This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1. Embryoid Body (EB) Formation
<ol>
	<li>Gelatin-adapt ESCs that have been maintained on mouse embryo fibroblasts (MEFs). Passage ESCs 3 times on 6 well tissue culture plate coated with 0.1% gelatin to remove MEFs.
	<ol>
		<li>Grow cells in ESC maintenance media with LIF to keep the cells undifferentiated (Table 1). Make sure cells never exceed 80% confluence. Use low passage (10 or less passages after removing from MEFs) cells for differentiation.</li>
	</ol>
	</li>
	<li>On the day before ESCs are cultured for EB differentiation, passage the cells so they will be approximately 50-70% confluent the next day. Continue culturing cells in ESC maintenance medium to retain cell pluripotency.</li>
	<li>On the day of the differentiation, aspirate ESC maintenance media and wash with 1 ml Phosphate Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphate buffer, pH 7.4) per well of a 6-well plate.
	<ol>
		<li>Add 200 &#181;l of 0.25% trypsin/EDTA to each well and incubate at 37 &#176;C for 3 min.</li>
		<li>Inactivate the trypsin/EDTA with 800 &#181;l of differentiation media. Pipet up and down in a 2 ml serological pipet with a p200 tip to break-up the cells into a single cell suspension. Ensure that the p200 tip fits snugly on the end so media does not go up between the pipet and tip interface.</li>
		<li>Pipet up and down approximately 5-10 times. Take care not to create bubbles during the pipetting.</li>
	</ol>
	</li>
	<li>Transfer cells to a 15 ml conical tube. Bring up the volume to 10 ml with PBS. Centrifuge at 315 x g for 5 min at room temperature.</li>
	<li>Remove supernatant. Wash twice with 5 ml PBS to remove remaining media containing LIF. Centrifuge as in step 1.4 for each wash.
	<ol>
		<li>Resuspend the final cell pellet in 2 ml of differentiation media (Table 2).</li>
	</ol>
	</li>
	<li>Count cells with a hemocytometer or cell counter and plate 6,000-10,000 cells/ml in a 10 cm Petri plate (non-tissue culture treated).
	<ol>
		<li>Determine the exact cells/ml empirically for each ESC line. Use sterile Petri plates normally used for bacterial work or low adherence plates. 
		NOTE: For best differentiation results, the EBs should form spheres that remain in suspension without attaching to the plate. Test several brands of plates to find ones with the least adherence.</li>
		<li>Incubate the differentiating ESCs in a 37 &#176;C tissue culture incubator with 5% CO2.</li>
	</ol>
	</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col span="2" />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock</td>
			<td>Final Concentration</td>
			<td>Volume</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>1x</td>
			<td></td>
			<td>410 ml</td>
			<td>Sigma/Aldrich</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>FBS (ES Screened)</td>
			<td>100%</td>
			<td>15%</td>
			<td>75 ml</td>
			<td>Hyclone</td>
			<td>SH30070.03E</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>35050</td>
		</tr>
		<tr>
			<td>Penncillin/Streptomycin</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>15070</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>114 &#181;M</td>
			<td>4 &#181;l</td>
			<td>Sigma/Aldrich</td>
			<td>M3148</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor (LIF)</td>
			<td>107 units/ml</td>
			<td>1,000 units/ml</td>
			<td>50 &#181;l</td>
			<td>Millipore</td>
			<td>ESG1107</td>
		</tr>
	</tbody>
</table>
Table 1: ESC Maintenance Media.
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col span="2" />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock</td>
			<td>Final Concentration</td>
			<td>Volume</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>1x</td>
			<td></td>
			<td>410 ml</td>
			<td>Sigma/Aldrich</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>FBS (User Screened for optimal differentiation)</td>
			<td>100%</td>
			<td>15%</td>
			<td>75 ml</td>
			<td>Hyclone</td>
			<td>SH30070.03E</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>35050</td>
		</tr>
		<tr>
			<td>Penncillin/Streptomycin</td>
			<td>100x</td>
			<td>1x</td>
			<td>5 ml</td>
			<td>Life Technologies</td>
			<td>15070</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>114 &#181;M</td>
			<td>4 &#181;l</td>
			<td>Sigma/Aldrich</td>
			<td>M3148</td>
		</tr>
	</tbody>
</table>
Table 2: Differentiation Media.
2. Preparing EB Cells for Virus Infection
<ol>
	<li>At the desired stage of development (between day 2 and day 3), transfer the EBs from the 10 cm Petri dish to 15 ml conical tube. Wash the plate with 5 ml PBS and add the wash to the conical tube.</li>
	<li>Pellet the EBs at 315 x g at room temperature for 5 min. Wash the EB cells with 5 ml PBS.</li>
	<li>Add 1 ml of thawed cell detachment solution (Containing proteolytic and collagenolytic enzymes) and place the tubes in a 37 &#176;C water bath (or incubator) for 30 min with occasional agitation (from flicking or light vortexing).</li>
	<li>Add 1 ml of differentiation media. Pipet up and down with a 2 ml pipet with a p200 pipet tip. Place the tube in 37 &#176;C water bath again for 30-60 min.</li>
	<li>Spin down the cells at 315 x g for 5 min and wash the cells with PBS.</li>
</ol>
3. Virus Infection
<ol>
	<li>Resuspend the cells in 1.5 ml of differentiation media. Transfer the cell suspension to a 6-well non-tissue culture treated plate.</li>
	<li>Prepare the retrovirus by standard techniques ahead of time. Add 5 to 100 &#181;l of viral supernatant to cells depending on viral titer. Do not add polybrene to enhance infection as this inhibits the subsequent reformation of EBs.</li>
	<li>Spinoculate the cells with virus by centrifuging at 2,120 x g for 90 min at room temperature.</li>
</ol>
4. Differentiating Viral Infected EB Cells in Hanging Drops
<ol>
	<li>Add 2 ml of differentiation media to each well of infected EB cell suspension.</li>
	<li>Pipet 3.5 ml of infected EB cell suspension into a sterile reagent reservoir for multipipettors. Use the multipipettor to pipet 15-20 rows of eight 20 &#181;l drops onto the inverted lid of the 15 cm Petri plate.</li>
	<li>Add 10 ml of sterile PBS or water to the bottom half of the Petri plate. Then invert the lid with the drops and place onto the Petri plate. Ensure that the drops are hanging upside down from lid with PBS or water below to keep the chamber humidified.</li>
	<li>Place the dishes in the 37 &#176;C tissue culture incubator with 5.0% CO2.</li>
	<li>After 2 days, collect EBs formed in the hanging drops by inverting lid and washing with 4.5 ml of differentiation media.
	<ol>
		<li>Transfer media and EBs to a new 10 cm non-tissue culture Petri plate. Wash the lid once more with 3 ml of differentiation media and transfer to the 10 cm plate.</li>
	</ol>
	</li>
	<li>Harvest cells for analysis by flow cytometry after a total of 8 days culture starting from the initial transfer of cells to differentiation media (-LIF).</li>
</ol>
5. Preparing Cells for Flow Cytometer Analysis
<ol>
	<li>Transfer cells to a 15 ml conical tube with a 10 ml pipet. Wash the plate with 5 ml PBS and transfer to the same conical tube.</li>
	<li>Pellet cells at 315 x g. Wash pellets with 10 ml PBS.</li>
	<li>Add 1 ml of thawed cell detachment solution and place the tubes in a 37 &#176;C water bath for 30 min with occasional agitation. For detection of some cell surface antigens, trypsin may be used instead.</li>
	<li>Add 1 ml differentiation media and pipet up and down with a 2 ml pipet. Use a p200 tip to the end of the pipet to help break-up digested EB into single cell suspension.
	<ol>
		<li>Place the tube in a 37 &#176;C water bath again for 60 min in order for the integral membrane proteins to recycle to the cell surface.</li>
	</ol>
	</li>
	<li>Pellet cells at 315 x g for 5 min.</li>
	<li>Wash the cells with 0.1% Bovine Serum Albumin Fraction V (BSA) prepared in PBS (PBS/BSA) and incubate the cells with specific fluorescently tagged antibodies.</li>
</ol>
6. Flow Cytometry Analysis for Hematopoietic Progenitors
<ol>
	<li>Place 106 EB derived cells in a 5 ml 12 x 75 mm round bottom tube. For adjusting flow cytometer compensation settings, use compensation beads according to manufacturer instructions.</li>
	<li>Pellet cells at 315 x g. Pour off supernatant and resuspend pellet in residual PBS/BSA solution in the tube.</li>
	<li>For detecting ESC HPCs, incubate the cells with fluorescent labeled antibodies: anti-mouse CD41-PE (R-Phycoerytherin) and anti-mouse CD117 (cKit)-APC/CY7 (Allophycocyanin Cyanine 7).
	<ol>
		<li>Dilute antibodies 1:100 in PBS/BSA. Add 100 &#181;l of diluted antibody to each sample. Determine the correct dilution of antibody for each vendor, clone, and lot or antibody. Determine optimal dilution for labeling cells individually.</li>
	</ol>
	</li>
	<li>Incubate samples on ice in the dark for 30 min.</li>
	<li>Add 2 ml PBS/ BSA and pellet cells at 315 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend pellet in 300 &#181;l of PBS or another isotonic buffer.</li>
	<li>Filter resuspended cells through a cell strainer cap (0.35 &#181;M) to remove large cell aggregates.</li>
	<li>Analyze cells on a flow cytometer.</li>
</ol>

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The authors have no competing financial interests to disclose.

As discussed above, ESC clones that inducibly express a gene of interest can be generated using doxycycline systems, however, generation of these lines is time-consuming and labor intensive. Described in this protocol is a method to express a gene of interest in single cell suspension prepared from ESC derived EBs. These infected cells are then reformed into EBs by hanging drop to examine subsequent differentiation. In the example (Figure 3), it is shown that expression of the mirn23a cluster en...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

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retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

In these studies gelatinized RW4 (Derived from 129X1/SvJ mouse strain) ESCs were used. EBs were isolated at 3 days of differentiation. For best results the EBs should be spherical and non-adherent (Figure 1A, B). After spinoculation with virus co-expressing the fluorescent marker GFP along with a gene of interest, EBs were reformed by the hanging drop method. EBs were successfully reformed from cell suspensions prepared from 2.0, 2.5, and 3.0 day EBs. However, EBs could not be reformed from cell suspensi...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

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8.4K 조회수.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

비디오: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...