The authors thank Janet Rossant for the original protocol. This study was supported by National Institute of Health grant CA106308 to WH.

In this protocol, we describe the preparation of mouse blastocysts and the establishment of TS cell lines. General mouse manipulations prior to the collection of blastocysts, including the set up for natural mating and the induction of super ovulation, basically follow the standard protocol illustrated by Nagy et al. 2003 (pp146-150). 
Derivation and maintenance of TS cells
<ol>
 <li>Setup breeding cross between mice of interest. </li>
 <li>Prepare mouse embryonic fibroblasts (MEFs) as feeder cells. Two days before the collection of blastocysts, MEFs (2 x 106 cells) are plated in 100 mm dishes. Next day, these cells are treated with 10 ml of TS medium containing mitomycin C (20 mg/ml) for a 2 hour-incubation at 37&deg;C, 5% CO2. This is followed by washing cells twice with PBS. The mitomycin C treated cells are then seeded in 12-well plates (1 x 105 cells/well). </li>
 <li>On the day of blastocyst collection (3.5 dpc, referred to as day one), MEF culture medium is replaced by TS medium plus 1x FGF4/Heparin (0.5 ml/well). </li>
 <li>Preparation for collection of the uterus: euthanize the mouse and locate the uterus (Figure 1A).</li>
 <li>Remove the uterus by grasping it with forceps at the cervix (located behind the bladder) and cut across the junction of uterus and cervix (Figure 1B). Pull the uterus, trim the membrane and fat tissues away and cut the uterus below the junction with the oviduct (Figure 1C). Place the uteri (Figure 1D) in a small volume (0.2 ml) of TS medium in a 60 mm plate. </li>
 <li>Flush each uterus horn with 1 ml of TS medium using a 1-ml syringe with a 26-gauge needle. Insert the needle into the base of uterus and flush slowly into a new 60 mm plate with 1 ml TS medium. The uterus should be inflated and extended fully during flushing. </li>
 <li>Collect and transfer the blastocysts carefully into a clean 12 well plate containing TS medium using a mouth-controlled pipette. Wash blastocysts three times by serial transfer through TS medium in 12-well plates. Place one blastocyst per well (Figure 2A) in a 12-well plate containing the mitomycin C-treated MEF feeders and culture at 37&deg;C, 5% CO2. </li>
 <li>The blastocysts will hatch from zona pellucida and attach to the wells in 24 to 36 hours. A small outgrowth will be readily observed on day 3. Feed the cultures with 500 &mu;l fresh TS medium plus 1x FGF4/Heparin.</li>
 <li>On day 4 or 5, the blastocyst outgrowth should be ready for disaggregation depending on its size. The ideal size for TS cell disaggregation is illustrated in Figure 3B. Wash the cultures once with 0.5 ml PBS. Add 100 ml 0.25% trypsin/EDTA and incubate for 5 minutes at 37&deg;C, 5% CO2. Break the outgrowth into small clumps by pipetting up and down vigorously with a P100 pipetteman. Stop the trypsinization by adding TS-70CM-1.5F4H (30% TS medium, 70% MEF-CM plus 1.5x FGF4/Heparin) into the well. Change medium 8 hours after disaggregation and continue to feed cells every 2 days.</li>
 <li>Between days 6 and 10 (highly variable) TS cell colonies can be observed. They have flat, epithelial sheet like morphology with a clear colony boundary (Figure 2C). Continue to feed the cultures every 2 days </li>
 <li>When TS cells reach 50% confluency (usually between days 15 and 20), wash once with PBS and add 100 ml 0.25% trypsin/EDTA. Pipette up and down during trypsinization to ensure a near single-cell suspension. Stop the trypsinization by adding TS-70CM-1.5F4H and transfer to new 6-well or 60mm plates containing the mitomycin C-treated MEF feeders (2.5 x 105 cells/6-well or 5 x105 cells/60mm plate). </li>
 <li>Change medium 8 hours after passage and continue to feed the cultures every 2 days. </li>
 <li>After one or two more passages (3-5 days between passages) on the MEF feeders, TS cells may be cultured without them. Because MEFs attach to the culture plate much faster than TS cells after trypsinization, such difference in the adherent rate can be utilized to separate TS cells from the MEF feeders. After passing the cultured cells, incubate them at 37&deg;C, 5% CO2 for 30 min. &nbsp;Most MEFs will attach to the plate and the TS population remains in suspension. Transfer the supernatant to a new plate. Repeat this process for several passages to obtain a pure TS population. </li>
 <li>Maintain TS cells in TS-70CM-1.5F4H and pass them every 4 days (1:10 to 1:20 split) or when the culture reaches 70% confluency. Passing the cells with high density (higher than 1:5 split) or culturing them when overly confluent may cause their differentiation. </li>
 <li>The identity of TS cells can be confirmed by immunostaining of a cell marker, Cdx2 (Figure 3). </li>
</ol>
Freezing TS cells
<ol>
 <li>Prepare 2x freezing medium (50% FBS, 30% TS medium and 20% DMSO). </li>
 <li>Collect TS cells by trypsinization, followed by centrifugation at 450g for 6 min. Resuspend them in TS medium with equal volume of 2x freezing medium. </li>
 <li>Freeze the cells slowly using an isopropyl alcohol chamber at -70&deg;C and transfer to liquid nitrogen after 24-48 hours. </li>
 <li>A 60 mm plate (70% confluent) is usually kept in 2 vials. </li>
</ol>
Thawing TS cells
<ol>
 <li>Quickly thaw the vial in 37&deg;C, collect them in 10 ml TS media by centrifugation (450g, 6 min), and resuspend in 30% TS media, 70CM-1.5F4H. </li>
 <li>One vial of cells is seeded in a 60 mm plate. </li>
</ol>
TS cell differentiation into TGC
Differentiation of TS cells into trophoblast giant cells (TGCs) can be induced by the withdrawal of MEF-CM, FGF4 and heparin. The TGC phenotype (large nuclei) can be detected four days after the induction (Tanaka et al., 1998 and Chiu et al. 2008). Immunostaining of p450scc detect the expression of this TGC marker in the differentiated culture (Figure 4A). Flow cytometric analysis of PI (propidium iodide) stained cells further shows the presence of differentiated cells containing higher DNA content (Figure 4B). These results suggest that TS cells undergo endoreduplication to become polyploid TGCs.
Representative Results
<img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Figure 1" /> 
 Figure 1. Dissection of mouse uteri. (A) Location of the uteri is indicated by arrows. (B) Cut across the junction of uterus and cervix as shown. (C) Cut the uterus just below the oviduct (OD) underneath the ovary (OV). (D) Image of the uteri. 
<img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Figure 2" /> 
 Figure 2. Representative pictures illustrate mouse blastocyst, blastocyst outgrowth and trophoblast colony. (A) A mouse blastocyst collected at E3.5 is shown. ICM, inner cell mass; MT, mural trophectoderm; PT, polar trophectoderm. (B) A trophoblast outgrowth on MEF feeders is shown. TS, trophoblast stem cell. (C) A trophoblast stem cell colony shows a clear edge on MEF feeders. Arrows indicate the cell boundary.
<img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Figure 3" /> 
 Figure 3. Identification of TS cells by immunostaining analysis. TS (A-D) and ES (control, E-H) cells are subject to marker identification. Cells are immunostained with antibodies to recognize Oct4 (A, E) or Cdx2 (B, F), and counterstained by DAPI (C, G) presented in merged images (D, H). Scale bar, 50 &mu;m. 
<img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Figure 4" /> 
 Figure 4. Differentiation of TS cells in vitro. (A) Undifferentiated (Undiff) or differentiated (Diff) cells in culture were examined for the expression of a TGC marker, p450scc (green), counterstained with DAPI (red). (B) Flow cytometric analysis of the differentiated cells, stained with PI, measures the DNA contents (M1, two to four copies; M2, more than four copies). The M2 population represents the polyploid trophoblast cells.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

No conflicts of interest declared.

In this video, we demonstrate the process to collect E3.5 blastocysts from uteri and experimental procedures to establish TS cell lines. We also describe the condition to maintain the stemness of TS cells and to induce their differentiation into differentiated cells. Two critical steps to obtain pure TS cell lines are the timing for outgrowth disaggregation (step 9) and processing the first passage (step 11). It has been reported that primitive endoderm-derived cells can arise in the culture after extensive blastocyst ou...

Reagents TS medium: RPMI-1640, 20% FBS, 1 mM sodium pyruvate, 100 mM beta-mercaptoethanol and 100 mg/ml penicillin-streptomycin. Mitomycin C: Dissolve 2 mg mitomycin C (Sigma M-0503) in 2 ml PBS and add this 2 ml mixture into 200 ml TS medium (10 ng/ml). Make 20 aliquots (10 ml) and store at -20&deg;C until use. Add one aliquot per 100 mm plate. Mouse embryonic fibroblast-condition medium (MEF-CM): Plate MEFs in 100 mm plates (1 x 106/plate). Next day, add 10 ml TS medium per dish and continue to culture for 48 hours. Collect the culture medium, filter them (0.45 mm) and store at -20&deg;C in 35 ml aliquots. Thaw each aliquot when needed which can be stored at 4&deg;C. The MEFs can be used to prepare for two more batches of CM before they become confluent. PBS/BSA: Dissolve BSA, fraction V (Sigma A3311, 0.1% (w/v)) in PBS (10 ml), filter through a 0.45 mm syringe filter and make 1 ml aliquots in 1.5 ml tubes. Store at -70&deg;C and thaw one tube when needed to prepare FGF4. FGF4 (1000x): Resuspend lyophilized FGF4 (Sigma F8424, 25 mg) with 1 ml of the PBS/BSA solution. Mix well and make 10 aliquots (100 ml) into 1.5 ml tubes and store at -70&deg;C. Thaw each tube when needed and store the remaining at 4&deg;C, do not re-freeze. Dilute 1000 times in TS medium to obtain 1x FGF4 (25 ng/ml). Heparin (1000x): Resuspend heparin (Sigma H3149, 10,000 units) in PBS to a final concentration of 1 mg/ml (1000x). Make 100 ml aliquots into 1.5 ml tubes and store at -70&deg;C. Thaw aliquots when needed and store the remaining at 4&deg;C, do not re-freeze. Dilute 1000 time in TS medium to obtain 1x heparin (1 ng/ml).

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.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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 ...

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18.1K Views.  University of Rochester. 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.

Video: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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 ...

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.

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 of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...