We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

This protocol requires the use of live, adult zebrafish to produce embryos. The embryos are harvested for tissue collection. It is essential to obtain approval from appropriate ethics review boards to conduct this experiment.
1. Obtain Staged Embryos
<ol>
	<li>The day before the experiment, prepare healthy, adult zebrafish for breeding. Place one male and one female on opposite sides of a clear divider in a breeding tank.</li>
	<li>Repeat 1.1 for as many breeding tanks as necessary for sufficient embryo production for the downstream application. Obtain embryos from both wild type fish and transgenic fish that express fluorescent proteins in the cell type of interest. &#160; 
	NOTE: The number of embryos needed for downstream applications in Steps 2-8 depends on the relative abundance of the cells of interest at the time point of interest. Though this may vary by cell type, 200 embryos produce 2,000-5,000 sorted cells when the cells of interest represent &#60;1.0% of the total cells at 24 hpf (hours post-fertilization).</li>
	<li>The next morning, change the water in the breading tank by transferring fish to a fresh breeding tank and remove the divider. Tilt the tank at an angle to encourage breeding.</li>
	<li>Collect staged embryos.
	<ol>
		<li>Every 15 min, collect embryos by transferring the adults to a fresh breeding tank and passing the eggs which are left behind through a tea strainer. 
		NOTE: Zebrafish embryos develop synchronously when maintained at comparable densities and temperatures.</li>
		<li>Rinse the eggs with Egg Water (0.21 g/L Instant Ocean salts in 1 L double distilled water) and transfer to a petri dish. Transfer the petri dish to a humid incubator at 28.5 &#176;C with air circulation.</li>
	</ol>
	</li>
	<li>Two hours after the last collection, sort fertilized, multicellular embryos into 10 cm petri dishes and reduce density to 50 embryos per dish. Select embryos from a single, 15 min time window of collection for downstream application. Incubate embryos at 28.5&#176;C. &#160; 
	NOTE: For example, collect embryos at 8:30, 8:45, 9:00, 9:15, 9:30, 9:45, 10:00 and 10:15 AM. Comparing across time points, if the largest number of fertilized embryos are from the clutches collected at 9:00, then use only these embryos for downstream applications.</li>
</ol>
2. Set Up for Single Cell Dissociation
<ol>
	<li>Approximately 30 min prior to the time point of interest (18 hpf) remove embryos from their chorion manually with fine forceps.</li>
	<li>Collect and label the following for each condition: two 2 ml microcentrifuge tubes, one 40 &#181;m cell strainer, one 35 mm cell culture dish, and two FACS tubes topped with a 35 &#181;m cell strainer.</li>
	<li>Chill the following reagents on ice: Egg Water containing 0.21g/L Instant Ocean salts in 1L double distilled water; De-yolking Buffer containing 55 mM NaCl, 1.8 mM KCl, and 1.25 mM NaHCO3; and FACS Buffer containing Leibovitz&#39;s L-15 media supplemented with 5% heat-inactivated fetal bovine serum.</li>
	<li>Bring 1 ml per sample of Cell Dissociation Reagent 1 to RT. Thaw 1 ml Cell Dissociation Reagent 2 per sample on ice. Bring to RT immediately before use.</li>
</ol>
3. Single Cell Dissociation
<ol>
	<li>Using a wide bore glass pipette, transfer 100-300 embryos to a 2 ml micro centrifuge tube in a minimum volume of Egg Water.</li>
	<li>Euthanize embryos by replacing water with 1 ml ice-cold Egg Water and submerging tube in ice for 20 min. 
	CAUTION! It is critical to obtain approval from proper institutional animal care oversight committee for this euthanasia method (chilling on ice followed by cell dissociation as secondary euthanasia). Standard euthanasia typically requires that embryos &#60;3 days post-fertilization are bleached after they are chilled, which is not appropriate for obtaining viable cells.</li>
	<li>Wash the embryos two times with 1 ml ice-cold Egg Water. To wash, use a wide bore glass pipette to remove Egg Water and a P1000 to add Egg Water.</li>
	<li>Remove the yolk by replacing the embryo water with 1 ml Deyolking Buffer and triturating 8-12 times with a P1000 tip, or until the yolk is dissolved and only the bodies of the embryos are visible.</li>
	<li>Collect the tissue by centrifugation at 300 x g for 1 min. Use a pipette to gently remove the supernatant without disrupting the tissue pellet. Re-suspend in 1 ml Egg Water.</li>
	<li>Repeat step 3.5 for a total of three washes, but on the final wash, re-suspend in 1 ml RT Cell Dissociation Reagent 1.</li>
	<li>Incubate in Cell Dissociation Reagent 1 for 10 min at RT with tubes placed horizontally. Every 2-3 min, gently triturate with a P1000 pipette to prevent clumping. 
	NOTE: Handle samples gently during trituration steps. Sometime a single, large clump will form in the tube from a tangle of embryo bodies. This will disperse with additional digestion aided by gentle trituration. DO NOT VORTEX.</li>
	<li>Collect tissue by centrifugation at 300 x g for 3 min. Remove supernatant and resuspend in 1 ml Cell Dissociation Reagent 2.</li>
	<li>Incubate for 5-15 min at RT. Place the tubes horizontally. Every 2-3 min, gently triturate to prevent clumping. Every 5 min, assess digestion progress.
	<ol>
		<li>To assess digestion progress, dilute 2 &#181;l supernatant into 18 &#181;l FACS Buffer and pipette as a droplet onto a cell culture dish.</li>
		<li>Place a coverslip over the sample and observe under a tissue culture microscope at 10X and 20X magnifications. 
		NOTE: The preparation should appear as a mix of single cells, small clusters, large clusters, and occasional mostly-intact embryo body.</li>
		<li>Visually assess the proportion of the preparation that is single cells and small clusters. &#160; 
		NOTE: Over-digestion will reduce viability; under-digestion will reduce single cell yield.</li>
	</ol>
	</li>
	<li>Collect the cell preparation by centrifugation at 300 x g for 5 min. Discard the supernatant and re-suspend in 1 ml cold FACS Buffer.</li>
	<li>Moisten a 40 &#181;m cell strainer with FACS Buffer. Pass the cell suspension through the 40 &#181;m cell strainer onto a 35 mm cell culture dish. Wash the cell strainer one time with 1 ml FACS sorting buffer.</li>
	<li>Transfer the flow-through to a 2 ml microcentrifuge tube. Collect cells by centrifugation at 300 x g for 5 min and re-suspend in 100 &#181;l FACS Buffer.</li>
	<li>Count cell yield using a hemocytometer. Further dilute samples to 5x106 cells per ml, or optimal concentration recommended for FACS machine of choice. Optional-When counting cell yield on the hemocytometer, counterstain dead cells with trypan blue to confirm viability of the cell preparation prior to FACS sorting. &#160; 
	NOTE: Preparations that are too dilute will take excess amount of time to sort. Preparations that are too concentrated tend to clump into multimers and are suboptimal for sorting a pure population.</li>
	<li>Reserve 10-20% of each sample to use as unstained controls. For remaining samples, stain dead cells by adding a fluorescent live/dead (L/D) discrimination dye into each sample. DO NOT wash. &#160; 
	NOTE: Any fluorescent dye that penetrates cells with compromised cell membranes and stains nucleic acids may be used as the L/D dye, provided that the fluorescence spectra is compatible with the FACS machine of choice and fluorophore labeling cells of interest.
	<ol>
		<li>Do not wash the preparation after adding the dye as some cells will die in transit to FACS purification and should be excluded from the sorted population.</li>
	</ol>
	</li>
	<li>Moisten the 35 &#181;m cell strainer capped FACS tube with 20 &#181;l FACS buffer. Add cells to the strainer and collect by gravity. Store on ice.</li>
</ol>
4. FACS Enrichment
<ol>
	<li>Use FACS to enrich for single, live cells expressing the fluorescent marker.
	<ol>
		<li>Set a gate to distinguish cells from debris on a scatter plot of forward scatter (FSC-A) amplitude vs side scatter amplitude (SSC-A), both with linear scaling. 
		Caution! Zebrafish cells are typically smaller than mouse or human cells. This is reflected in a lower basal separation between cells and debris.</li>
		<li>From the gate set in 4.1.1, set a gate to enrich for single cells and exclude multimers using a scatter plot of forward scatter height (FSC-H) and low side scatter height (SSC-H), both with linear scaling. 
		NOTE: Cells with disproportionately high FSC-H and low SSC-H are likely multimers and are excluded from sorting.</li>
		<li>From the gate set in 4.1.2, using single color controls, set a gate to include only live cells using a scatter plot of FSA-A with linear scaling and amplitude of the channel used to detect L/D stain with log scaling. Include L/D negative cells.</li>
		<li>From the gate set in 4.1.3, using single color controls, set a gate to include only live cells with positive fluorescence using a scatter plot of FSA-A with linear scaling and amplitude of the channel used to detect fluorescent protein cell marker with log scaling. Include positive cells.</li>
		<li>Set any compensation controls, if necessary, to account for interference between the L/D stain and fluorescent protein spectra.</li>
	</ol>
	</li>
	<li>Set sort logic to cells that fall into all of the gates set in step 4.1.
	<ol>
		<li>Using double labeled cells, verify gating for cell sorting. &#160; 
		NOTE: Cells for sorting will be cells rather than debris, single cells rather than multimers, L/D negative and fluorescence positive cells.</li>
	</ol>
	</li>
	<li>Sort 2,000-4,000 cells from the population of interest into 5 &#181;l of cold FACS Buffer in a microcentrifuge tube on ice. To minimize shearing and strain on cells during sorting, use the lowest pressures possible for the cell sorter. &#160; 
	NOTE: The 5 &#181;l droplet of FACS Buffer serves as a cushion for cells exiting the FACS machine. Collecting 2,000-4,000 cells directly into FACS Buffer eliminates the need for centrifugation prior to loading onto the IFC chips. This strategy is recommended because centrifugation can lead to formation of multimers if cells adhere to one another in the pellet.</li>
	<li>Assess the post-sort viability analysis.
	<ol>
		<li>Transfer 1 &#181;l sorted cells to a fresh FACS tube with 100 &#181;l FACS sorting buffer and 1:1,000 dilution of L/D stain. Pass the cells through the FACS sorter with the gating strategy in Step 4.1. Use the proportion of L/D negative to positive to estimate viability of sorted cells.</li>
	</ol>
	</li>
</ol>
5. Load Cells onto Microfluidics Chip
<ol>
	<li>Using a hemocytometer, measure both the concentration and size of sorted cells.
	<ol>
		<li>Dilute sorted cells to at least 10 &#181;l. Dilute an aliquot of 5 &#181;l cells with 5 &#181;l trypan blue. Load onto hemocytometer and apply coverslip.</li>
		<li>Collect bright field images of all cells in 4 x 4 grids of hemocytometer. Count the number of live cells and calculate live cells/ml. Live cells do not take up trypan blue. &#160; 
		NOTE: Use any standard image analysis software to measure the diameter of all live cells. Calculate the average and standard deviation of the cell size, and select an IFC plate suitable for the cell size range of interest. If cell size range straddles an IFC plate cell size range, use multiple plates to capture the full range of cells of interest.</li>
	</ol>
	</li>
	<li>Load cells on to IFC plate according to manufacturer&#39;s instructions (see Materials).
	<ol>
		<li>Dilute cells to 1x106 cells/ml and perform buoyancy optimization as per manufacturer&#39;s instructions. &#160; 
		NOTE: Buoyancy optimization ensures that cells neither sink to the bottom nor float to the top of the loading well for optimal loading onto IFC chip. The ratio of buffer to cells may vary by cell type, but typically ranges 6:4-7:3 of cells:buffer.</li>
		<li>Add cells to primed-IFC plate. Load plate into compatible fluidics machine, and run a cell loading script to push cells through microfluidics circuit and into capture lanes.</li>
	</ol>
	</li>
	<li>Confirm that cells are lodged in capture sites on IFC plate.
	<ol>
		<li>Remove IFC plate from fluidics machine. Mount the IFC plate on a microscope equipped with a plate adaptor.</li>
		<li>Collect snapshots of bright field and fluorescence for each capture site at 10X magnification. &#160; 
		NOTE: Brightfield capture times may range from 10-50 ms, depending on lamp intensity. Fluorescence capture times may range from 250-750 ms, depending on fluorophore brightness. Avoid overexposing cells to prevent photodamage.</li>
	</ol>
	</li>
</ol>
6. cDNA Synthesis
<ol>
	<li>Perform cell lysis in situ according to IFC plate manufacturer&#39;s instructions.
	<ol>
		<li>Add lysis buffers to wells on IFC plate. Load plate onto compatible fluidics machine and run cell lysis script as per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Perform reverse transcription according to IFC plate manufacturer&#39;s instructions.
	<ol>
		<li>Add reverse transcription reagents to IFC plate. Load plate onto compatible fluidics machine. Run reverse transcription script. Sample cycling conditions: 1 cycle at 25&#176;C for 10 min, 1 cycle at 42&#176;C for 1 hr, then 1 cycle at 85&#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Perform pre-amplification with gene-specific probes according to IFC plate manufacturer&#39;s instructions. 
	NOTE: Due to their greater specificity with low-copy numbers, use fluorogenic-labeled probes rather than probes optimized for use with intercalator dyes.
	<ol>
		<li>Pool primers and dilute to final 180 nM each. Add pooled primers to IFC plate. Load plate onto compatible fluidics machine. Run preamplification script.</li>
	</ol>
	</li>
	<li>Perform pre-amplification with the following cycle conditions: 1 cycle at 95&#176;C for 10 min, then 18 cycles with denaturing at 95&#176;C for 15 sec then annealing and extension at 60&#176;C for 4 min. Store pre-amplified cDNA at 4&#176;C until harvesting. Harvest cDNA from microfluidic plate according to manufacturer&#39;s instructions and store at -20&#730;C until use.</li>
</ol>
7. Select cDNA from Single Cells for Single Target qRT-PCR
<ol>
	<li>Dilute cDNA in 25 &#181;l dilution reagent according to manufacturer&#39;s instructions. &#160; 
	NOTE: Approximate final yield is 28 &#181;l of pre-amplified cDNA per cell. Reserve an aliquot of diluent for use as a no-template control in qRT-PCR.
	<ol>
		<li>Referring to bright field and fluorescent images recorded in step 5.3.2, score each capture site. Manually count and record the number of cells. Assess and record whether each cell is fluorescent. &#160; 
		NOTE: Each individual site may contain 0, 1, or &#62;1 cell, where &#62;1 cell includes capture sites that contain multiple cells and/or unidentifiable debris. If the images are sufficient quality, a pixel value may also be assigned to quantify the intensity of the fluorescence signal.</li>
	</ol>
	</li>
	<li>Select samples for qRT-PCR analysis.
	<ol>
		<li>Select cDNA samples from capture sites identified in step 7.2 that contain exact 1 cell with positive fluorescence. Pool 1 &#181;l aliquots of cDNA from each sample. 
		NOTE: This is the &#34;Pool&#34; positive control used to determine whether genes of interest are expressed in any of the selected cells.</li>
		<li>Select cDNA from at least one capture site that contains exactly 0 cells as a negative control. Use diluent from step 7.1.2 as a no-template control.</li>
	</ol>
	</li>
	<li>Run qRT-PCR assay.
	<ol>
		<li>Use only probes used for pre-amplification in step 6.4.1. Load all samples in triplicate. Cycling conditions for a 10 &#181;l reaction on a 384 well plate: 1 cycle 50&#176;C 2 min, 1 cycle 95&#176;C for 10 min, 40 cycles with denaturing at 95&#176;C 15 sec then annealing and extension at 60&#176;C for 1 min.</li>
	</ol>
	</li>
</ol>
8. Data Analysis
<ol>
	<li>Validate qRT-PCR products by standard gel electrophoresis. Confirm product is a single band at expected molecular weight (varies for each probe). &#160; 
	NOTE: If a probe produces multiple bands or a single band at an inappropriate size, then specificity is questionable, and this excludes the gene from analysis.</li>
	<li>Examine all amplification curves and account for any abnormalities14. Calculate the average CT value for each sample/gene combination15. &#160; 
	NOTE: CT values for positive controls typically range from 10-30. CT values for negative controls typically range from 35-40 (no amplification). CT values 30-35 are considered very low expression and should be interpreted with caution14.</li>
	<li>Report data
	<ol>
		<li>If samples fall in the CT=10-30, data may be also reported as fold change in transcript abundance relative to a control sample.&#160;&#160; 
		NOTE: Fold change equals 2^(-&#916;&#916;CT) where &#916;CT is relative to a housekeeping gene by subtracting the average CT of the sample from the housekeeping gene and &#916;&#916;CT is the differences in &#916;CT between the sample and a positive control15.</li>
	</ol>
	</li>
</ol>

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N., Chen, J. N., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+in+the+heart+field+of+zebrafish.">Regulation in the heart field of zebrafish.</a> Development. 125, 1095-1101 (1998).</li><li>de Pater, E., 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=Distinct+phases+of+cardiomyocyte+differentiation+regulate+growth+of+the+zebrafish+heart.">Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.</a> Development. 136, 1633-1641 (2009).</li><li>Hami, D., Grimes, A. C., Tsai, H. J., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+development+requires+a+conserved+secondary+heart+field.">Zebrafish cardiac development requires a conserved secondary heart field.</a> Development. 138, 2389-2398 (2011).</li><li>Yelon, D., Horne, S. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Developmental biology. 214, 23-37 (1999).</li><li>Molina, N., 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=Stimulus-induced+modulation+of+transcriptional+bursting+in+a+single+mammalian+gene.">Stimulus-induced modulation of transcriptional bursting in a single mammalian gene.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 20563-20568 (2013).</li></ol>

The authors have nothing to disclose.

The method described herein uses expression of a fluorescent protein under control of a cell-type specific promoter to enrich a population of cardiac progenitor cells from zebrafish embryos for use in microfluidic assisted single cell capture system to assess expression of a subset of cardiac genes in single cells. Provided that FACS laser excitation and emission capabilities are compatible with the fluorophore(s) of choice, this method can be used for any fluorescent reporter line. Many zebrafish reporter lines are already in existence, and transgenic fish carrying novel reporters can be generated in as little as 3 months. Additionally, this work flow may be adapted to produce cDNA from the whole transcriptome for single cell-RNA sequencing. To do so, steps 5-7 will need to be modified slightly for use with chemistries optimized for preparing cDNA for generating libraries suitable for RNA sequencing. However, it is advisable to validate population heterogeneity by using the method described here prior to pursing high throughput sequencing applications.
It is important to note that there are some limitations to the described method. First, use of integrated microfluidic circuit (IFC) plates requires specialized, costly equipment. However, microfluidic chips offer substantial advantages over traditional 96 well and 384 well plate formats for assaying single cells. By flowing cells and reagents through micrometer scale lanes, single cells are positioned into single capture sites where they can be directly observed to confirm that they are single cells in good health. RNA extraction and cDNA synthesis occur in situ on the IFC chip using very low volumes for each cell. Integrated fluidic chips, at the time of this writing, are commercially available through only one source. Though non-commercial fabrication has been performed by several groups, it is outside of the capacity of most laboratories. Steps 5-7 may need to be modified for platforms produced by other companies or in-house fabrications. Second, though commercially available IFC plates can accommodate up to 96 genes, the genes selected are limited by the availability of fluorogenic-labeled gene probes validated for use in zebrafish. Third, zebrafish cells are typically smaller than mammalian cells, and this small size presents challenges to sample processing. A small change in cell diameter translates into a large change in cell volume, reducing available material and decreasing the likelihood of detecting low expressing genes.
There are several additional considerations for this protocol. In step 1, it is important to only use embryos fertilized within a short time window. Keeping temperature constant and without limiting oxygen conditions, zebrafish embryos develop synchronously. The ultimate readout of this protocol (relative gene expression from single cells) cannot discriminate the source of heterogeneity between individual cells. For this reason, interpretation of intercellular heterogeneity relies on carefully staged embryos and synchronous development. In steps 2-3, there is a tradeoff between dissociation and viability. Though step 4 selects for viable cells, and step 3 contains several filtering steps that help exclude multimers, over-digestion will reduce the viable material available for FACS and potentially reduce sorted cell yield. Steps to troubleshoot low viability include reducing digestion time, reducing trituration intensity, and optimizing the number of embryos in the starting material. FACS enrichment of the cell population of interest (step 4) is arguably the most critical step in this protocol. Stringent sorting criteria are essential to generate a single cell preparation containing only live cells expressing the fluorescent protein of interest. Contaminating cells from other populations could lead to false representation of intercellular heterogeneity. Notably, FACS enrichment of a population of interest is not restricted to the two color strategy shown in this protocol. More complex strategies can produce more refined cell populations and are a rapid way to extend the methodology reported in the proof of principle study. In step 5, cells may be directly observed to confirm the number of cells captured, cell health, and expression of fluorescence protein. But, depending on cell size and brightness, the fluorophore of interest may not be visually detectable and is not a reliable readout. Successful completion of steps 6-8 requires careful adherence to manufacturer&#39;s protocols.
As proof of principle, expression levels of a subset of known cardiac markers were assessed in single derived from a previously described BAC transgenic zebrafish line nkx2.5:ZsY to examine cardiac differentiation markers in individual nkx2.5:ZsY positive cells at 18 hr post fertilization (hpf). To explore the heterogeneity of differentiation markers expressed in captured nkx2.5:ZsYexpressing cells, a suite of genes were assayed including housekeeping genes (ef1a, gapdh), transcription factors known to be turned on early in cardiac specification (gata4, nkx2.5), a second heart field progenitor marker (isl1), and genes known to be turned on later during cardiac differentiation (myl7, vmhc). The relative abundance of each gene was measured by qRT-PCR by calculating cycle threshold (CT). The lowest CT value calculated was 17.9 and the highest was 40, representing no amplification (Table 1). CT values of &#62;35.0 were considered below detection.
Of the 46 cells in the data set, 6 were excluded due to failed amplification of any genes. The remaining cells were sub-categorized cells into 5 types based on expression of gata4, nkx2.5, myl7, and vmhc, and were sorted by ef1a value within groups. Though gapdh was originally included as a possible housekeeping gene, expression was highly variable across cells, making it unsuitable as a housekeeping gene. Gapdh expression likely better represents changes to energy metabolism associated with cardiac differentiation in this cell type. Interestingly, there was one cell in which ef1a was un-detectable but another gene was detectable (gata1 in cells 26). This suggests that while ef1a is generally an adequate housekeeping gene to differentiate between successful and unsuccessful single cell reverse transcription reactions, it may not be ideal for all applications. The lack of ef1a expression in cell 26 could be due to single cell transcriptional dynamics. Recent single-cells studies demonstrate that gene transcription is fundamentally stochastic, alternating between phases of rapid and negligible transcriptional activity26. This transcriptional bursting model suggests that use of a housekeeping gene for quantitative comparisons between single cells may be altogether inappropriate.
In the Type 1 group, ef1a is the only gene detectable. These cell may comprise either nkx2.5:ZsY negative cells or cells with very low expression of the other genes of interest. Type 2 cells, with the exception of Cell-26, expressed both ef1a and gata4 with no correlation in the relative expression levels of these genes. Notably, nkx2.5 expression in Type 1 and Type 2 cells may be due to sub-optimal FACS stringency, transcriptional bursting, transgene leakiness, or sub-threshold expression levels. Type 3 cells expressed detectable levels of ef1a, gata4, and nkx-2.5. These cells are likely cardiac progenitor cells and may include differentiating atrial cardiomyocytes. Type 4 cells expressed detectable levels of ef1a, gata4, nkx2.5, myl7 and vmhc and are likely cells differentiating into ventricular cardiomyocytes. Type 5 cells expressed ef1a, gata4, nkx2.5, myl7, vmhc, and gapdh. The addition of detectable levels of gapdh suggests that these cells have the capacity for enhanced glycolytic metabolism found in differentiated cardiomyocytes. It was intriguing that, despite sorting for ZsYellow positive cells from Tg(nkx2.5:ZsYellow), embryos, 21/40 cells had sub-threshold levels of nkx2.5 in our qRT-PCR analysis. This could be due to transcriptional bursting or could reflect differences in transcript processing between endogenous nkx2.5 and ZsYellow. Importantly, the second heart field marker isl1 was undetectable in any cells or cDNA pooled from all of our capture sites. In sum, comparing across samples, substantial heterogeneity was evident at the single cell level, suggesting that at 18 hpf, nkx2.5:ZsY+ cells comprise cardiac progenitors as well as progeny at different stages of differentiation.

Most current studies of cell and molecular biology are based on population averages. However, important biological events may be masked by these traditional population-based analyses since minor populations can play major roles in biological processes and disease outcome. Understanding gene expression in heterogeneous populations at the single cell level can (and has) lead to relevant biological and clinical insights1,2. Of concern to embryonic development studies, in a larger population of cells, progenitor cells are often underrepresented, making it challenging to detect subtle changes in gene expression that ultimately initiate cell fate decisions3. Similarly, a single cell type may have different expression profiles in response to the microenvironment4. For example, resident endothelial cells in the same organ or in different organs (e.g., aorta or kidney) exhibit significant heterogeneity despite sharing common morphological and functional features5. In addition, cancer cells populating the same tumor can also have varying molecular profiles or mutations at the single cell level6.
In model systems, transcriptomics in single cells has successfully identified new cell populations, characterized intermediate states that occur during cell differentiation, and revealed differential cellular responses to stimuli7,8,9. Such insights would have been masked in conventional population-based studies. Zebrafish embryos are a tremendously under-utilized source of stem, progenitor, and differentiating cells for exploring questions of single cell heterogeneity and molecular regulation of cellular identities during development. Their highly stereotyped, ex vivo development and ease of genetic manipulation make them an excellent model system for this approach10,11. Specifically, a major limitation to interpretation of single cell gene expression data is that reliable identification of novel intermediate cell states during development requires very careful timing of tissue collection9. This is necessary to ensure that heterogeneity between captured cells represents heterogeneity within a tissue at a single time point rather than heterogeneity in gene expression presented by age-dependent cell differentiation. Compared to mice, zebrafish embryo development may be precisely synchronized across a large number of embryos12. Additionally, with large clutch sizes, zebrafish embryos can be used as an abundant source of stem and progenitor cells.
This protocol describes a method to isolate cells from zebrafish embryos and capture single cells using a commercially available integrated microfluidics circuit (IFC) chip and autoprep system for qRT-PCR gene expression analysis. This protocol can be rapidly transferrable to any high throughput multiplexing assays including whole transcriptome sequencing that allows more comprehensive analysis of cellular heterogeneity13. It also offers several advantages to traditional gene expression assays. The single cell isolation protocol yields high viability after FACS, which decreases the proportion of compromised cells that are included in downstream applications. By using an IFC, captured cells may be directly observed to evaluate capture rates and assess cell health morphologically. In addition, this protocol is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidic cell capture technologies.
As proof of principle, single cells derived from cardiac progenitors were isolated and captured on an IFC chip, and then the relative abundance of cardiac differentiation markers was measured by qRT-PCR. Gene expression analysis at the single cell level demonstrates that cardiac progenitors coexist with their differentiating progeny. The insight gained from single-cell profiling of cardiac progenitors may shed light on the heterogeneity in gene expression patterns among cardiac progenitor cells during vertebrate development, which may have been masked in traditional population-based analyses.

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplies</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11254-20</td>
			<td>For removing the chorion from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube 2 mL</td>
			<td style="width:111px;">GeneMate</td>
			<td style="width:103px;">C-3261-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um cell strainer</td>
			<td style="width:111px;">Biobasic</td>
			<td style="width:103px;">SP104151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm culture dish</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">351008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS tubes topped with 35 um cell strainer</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005091</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IFC chip manufacturer&#39;s protocol</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-6117</td>
			<td>Version 100-6117 E1 was used in representative experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wide bore pastuer glass pippette</td>
			<td style="width:111px;">VWR</td>
			<td style="width:103px;">14673-010</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult wild type zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used AB line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult transgenic zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used Tg(nkx2.5:ZsYellow)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents for cell dissociation</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double distilled water</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:111px;">Instant Ocean</td>
			<td style="width:103px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">S271-3</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">P5405</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">S6014</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leibovitz&#39;s L-15</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">03-600-511</td>
			<td>Heat inactivate; any brand of FBS should be fine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 1 -TrypLE</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">12605-010</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 2 - FACSmax</td>
			<td style="width:111px;">Genlantis</td>
			<td style="width:103px;">T200100</td>
			<td>Store -20; thaw on ice; bring to room temp before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pronase (optional)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">P5147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L/D Dye - Sytox Blue</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">S34857</td>
			<td>Any live/dead stain suitable for flow cytometry will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents for IFC plate use and qRT-PCR</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene-specific probes</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>Probes will vary by experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe ef1a</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03432748_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe gata4</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03443262_g1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe nk2-5</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03074126_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe myl7</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03105700_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe vmhc</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03431136_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe isl1</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03425734_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4369016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents listed in IFC manufacturer&#39;s protocol</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 Reagent Kit</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5319</td>
			<td>Reagents for loading cells onto IFC plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ambion Single Cell-to-CTTM</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4458237</td>
			<td>Reagents for reverse transcription and pre-amplification steps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Molecular Biology Quality Water</td>
			<td style="width:111px;">Corning</td>
			<td style="width:103px;">46-000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 IFC for PreAmp (5-10 um)</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5757</td>
			<td>IFC plate for small cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 AutoPrep machine</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5477</td>
			<td>For IFC plate use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">Z359629</td>
			<td>For counting cells and assessing cell size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For removing embryos from chorion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue culture microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For assessing single cell digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS machine</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For isolating cells of interest</td>
		</tr>
	</tbody>
</table>

isolation and characterization of single cells from zebrafish embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

As proof of principle, gene expression was assessed to explore differentiation dynamics during cardiac development. In zebrafish, cardiac progenitors arise from a mesodermal population of cells that migrate to the anterior lateral plate mesoderm where they fuse to form the linear heart tube. Prior to fusion, cardiac progenitors begin to express the transcription factor nkx2.5 (NK2 homeobox 5), which is thought to be the earliest specific marker of cardiac progenitors16,17. Here, a previously described BAC transgenic fish Tg(nkx2.5:ZsYellow)18, abbreviated nkx2.5:ZsY was used to examine cardiac differentiation markers in single cells at the 18 somite stage, 18 hr post fertilization (hpf)12. This was the earliest time point at which ZsYellow signal was visually detectable. As previously described for this transgenic line, ZsYellow labels cardiac progenitors, as well as a few extra-cardiac cells that give rise to the pharyngeal arch endothelial cells at 28 hpf19 (Figure 1A-B, data not shown). At 18 hpf, nkx2.5:ZsY embryos were dissociated into a single cell suspension then stained with Sytox Blue to exclude dead cells. Live, ZsYellow positive, Sytox Blue negative cells were FACS sorted using either a MoFloXDP or Sony SH800Z sorter equipped with 100 &#181;m nozzle (Figure&#160;1C-F). To assess purity of the sorted population and post-sort viability, an aliquot of sorted cells were stained with Sytox Blue and evaluated the percentage of events that fell within the original sorting gate (Figure 1G-J).
After sorting, cells were entered into an integrated microfluidic circuit (IFC) chip work flow (Figure 2A) according to manufacturer&#39;s instructions. A hemocytometer, combined with trypan blue exclusion was used to measure cell diameter, concentration, and viability (Figure 2B, and data not shown). Cell buoyancy was optimized (6.5:3.5 cells:buffer) according to manufacturer&#39;s instruction, and cells were loaded onto an IFC to capture 5-10 &#181;m diameter cells. To assess capture efficiency, fluorescence and/or bright field signals were imaged at all capture sites, a tiling function in FIJI was used to stitch a single picture of the microfluidics plate. Each capture site contained 0, 1, or &#62;1 individual cells (Figure 2C-F). As expected from FACS enrichment, captured cells expressed ZsYellow (Figure 2G). Capture efficiency exceeded 90% in 5 individual experiments using nkx2.5:ZsY sorted cells, and at least 70% of capture sites were occupied by a single cell (data not shown). Cells were lysed, RNA isolated, cDNA synthesized and specific target genes were amplified, all according to manufacturer&#39;s instructions.
A subset of cell capture sites were selected for qRT-PCR analysis as described in the protocol above. Specifically, elongation factor 1a (efl1a)20 and glyceraldehyde-3 phosphate (gapdh)21 were assayed as housekeeping genes expected to be expressed in every cell; GATA binding protein 4 (gata4)22 and NK2 homeobox 5 (nkx2.5) as early cardiac progenitor markers; ISL LIM homeobox 1 box 1 (isl1) as a second heart field marker23,24; and myosin light chain (myl7) and ventricular myosin heavy chain (vmhc) 25 to mark ventricular cardiomyocytes. Gene-specific probes were validated using cDNA from 48 hpf embryos (Figure 3). qRT-PCR was used to assess relative gene expression of these genes in 45 single cells from 18 hpf embryos, a no-template negative control, and a pooled population positive control containing cDNA from all 96 capture sites. Raw CT values for 40 representative cells and controls are shown in Table 1. Notably, from the pool of 46 cells examined, 6 cells were excluded from analysis because all gene expression CT values exceeded CT=35.0, and it is unknown whether these high CT values are attributable to true, very low expression levels or sample degradation. Since the range of CT values for the housekeeping gene, ef1a, was too broad to compare gene expression between samples, and many CT values exceeded 30, CT values were visualized as a heat map (Figure 3). Comparing across samples, substantial heterogeneity in gene expression was observed, and cells were classified cells as Type 1-5 based on expression pattern (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53877/53877fig1.jpg" /> 
Figure 1.&#160;Single cell isolation of zebrafish nkx2.5:ZsY positive cells at 18 hpf. Whole mount images of representative Tg(nkx2.5:ZsYellow) embryo at 18 hpf with (A) ZsYellow fluorescence alone or (B) merged with bright field image. (C-F) Representative FACS gating strategy to enrich for ZsYellow positive cells and (G-J) post-sort analysis with (C,G) FSC/SSC size gating, (D,H) doublet discrimination, (E,I) Live/Dead gating, and (F,J) sorted population. Scale bar is 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53877/53877fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53877/53877fig2.jpg" /> 
Figure 2. Single cell capture of zebrafish nkx2.5:ZsY positive cells. (A) Work flow. (B) Cell size distribution for sorted cells from Tg(nkx2.5:ZsYellow) embryos isolated at 18 hpf. (C-F) Representative cell capture events on IFC plate where (C) is an empty well, (D) contains two cells, (E) has a single cell lodged in the fluidics channel as a &#34;channel capture&#34;, and (F) is a single captured cell. Purple boxes mark inset for magnified view of capture sites. (G) Single cell capture with brightfield, ZsYellow and merged images. (C-F) White scale bar is 50 &#181;m; yellow scale bar is 10 &#181;m. (G) Scale bar is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53877/53877fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53877/53877fig3.jpg" /> 
Figure 3.&#160;Gene expression analysis of capture single zebrafish nkx2.5:ZsY positive cells. Gene expression by qRT-PCR in positive controls (embryos at 2 days-post-fertilization, pooled cDNA from single cells), negative control (no-template) and 40 single cells (Cell 01-40). Raw CT values were color coded based as described in the Key. Ef1a = elongation factor 1a, gata4 = GATA binding protein 4, nkx2.5 = NK2 homeobox 5, myl7 = myosin light chain 7, vmhc = ventricular myosin heavy chain, gapdh = glyceraldehyde-3 phosphate, and isl1 = ISL LIM homeobox 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53877/53877fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="7">Cycle Threshold (CT)&#160;&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>ef1a</td>
			<td>gata4</td>
			<td>nkx25</td>
			<td>myl7</td>
			<td>vmhc</td>
			<td>gapdh</td>
			<td>isl1</td>
		</tr>
		<tr>
			<td></td>
			<td>Embryo</td>
			<td>20.69</td>
			<td>20.92</td>
			<td>28.59</td>
			<td>32.87</td>
			<td>26.30</td>
			<td>27.00</td>
			<td>33.57</td>
		</tr>
		<tr>
			<td></td>
			<td>Pool</td>
			<td>26.4</td>
			<td>22.4</td>
			<td>27.7</td>
			<td>27.4</td>
			<td>24.5</td>
			<td>29.2</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td></td>
			<td>NTC</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td rowspan="9">Type 1</td>
			<td>Cell-01</td>
			<td>25.1</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-02</td>
			<td>25.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-03</td>
			<td>26.3</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-04</td>
			<td>27.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-05</td>
			<td>28.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-06</td>
			<td>28.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-07</td>
			<td>29.3</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-08</td>
			<td>30.7</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-09</td>
			<td>34.3</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td rowspan="17">Type 2</td>
			<td>Cell-10</td>
			<td>23.5</td>
			<td>28.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>26.3</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-11</td>
			<td>23.5</td>
			<td>27.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-12</td>
			<td>23.7</td>
			<td>17.9</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-13</td>
			<td>24.1</td>
			<td>32.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-14</td>
			<td>24.9</td>
			<td>27.5</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-15</td>
			<td>25.9</td>
			<td>28.6</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-16</td>
			<td>26.0</td>
			<td>28.1</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-17</td>
			<td>27.0</td>
			<td>27.7</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>38.5</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-18</td>
			<td>27.0</td>
			<td>27.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-19</td>
			<td>27.0</td>
			<td>30.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>38.2</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-20</td>
			<td>27.0</td>
			<td>28.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>39.2</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-21</td>
			<td>27.7</td>
			<td>30.3</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>38.6</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-22</td>
			<td>30.4</td>
			<td>26.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>38.3</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-23</td>
			<td>31.3</td>
			<td>22.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>39.7</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-24</td>
			<td>31.5</td>
			<td>28.8</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>39.5</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-25</td>
			<td>33.8</td>
			<td>27.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>37.3</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-26</td>
			<td>40.0</td>
			<td>27.4</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>36.6</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td rowspan="9">Type 3</td>
			<td>Cell-27</td>
			<td>24.7</td>
			<td>19.9</td>
			<td>28.8</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-28</td>
			<td>24.8</td>
			<td>28.4</td>
			<td>26.3</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-29</td>
			<td>25.1</td>
			<td>23.5</td>
			<td>27.6</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>32.5</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-30</td>
			<td>26.3</td>
			<td>21.4</td>
			<td>27.5</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>39.9</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-31</td>
			<td>26.9</td>
			<td>24.8</td>
			<td>28.2</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-32</td>
			<td>27.0</td>
			<td>22.5</td>
			<td>23.8</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-33</td>
			<td>27.8</td>
			<td>27.6</td>
			<td>27.7</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-34</td>
			<td>28.1</td>
			<td>21.9</td>
			<td>26.6</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>37.7</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-35</td>
			<td>29.3</td>
			<td>26.5</td>
			<td>28.1</td>
			<td>40.0</td>
			<td>40.0</td>
			<td>38.3</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td rowspan="2">Type 4</td>
			<td>Cell-36</td>
			<td>24.8</td>
			<td>20.3</td>
			<td>26.0</td>
			<td>27.4</td>
			<td>26.6</td>
			<td>40.0</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-37</td>
			<td>28.7</td>
			<td>22.3</td>
			<td>25.9</td>
			<td>28.9</td>
			<td>22.9</td>
			<td>38.5</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td rowspan="3">Type 5</td>
			<td>Cell-38</td>
			<td>25.5</td>
			<td>20.0</td>
			<td>29.3</td>
			<td>23.0</td>
			<td>19.9</td>
			<td>25.9</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-39</td>
			<td>25.9</td>
			<td>21.8</td>
			<td>28.6</td>
			<td>25.1</td>
			<td>21.7</td>
			<td>25.7</td>
			<td>40.0</td>
		</tr>
		<tr>
			<td>Cell-40</td>
			<td>28.9</td>
			<td>22.0</td>
			<td>27.0</td>
			<td>23.0</td>
			<td>21.3</td>
			<td>27.6</td>
			<td>40.0</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Raw CT values.

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Isolation and Characterization of Single Cells from Zebrafish Embryos

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

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14.4K Views.  University of North Carolina at Chapel Hill. This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells. 

Video: Isolation and Characterization of Single Cells from Zebrafish Embryos

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

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 ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...