Name: collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach
Uploaded: 2017-01-08T15:00:00
Description: Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

This research was supported by the Basic Science Research Program (2013R1A1A2060930) and the Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning. This research is also supported by a Start-up Operating Grant from The Hospital for Sick Children (H. K. Sung). We would like to thank Laura Barwell and Sarah J. S. Kim for their excellent help in editing this manuscript and Dr. Andras Nagy for providing the G4 mESC line.

NOTE: A schematic of the serum- and feeder-free CM collection protocol is shown in Figure 1.
1. Materials (Preparation of MEFs, Medium, Plates, and Solutions)
<ol>
	<li>Prepare 500 ml of medium to culture the MEFs. Supplement Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% FBS (ESC quality), 50 units/ml penicillin, and 50 mg/ml streptomycin.</li>
	<li>Isolate MEFs from embryos following an established routine protocol13 and maintain them in MEF medium.</li>
	<li>Prepare 500...

<ol>
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For the successful collection of serum- and feeder-free mESC-CM, the following suggestions should be taken into consideration. The most critical factor is using early passage mESCs for the collection of mESC-CM. Previously, it has been shown that early passage mESC-CM has better anti-aging effects compared to late passage mESCs. The passage number of mESCs has been reported to affect their developmental potential16 and pluripotency17.
While additional research is needed t...

The goal of this protocol is to collect mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) from serum- and feeder-free culture conditions and to characterize its biological functions.
In general, embryonic stem cells (ESCs) have great potential for regenerative medicine and cell therapy due to their pluripotency and capacity for self-renewal1-3. However, the direct transplantation of stem cells has several limitations, such as immune rejection and tumor formation4,5. Therefore, a cell-free approach may provide an alternate therapeutic strategy for regenerative medicine and aging interventions6,7.
Senescence is viewed as a cellular counterpart to the aging of tissues and organs, characterized by a permanent state of growth arrest, altered cell physiology, and behaviors. Aging is the main risk factor for prevalent diseases including cancer, cardiovascular disease, type 2 diabetes, and neurodegeneration8. One of the obvious characteristics of aging is the decline in the regenerative potential of tissues, which is caused by stem cell aging and exhaustion9. Many significant studies have shown pharmacological molecules, such as rapamycin9, resveratrol10, and metformin11, and blood-borne systemic factors, namely GDF1112, that have the ability to consistently delay aging and extend life span.
In the present study, mESC-CM has been harvested without serum (fetal bovine serum, FBS) and feeder (mouse embryonic fibroblasts, MEFs) layers to exclude the contamination of serum factors and secretory factors from MEFs. These conditions allowed for a serum- and feeder-free CM that consequently enabled the accurate identification of mESC-specific secretory factors.
This proposed protocol is highly efficient, relatively cost effective, and easy to operate. This technique provides insights into the characterization of mESC-derived soluble factors that can mediate an anti-senescence effect, which may be used for the development of a safe and potentially advantageous cell-free therapeutic approach toward interventions for aging-associated diseases and other regenerative treatments.

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		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">DMEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11960-044</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">FBS</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#30044333</td>
			<td style="width:64px;">20%, ES cell quality</td>
		</tr>
		<tr height="126">
			<td height="148" rowspan="2" style="height:148px;width:64px;">Penicillin and streptomycin&#160;</td>
			<td rowspan="2" style="width:64px;">Invitrogen</td>
			<td rowspan="2" style="width:64px;">#15140</td>
			<td style="width:64px;">50units/ml penicillin and 50mg/ml strepto</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">-mycin.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">L-glutamine&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#25030</td>
			<td style="width:64px;">2mM</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Nonessential amino acids (NEAA)&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11140</td>
			<td style="width:64px;">100uM</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M3148</td>
			<td style="width:64px;">100uM</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Leukemia inhibitory factor&#160;</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:64px;">#ESG1107</td>
			<td style="width:64px;">100units/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">OPTI-MEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#22600</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">X-gal&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#B4252</td>
			<td style="width:64px;">1mg/ml</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Paraformaldehyde (PFA)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">P6148</td>
			<td style="width:64px;">3.70%</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Dimethylformamide (DMF)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#D4551</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Potassium ferricyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455946</td>
			<td style="width:64px;">5mM</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">potassium ferrocyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455989</td>
			<td style="width:64px;">5mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">NaCl&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#S7653</td>
			<td style="width:64px;">150mM</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:64px;">MgCl2&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M2393</td>
			<td style="width:64px;">2mM</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Mytomycin C&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M4287</td>
			<td style="width:64px;">10ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Propidium iodide&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#P4170</td>
			<td style="width:64px;">50ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">TRIzol</td>
			<td style="width:64px;">Ambion</td>
			<td style="width:64px;">#15596018</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">M-MLV reverse transcript-tase</td>
			<td style="width:64px;">Promega</td>
			<td style="width:64px;">#M170B</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">Power SYBR Green PCR master mix&#160;</td>
			<td style="width:64px;">Applied Biosystems</td>
			<td style="width:64px;">#4367659</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">HDFs, NHDF-Ad-Der-Fibroblast&#160;</td>
			<td style="width:64px;">LONZA</td>
			<td style="width:64px;">#CC-2511</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Bottle top filter,&#160;</td>
			<td style="width:64px;">Corning</td>
			<td style="width:64px;">#430513</td>
			<td style="width:64px;">0.2&#956;m</td>
		</tr>
	</tbody>
</table>

collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

Originally, mESCs are maintained on an MEF feeder in mESC medium with FBS and other supplements (Figures 1A and 2A). CM was collected from mESCs in Reduced Serum Media without a feeder layer, FBS, or other supplements (Figures 1B and 2B). This culture condition allows us to collect mESC-specific conditioned medium without potential contamination by the factors from the feeder, FBS, or other supplements. The control medium...

Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

The overall goals of this protocol are to collect conditioned medium from mouse embryonic stem cells under serum and feeder free culture conditions and to characterize the biological functions of this medium. This procedure provides insights into the anti senescence activity of soluble factors secreted from mouse embryonic stem cells for the development of safe and cell free therapeutic approach for age related diseases. Demonstrating this procedure will be Dr.Yun-Ui Bae a research professor of my laboratory.In a biological safety hood, treat a mouse embryonic fibroblast or MEF cell culture with 20 milliliters of MEF medium supplemented with mitomycin C in a 15 centimeter cell culture dish for two hours at 37 degrees celsius and 5%carbon dioxide. At the end of the incubation, wash the cells three times with PBS and detach them with three milliliters of trypsin EDTA for three minutes at 37 degrees celsius. Neutralizing the trypsin with six milliliters of fresh MEF medium when the cells have lifted from the bottom of the plate.After collecting the cells by centrifugation, re-suspend the pellet in five milliliters of MEF medium and plate two times 10 to the sixth inactivated MEFs per 10 centimeter culture dish in fresh MEF medium. Culture the feeder cells for 24 hours. Then replace the MEF medium with mouse embryonic stem cell medium and seed two times 10 to the sixth mouse embryonic stem cells onto the feeders for at least 48 hours of co culture.In a biological safety hood, rinse a plate of mouse embryonic stem cells with five milliliters of PBS and detach the cells with trypsin plus EDTA as just demonstrated. After collecting the cells by centrifugation, re-suspend the stem cell pellet in five milliliters of fresh stem cell medium and plate one milliliter of cells per dish in fresh embryonic stem cell medium onto five gelatin coated cell culture dishes. Return the cells to the incubator until the cultures reach 80-85%confluency.Then rinse the cells in at least eight milliliters of PBS per plate for three 10 minute washes followed by a 24 hour incubation in reduced serum medium. After no more than 24 hours, transfer the supernatant into a 50 milliliter conical tube for centrifugation. Followed by filtration through a 0.2 micrometer bottle top strainer.To test the effects of the embryonic stem cell conditioned medium by a senescence associated beta galactosidase assay, seed human dermal fibroblasts in a six well plate in a human dermal fibroblast medium. After an overnight incubation at 37 degrees celsius and 5%carbon dioxide, replace half of the medium with fresh mouse embryonic stem cell conditioned medium for another 72 hour culture. On the third day of culture, rinse the cells in two milliliters of PBS per well for two 30 second washes.Then, fix the cells in 3.7%paraformaldehyde for five minutes at room temperature in a fume hood. After fixing, wash the cells two times with two milliliters of PBS, as just demonstrated and label the cells with one to two milliliters of SA beta gal solution for 17 and half hours at 37 degrees celsius without carbon dioxide. At the end of the staining period, wash the cells two times with PBS as just demonstrated.Then, counter stain with Eosen for five minutes at room temperature. After another set of PBS washes, image the cells at a 100 times magnification by light microscopy. For cell cycle analysis, seed eight times 10 to the fourth human dermal fibroblast per well in a six centimeter cell culture dish in fresh human dermal fibroblast medium for an overnight incubation at 37 degrees celsius and 5%carbon dioxide.The next morning, replace half of the medium with mouse embryonic stem cell conditioned medium and return the cells to the incubator. After 24 hours, harvest the cells by trypsin EDTA dissociation followed by two washes in one milliliter of PBS solution. Re-suspend the pellet in 100 microliters of cold PBS solution after the second wash.Then, fix the cells with the drop wise application at 200 microliters of cold ethanol while vortexing. Store the cells at four degrees celsius for at least an hour. Then, wash them in fresh PBS solution.After the second centrifugation, treat the pellet with 250 microliters of sodium citrate buffer supplemented with RNase for 30 minutes at 37 degrees celsius without carbon dioxide. At the end of the incubation, label the cells with 250 microliters of sodium citrate buffer containing propidium iodide for 20 minutes. Then, assess the cell cycle stages of each sample by flow cytometry.For quantitative reverse transcriptase PCR analysis, use an RNA extraction kit to isolate the total RNA from mouse embryonic stem cell conditioned medium treated human dermal fibroblasts according to the manufacturer's protocol. Use a spectrophotometer to quantify the total extracted RNA. Then, formulate reaction mixture containing the oligo deoxythymidine primers and M-MLV reverse transcriptase according to the manufacturer's protocol.Then add one microgram of the total RNA to the PCR tube containing the reaction mixture. Finally, measure the amplification of the CDNA with a real time PCR machine using the green PCR master mix and the appropriate gene primers. Mouse embryonic stem cells grown under normal mouse embryonic stem cell culture conditions on a MEF feeder cell layer display an oval shape and a shiny appearance.While mouse embryonic stem cells grown under serum and feeder free culture conditions demonstrate a flattened and irregular morphology. The treatment of the senescent human dermal fibroblast with mouse embryonic stem cell conditioned medium decreases the number of senescent associated betagalactosidase positive cells. Further, cell cycle analysis of these cells reveals that mouse embryonic stem cell conditioned medium treatment dramatically increases the number of cells in the S and G2/M phases while reducing the number of cells in the G0/G1 phase.In addition, mouse embryonic stem cell conditioned medium treatment decreases the expression of senescence associated genes and of secretary cytokines associated with the senescent phenotype. While attempting this procedure, it is very important to remember to collect the serum and feeder free medium after 24 hours, as a long incubation increases the possibility of cell autolysis or apoptosis by starvation. After watching this video, you should have a good understanding of how to collect serum and feeder free mouse embryonic stem cell conditioned medium for a cell free approach.Don't forget that working with reagent such as paraformaldehyde can be extremely hazardous, and that precaution, such as handling this reagent in a fume hood, should always be taken while performing this procedure.

collection-serum-feeder-free-mouse-embryonic-stem-cell-conditioned

Research

JoVE Journal

Developmental Biology

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 ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...