This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

All mice were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and enrolled in an NHLBI Animal Care and Use Committee&#8211;approved protocol. Female B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) and C57Bl/6 mice, 6-12 weeks old, were used as donor and recipient, respectively.
A list of materials, reagents, and equipment is provided in Table 1.
1. LeGO Transduction of Mouse HSPCs and Bone Marrow Transplantation
Perform procedures described below in a certified biosafety level 2 (BSL-2) cabinet (tissue culture hood).
<ol>
	<li>Production of LeGO Vectors Encoding 5FPs
	<ol>
		<li>Use the lentiviral &#8220;Gene Ontology&#8221; (LeGO) vector plasmids, each encoding a different FP from a strong constitutive SFFV promoter, including LeGO-Cer2 (Cerulean), LeGO-G2 (EGFP), LeGO-V2 (Venus), LeGO-T2 (tdTomato), and LeGO-C2 (mCherry) kindly provided by Dr. Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany); now also available from Addgene5-7.</li>
		<li>To produce LeGO vectors, seed 5 x 106 293T cells in 10 cm dishes (use 12 dishes for each vector) 24 hr prior to transfection in 8 ml of I10 media (IMDM supplemented with 10% FCS and glutamine /penicillin /streptomycin).</li>
		<li>Transfect the cells with Calcium Phosphate using slightly modified CAPHOS kit recommendations:
		<ol>
			<li>For each plate mix 15 &#181;g LeGO vector, 12 &#181;g pCDNA3.HIVgag/pol.4xCTE, 4 &#181;g pRev-TAT, and 1 &#181;g pMD2.G-VSV-G plasmids in presence of 50 &#181;l&#160;2.5 M CaCl2, and add water to yield a 500 &#181;l&#160;total volume.</li>
			<li>Precipitate DNA plasmids with 500 &#181;l&#160;of 2x HeBS (HEPES-Buffered Saline) and incubate for 20 min at room temperature prior to adding to each plate of 293T cells.</li>
			<li>Incubate cells for 6 hr in a 37 &#176;C 5% CO2 incubator; remove media and add fresh culture media 6 hr post transfection.</li>
		</ol>
		</li>
		<li>Collect culture supernatants containing lentiviral particles from each plate, daily, for 3 days. Each day following supernatant collection add fresh I10 media to each plate.
		<ol>
			<li>Filter through 0.22 &#181;m pores, pool and concentrate supernatants by ultracentrifugation on an SW28 rotor for 2 hr at 71,900 x g and 4&#160;&#176;C.</li>
			<li>Resuspend the supernatants in 1 ml of StemSpan media. Aliquot the lentiviral stocks in 200 &#181;l&#160;and store at -80 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Titration of Lentiviral Particles 
	Determine the titer of LeGO vector particles in collected supernatants via quantitation of transducing units/ml using NIH3T3 cells. Titers allow calculation of the volume of each vector supernatant needed to result in the desired relative multiplicity of infection (MOI) for experimental target cells.
	<ol>
		<li>Plate 5 x 104 NIH3T3 cells per well (12-well plate) in 2 ml of I10 media one day before the titration.</li>
		<li>Prepare 5-fold serial dilutions of vector supernatants in I10 media supplemented with protamine sulfate (4 mg/ml&#160;is 1,000x).</li>
		<li>Remove the media from the cells, and replace with 500 &#181;l of supernatant dilutions containing the equivalent of 2, 0.8, 0.32, and 0.128 &#181;l of concentrated virus.</li>
		<li>Determine the number (No) of cells present per well at the time of transduction, counting one control well.</li>
		<li>6 hr after transduction, add 2 ml of I10 media and incubate for 3 days at 37 &#176;C, 5% CO2.</li>
		<li>Harvest the cells from each well using Trypsin-EDTA, and determine the percentage of fluorescent cells (%FP) via flow cytometry.</li>
		<li>Calculate the titer as follow, averaging the titers obtained for each well (dilution) resulting in a %FP between 1 and 50%: Titer (vector particles/ml) = No x %FP/ Volume of vector supernatant (ml). Average the titers obtained for each dilution if the %FP is between 1 and 50%.</li>
	</ol>
	</li>
	<li>Mouse Cell Collection, Purification, Transduction, and Transplantation (schematically shown in Figure 1A adapted from Malide et al.4).
	<ol>
		<li>Sacrifice donor mice by an approved procedure. Harvest bone marrow from anterior and posterior limbs: dissect humeri, femurs and tibias, thoroughly remove all muscle, ligaments and excess tissue from bones, cut across the end of each bone as close as possible to the joint, and flush the bone marrow out of the bone using a 27-0 G&#160;needle on a syringe containing I10 media.</li>
		<li>Pellet the whole bone marrow cells for 10 min at 500 x g, 4 &#176;C and lyse red cells twice with 50 ml ACK buffer. Resuspend cells in 10 ml I10 media.</li>
		<li>Purify lineage negative (Lin-) progenitor cells with the MACS lineage depletion kit according to a modified kit protocol:
		<ol>
			<li>Use 30 &#181;l of Anti-Biotin MicroBeads instead of 10 &#181;l/ 107 cells; complete the washing steps with I2 media (IMDM supplemented with 2% FCS) instead of buffer.</li>
			<li>Insert one washing step between the incubation with Biotin-Antibody Cocktail and the incubation with Anti-Biotin MicroBeads; and equilibrate the column with I10 instead of buffer.</li>
		</ol>
		</li>
		<li>Culture Lin- cells for 48 hr at a starting density of 5 x 105 cells/ml in StemSpan media supplemented with 10 ng/ml murine IL-3, 100 ng/ml murine IL-11, 100 ng/ml human Flt-3 ligand, and 50 ng/ml murine stem cell factor.</li>
		<li>Transfer 1 to 4 x 105 cells to 12-well plates coated with RetroNectin and transduce with LeGO supernatants, each at a multiplicity of infection (MOI) of 6-7 to achieve 50% expression efficiency for each FP, in the presence of StemSpan media and cytokines detailed above and 4 &#181;g/ml protamine sulfate.</li>
		<li>Transduce cells with each LeGO vector individually as described in step 1.3.5 and mix cells following transduction (termed Mix) or co-transduce cells with all 5 LeGO vector supernatants simultaneously (termed Co). Remove cells 24 hr later, wash once with StemSpan media without cytokines, and resuspend them in StemSpan media without cytokines for transplantation, or transfer them to fresh media with cytokines, and culture for additional 96 hr before confocal microscopy and flow cytometry.
		<ol>
			<li>Perform bone marrow transplant by infusing transduced Lin- HSPCs via tail vein injection into 11 Gy single dose pre-irradiated recipients (6-18 hr&#160;prior to transplant)&#160;recipients, in a volume of 100 &#181;l, at a cell dose per mouse corresponding to 1-4 x 105 Lin- cells originally plated for transduction. In each experiment, transplant an entire cohort of animals with the same population of transduced donor BM cells.</li>
			<li>For in vitro characterization of transduced HSPCs, culture the cells for an additional 4-5 days in StemSpan supplemented with cytokines and analyze by flow cytometry for transduction efficiency, and by microscopy for color diversity.
			<ol>
				<li>Sacrifice individual mice&#160;and retrieve tissues and organs for imaging, at various time points up to 120 days post-transplantation.
				<ol>
					<li>Spray with&#160;ethanol the fur of the mouse and incise the skin on the ventral side avoiding hair dissemination.</li>
					<li>Dissect and excise according to standard procedures the spleen, popliteal lymph nodes, lung, heart, skin, adipose tissue, skeletal muscle, kidney, liver, as well as the head to access to calvaria.</li>
					<li>Alternatively, use calvaria or set up a bone window for live imaging repeatedly over time2,8,9 or image superficial organs such as the skin and lymph nodes in the live animal.</li>
				</ol>
				</li>
				<li>Place individual organs in 35 mm number 0 cover glass culture dishes in 50-100 &#956;l PBS&#160;for imaging them intact without sectioning, fixation or further processing as described in step 2.5.</li>
				<li>For volumetric imaging, keep the organs in in cold PBS until use. For time-lapse imaging proceed immediately to imaging organs in DMEM, 10% FBS containing 20 mM HEPES at 37 &#176;C</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Confocal and Two-photon Microscopy and Image Analysis
<ol>
	<li>Perform microscopy imaging using a Leica SP5 five channels confocal and multiphoton system equipped with multi-line Argon, diode 561 nm, HeNe 594 nm, and HeNe 633 nm visible lasers. Use in 2-photon mode, a pulsed femtosecond Ti:Sapphire (Ti-Sa) laser, tunable for excitation from 680 to 1,080 nm with dispersion correction. Use in experiments involving red-shifted FPs (tdTomato and mCherry), an optical parametric oscillator (OPO) laser to extend the output wavelength range as far as 1,030-1,300 nm, sequentially or simultaneously with the Ti-Sa laser.</li>
	<li>Image various tissues using a HCX-IRAPO-L 25X/0.95 NA water dipping objective (WD = 2.5 mm), a HC-PLAPO-CS 20X/0.70 NA dry objective (WD = 0.6 mm), or HC-PL-IRAPO 40X/1.1 NA water immersion objective (WD = 0.6 mm).</li>
	<li>Obtain confocal fluorescence spectra collecting lambda stacks (xy&#955;) of emitted light in 5 nm-bandwidths of the visible spectrum from BM cells transduced with a single FP LeGO vector as well as of control, not-transplanted BM.
	<ol>
		<li>Process images with the Leica LAS-AF v2.4.1 software to create reference spectra of all 5 FPs as well as of autofluorescence (Figure 2A). Using the individual FP spectra and the difference in fluorescence brightness (expressed as % of EGFP) of Cerulean (79%), Venus (156%), tdTomato (283%) and mCherry (47%) set 5 sequential imaging channels as follows: Cerulean (458/468-482 nm), EGFP (488/496-514 nm), Venus (514/523-558 nm), tdTomato (561/579-597 nm), and mCherry (594/618-670 nm) (Figure 2B).</li>
		<li>Validate accurate FPs separation, as cells transduced with each individual FP are visible only in the corresponding channel and absent from the rest (Figure 2C). In addition, this defined multi-channel approach creates an unique 5-channels &#8220;colorprint&#8221; that enables subsequent color analysis and clonal tracking and has the advantage of reducing acquisition times compared to spectral imaging.</li>
	</ol>
	</li>
	<li>Separate fluorescence emission, in 2-photon mode, by high-efficiency custom dichroic mirrors and collect with a four channels non-descanned detector (NDD) as follows: a dichroic mirror at 568 nm followed by a dichroic mirror at 465 nm, followed by a 452/45 nm emission filter for SHG or autofluorescence and Cerulean, a 525/40 nm emission filter for EGFP or Venus, and a 648 nm dichroic mirror and 620/60 nm emission filter for tdTomato or mCherry.
	<ol>
		<li>Reveal structural information by two-photon microscopy intrinsic contrast imaging (as described above) of tissue autofluorescence from elastin, NADH, (excited at 780 nm), and second harmonic generated signal (SHG) from bone and fibrillar collagen (excited at 920 nm or 860 nm collected in a back-scattered direction)10,11.</li>
	</ol>
	</li>
	<li>For 3D volume rendering, collect series of x-y-z images (typically 1 x&#160;1 x&#160;4 &#956;m3&#160;voxel size) along the z-axis at 5 &#956;m intervals over a range of depths (150-300 &#956;m) throughout the bone marrow and other tissues, over large regions using the tile function of the software to automatically generate stitched volumes comprising an entire sternum fossae, approximately 2.5 x&#160;1.2 mm2 (x-y) and 300 &#956;m (z) (Figures 2D-2E) and Movie&#160;1.
	<ol>
		<li>Image calvarial marrow directly through the skull without sectioning2, 8.</li>
		<li>Section sternum along the sagittal plane (bisection) and then transversally at the joint between 2 sternal fosse and image through the cut face3,4,12 (Overview in Figure 1B adapted from Takaku et al3).</li>
	</ol>
	</li>
	<li>For 4D time-lapse imaging, place freshly excised popliteal lymph node, spleen, lung, or calvarial bone samples uncut onto 35 mm number 0 coverglass culture dishes, in 50-100 &#956;l&#160;DMEM, 10% FBS containing 20 mM HEPES at 37 &#176;C and use 2-photon Ti-Sa excitation (860 nm) combined with confocal excitation (458 nm, 488 nm, 514 nm, 561 nm, 594 nm) and Leica resonant scanner (8000 Hz/sec), taking z-stacks (~80 &#956;m) at 20 sec intervals for up to 1 hr.
	<ol>
		<li>For simultaneous three-color 2P imaging use Ti-Sa tuned at 860 nm simultaneously with the OPO laser (tuned at 1,130 nm&#160;for tdTomato; or 1,140 nm mCherry) (Movie&#160;2).</li>
	</ol>
	</li>
	<li>Reconstruction and Analysis of 3D Volumes and 4D time sequences
	<ol>
		<li>Use Imaris x64 software version 7.6, or alternative software packages to transform the tiled-stacks of raw images into volume-rendered (3D) data of fluorescent cells inside bone marrow and different tissues and to export them as 3D-rotation movies, as previously described3,4 (Movie&#160;1) step-by-step example:
		<ol>
			<li>In Imaris, Open the z-stack image; Select &#8220;Surpass&#8221; in order to display the 3D volume visualization.</li>
			<li>Use &#8220;navigate&#8221; to turn the volume around 360&#176;.</li>
			<li>Select &#8220;Animation&#8221; to create an animation; use key frames to add selected views, zoom, rotations of the volume; Preview the movie, edit as necessary and save the file in a desired movie format (ex: avi, mov).</li>
		</ol>
		</li>
		<li>For quantitative assessment of the 3D cell and clone frequency, positions, sizes and distribution, further process the z-stacks using the Imaris XT modules which integrate MATLAB applications performing distance measurements (using a distance transformation algorithm) and clusters analysis, as previously described3,4. Transform the sequences of image stacks over time into volume-rendered four-dimensional (4D) movies with Imaris x64 software, and use spot and surface analysis for semiautomated tracking of cell motility in three dimensions using autoregressive motion algorithms.</li>
		<li>Perform multispectral analysis of cells as follows: extract the multi-color labeled spots by adding all 5 channels through channel arithmetic of Imaris XT into a new channel; determine for all spots (cells) the mean intensity fluorescence in each of the 5 component channels. Export the values in Excel software to plot graphs displaying the unique&#8221; colorprint&#8221; i.e. the relative % of each FP in individual cell (Figure 2E graph). Correct the data set for tissue drift, and calculate cell velocity and displacement over time, track lengths, paths, and distances; export values in Excel software to plot graphs. Assemble composite figures with Adobe Photoshop CSS 5 software.</li>
	</ol>
	</li>
</ol>

<ol>
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T., Scadden, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124, 3529-3535 (2011).</li><li>Lo Celso, C., 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=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Takaku, T., 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=Hematopoiesis+in+3+dimensions+human+and+murine+bone+marrow+architecture+visualized+by+confocal+microscopy.">Hematopoiesis in 3 dimensions human and murine bone marrow architecture visualized by confocal microscopy.</a> Blood. 116, e41-e55 (2010).</li><li>Malide, D., Metais, J. Y., Dunbar, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clonal+analysis+of+murine+hematopoietic+stem+and+progenitor+cells+marked+by+5+fluorescent+proteins+using+confocal+and+multiphoton+microscopy.">Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.</a> Blood. 120, e105-e116 (2012).</li><li>Weber, K., Mock, U., Petrowitz, B., Bartsch, U., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+ontology+(LeGO)+vectors+equipped+with+novel+drug-selectable+fluorescent+proteins+new+building+blocks+for+cell+marking+and+multi-gene+analysis.">Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins new building blocks for cell marking and multi-gene analysis.</a> Gene Ther. 17, 511-520 (2010).</li><li>Weber, K., Bartsch, U., Stocking, C., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolor+panel+of+novel+lentiviral+'gene+ontology'+(LeGO)+vectors+for+functional+gene+analysis.">A multicolor panel of novel lentiviral 'gene ontology' (LeGO) vectors for functional gene analysis.</a> Mol Ther. 16, 698-706 (2008).</li><li>Weber, K., 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=RGB+marking+facilitates+multicolor+clonal+cell+tracking.">RGB marking facilitates multicolor clonal cell tracking.</a> Nat Med. 17, 504-509 (2011).</li><li>Lo Celso, ., Lin, C. P., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transplanted+hematopoietic+stem+and+progenitor+cells+in+mouse+calvarium+bone+marrow.">In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow.</a> Nature protocols. 6, 1-14 (2011).</li><li>Kohler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cellular+dynamics+and+endosteal+location+of+aged+early+hematopoietic+progenitor+cells+revealed+by+time-lapse+intravital+imaging+in+long+bones.">Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones.</a> Blood. 114, 290-298 (2009).</li><li>Campagnola, P. 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The authors declare no competing financial interests.

We describe here the details of a powerful methodology recently devised for clonal cell tracking, combining the large diversity generated by combinatorial marking with 5FP-encoding LeGO vectors and imaging with tandem confocal and 2-photon microscopy to achieve volumetric and dynamic imaging in live tissues. This extended the use of FP-based color marking by recording confocal spectral identity in 5 &#8220;distinct&#8221; 8-bits channels. Thus the relative ratio of Cerulean, EGFP, Venus, tdTomato, mCherry within each cell creates a unique &#8220;colorprint&#8221; for identification of clonal progeny. The multicolor labeling by 5FPs increases the label palette with more diversity, which was shown particularly useful when studying the distribution, contacts, tiled arrangements and competitive interactions among cells or group of cells of the same type in dense tissue samples. Combined with 2-photon excitation of intrinsic structural features of various tissues and organs, we can track color marked clonal cells in their native environment without the need for fixation and physical sectioning. The demonstration that daughter cells derived from individual progenitors retain a distinctive &#8220;color-print&#8221; despite differentiation and proliferation was demonstrated in our published study, with hundreds of differentiated cells found in individual in vitro colonies (CFU) showing the same &#8220;colorprint.&#8221;
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
Although it is currently technically impossible to image the sternal bone marrow in the same mouse repeatedly over time, valuable information can be obtained by studying individual mice at different time points from a cohort originally transplanted with the same population of LeGO-transduced Lin- cells. The sternum is an excellent location to study bone marrow regeneration due to its early and complete hematopoietic reconstitution, stability, easy sectioning, reproducibility, and large volume. It is critical to reach close to 50% transduction efficiency of Lin- cells for each individual FP vector, before proceeding with a transplantation and imaging experiment, in order to have sufficient color diversity and FP-expressing cells to make the experiment informative. There will always be a caveat that more than one clone may by chance end up with similar or identical colorprints, particularly if transduction is inefficient and many cells have only one or two insertions. It is also important to be confident that each individual tail vein injection delivered close to 100% of the cell dose.
This proves the feasibility of high-resolution 4D live dynamic studies by combining resonant scanning multiphoton and confocal time-lapse imaging. It allows not only static characterization of various clones but also analysis of kinetic differences among cells marked by same color. Moreover demonstrates video-rate 4D imaging of three FPs labeled samples employing successfully in tandem TiSa and OPO lasers for higher efficiency, less harmful, deeper penetration of live tissue, paving the way for 2-photon intravital imaging. In addition, establishes a valuable option to track cells over prolonged periods (months) overcoming current dye-based limitations. This approach uses commercially available confocal and two-photon laser microscope and automated user-interactive image analysis methods based on a commercially available software package allowing easy implementation in usual microscopy facilities.
Finally, this methodology is not limited to the analysis of HSPCs. Many biologic systems could benefit from the ability to resolve spatiotemporal arrangements of clonally complex cellular and structural elements via multicolor labeling and confocal and 2-photon imaging. Organ regeneration, immune responses, and tumor metastatic patterns could be interrogated, to suggest just a few potential applications.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

The production of blood cells, termed hematopoiesis, is maintained by a small population of hematopoietic stem and progenitor cells (HSPCs). These cells reside within the bone marrow (BM) in a complex microenvironmental niche consisting of osteoblasts, stromal cells, adipose tissue, and vascular structures, all implicated in the control of self-renewal and differentiation1,2. As intact BM has been traditionally inaccessible to direct observations, the interactions between HSPC and their microenvironment remains largely uncharacterized in vivo. Previously, we established a methodology to visualize the 3D architecture of intact BM using confocal fluorescence and reflection microscopy3. We characterized expansion of EGFP-marked HSPCs in the BM, but the use of only a single FP precluded analysis of regeneration at a clonal level. Very recently we took advantage of the Lentiviral Gene Ontology (LeGO) vectors constitutively expressing fluorescent proteins (FPs) to efficiently mark cells4,5. Co-transduction of HSPC with 3 or 5 vectors generates a diverse palette of combinatorial colors, allowing tracking of multiple individual HSPC clones.
Marked HSPC combined with new imaging technology permitted to trace individual HSPC homing and engraftment in the BM of irradiated mice. LeGO vectors were used to mark HSPC with 3 or 5 different FPs (from Cerulean, eGFP, Venus, tdTomato, and mCherry) and follow their engraftment over time in the BM by confocal and 2-photon microscopy, allowing clear visualization of bone and other matrix structures, and the relation of HSPC clones to components of their microenvironment. HSPC were purified as the lineage negative cells from C57Bl/6 mice BM, transduced with LeGO vectors, and reinfused by tail vein injection into myelo-ablated congenic mice. By monitoring large volumes of the sternum BM tissue we visualized endosteal engraftment occurring early in BM regeneration. Cell clusters with diverse color spectra appeared initially in close proximity to the bone and progressed centrally over time. Interestingly, after more than 3 weeks the marrow consisted of macroscopic clonal clusters, suggesting spread of hematopoiesis derived from individual HSPCs contiguously in the marrow, rather than widespread dissemination of HSPCs via the bloodstream. There was less color diversity at late time points, suggesting difficulty in transducing long-term repopulating cells with multiple vectors.
In addition, HSPCs formed clusters in the spleen, an organ also responsible for hematopoiesis in mice. HPSC-derived individual cells could be resolved in the thymus, lymph nodes, spleen, liver, lung, heart, skin, skeletal muscle, adipose tissue, and kidney as well. The 3D images can be assessed qualitatively and quantitatively to appreciate the distribution of cells with minimal perturbations of the tissues. Finally, we illustrated the feasibility of live dynamic studies in 4D by combining resonant scanning multiphoton and confocal time-lapse imaging. This methodology enables non-invasive high resolution, multidimensional cell-fate tracing of spectrally marked cells populations in their intact 3D architecture, providing a powerful tool in the study of tissue regeneration and pathology.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Production of Viruses&lt;/strong&gt;</td></tr><tr><td>LeGO-Cer2</td><td>Addgene</td><td>27338</td><td>Expression of Cerulean</td></tr><tr><td>LeGO-G2</td><td>Addgene</td><td>25917</td><td>Expression of eGFP</td></tr><tr><td>LeGO-V2</td><td>Addgene</td><td>27340</td><td>Expression of Venus</td></tr><tr><td>LeGO-T2</td><td>Addgene</td><td>27342</td><td>Expression of tdTomato</td></tr><tr><td>LeGO-C2</td><td>Addgene</td><td>27339</td><td>Expression of mCherry</td></tr><tr><td>Calciumm Phosphate Transfection Kit</td><td>Sigma</td><td>CAPHOS-1KT</td><td></td></tr><tr><td>Tissue culture dish</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>IMDM</td><td>Gibco, Life Technology</td><td>12440-053</td><td></td></tr><tr><td>Fetal Bovine Serum Heat Inactivated</td><td>Sigma</td><td>F4135-500ML</td><td></td></tr><tr><td>Pen Strep Glutamine</td><td>Gibco, Life Technology</td><td>10378-016</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>SW28 Ultracentrifuge</td><td>Beckman Coulter</td><td>L60</td><td></td></tr><tr><td>Millex Syringe Driven Filter Unite (0.22 &amp;micro;m)</td><td>Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Mouse Cell Collection, Purification, Transduction, and Transplantation&lt;/strong&gt;</td></tr><tr><td>ACK lysing buffer</td><td>Quality Biologicals Inc.</td><td>118-156-101</td><td></td></tr><tr><td>Lineage Cell Depletion Kit (mouse)</td><td>Miltenyi Biotec Inc. USA</td><td>130-090-858</td><td></td></tr><tr><td>LS columns + tubes</td><td>Miltenyi Biotec Inc. USA</td><td>130-041-306</td><td></td></tr><tr><td>Pre-Separation Filters (30 &amp;micro;m)</td><td>Miltenyi Biotec Inc. USA</td><td>130-041-407</td><td></td></tr><tr><td>StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500 ml)</td><td>StemCell Technologies Inc&amp;nbsp;</td><td>9650</td><td></td></tr><tr><td>Murine IL-11 CF</td><td>R &amp;amp; D Systems Inc</td><td>418-ML-025/CF</td><td>100 &amp;micro;g/ml is 1,000x</td></tr><tr><td>Recombinant murine SCF</td><td>RDI Division of Fitzgerald Industries Intl&amp;nbsp;</td><td>RDI-2503</td><td>100 &amp;micro;g/ml is 2,000x</td></tr><tr><td>Recombinant murine IL-3</td><td>R &amp;amp; D Systems Inc</td><td>403-ML-050</td><td>20 &amp;micro;g/ml is 2,000x</td></tr><tr><td>FLT-3 Ligand</td><td>Miltenyi Biotec Inc. USA</td><td>130-096-480</td><td>100 &amp;micro;g/ml is 1,000x</td></tr><tr><td>12-well plates, Costar</td><td>Corning Inc.</td><td>3527</td><td></td></tr><tr><td>Retronectin</td><td>Takara Bio Inc</td><td>T100A/B</td><td>Resuspend at 50 &amp;micro;g/ml</td></tr><tr><td>Protamine Sulfate</td><td>Sigma</td><td>P-4020</td><td>4 &amp;micro;g/ml is 1,000x</td></tr><tr><td colspan="4">&lt;strong&gt;Confocal and Two-photon Microscopy&lt;/strong&gt;</td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>1 M Hepes</td><td>Cellgro, Mediatech Inc.</td><td>25-060-CI</td><td></td></tr><tr><td>Glass bottom culture dish P35G-0-20-C</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td></td></tr><tr><td>Glass bottom culture dish P35G-0-14-C</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td><td>Thermo Scientific</td><td>155383</td><td></td></tr><tr><td>Coverslips 22 mm No 2</td><td>Thomas Scientific</td><td>6662-Q55</td><td></td></tr><tr><td>Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Chameleon Vision II -TiSaph laser range 680-1,080 nm</td><td>Coherent</td><td></td><td></td></tr><tr><td>Chameleon Compact OPO laser range 1,030-1,350 nm</td><td>Coherent</td><td></td><td></td></tr><tr><td>HCX-IRAPO-L 25X/0.95 NA water dipping objective (WD=2.5 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>HC-PLAPO-CS 20X/0.70 NA dry objective (WD=0.6 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>HC-PL-IRAPO 40X/1.1 NA water immersion objective (WD=0.6 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Imaris software version 7.6</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

Whole-mount 3D confocal/2-photon microscopy reconstructions of the sternal bone marrow time course revealed the engraftment and expansion of transplanted co-5FPs in a pattern with remarkable characteristics: clones appeared clearly delineated, homogenously marked with wide palette of colors initially and progress over time to preferentially contain cells of mostly one color. Confocal microscopy setup and representative examples of imaging 5FPs-marked HSPC in the sternal bone marrow are illustrated in Figure 2 and Movies&#160;1 and&#160;2.
Whole-mount 3D confocal/2-photon microscopy reconstructions of intact tissues demonstrate the possibility to trace the fate of color marked BM-derived cells for extended time periods in the live tissues. Representative examples of bone-marrow derived cells in hematopoietic and non-hematopoietic organs following transplantation are shown in Figure 3.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51669/51669fig1highres.jpg" width="500" /> 
Figure 1. Overview of the experimental procedures. A)&#160;The schematic steps illustrate isolation of Lin- BM cells, transduction with LeGO vectors encoding a variety of FPs color variants, and reinfusion of transduced cells into-myelo-ablated mice (bone marrow transplant). B) Bone marrow (sternum, calvaria), as well as various organs/tissues were examined by confocal and two-photon microscopy at different time points following transplant. Panel A is adapted from Figure 1 in Malide et al.4 and Panel B adapted from Figure 1 in Takaku et al3.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51669/51669fig2highres.jpg" width="600" /> 
Figure 2. Confocal microscopy setup&#160;and representative example for imaging 5FPs-marked HSPC in the bone marrow. A)&#160;Spectral (xy&#955;) imaging used to record reference emission spectra for each FP, illustrated in normalized histograms pseudocolored with cyan (Cerulean), green (EGFP), yellow (Venus), magenta (tdTomato), and red (mCherry): excited by 594 nm- (solid red line) compared to 561 nm- (dashed red line) wavelength. B) Five channels set to image sequentially emission of: Cerulean (468-482 nm), EGFP (496-514 nm), Venus (523-558 nm), tdTomato (579-597 nm) and mCherry (618-670 nm). Panels A and B are adapted from Figure 1 in Malide et al.4 C) Imaging of sternal BM from mice transplanted with transduced with individual FP vectors. Each FP was visible only in the cells transduced with the corresponding vector imaged in the appropriate channel, and absent from the others (no cross-talk). D,E) Engraftment and expansion of transplanted co-5FP marked cells in the bone marrow in vivo. At day 4 post-transplant (D) clusters of cells marked in wide variety of colors were visible close proximity to the bone edge (SHG, white), preferentially located adjacent to the joint between two fossae. By day 30 (E) one large clone of unique color (green - yellow) has expanded to occupy the entire fossae as illustrated in the merged as well as single channels images. FPs content analysis demonstrates homogeneous marking by 2 FPs variants EGFP and Venus.
<img alt="Figure 3" fo:content-width="5.25in" src="/files/ftp_upload/51669/51669fig3highres.jpg" width="650" /> 
Figure 3. Examples of bone-marrow derived cells in hematopoietic and non-hematopoietic organs following transplantation. Various tissues and organs were imaged intact through depths of 150-200 &#181;m and large surface areas, computationally stitched and rendered in 3D as shadow reconstructions. A) Popliteal lymph node at 120 days post-transplant demonstrates scattered cells mostly marked in yellow (Venus) and red (mCherry) peripherally surrounded by collagen fibers (white, SHG). B) Similarly, the spleen at the same time, displays small clusters and mostly scattered cells of various colors intertwined by collagen fibers network. C) In the liver fluorescent cells with morphology suggestive of stellate cells, or macrophages (Kupffer cells) of various colors were aligned along collagen fibers network (SHG white) delineating hepatic lobular structures. D) In an skin flap imaged from dermal side fluorescent cells of diverse colors and morphologies were seen, most with large size and morphology suggesting Langerhan&#8217;s or dendritic-like identity, lying under elastin fibers (autofluorescence at 780 nm, white) and along collagen (SHG at 920 nm, white) muscle fibers and hair follicles. E) In the heart viewed from the epicardial side numerous individual large fluorescent cells with macrophage-like morphology originating from multiple clones based on the diverse colors seen are visible superficially and also interspersed deeper between cardiomyocytes (white) visualized by their intrinsic two-photon autofluorescence (at 780 nm). No FP fluorescent cardiomyocytes were observed. Nearby cells (C-E) of the same color suggest in situ proliferation and short-distance migration. F) In the lung numerous fluorescent cells with a diverse palette of colors were scattered throughout the lace-like structure of collagen fibers (SHG) at all depth levels, with variable morphologies suggesting dendritic cell, macrophage, and type 2 pneumocyte identities.
Movie 1. 3D reconstruction of sternal BM at 4 days post-transplant Co 5FPs. Clusters of cells marked in wide variety of colors were visible in close proximity to the bone edge (SHG, white), preferentially located adjacent to the joint between two fossae. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51669/Malide-Metais-Video1-BMday_4-5FPs-3D.avi" target="_blank">Please click here to view this movie.</a>
Movie 2. 4D time lapse sternal BM two-photon and OPO microscopy at day 39 post transplant Co 3FPs (Cerulean - white, Venus - yellow, tdTomato - magenta) bone (SHG in white). Note several static clones marked in yellow or in yellow-magenta combination adjacent to bone in contrast to highly motile individual myeloid progenitor cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51669/Malide-Metais-Video2-BMday39-3FPs-4D.avi" target="_blank">Please click here to view this movie.</a>

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

in-vivo-clonal-tracking-hematopoietic-stem-progenitor-cells-marked

Nanyang Technological University

Research

JoVE Journal

Biology

13.1K Views.  NHLBI/NIH.  Combinatorische 5 fluorescerende eiwitten markering van hematopoietische stamcellen en voorlopercellen maakt in vivo klonale volgen via confocale en twee-foton microscopie, het verstrekken van inzicht in het beenmerg hematopoietische architectuur tijdens de regeneratie. Deze methode maakt het mogelijk niet-invasieve lot in kaart brengen van spectraal-gecodeerde HSPCs-afgeleide cellen in intacte weefsels voor langere perioden na de transplantatie. 

Video: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Immunology and Infection

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Developmental Biology

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Bioengineering

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Medicine

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Neuroscience

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Samenvatting

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