PWD is supported by a Canadian Institutes of Health Research fellowship (CIHR) and a CIHR grant to Gerald M. Kidder at Western University. The author would also like to acknowledge Julang Li at the University of Guelph for help with the development of the protocol presented here.

1. Media Preparation	
	<ol>
		<li>Prepare media 2-3 hr in advance of use and place with a loosened cap in the cell culture incubator where the experiment will be taking place. Media used in the present protocol:
			<ol>
				<li>Prepare stem cell medium consisting of DMEM/F12 supplemented with 1 x B27, 40 ng/ml bFGF, and 20 ng/ml EGF.</li>			 
				<li>Prepare germ cell differentiation medium consisting of m199 supplemented with 0.05 IU FSH, 0.03 IU LH, 3 mg/ml BSA, 5 &mu;l/ml ITS, 0.23 mM sodium pyruvate, 1 mg/ml Fetuin, and 1 ng/ml EGF. A base medium consisting of all additives but omitting FSH, LH and EGF can be prepared and stored at 4 &deg;C for up to one week.</li>			
			</ol>
		</li>	
	</ol>
	2. Stem Cell Culture Preparation	
	<ol>
		<li>Isolate stem cells from newborn mouse pups as previously described12. Utilize stem cells at passage 2-4 for in vitro germ cell differentiation. The efficiency of OLC formation is directly related to the quality of the starting stem cells. It is best to start the differentiation as soon as the population appears clean and healthy which is generally at around passage 2. Further culturing the stem cells past passage 2 negatively affects the OLC formation efficiency (unpublished results).</li>	 
	 <li>48 hr prior to initiating differentiation sub-culture the stem cells. Remove all the suspended spherical cell aggregates and spent media from the culture dish, using a serological pipette, and place in a 15 ml tube. It is important to only collect the suspended spherical aggregates of cells and not the cells attached to the bottom of the dish that are spontaneously differentiating.</li>	 
	 <li>Pellet the cells at 500 x g for 5 min., discard the supernatant, and resuspend in 500 &mu;l of fresh stem cell media warmed to 37 &deg;C. Gently pipette the aggregates to partially dissociate the cells. Do not aggressively pipette the cells as this will reduce cell viability. Wash the partially dissociated cells into 9.5 ml of fresh pre-warmed stem cell medium on a low attachment 10 cm cell culture dish.</li>	 
	 <li>Return the cells to a 37 &deg;C, 5% CO2 cell culture incubator for 48 hr prior to initiation of differentiation.</li>	
	</ol>
	3. OLC Differentiation	
	<ol>
		<li>Using a serological pipette remove the stem cells from the culture dish and place in a 15 ml tube, leave behind any attached cells. Pellet the cells at 500 x g and resuspend in 500 &mu;l sterile PBS. Vigorous pipette the cells using a wide bore 1,000 &mu;l tip into clumps not larger than 10-20 cells. Periodically remove a small amount of cells, during dissociation, and check under a white light microscope to confirm aggregated cells are in clumps of 10-20 individual cells. Over dissociation will result in reduced cell viability.</li>	 
		<li>Add 9.5 ml PBS to the dissociated cells and add 15 &mu;l of this cell suspension to a hemocytometer for cell counting. Pellet the cells at 500 x g for 5 min.</li>	 
	 <li>Re-suspend the cells at a concentration of 1.32X106 cells/ml in differentiation medium.</li>	 
	 <li>Plate the cells by adding 500 &mu;l per well into a flat bottom 24 well suspension dish.</li>	 
	 <li>Place the 24 well dish into a cell culture incubator at 37 &deg;C in 5% CO2 for 48 hr. Remove 250 &mu;l medium from each well and place in a correspondingly labeled 1.5 ml tube. Add 250 &mu;l fresh differentiation medium to each well. Centrifuge the spent medium at 500 x g for 5 min. and discard the supernatant. Resuspend any pelleted cells in 50 &mu;l fresh differentiation medium and return to the corresponding culture well on the differentiation plate.</li>	 
	 <li>Every 48 hr change half the medium up to day 12 of differentiation.</li>	
	</ol>

<ol>
	<li>van den Hurk, R., Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+mammalian+oocytes+and+their+growth,+differentiation+and+maturation+within+ovarian+follicles.">Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.</a> Theriogenology. 63, 1717-1751 (2005).</li><li>Molyneaux, K. A., Stallock, J., Schaible, K., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+living+mouse+germ+cell+migration.">Time-lapse analysis of living mouse germ cell migration.</a> Dev. Biol. 240, 488-498 (2001).</li><li>Lawson, K. A., Hage, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+origin+of+primordial+germ+cells+in+the+mouse.">Clonal analysis of the origin of primordial germ cells in the mouse.</a> Ciba Found Symp. 182, 68-91 (1994).</li><li>Ginsburg, M., Snow, M. H., McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cells+in+the+mouse+embryo+during+gastrulation.">Primordial germ cells in the mouse embryo during gastrulation.</a> Development. 110, 521-528 (1990).</li><li>Hubner, K., Fuhrmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocytes+from+mouse+embryonic+stem+cells.">Derivation of oocytes from mouse embryonic stem cells.</a> Science. 300, 1251-1256 (2003).</li><li>Toyooka, Y., Tsunekawa, N., Akasu, R., Noce, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+can+form+germ+cells+in+vitro.">Embryonic stem cells can form germ cells in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 11457-11462 (2003).</li><li>Wang, L., Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-like+Cells+Induced+from+Mouse+Spermatogonial+Stem+Cells.">Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells.</a> Cell Biosci. 2, 27 (2012).</li><li>Dyce, P. W., Wen, L., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+germline+potential+of+stem+cells+derived+from+fetal+porcine+skin.">In vitro germline potential of stem cells derived from fetal porcine skin.</a> Nat. Cell Biol. 8, 384-390 (2006).</li><li>Danner, S., Kajahn, J., Geismann, C., Klink, E., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocyte-like+cells+from+a+clonal+pancreatic+stem+cell+line.">Derivation of oocyte-like cells from a clonal pancreatic stem cell line.</a> Mol. Hum. Reprod. 13, 11-20 (2007).</li><li>Cheng, X., Chen, S., Yu, X., Zheng, P., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP15+gene+is+activated+during+human+amniotic+fluid+stem+cell+differentiation+into+oocyte-like+cells.">BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells.</a> DNA Cell Biol. 31, 1198-1204 (2012).</li><li>Song, S. H., Kumar, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+porcine+multipotent+stem/stromal+cells+derived+from+skin,+adipose,+and+ovarian+tissues+and+their+differentiation+in+vitro+into+putative+oocyte-like+cells.">Characterization of porcine multipotent stem/stromal cells derived from skin, adipose, and ovarian tissues and their differentiation in vitro into putative oocyte-like cells.</a> Stem Cells Dev. 20, 1359-1370 (2011).</li><li>Dyce, P. W., Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+germ+line+potential+of+stem+cells+derived+from+newborn+mouse+skin.">In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin.</a> PLoS One. 6, e20339 (2011).</li><li>Linher, K., Dyce, P., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cell-like+cells+differentiated+in+vitro+from+skin-derived+stem+cells.">Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells.</a> PLoS One. 4, e8263 (2009).</li><li>Hayashi, K., Ogushi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offspring+from+Oocytes+Derived+from+in+Vitro+Primordial+Germ+Cell-Like+Cells+in+Mice.">Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-Like Cells in Mice.</a> Science. , (2012).</li></ol>

No conflicts of interest declared.

To date functional OLCs have not been developed using a completely in vitro assay. Recently a study by Hayashi et al. was able to produce offspring from PGC-like cells formed in vitro and transferred in vivo for further development14. Starting with ES cells or induced pluripotent cells they were able to form PGC like cells which, when recovered following in vivo transplantation were able to be matured, fertilized, and used to generate offspring 14. Th...

During early embryogenesis, gametogenesis occurs through a series of stages including primordial germ cell (PGC) formation, migration, and finally colonization of the gonadal ridges. During this time PGCs undergo proliferation and differentiation into increasingly more mature gametes 1. The fact that PGCs migrate from the base of the allantois into the embryo hindgut and finally along the dorsal wall eventually colonizing the gonadal ridges makes them exceedingly difficult to study 2. Despite advances in the field, studies attempting to understand PGC formation and differentiation have been impeded by their limited number, location, and migratory nature 3,4. 	
	In recent years embryonic stem cells have been shown to have the potential to form germ cells in vitro 5,6. Similarly, several somatic stem cells have also been shown to have the potential to form germ cells following in vitro culturing 7-11. Recently stem cells isolated from newborn mouse skin were differentiated in vitro into germ-like cells and early stage oocytes 12. During differentiation the subset of the stem cells expressing Oct4 increased and structures resembling cumulus-oocyte complexes were formed. The oocyte-like cells (OLCs) can be picked from the cultures and compared to natural oocytes. Using this culture method OLCs measuring 40-45 &mu;m are obtained that express similar markers to oocytes such as Gdf9b, VASA, and DAZL. To date the resulting OLCs remain unable to mature or be fertilized 12.	
	Although the OLCs remain unable to function the protocol used to form these OLCs may still have utility as an assay to study the formation and development of earlier stage germ cells within a closed in vitro system. The original publication discussing the formation of OLCs from somatic stem cells utilized fetal porcine skin as a stem cell source 8. Expanding on that study the PGC-like cells formed early during induced differentiation were shown to be the precursors to the OLCs and express such PGC markers as OCT4, VASA, STELLA, C-KIT, and DAZL 13. This data supports the potential of utilizing this system to study germ cell formation and differentiation in vitro. The protocol utilized to form OLCs from newborn mouse skin will be demonstrated in this video article. This protocol offers an in vitro model to study germ cell development that may provide a means to elucidate factors important for their proliferation and differentiation.

<table><tbody><tr><td>DMEM/F12</td><td>Invitrogen</td><td>12500</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504044</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>PHG0311</td><td></td></tr><tr><td>bFGF</td><td>Invitrogen</td><td>PHG0266</td><td></td></tr><tr><td>ITS</td><td>Invitrogen</td><td>41400-045</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-6003</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen</td><td>11360-070</td><td></td></tr><tr><td>Fetuin</td><td>Sigma</td><td>F3385</td><td></td></tr><tr><td>FSH</td><td>Sioux Biochemicals</td><td>915</td><td></td></tr><tr><td>LH</td><td>Sioux Biochemicals</td><td>925</td><td></td></tr><tr><td>M199</td><td>Invitrogen</td><td>31100-035</td><td></td></tr><tr><td>24 well plates</td><td>Nunc</td><td>144530</td><td></td></tr><tr><td>10 cm dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>15 ml tubes</td><td>BD</td><td>352095</td><td></td></tr></tbody></table>

differentiation of newborn mouse skin derived stem cells into germ-like cells in vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Initially the cells attach to the culture dish bottom and spread out. At 42-72 hr into differentiation small suspended OCT4 positive cells form and proliferate (Figure 1A). Shortly after the visualization of these cells the majority will disappear and the attached cells will form dense colony aggregates on the culture bottom. Following a few days these aggregates will detach from the culture surface. In some of these aggregates a large cell can be observed (Figure 1b and 1c</stro...

Watch this Scientific Journal Video about Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro at JoVE.com

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

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9.2K Views.  The University of Western Ontario. In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Video: Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Developmental Biology

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

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 ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Neuroscience

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Source: Edamakanti, C. R., et al. <a href="https://app.jove.com/v/60554">Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum.</a> J. Vis. Exp. (2020).
This video demonstrates a method for isolating stem cells from the postnatal mouse cerebellum and differentiating them into neural cells. Stem cells from the isolated cell suspension are purified using antibody-coated magnetic beads and are cultured to form neurospheres. The neurospheres are then maintained in a differentiation medium to generate neural cells.

Izolacja komórek macierzystych z poporodowego móżdżku myszy i różnicowanie w komórki nerwowe

1. Preparation of Solutions

	Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco&#39;s phosphate-buffered saline (DPBS) ...

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Neuronal Culture Techniques

Advanced Neuronal Models

Differentiation of Neural Progenitor Cells into Neurons

Seed about 500,000 mouse embryonic stem cell derivatives in 2 milliliters of basal differentiation medium per well onto the 0.1% gelatin-coated plates. Randomly divide the derivatives into three groups in order to test three phase II differentiation protocols. Incubate the plate for six hours. Then, wash the cells twice with 2 milliliters of PBS and add 2 milliliters of basal differentiation medium, N2 B27 medium 1, or N2 B27 medium 2 to each well, depending on the protocol. Place the plates into the incubator, and allow the cells to differentiate for another 10 days, changing the corresponding medium every two days.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

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Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Isolation and Derivation of Mouse Embryonic Germinal Cells

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Izolacja komórek macierzystych z poporodowego móżdżku myszy i różnicowanie w komórki nerwowe

Różnicowanie nerwowych komórek progenitorowych w neurony