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 collecting tubes (1.5 mL tubes) with Fetal bovine serum (FBS) at 4 &#176;C overnight.</li>
	<li>On the day of the experiment: Set the water bath to 37 &#176;C.</li>
	<li>Pre-warm Dulbecco&#39;s Modified Eagle Medium (DMEM) at 37 &#176;C and prepare 2 mL of 1x dissociation buffer for each sample right before use (Table 1) in 15 mL centrifuge tube. 
	NOTE: 2 mL dissociation buffer is prepared for two testes. For the dissociation buffer recipe, please refer to Table 1.</li>
</ol>
2. Animal dissection and preparation of testicular cell suspension
<ol>
	<li>Sacrifice an 8-week-old male mouse by leaving in a carbon dioxide chamber for at least 10 min.</li>
	<li>Remove both testes and place on a 60 mm Petri dish containing 2 mL of ice-cold phosphate-buffered saline (PBS).</li>
	<li>Remove the tunica albuginea from the testes. Slightly disperse the seminiferous tubules by gently separating with forceps.</li>
	<li>Transfer the seminiferous tubules to a 100 mm Petri dish with a new drop of ice-cold PBS and untangle seminiferous tubules gently with forceps. Repeat this wash 3 times to remove interstitial cells as much as possible.</li>
	<li>Incubate untangled seminiferous tubules in a 15 mL tube containing 2 mL of dissociation buffer at 37 &#176;C for 20 min.</li>
	<li>Gently pipette the tubules 20 times using a 1000 &#181;L micropipette.</li>
	<li>Incubate for 6 min. Repeat gentle pipetting 20 times.</li>
	<li>Incubate for 3 min. Repeat gentle pipetting 10 times until no visible chunks remain.</li>
	<li>Add 10 mL of FACS buffer to the suspension. Centrifuge at 300 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Repeat step 2.9 twice to remove the spermatozoa and as much debris as possible.</li>
</ol>
3. Cell staining
<ol>
	<li>Resuspend the cell pellet with 3 mL of FACS buffer (Table 1) and filter the cell suspension through a 70 &#181;m nylon cell strainer into a 50 mL tube. 
	NOTE: The expected cell yield is approximately 100 million cells per two testicles (from an 8-week-old B6 wild-type mouse).</li>
	<li>Count the cell number and split 10% of the cells into a new tube as the unstained negative control and leave on ice.</li>
	<li>Add 6 &#181;L of DCV stain (the original concentration is 5 mM) to the remaining cell suspension and mix well. The final concentration is 10 &#181;M, and the capacity is approximately 100 million cells.</li>
	<li>Incubate at 37 &#176;C for 30 min in the dark. Gently shake the cell suspension every 10 min.</li>
	<li>After incubation, without a wash step, directly add 5 &#181;L of DNase I (the stock concentration is 10 mg/mL) to the cell suspension and filter the cells into a 5 mL FACS tube through the 35 &#181;m nylon mesh cap. Keep the samples on ice until sorting.</li>
</ol>
4. Flow cytometry and experimental gates
<ol>
	<li>Prepare the FACS cell sorter. Ensure that FACS cell sorter is equipped with Excitation optics: Violet (405-nm) laser; Detection optics: &#64257;lter combination of 450/50 bandpass [same filter set of 4&#8242;,6-diamidino-2-phenylindole (DAPI) for DCV-blue detection] and 665/30 bandpass [same filter set of Allophycocyanin (APC) for DCV-red detection]. Here we use Sony SH800S cell sorter as an example.</li>
	<li>Create a new experiment and set up the following working plots displaying parameters to be optimized: Click &#8220;New Density&#8221; on the &#8220;Worksheet Tools&#8221; menu bar to create a forward scatter - area (FSC-A) vs. back scatter &#8211; area (BSC-A) density plot on a linear scale. Click &#8220;New Histogram&#8221; to create a DCV-blue histogram plot on logarithmic scale.</li>
	<li>Briefly vortex the unstained negative control (from step 3.2) and load the sample.</li>
	<li>Click &#8220;Start&#8221; and &#8220;Record&#8221; to begin processing the unstained sample. While the sample is running, click &#8220;Detector &#38; Threshold Settings&#8221; to optimize FSC and BSC voltages by adjusting both the photomultiplier tube (PMT) voltages up or down to place the unstained cells on the scale of the FSC/BSC plot.</li>
	<li>Adjust the PMT voltage up and down on &#8220;FL1: DAPI&#8221; while the sample is running to locate the position of the DCV-negative population in the &#64257;rst decade of the DCV-blue histogram logarithmic plot (Figure 2A). After completing the PMT voltage adjustment, click &#8220;Stop&#8221; to unload the unstained sample.</li>
	<li>Briefly vortex the sample (from step 3.5) and load the sample.</li>
	<li>Click &#8220;Next Tube&#8221; to create a new worksheet for the DCV-stained sample; and click &#8220;Start&#8221; and &#8220;Record&#8221; to acquire the DCV-stained sample, record &#8805; 1 x 106 events. Add the following working plots: Click &#8220;New Density&#8221; on the &#8220;Worksheet Tools&#8221; menu bar to create an FSC-H (height) vs. FSC-W (width) density plot on a linear scale; Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale. After recording &#8805; 1 x 106 events, click &#8220;Stop&#8221; and unload the sample.</li>
	<li>On the FSC-A vs. BSC-A density plot, click &#8220;Polygon&#8221; on the &#8220;Plot Tools&#8221; menu bar to draw a big gate &#8220;Cells&#8221; to include most cells and exclude small debris (Figure 2B). Apply the gate &#8220;Cells&#8221; to FSC-H vs. FSC-W density plot. Click &#8220;Rectangle&#8221; to draw a &#8220;Single Cells&#8221; gate to exclude non-single cells (Figure 2C).</li>
	<li>Apply &#8220;Single Cells&#8221; gate to DCV-blue vs. DCV-red density plot and adjust the scale to capture an extended profile as shown in Figure 2D. Click &#8220;Polygon&#8221; on the &#8220;Plot Tools&#8221; menu bar to draw a &#8220;DCV&#8221; gate to exclude the unstained cells and side population (Figure 2D).</li>
	<li>Apply &#8220;DCV&#8221; gate to DCV-blue histogram plot on a linear scale. The three major peaks refer to different DNA content: 1C, 2C, and 4C (Figure 2E).</li>
	<li>Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale and apply &#8220;DCV&#8221; gate to perform back gating from Figure 2E to locate the 1C and 4C populations (Figure 2F). Click &#8220;Ellipse&#8221; to draw a gate on the 1C population, which is within a condensed area (gate 1C). Click &#8220;Polygon&#8221; to draw a gate on the 4C population which is a continuous curve (gate 4C).</li>
	<li>Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale and apply &#8220;4C&#8221; gate; adjust the scale to zoom in and click &#8220;Polygon&#8221; to draw a &#8220;4C_1&#8221; gate for more precise selection (Figure 2G).</li>
	<li>Click &#8220;New Density&#8221; to create a new FSC-A vs. BSC-A density plot on a linear scale and apply &#8220;4C_1&#8221; gate; there will be three enriched populations separated by size corresponding to leptotene (L) /zygotene (Z), pachytene (P) and diplotene (D) spermatocytes. Click &#8220;Polygon&#8221; or &#8220;Ellipse&#8221; to draw 3 gates: &#8220;L/Z&#8221;, &#8220;P&#8221;, and &#8220;D&#8221; based on the growing size (Figure 2H).</li>
	<li>Click &#8220;New Dot Plot&#8221; to create a DCV-blue vs. DCV-red color dot plot on a linear scale to apply gate and ensure the three populations are in a continuous order within the &#8220;4C_1&#8221; gate (Figure 2I).</li>
	<li>Similarly, for the 1C population, click &#8220;New Density&#8221; to create a new FSC-A vs. BSC-A density plot on a linear scale and apply &#8220;1C&#8221; gate, select the unified size of cells as pure round spermatid population, and click &#8220;Ellipse&#8221; to draw an &#8220;RS&#8221; gate (Figure 2J).</li>
</ol>
5. Sort male germ cell subpopulations
<ol>
	<li>Prepare 1.5 mL tubes containing 500 &#956;L 50% FBS for cell collection and load the collection tube into the collector and click &#8220;Load Collection&#8221;.</li>
	<li>Click &#8220;Next Tube&#8221;, &#8220;Start&#8221;, &#8220;Record&#8221; and &#8220;Sort Start&#8221;. For using a two-way system that allows two populations of interest to be sorted at the same time into the collection device, follow pairwise combinations: leptotene (L) /zygotene (Z) and pachytene (P) spermatocytes based on L/Z and P back-gates; Round spermatids (RS) and diplotene (D) spermatocytes based on RS and D back-gates.</li>
	<li>While the sample is running, adjust the flow rate to ~3000 events/s to get the most efficient sorting.</li>
</ol>
6. Purity analysis of sorted cells
<ol>
	<li>Collect &#8805; 10,000 cells/each population. Perform cell immunostaining to confirm the substage.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C and carefully discard the supernatant, keeping around 110 &#181;L of the liquid in the bottom of the tube.</li>
	<li>Take a 10 &#181;L drop of cell suspension to observe under microscope and evaluate the overview cell morphology and number.</li>
	<li>Apply 100 &#181;L of cell suspension to each of the sample chamber slides (see Table of Materials), and load the chambers to the Cytospin.</li>
	<li>Spin the samples at 30 x g for 5 min at room temperature. Draw a circle around the cell with a hydrophobic pen. Dry slides on lab bench for a few minutes at room temperature.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off.</li>
	<li>Add primary antibody solution (Dilute primary antibodies with 5% Donkey Serum in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at 4 &#176;C overnight. 
	NOTE: To judge the substage of meiotic spermatocytes, SYCP3 was detected, which is a marker of meiotic chromosome axes, and &#947;H2AX, which is a marker of DNA damage response. (For the antibody working concentration, please refer to the Table of Materials).</li>
	<li>Tap off the primary antibody solution from the slides.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off. Repeat one time.</li>
	<li>Add secondary antibody solution (dilute secondary antibodies in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at room temperature for 1 h in dark.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off. Repeat one time.</li>
	<li>Add 50 &#181;L of DAPI (stock concentration is 0.1 &#956;g/mL) stain for 5 min and tap off.</li>
	<li>Add 1-2 drops of mounting media on the slides. Carefully cover the mounting media with a cover glass and gently press the cover glass to remove extra mounting media and air bubble. The slides are ready for microscopy evaluation.</li>
</ol>

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The authors declare that they have no competing interests.

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 ...

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Developmental Biology

5.7K Views.  Cincinnati Children's Hospital Medical Center. 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.

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

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, ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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 ...

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 ...