal erythropoiesis, TER119 negative erythroblasts were cultured in Epo containing medium immediately after transduction. Therefore, one can analyze the functions of the genes of interest in the late stage of terminal erythropoiesis. In summary, a broad application of this system would help dissect gene functions in different stages of terminal erythropoiesis.

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s MediumIMDM (IMDM)</td><td>Gibco</td><td>12440-053</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GEMINI Bio-product</td><td>700-102P</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Hyclone</td><td>SV30010</td><td>100x</td></tr><tr><td>L-Glutamine</td><td>Hyclone</td><td>SH30034.01</td><td>200 mM</td></tr><tr><td>Mouse FIT3 Ligand (Flt-3L)</td><td>BD Biosciences&amp;nbsp;</td><td>14-8001-80</td><td></td></tr><tr><td>Mouse Recombinant Interleukin-6 (IL-6)&amp;nbsp;</td><td>GEMINI Bio-product</td><td>300-327P</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences&amp;nbsp;</td><td>354008</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>STEMCELL TECH</td><td>9300</td><td>10% stock in IMDM</td></tr><tr><td>Erythropoietin(Epo)</td><td>PROCRIT</td><td>NOC 59676-303-00</td><td>3,000 U/ml stock</td></tr><tr><td>Streptavidin particles Plus</td><td>BD Pharmingen</td><td>557812</td><td></td></tr><tr><td>EasySep Magnet</td><td>STEMCELL TECH</td><td>18000</td><td></td></tr><tr><td>Polypropylene Round-Bottom Tube, 5 ml</td><td>FALCON</td><td>352063</td><td></td></tr></tbody></table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

Figure 1 outlines the experimental strategies. The protocol consists of two independent conditions for targeting the functions of the signaling molecules in the early and late stages of terminal erythropoiesis. TER119 negative fetal liver erythroblasts were purified from E13.5 mouse fetus. Flow cytometric analysis of fetal liver erythroid cells before and after purification demonstrated that the purification was efficient (Figures 2A and 2B). For the early stage of terminal erythropoiesis (Figure 1, dashed lines), after viral transduction, the cells were cultured in Epo-free medium (SCF medium) for 12 hr. During this period, the cells showed minimal differentiation (Figure 2C) with no significant apoptosis observed in the transduced cells (GFP positive) (Figure 3). The cells started to differentiate after transferring to Epo-containing medium (Figure 4), which was similar to the cells cultured in Epo containing medium immediately after transduction (Figure 1, solid lines and Figure 5). During the 2 day culture in Epo containing medium in both methods, most of the erythroid progenitor cells (CD71low TER119low) exhibited induction of the transferrin receptor (CD71) and TER119 on day 2 (CD71high TER119high) (Figure 4A and Figure 5A). Enucleation occurred on day 2, as observed by HoechsthighTER119high (erythroblasts) and HoechstlowTER119high (reticulocytes) (Figure 4B and Figure 5B). It is notable that cells immediately culture in Epo (solid line) was relatively more efficient for differentiation and enucleation than cells cultured first in SCF medium (dashed line).
<img alt="Figure 1" fo:content-width="5in" width="500" src="/files/ftp_upload/51894/51894fig1highres.jpg" /> 
Figure 1. Schematic overview of the experimental strategy. Dashed and solid lines represent methods for studying signaling molecules in the early and late stages of terminal erythropoiesis, respectively.
<img alt="Figure 2" fo:content-width="5in" width="500" src="/files/ftp_upload/51894/51894fig2highres.jpg" /> 
Figure 2. Flow cytometric analysis of mouse fetal liver erythroid cells before and after purification of TER119 negative erythroblasts. (A) Total fetal liver cells stained with TER119 and CD71. (B) Purified TER119 negative cells stained with TER119 and CD71. (C) Purified TER119 negative cells after 12 hr culture in SCF medium. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51894/51894fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" width="500" src="/files/ftp_upload/51894/51894fig3highres.jpg" /> 
Figure 3. Flow cytometric analysis of apoptosis after culture in Epo-free medium (SCF medium) for 12 hr. (A-B) The TER119 negative erythroblasts were transduced with control lentiviruses (A) or lentiviruses encoding green fluorescent protein (GFP) (B). The percentage of the GFP positive transduced cells was presented after 12 hr of culture in SCF medium. (C-D) Analysis of apoptosis in GFP negative (C) and positive (D) cells from (B).
<img alt="Figure 4" fo:content-width="5in" width="500" src="/files/ftp_upload/51894/51894fig4highres.jpg" /> 
Figure 4. Flow cytometric analysis of differentiation and enucleation of the transduced erythroblasts cultured in SCF medium followed by Epo medium. (A) As in Figure 3, GFP negative and positive cells were gated for differentiation analysis using CD71 and TER119 after the cells were changed to Epo medium and cultured for an additional 48 hr. (B) Enucleation assay using Hoechst and TER119 of the indicated cells after 48 hr in Epo medium.
<img alt="Figure 5" fo:content-width="5in" width="500" src="/files/ftp_upload/51894/51894fig5highres.jpg" /> 
Figure 5. Flow cytometric analysis of differentiation and enucleation of the transduced erythroblasts directly cultured in Epo medium. (A) The TER119 negative cells were transduced with lentiviruses encoding GFP. The cells were immediately cultured in Epo medium after transduction. Cell differentiation analysis of GFP negative and positive cells was performed using CD71 and TER119 after 48 hr in culture. (B) Enucleation assay of the indicated cells using Hoechst and TER119 after 48 hr in culture.
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		</tr>
		<tr >
			<td>IMDM</td>
			<td>41.385 ml</td>
		</tr>
		<tr >
			<td>Fetal bovine serum (FBS, 15%) </td>
			<td>7.5 ml</td>
		</tr>
		<tr >
			<td>b-mercaptoethanol (10-4 M)</td>
			<td>50 &#181;l (10-1 M stock in IMDM)</td>
		</tr>
		<tr >
			<td>Penicillin-Streptomycin (1%)</td>
			<td>500 &#181;l</td>
		</tr>
		<tr >
			<td>Glutamine (2 mM)</td>
			<td>500 &#181;l</td>
		</tr>
		<tr >
			<td>Stem Cell Factor (50 ng/ml)</td>
			<td>25 &#181;l from 100 ng/ml stock</td>
		</tr>
		<tr >
			<td>FLT3 Ligand (FLT-3L) (30 ng/ml)</td>
			<td>30 &#181;l from 50 ng/ml stock</td>
		</tr>
		<tr >
			<td>IL-6 (20 ng/ml)</td>
			<td>10 &#181;l from 100 ng/ml stock</td>
		</tr>
	</tbody>
</table>
Table 1. Ingredient for SCF medium (Epo free medium).
<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td>Ingredient</td>
			<td >Quantity</td>
		</tr>
		<tr >
			<td>IMDM</td>
			<td>36.3 ml</td>
		</tr>
		<tr >
			<td>FBS (15%)</td>
			<td>7.5 ml</td>
		</tr>
		<tr >
			<td>Bovine Serum Albumin (BSA, 10%)</td>
			<td>5 ml (10% stock in IMDM)</td>
		</tr>
		<tr >
			<td>Insulin (10 mg/ml)</td>
			<td>50 &#181;l from 4 &#176;C stock</td>
		</tr>
		<tr >
			<td>Holo-transferrin (200 mg/ml)</td>
			<td>200 &#181;l (50 mg/ml in Water, -20 &#176;C stock)</td>
		</tr>
		<tr >
			<td>b-mercaptoethanol (10-4 M)</td>
			<td> 50 &#181;l (10-1 M stock in IMDM)</td>
		</tr>
		<tr >
			<td>Penicillin-Streptomycin (1%)</td>
			<td>500 &#181;l </td>
		</tr>
		<tr >
			<td>2 mM Glutamine</td>
			<td>500 &#181;l </td>
		</tr>
		<tr >
			<td>Epo (2 U/ml)</td>
			<td>33 &#181;l (3,000 U/ml stock)</td>
		</tr>
	</tbody>
</table>
Table 2. Ingredient for Epo containing medium.

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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14.9K Views.  Northwestern University. We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages. 

Video: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

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, ...

Developmental Biology

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 ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

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 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 ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

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Chapters in this video

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Cell Type-specific Gene Expression Profiling in the Mouse Liver