The authors would like to acknowledge the invaluable help of:
Dr Neal First and Kourtney Wilkinson for manuscript editing; Dr Lucy Senter, Dr. Brigit Willeford and Mike Basett for assistance with training and animal care at the MSU ALAC accredited mouse facility; Dr Dwayne Wise for his assistance with microscopy and image capture; Cesar Monroy, Hannah Swoope, and Bobbie Huddleston for their assistance with video production at MSU. This research was funded by Office of Institutional Research and the Department of Biological Sciences at Mississippi State University.

Part 1: Pregnant Mouse Laparotomy
<ol>
 <li>Using cervical dislocation, euthanize a pregnant C57BL6J female mouse at 10.5-11.5 dpc.</li>
 <li>Clean the abdomen with antimicrobial soap, and then, shave it.</li>
 <li>After shaving, wash the abdomen with a saline solution. </li>
 <li>Then, dry the abdomen with a sterile gauze.</li>
 <li>Cover the mouse with a new sterile field.</li>
 <li>Make a ventral incision using forceps and dissection scissors.</li>
 <li>Identify and remove the entire uterus from the abdominal cavity. Mouse embryos will be visible inside the uterus. </li>
 <li>Transfer the uterus into a petri dish filled with D-PBS, and keep it on ice.</li>
 <li>Using a sterile scalpel and forceps, remove the placenta and extra embryonic tissues of each embryo.</li>
 <li>Transfer embryos into a fresh petri dish filled with D-PBS.</li>
 <li>Measure the length of the mouse embryos. We found that the sizes differed according to the embryo&#x2019;s age. For example 8.5 dpc measured at an average of 6mm, 10.5 dpc measured at an average of 11mm, and 12.5 dpc measured at an average of 16 mm. </li>
 <li>Then, remove their tails for DNA extraction.</li>
</ol>
Part 2: Gonadal Ridge Dissection
Under stereomicroscope and Light source Schott Fostec
<ol>
 <li>Place a filter paper in a Petri dish. Then, place the mouse embryo on top of the filter paper to dry the embryo and immobilize for dissection.</li>
 <li>Using a sterile scalpel, make a transverse cut of the mouse embryo above the origin of the umbilical cord.</li>
 <li>Under a stereomicroscope, using sterile fine forceps and a sterile fine teasing needle, remove the intestines and liver so that the urinary system is visible. 
 The kidneys are located laterally in the abdominal cavity, and the bladder is located medial and caudal in comparison to the kidneys. The embryo can be identified as male because the gonadal ridges are located on either side of the bladder. In the female, the gonadal ridges are firmly attached to the caudal lateral end of the kidneys.</li>
 <li>Peel the gonadal ridge out by sliding a needle behind it. The gonadal ridge is removed by cutting away the tissues that support it.</li>
 <li>Transfer the gonadal ridges to a new petri dish filled with fresh D-PBS. 
 Microscopically, the male gonadal ridge should be stripped, large and oval in shape. In contrast, the female gonadal ridge should be spotted, have elongated shape and be smaller in comparison to the male gonadal ridge.</li>
 </ol>
Part 3: Gonadal Ridge Digestion
 Under stereomicroscope:
<ol>
 <li>Mesonephros Dissection: Separate the gonad from the mesonephros and ridge using a fine and sharp needle.
<ul>
 <li>It is important to remove the mesonephros from the gonadal ridge because it is a somatic tissue that will overgrow in the PGC derivation process.</li>
</ul>
</li>
 <li>Gonadal Ridge Digestion: Collect clean gonadal ridges in a 0.5ul drop of fresh D-PBS. Add 20 ml of Collagenase/Dispase Solution (Final concentration at 1mg/ml).<ul>
 <li>Embryos should be processed individually. Each embryo should have its own separate and labeled petri dish. </li>
</ul>
</li>
<li>Gonadal Ridge Mincing: Cut the gonadal ridge into small pieces using a sterile needle and sterile fine forceps No. 4.</li>
<li>Incubation: Petri dishes are transferred to an incubator at 37&deg;C for 15 minutes.
 <ul>
 <li>The incubation time could vary between tissues.</li>
 </ul>
</li>
<li>Pipetting: After 15 minutes, the tissue can be dissociated by pipetting. Using pulled glass capillary pipettes with diameters between 40 -100 mm Break up the pieces by pipetting up and down. This will form small clumps. Transfer the clumps to 0.5ml of a pre warmed D-PBS drop for a final wash.</li>
 <li>After washing, transfer the clumps to eppendorf tubes.
 <ul>
 <li>Do not over expose the tissue to collagenase.</li>
 <li>Do not make a single cell suspension. This is important for colony formation. If it is a single cell suspension, PGCs tend to differentiate and migrate.</li>
 <li>This process should not be longer than 20 minutes, or your cells will lose viability.</li>
</ul>
</li>
 <li>Centrifuge at 2000 rpm for 5 minutes at room temperature. </li>
 <li>After centrifuging, remove the supernatant, and resuspend the pellet in 0.5 ml of supplemented knock out media (Pre-warmed at 37&deg;C).</li>
</ol>
Part 4: Primordial Germinal Cells Culture
"You should perform the next steps quickly to preserve the viability of PGCs for isolation and derivation.
<ol>
 <li>24 hours before this process you should prepare a mitotically inactivated MEF-CF1 feeder layer following the protocol from Zhang J. et al 2008.
<ul>
 <li>Prepare 20 dishes for one pregnant female mouse (6-8 embryos).</li>
 <li>MEFs lose the ability to be a feeder layer (promote growth and prevent differentiation) after 5-6 passages.</li>
 <li>Do not use MEFs older than 48 hours.</li>
</ul>
</li>
 <li>Immediately before plating PGCs, the MEF medium must be slowly removed from the MEF feeder layer.</li>
 <li>Then, add 0.5 ml/well of pre-warmed supplemented knockout medium onto the MEF feeder layer.
<ul>
 <li>MEF media contains fetal bovine serum that induces differentiation of PGCs.</li>
 <li>Knockout medium must be supplemented with specie specific growth factors immediately before use to ensure the activity of the grow factor.</li>
</ul>
</li>
 <li>Using symmetrical distribution, plate PGCs over the MEF feeder layer.
<ul>
 <li>If these pieces are too close, they tend to aggregate and make dense colonies that attach poorly or begin differentiating.</li>
 <li>Culture dishes must be labeled with Embryonic Germinal cell line name, passage number, and date.</li>
</ul>
</li>
 <li>Carefully, transfer the culture dishes into an incubator at 37&deg;C and 5% CO2.</li>
 <li>Monitor the cells every 24 hours.</li>
 <li>After 48 hours, remove 250 ml of media from the very top of the culture to avoid disturbing the colony's attachment. Then, add 250 ml of fresh supplemented knock-out media (pre-warmed at 37&deg;C).</li>
 <li>After step 7, wait another 48 hours, and then, completely remove all media and replace it with fresh supplemented knock-out media (pre-warmed at 37&deg;C). Do this every 2 days for 8-10 days until PGC colonies form.</li>
</ol>
Part 5: Embryonic Germinal Cells 
<ol>
 <li>After 10 days of PGC isolation, embryonic germinal cell colonies are formed. </li>
 <li>Embryonic germinal cell lines are kept alive by manual passages every 8-10 days. They continue to present an undifferentiated morphology and express pluripotency markers such as alkalyne phosphatase, Oct-4, SSEA-1, and SOX-2</li>
</ol>
Notes
<ul>
 <li>Sterile/aseptic conditions are essential in the culture room. </li>
 <li>For derivation and culture, PGCs are processed in a Purifier Class II Bio safety Cabinet.</li>
 <li>Incubations are in a humidified 37&deg;C and 5% CO2 incubator.</li>
 <li>All media and reagents are filtered prior to use in a 0.2 um filter, stored at 4&deg;C, and pre-warmed at 37&deg;C before use.</li>
 <li>During the dissection steps, all embryos and tissues must be kept on ice.</li>
 <li>All reagent flasks are decontaminated with ethanol before placing in a cabinet.</li>
 <li>Use of gloves, lab coat, and nurse caps are mandatory.</li>
</ul>
Conclusion
We have provided a video that shows you how to isolate, derive, and culture embryonic germinal cells from gonadal ridges of 10.5-11.5 dpc mouse embryos. The reproducible isolation and long term culture of Embryonic germinal cell lines provides a critical foundation for study of early embryonic development, genetic and epigenetic germinal patterns, gonad formation, environmental effects of surrounding organs during the developmental process of gametogenesis in the male and female embryos, and determination of pathways that leads to cancer and infertility.
<img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Figure 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

This research was conducted after review and Institutional Animal Care and Use Committee (IACUC) approval of our mouse protocol #08030 (The Role of Reactive Oxygen Species -ROS in germ line development and tumor formation: Holding and breeding protocol) at Mississippi State University.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>10.5 - 11.5 dpc pregnant C57BL6J female mouse crossed with the same background male mouse.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Fibroblast MEF-CF1 ATCC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>SCRC-1040</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190/086</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase /Dispase Solution</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>269638</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
<tr>
<td>Mitomycin C</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4287</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>MEF Medium: Dulbecco Medium Eagle Modified (D-MEM) GLUTAMAX High glucose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10566024</td>
<td>&nbsp;</td>
<tr>
<td>10% Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000/044</td>
<td>&nbsp;</td>
<tr>
<td>1% of antibiotic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420/096</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Media: 80% knockout D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829/018</td>
<td>&nbsp;</td>
<tr>
<td>20% Knockout serum Invitrogen Cat. No. 1028/028, 200mM L-glutamine Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>25130/081</td>
<td>with &beta;-mercaptoethanol Sigma Catalogue Number: M7522</td>
</tr>
<tr>
<td>1X non essential amino acid solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140/050</td>
<td>&nbsp;</td>
<tr>
<td>1% antibiotic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420/096</td>
<td>supplemented with grow factor as 2500 U of Leukemia inhibitory factor (mLIF-ESGRO) Millipore Catalogue Number: ESG1106</td>
</tr>
<tr>
<td>40 ng/ml of Recombinant Murine Stem Cell Factor (SCF) ReproTech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>250-03</td>
<td>&nbsp;</td>
<tr>
<td>20 ng/ml of Basic Fibroblast Growth factor (bFGF)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Corning center well culture dish 60mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>07-200-79</td>
<td>1.5 ml eppendorf tube, and 15 ml falcon tubes</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			Forceps No. 14 and 15, scalpel 35 mm, dissection scissors, surgical fields, antimicrobial soap, saline solution 0.9 %, gauze, ice foam container, razor blade, scalpel 35 mm, fine forceps No. 4 and 5, fine teasing needle with handle, filter paper sheets, and pulled glass pipettes. Equipment: dissecting stereomicroscope, light source Schott Fostec, inverted microscope, micropipette 10 and 200ul, centrifuge, bio safety Cabinet.		</div>

isolation and derivation of mouse embryonic germinal cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

isolation-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.0K 조회수.  Mississippi State University. 초기 개발 단계 동안 원시 종자 세포로 차별화하는 배아 새싹 세포의 능력은 암, 불임에 관한 우리의 가설을 해결하기 위해 완벽한 모델​​입니다. 이 프로토콜은 10.5-11.5 일 게시 coitum 마우스 배아의 개발 생식선에서 원시 종자 세포를 분리하는 방법을 보여줍니다.

비디오: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...