This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

Mice were housed in pathogen-free conditions in compliance with the institutional guidelines until euthanization (The Institutional Animal Care and Use Committee (IACUC) guidelines at the National University of Singapore and the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines).
1. Mice
<ol>
	<li>Order one mouse of the appropriate genetic background. This protocol is based on tissue derived from one C57BL/6 mouse.</li>
</ol>
2. Preparation of Complete Medium
<ol>
	<li>Prepare complete medium by adding the following components to Roswell Park Memorial Institute (RPMI) 1640 medium: 10% fetal calf serum (FCS), 50 &#181;M 2-mercaptoethanol, 100 &#181;M asparagine, 2 mM glutamine, 1% penicillin-streptomycin solution.</li>
</ol>
3. Preparation of Enzyme Solutions
<ol>
	<li>Prepare collagenase D solution in a 15 ml conical bottom tube.
	<ol>
		<li>Weigh 10 mg of collagenase D. Dissolve collagenase D in 4 ml complete medium.</li>
	</ol>
	</li>
	<li>Prepare pronase solution in a 1.7 ml microcentrifuge tube.
	<ol>
		<li>Weigh 10 mg of pronase.</li>
		<li>Add 5 &#181;l&#160;of 1 M Tris buffer (pH 8.0). Add 1 &#181;l&#160;of 0.5 M EDTA (pH 8.0).</li>
		<li>Top up with 494 &#181;l&#160;of sterile water.</li>
		<li>Incubate the pronase solution at 37 &#176;C in a water bath for 30 min.</li>
	</ol>
	</li>
</ol>
4. Preparation of Collagenase D-pronase Mix (&#8804;2 Tails)
Note: Perform the subsequent steps in a sterile cell culture hood.
<ol>
	<li>Add 250 &#181;l&#160;of pronase solution to 4 ml of collagenase D solution.</li>
	<li>Pass the collagenase D-pronase mixture through a 0.2 &#181;m syringe filter into a sterile 15 ml conical bottom tube.</li>
</ol>
5. Extraction of Fibroblasts from Ear and Tail Tissues
<ol>
	<li>Euthanize mice according to the appropriate institutional guidelines.</li>
	<li>Place autoclaved surgical instruments (scissors and forceps) in the cell culture hood.</li>
	<li>Add 10 ml complete medium into two 10 cm cell culture dishes each.</li>
	<li>Cut ears (~1 cm radius) and 5 cm of tail (from the tip of the tail) of a mouse with scissors and incubate for 5 min&#160;in 40 ml 70% ethanol in a sterile 50 ml conical bottom tube.</li>
	<li>Air-dry ears and tail by placing them in an open 10 cm cell culture dish in the hood for 5 min. Once dried, transfer ear and tail pieces to two culture dishes labelled &#34;ears&#34; and &#34;tail&#34; containing 10 ml complete medium as described in step 5.3.</li>
	<li>Remove hair from the ears and tail using scissors.</li>
	<li>Cut ears and tail into pieces smaller than 3 mm in size using scissors.</li>
	<li>Transfer the cut tissues into 1.8 ml cryotube vials labelled &#34;ears&#34; and &#34;tail&#34; and add sufficient collagenase D-pronase solution for the volume to reach the 1.8 ml mark on the vial.</li>
	<li>Place the cryotube vials horizontally on a shaker and shake the samples at 200 rpm for 90 min&#160;at 37 &#176;C.</li>
	<li>After 90 min&#160;incubation, remove the cryotube vials from the shaker and place the vials in the hood.</li>
	<li>Add 10 ml complete medium into two 10 cm cell culture dishes, labelled &#34;ears&#34; and &#34;tail&#34; each, and put a 70 &#181;m cell strainer in each dish.</li>
	<li>Place the digested ear and tail tissues in the 70 &#181;m cell strainer in the accordingly labelled dishes prepared in step 5.11 and forcefully grind the tissues using a 10 ml syringe plunger for &#62;5 min. Shake the cell strainer occasionally in the medium to wash cells out of the cell strainer.</li>
	<li>Pipette the cell suspension from each dish into two 15 ml conical bottom tubes labelled &#34;ears&#34; and &#34;tail&#34;. Wash the dish and strainer with additional 10 ml complete medium and add the medium to the appropriate 15 ml conical bottom tubes.</li>
	<li>Spin down the cell suspension for 7 min&#160;at ~580 x g and 4 &#176;C using a refrigerated cell centrifuge.</li>
	<li>Remove supernatant, add 10 ml complete medium to the cell pellet in the 15 ml conical bottom tube and resuspend the cells.</li>
	<li>Repeat step 5.14 and 5.15.</li>
</ol>
6. Culturing of Cell Mixture
<ol>
	<li>Remove supernatant. Ensure that the cell pellet remains undisturbed.</li>
	<li>Re-suspend cells in 10 ml complete medium and add the respective mixture to two 10 cm cell culture dishes labelled &#34;ears&#34; and &#34;tail&#34;.</li>
	<li>Add 10 &#181;l amphotericin B solution (stock solution: 250 &#181;g/ml) to the culture.</li>
	<li>Incubate cells at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>On the third day, replace the medium with 10 ml fresh complete medium containing 10 &#181;l of amphotericin B to remove debris (Figures 1 and 2).</li>
</ol>
7. Sub-culture of Fibroblasts
<ol>
	<li>When culture reaches around 70% confluency (day 3-4 of culture) remove the medium and wash the cells with 5 ml sterile 1x phosphate buffered saline (PBS).</li>
	<li>Remove PBS and add 2 ml sterile 1x trypsin-EDTA solution to the cells.</li>
	<li>Incubate the cells for 5 min&#160;at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>After 5 min, gently tap the culture dish and add 6 ml complete medium to the cells.</li>
	<li>Transfer the cell suspension to a 15 ml conical bottom tube and spin the tube for 5 min&#160;at ~450 x g and 4 &#176;C using a refrigerated cell centrifuge.</li>
	<li>Remove supernatant, and gently re-suspend the cell pellet in 5 ml complete medium.</li>
	<li>Seed 2 x 105 cells in a 10 cm cell culture dish and incubate the cells at 37 &#176;C in a humidified 5% CO2 incubator for 3-4 days before repeating steps 7.1 to 7.6.</li>
</ol>
8. Preparation of Ears for Shipment
Note: Perform the subsequent steps in a sterile cell culture hood.
<ol>
	<li>Euthanize mice according to the appropriate institutional guidelines.</li>
	<li>Place autoclaved scissors and forceps into the cell culture hood.</li>
	<li>Add 50 ml complete medium into a 50 ml conical bottom tube.</li>
	<li>Cut ears (~1 cm radius) of a mouse with scissors.</li>
	<li>Transfer the ears into the 50 ml conical bottom tube (as prepared in step 3) using forceps.</li>
	<li>Seal the 50 ml conical bottom tube with parafilm before shipping at RT in an appropriate box.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+matrices+for+tissue+engineering+and+regenerative+medicine+applications.">Cell-derived matrices for tissue engineering and regenerative medicine applications.</a> Biomater Sci. 3 (1), 12-24 (2015).</li><li>Elenbaas, B., 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=Human+breast+cancer+cells+generated+by+oncogenic+transformation+of+primary+epithelial+cells.">Human breast cancer cells generated by oncogenic transformation of primary epithelial cells.</a> Genes Dev. 15 (1), 50-65 (2001).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> J Neuroinflammation. 9 (1), 115 (2012).</li><li>Lim, J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+transfection+of+HUVEC+and+MEF+cells+using+DNA+complexes+with+magnetic+nanoparticles+in+an+oscillating+field.">Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field.</a> J Genet. 91 (2), 223-227 (2012).</li><li>Li, 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=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21 (11), 1527-1543 (2010).</li><li>Patel, M., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+reprogramming+somatic+cells+to+induced+pluripotent+stem+cells.">Advances in reprogramming somatic cells to induced pluripotent stem cells.</a> Stem Cell Rev. 6 (3), 367-380 (2010).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> J Invest Dermatol. 127 (5), 998-1008 (2007).</li><li>Shen, Y. 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The authors declare no conflict of financial interests.

Here we provide a simple, inexpensive and fast experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The extraction should result in adherent and rapidly dividing fibroblasts within 3 days post-isolation of the tissue. An important limitation of primary cells is senescence, a permanent growth arrest15. Using the protocol, fibroblast cultures can be passaged for 5 to 6 times before fibroblasts become senescent, indicated by the flattening of cells, increase in size (2-3 ti...

Primary cells are derived from living tissue and cultured under in vitro conditions. It is generally assumed that primary cells more closely resemble the physiological state and genetic background of the tissue from which they originated than immortalized or tumor cell lines1. For that reason, primary cells represent a useful model for studying biological questions2,3. However, unlike established cell lines that grow indefinitely, primary cells eventually undergo senescence in culture and need to be frequently re-established.
Commonly used primary cells include fibroblasts, epithelial cells, endothelial cells, T cells, B cells, bone marrow-derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC). Fibroblasts are often utilized as primary cell culture model. They offer key advantages over other primary cells. Cell cultures are easily established, readily maintained and require no purification of cells prior to culture. They have rapid initial proliferation and no requirement for specialized medium or activation protocols. Fibroblasts can be efficiently transfected using biological, chemical, and physical protocols4,5. There is a possibility to store ears for up to 10 days at RT prior to establishing cell cultures. Fibroblast cultures are conducive to visualization of cytoplasmic processes and suitable for reprogramming into induced pluripotent stem (iPS) cells6.
Fibroblasts are important cells of the connective tissue that secrete collagen proteins and extracellular matrix7. They provide the structural framework in many tissues8 and play an essential role in wound healing and tissue repair9,10.
Here, we describe a simple and quick (&#60;4 hr) protocol to establish fibroblast cultures from ears and tails of mice11. The protocol requires minimal mouse experience to harvest the tissues (in contrast to other protocols12,13) and can be used to establish cultures from ears stored in medium at RT for up to 10 days.

<table><tbody><tr><td>RPMI-1640</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Asparagine</td><td>Sigma-Aldrich</td><td>A4159</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Ethanol</td><td>Merck Millipore</td><td>107017</td><td>Absolute for analysis</td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics</td><td>11088866001</td><td>From &lt;em&gt;Clostridium histolyticum&lt;/em&gt;, lyophilized, non-sterile</td></tr><tr><td>Pronase protease</td><td>Merck Millipore</td><td>53702</td><td>From &lt;em&gt;Streptomyces griseus&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Tris buffer (pH 8)</td><td>1st BASE</td><td>1415</td><td>Ultra pure grade</td></tr><tr><td>0.5M EDTA (pH 8)</td><td>1st BASE</td><td>BUF-1053</td><td>Biotechnology grade</td></tr><tr><td>10X Phosphate Buffered Saline (PBS)</td><td>1st BASE</td><td>BUF-2040-10X4L</td><td>Ultra pure grade</td></tr><tr><td>Trypsin-EDTA solution 10X</td><td>Sigma-Aldrich</td><td>59418C-100ML</td><td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2492-20ml</td><td>250 &amp;mu;g/ml in deionized water, sterile-filtered</td></tr><tr><td>Scissors</td><td>Aesculap</td><td></td><td></td></tr><tr><td>Forcep</td><td>Aesculap</td><td>AE-BD312R</td><td></td></tr><tr><td>0.2 &amp;mu;M syringe filter</td><td>Sartorius Stedim</td><td>16534</td><td></td></tr><tr><td>70 &amp;mu;M cell strainer</td><td>SPL</td><td>93070</td><td></td></tr><tr><td>Syringe plunger</td><td>Terumo</td><td>SS+10L</td><td></td></tr><tr><td>Cryovial tube</td><td>NUNC</td><td>368362</td><td></td></tr><tr><td>1.7 ml microcentrifuge tube</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Greiner</td><td>664160</td><td>Cell culture treated dish&amp;nbsp;</td></tr><tr><td>15 ml conical bottom tube</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>50 ml conical bottom tube</td><td>Greiner</td><td>227261</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1002</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Incubation shaker</td><td>Sartorius Stedim</td><td></td><td>Certomat-BS1</td></tr><tr><td>Zeiss Axiovert 25 light microscope</td><td>Carl Zeiss AG</td><td></td><td></td></tr></tbody></table>

generating primary fibroblast cultures from mouse ear and tail tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

Extraction of fibroblasts from tissue results in a significant amount of tissue debris (Figure 1). In contrast to tissue debris, fibroblasts adhere to tissue culture plastic surfaces between day 1 and 3 of culture. The medium of fibroblast cultures can be safely changed on day 3 of culture, which should significantly decrease the levels of debris present in the culture (Figure 2). Fibroblasts display an elongated morphology and a clearly visible cytoplasm (Figures 1 and 2</strong...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Research

JoVE Journal

Developmental Biology

33.8K Views.  Yong Loo Lin School of Medicine, National University of Singapore.  We beschrijven een eenvoudige en snelle experimentele procedure voor het genereren van primaire fibroblasten uit de oren en staarten van muizen. De werkwijze vereist geen speciale dierendressuur en kan worden gebruikt voor het genereren van fibroblast cultures van oren opgeslagen bij kamertemperatuur gedurende maximaal 10 dagen. 

Video: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Cancer Research

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioengineering

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Biology

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

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

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

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

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

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