Name: isolation of murine spermatogenic cells using a violet-excited cell-permeable dna binding dye
Uploaded: 2021-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

We thank members of the Namekawa, Yoshida, and Maezawa laboratories for their help; Katie Gerhardt for editing the manuscript; Mary Ann Handel for sharing the H1T antibody, the Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Research Flow Cytometry Core for sharing the FACS equipment supported by NIH S10OD023410; Grant-in-Aid for Scientific Research (KAKENHI; 17K07424) to T.N.; Lalor Foundation Postdoctoral Fellowship to A.S.; AMED-CREST (JP17gm1110005h0001) to S.Y.; the Research Project Grant by the Azabu University Research Services Division, Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Private University Research Branding Project (2016&#8211;2019), Grant-in-Aid for Research Activity Start-up (19K21196), the Takeda Science Foundation (2019), and the Uehara Memorial Foundation Research Incentive Grant (2018) to S.M.; National Institute of Health R01 GM122776 to S.H.N.

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (protocol no. IACUC2018-0040) at Cincinnati Children&#8217;s Hospital Medical Center.
1. Equipment and reagent setup for the preparation of testicular cell suspension
<ol>
	<li>Prepare each enzyme stock in 1x Hanks&#8217; Balanced Salt Solution (HBSS) and store at -20 &#176;C. (Table1). 
	NOTE: Prepare any time before the experiment.</li>
	<li>One day before the experiment: Coat coll...

<ol>
	<li>Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis:+The+Commitment+to+Meiosis.">Spermatogenesis: The Commitment to Meiosis.</a> Physiological Reviews. 96 (1), 1-17 (2016).</li><li>Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Spermatogenesis+Reflects+the+Unity+and+Diversity+of+Tissue+Stem+Cell+Niche+Systems.">Mouse Spermatogenesis Reflects the Unity and Diversity of Tissue Stem Cell Niche Systems.</a> Cold Spring Harbor Perspectives in Biology. , 036186 (2020).</li><li>Rathke, C., Baarends, W. M., Awe, S., Renkawitz-Pohl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+dynamics+during+spermiogenesis.">Chromatin dynamics during spermiogenesis.</a> Biochimica et Biophysica Acta. 1839 (3), 155-168 (2014).</li><li>Maezawa, 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=SCML2+promotes+heterochromatin+organization+in+late+spermatogenesis.">SCML2 promotes heterochromatin organization in late spermatogenesis.</a> Journal of Cell Science. 131 (17), 217125 (2018).</li><li>Maezawa, S., Yukawa, M., Alavattam, K. G., Barski, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+reorganization+of+open+chromatin+underlies+diverse+transcriptomes+during+spermatogenesis.">Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis.</a> Nucleic Acids Research. 46 (2), 593-608 (2018).</li><li>Bellve, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+culture,+and+fractionation+of+spermatogenic+cells.">Purification, culture, and fractionation of spermatogenic cells.</a> Methods in Enzymology. 225, 84-113 (1993).</li><li>Bryant, J. M., Meyer-Ficca, M. L., Dang, V. M., Berger, S. L., Meyer, R. 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F., 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=Revealing+stage-specific+expression+patterns+of+long+noncoding+RNAs+along+mouse+spermatogenesis.">Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis.</a> RNA Biology. 17 (3), 350-365 (2020).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Alavattam, K. G., Abe, H., Sakashita, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+Spread+Analyses+of+Meiotic+Sex+Chromosome+Inactivation.">Chromosome Spread Analyses of Meiotic Sex Chromosome Inactivation.</a> Methods in Molecular Biology. 1861, 113-129 (2018).</li><li>Maezawa, 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=Super-enhancer+switching+drives+a+burst+in+gene+expression+at+the+mitosis-to-meiosis+transition.">Super-enhancer switching drives a burst in gene expression at the mitosis-to-meiosis transition.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Sakashita, A., 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=Endogenous+retroviruses+drive+species-specific+germline+transcriptomes+in+mammals.">Endogenous retroviruses drive species-specific germline transcriptomes in mammals.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Agrimson, K. 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=Characterizing+the+Spermatogonial+Response+to+Retinoic+Acid+During+the+Onset+of+Spermatogenesis+and+Following+Synchronization+in+the+Neonatal+Mouse+Testis.">Characterizing the Spermatogonial Response to Retinoic Acid During the Onset of Spermatogenesis and Following Synchronization in the Neonatal Mouse Testis.</a> Biology of Reproduction. 95 (4), 81 (2016).</li><li>Hogarth, C. A., 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=Turning+a+spermatogenic+wave+into+a+tsunami:+synchronizing+murine+spermatogenesis+using+WIN+18,446.">Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.</a> Biology of Reproduction. 88 (2), 40 (2013).</li><li>Patel, L., 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=Dynamic+reorganization+of+the+genome+shapes+the+recombination+landscape+in+meiotic+prophase.">Dynamic reorganization of the genome shapes the recombination landscape in meiotic prophase.</a> Nature Structural &amp; Molecular Biology. 26 (3), 164-174 (2019).</li><li>Alavattam, K. 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Here we present a practical and simple protocol to isolate subpopulations of spermatocytes and spermatids from a single adult male mouse. To ensure the reproducibility of this protocol, there are some critical steps that need attention. Before enzyme digestion, wash step aims to remove interstitial cells; after digestion, this step helps to remove spermatozoa and debris. Washing/centrifuging 3 times is important. In our dissociation buffer recipe, the combination of several different enzymes facilitates the dissociation ...

Spermatogenesis is a complex process wherein a small population of spermatogonial stem cells sustain continuous production of a large number of sperm throughout adult life1,2. During spermatogenesis, dynamic chromatin remodeling takes place when spermatogenic cells undergo meiosis to produce haploid spermatids3,4,5. Isolation of meiotic spermatocytes is essential for molecular investigation, and several different approaches to isolate meiotic spermatocytes have been established, including sedimentation-based separation6,7 and fluorescence-activated cell sorting (FACS)8,9,10,11,12,13,14,15,16,17. However, these methods have technical limitations. While sedimentation-based separation yields a large number of cells5,6,7, it is labor intensive. The established FACS-based method uses Hoechst 33342 (Ho342) to separate meiotic spermatocytes based on DNA content and light scattering properties and requires FACS cell sorters equipped with an ultraviolet (UV) laser8,9,10,11. Alternative FACS-based methods require transgenic mouse lines that express florescent proteins, synchronization of spermatogenesis12, or cell fixation and antibody labeling that is not compatible with isolation of live cells13. While there is another alternative method using a cell-permeable DNA binding dye, DyeCycle Green stain14,15,16,17, this method is recommended for the isolation of spermatogenic cells from juvenile testis. Therefore, there is a critical need to develop a simple and robust isolation method for live meiotic spermatocytes that can be applied to any mouse strain of any age and that can be performed using any FACS cell sorter.
Here we describe such a long-sought cell isolation protocol using the DyeCycle Violet (DCV) stain. DCV is a low cytotoxicity, cell-permeable DNA binding dye structurally similar to Ho342 but with an excitation spectrum shifted toward the violet range18. In addition, DCV has a broader emission spectrum compared to DCG. Thus, it can be excited by both UV and violet lasers, which improves the flexibility of equipment, allowing the use of an FACS cell sorter not equipped with a UV laser. The DCV protocol presented here uses two-dimensional separation with DCV blue and DCV red, mimicking the advantage of the Ho342 protocol. With this advantage, our DCV protocol allows us to isolate highly enriched germ cells from the adult testis. We provide a detailed gating protocol to isolate live spermatogenic cells from adult mouse testes of one mouse (from two testes). We also describe an efficient and quick protocol to prepare single-cell suspension from mouse testes that can be used for this cell isolation. The procedure requires a short time to complete (preparation of single cell suspension - 1 hour, dye staining - 30 min, and cell sorting - 2-3 hours: total - 4-5 hours depending on the number of needed cells; Figure 1). Following cell isolation, a wide range of downstream applications including RNA-seq, ATAC-seq, ChIP-seq, and cell culture can be completed.

<table>
	<tbody>
		<tr>
			<td>1.5 ml tube</td>
			<td>Watson</td>
			<td>131-7155C</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1332-015S</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene tube with cell strainer snap cap (35 &#181;m nylon mesh)</td>
			<td>Corning, Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1342-050S</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m nylon mesh</td>
			<td>Corning, Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>Sony</td>
			<td>SH800S</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, recombinant, Animal-derived-free</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-23141</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin 3</td>
			<td>Shandon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Fisher</td>
			<td>D1306</td>
			<td>working concentration: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma</td>
			<td>D5025-150KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>S30-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885076</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hostone H1T antibody</td>
			<td>gift from Mary Ann Handel</td>
			<td></td>
			<td>1/2000 diluted</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes</td>
			<td>Sigma</td>
			<td>H3506-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettemen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>rH2AX antibody</td>
			<td>Millipore</td>
			<td>05-635</td>
			<td>working concentration: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sperm Fertilization Protein 56 (Sp56) antibody</td>
			<td>QED Bioscience</td>
			<td>55101</td>
			<td>working concentration: 0.5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterilized forceps and scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SYCP3 antibody</td>
			<td>Abcam</td>
			<td>ab205846</td>
			<td>working concentration: 5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>TWEEN 20 (Polysorbate 20)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vybrant DyeCycle Violet Stain (DCV)</td>
			<td>Invitrogen</td>
			<td>V35003</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine spermatogenic cells using a violet-excited cell-permeable dna binding dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

A representative result of this sorting protocol is shown in Figure 3. The total sorting time of two testes (one mouse) is usually around 3 hours, which is dependent on the concentration of cell suspension and the sorting speed. After sorting, the purity of spermatocytes is confirmed by immunostaining of SYCP3 and &#947;H2AX&#160;(Figure 3A). The representative purity of sorted L/Z, P, D spermatocyte fractions are around 80.4%, 90.6%, and 87.6%, respectively (<s...

Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

This new efficient method for isolating subpopulations of spermatocytes and spermatids from adult mice can be used to investigate the molecular mechanisms underlying meiosis and spermatogenesis. This method uses a low cytotoxicity DNA binding dye with a wide excitation spectrum that can be applied to most currently used FACS orders. This method is very straightforward.The most challenging aspect is the flow cytometry gating but the detailed gating strategy should help with adapting the protocol to other cells sorters. Demonstrating the procedure will be Yu-Han Yeh, a research assistant from Satoshi Namekawa's Laboratory. After harvesting both testes from an eight week old male mouse, place the tissues in a 60 millimeter Petri dish containing two milliliters of ice cold PBS and remove the tunica albuginea.Gently separate the testes with forceps to slightly disperse the seminiferous tubules. And transfer the tubules into a drop of fresh ice cold PBS in a 100 millimeter Petri dish. Use the forceps to gently untangle the tubules and wash the tubules in three additional droplets of PBS as just demonstrated.After the last wash, place the untangled seminiferous tubules into a 15 milliliter tube, containing two milliliters of dissociation buffer for a 20-minute incubation at 37 degrees Celsius. At the end of the incubation, use a 1000 microliter pipette to gently pipette the tubules 20 times, before returning the tube to the incubator for an additional six minutes. At the end of the incubation, pipette the tissues an additional 20 times, before returning the tube to the incubator.After three minutes, gently pipette the tubules 10 times or until no visible tissue pieces remain, before stopping the dissociation with 10 milliliters of FACS buffer. Then, centrifuge to the tissue suspension two times using an additional 10 milliliters for fresh FACS buffer for the second wash to remove the spermatozoa and as much debris as possible. To stain the cells for flow cytometric analysis, re-suspend the pellet in three milliliters of FACS buffer.And filter the cell suspension through a 70 micron nylon cell strainer, into a 50 milliliter tube. After counting, transfer 10%of the cells into a new tube on ice as the unstained negative control. And thoroughly mix six microliters of DCV stain into the remaining cell suspension.After a 30-minute incubation at 37 degrees Celsius in the dark with gentle shaking every 10 minutes, add five microliters of DNase I to the cells. And filter the cells through a 35 micron nylon mesh strainer into a five milliliter FACS tube on ice. For flow cytometric analysis of the cells, create a new experiment in the flow analysis software, and under the worksheet tools menu, click new density to create a forward scatter area versus backscatter area density plot on a linear scale.Click new histogram to create a DCV blue histogram plot on a logarithmic scale. And click start and record to begin processing the unstained sample. While the sample is running, click detector and threshold settings to adjust both the forward and side scatter PMT voltages to place the unstained cells on the scale of the forward and back scatter plot.Adjust the PMT voltage for the jappy channel to locate the position of the DCV negative population in the first decade of the DCV blue histogram logarithmic plot. Then, click stop to unload the unstained sample. After briefly vortexing and loading the stained sample, click next tube to create a new worksheet for the sample.Set the cytometer to acquire at least one times 10 to the six events, and click start and record. Next, click new density to create forward scatter height versus forward scatter width. And DCV blue versus DCV red density plots on a linear scale.On the forward scatter area versus backscatter area density plot, use the polygon tool to draw a gate called cells to include most of the cells and to exclude small debris. Apply this gate to the forward scatter height versus forward scatter width plot and use the rectangle tool to draw a single cell's gate to exclude non single cells. Apply the single cell's gate to the DCV blue versus DCV red density plot and adjust the scale to capture an extended profile.Use the polygon tool to draw a DCV gate to exclude the unstained cells and side population, and apply the gate to a DCV blue histogram plot on a linear scale. The three major peaks refer to the different 1C, 2C and 4C DNA contents. Create a second DCV blue versus DCV red density plot on a linear scale.And back gate the DCV gate onto the plot to locate the 1C and 4C populations. Create another DCV blue versus DCV red density plot on a linear scale. And use the ellipse tool to draw a gate on the 1C population.Use the polygon tool to gate the 4C population with a continuous curve. And create another DCV blue versus DCV red density plot on a linear scale. Use the polygon tool to create a 4C one gate.In a new forward by side scatter plot, use the ellipse tool to create pachytene, diplotene and leptotene, zygotene spermatocyte gates. When all of the gates have been drawn, create a new DCV blue versus DCV red color dot plot on a linear scale to apply the for 4C gate to ensure that the three populations are in a continuous order within the gate. Next, create a new forward scatter area versus backscatter area density plot on a linear scale, and select the unified size of cells as a pure round spermatid population.After sorting, the representative purities of the separated leptotene, zygotene, pachytene and diplotene spermatocyte fractions are typically between 80%to 90%as confirmed by SYCP3 and Gamma H2AX immunostaining. The purity of the round spermatid faction can be confirmed by nucleus staining with DCV in combination with Sp56 and H1t staining, and is also typically around 90%The viability of the isolated cell populations is usually over 95%And the average total yields of each fraction from a single adult mouse is typically sufficient for various downstream analysis. The sorted cells can be used for various next generation sequencing analysis to better explore gene expression and regulation during spermatogenesis.

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Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...