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

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–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).

1. Production of LeGO Vectors Encoding 5FPs
   1. Use the lentiviral “Gene Ontology” (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 Addgene^5-7.
   2. 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).
   3. Transfect the cells with Calcium Phosphate using slightly modified CAPHOS kit recommendations:
      1. For each plate mix 15 µg LeGO vector, 12 µg pCDNA3.HIVgag/pol.4xCTE, 4 µg pRev-TAT, and 1 µg pMD2.G-VSV-G plasmids in presence of 50 µl 2.5 M CaCl₂, and add water to yield a 500 µl total volume.
      2. Precipitate DNA plasmids with 500 µl of 2x HeBS (HEPES-Buffered Saline) and incubate for 20 min at room temperature prior to adding to each plate of 293T cells.
      3. Incubate cells for 6 hr in a 37 °C 5% CO₂ incubator; remove media and add fresh culture media 6 hr post transfection.
   4. 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.
      1. Filter through 0.22 µm pores, pool and concentrate supernatants by ultracentrifugation on an SW28 rotor for 2 hr at 71,900 x g and 4 °C.
      2. Resuspend the supernatants in 1 ml of StemSpan media. Aliquot the lentiviral stocks in 200 µl and store at -80 °C.
2. 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.
   1. Plate 5 x 10⁴ NIH3T3 cells per well (12-well plate) in 2 ml of I10 media one day before the titration.
   2. Prepare 5-fold serial dilutions of vector supernatants in I10 media supplemented with protamine sulfate (4 mg/ml is 1,000x).
   3. Remove the media from the cells, and replace with 500 µl of supernatant dilutions containing the equivalent of 2, 0.8, 0.32, and 0.128 µl of concentrated virus.
   4. Determine the number (No) of cells present per well at the time of transduction, counting one control well.
   5. 6 hr after transduction, add 2 ml of I10 media and incubate for 3 days at 37 °C, 5% CO₂.
   6. Harvest the cells from each well using Trypsin-EDTA, and determine the percentage of fluorescent cells (%FP) via flow cytometry.
   7. 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%.
3. Mouse Cell Collection, Purification, Transduction, and Transplantation (schematically shown in Figure 1A adapted from Malide et al.⁴).
   1. 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 needle on a syringe containing I10 media.
   2. Pellet the whole bone marrow cells for 10 min at 500 x g, 4 °C and lyse red cells twice with 50 ml ACK buffer. Resuspend cells in 10 ml I10 media.
   3. Purify lineage negative (Lin-) progenitor cells with the MACS lineage depletion kit according to a modified kit protocol:
      1. Use 30 µl of Anti-Biotin MicroBeads instead of 10 µl/ 10⁷ cells; complete the washing steps with I2 media (IMDM supplemented with 2% FCS) instead of buffer.
      2. 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.
   4. 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.
   5. 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 µg/ml protamine sulfate.
   6. 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.
      1. Perform bone marrow transplant by infusing transduced Lin- HSPCs via tail vein injection into 11 Gy single dose pre-irradiated recipients (6-18 hr prior to transplant) recipients, in a volume of 100 µl, 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.
      2. 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.
         1. Sacrifice individual mice and retrieve tissues and organs for imaging, at various time points up to 120 days post-transplantation.
            1. Spray with ethanol the fur of the mouse and incise the skin on the ventral side avoiding hair dissemination.
            2. 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.
            3. Alternatively, use calvaria or set up a bone window for live imaging repeatedly over time^2,8,9 or image superficial organs such as the skin and lymph nodes in the live animal.
         2. Place individual organs in 35 mm number 0 cover glass culture dishes in 50-100 μl PBS for imaging them intact without sectioning, fixation or further processing as described in step 2.5.
         3. 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 °C

2. Confocal and Two-photon Microscopy and Image Analysis

1. 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.
2. 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).
3. Obtain confocal fluorescence spectra collecting lambda stacks (xyλ) 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.
   1. 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).
   2. 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 “colorprint” that enables subsequent color analysis and clonal tracking and has the advantage of reducing acquisition times compared to spectral imaging.
4. 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.
   1. 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.
5. For 3D volume rendering, collect series of x-y-z images (typically 1 x 1 x 4 μm³ voxel size) along the z-axis at 5 μm intervals over a range of depths (150-300 μm) 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 1.2 mm² (x-y) and 300 μm (z) (Figures 2D-2E) and Movie 1.
   1. Image calvarial marrow directly through the skull without sectioning^{2, 8}.
   2. Section sternum along the sagittal plane (bisection) and then transversally at the joint between 2 sternal fosse and image through the cut face^3,4,12 (Overview in Figure 1B adapted from Takaku et al³).
6. 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 μl DMEM, 10% FBS containing 20 mM HEPES at 37 °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 μm) at 20 sec intervals for up to 1 hr.
   1. For simultaneous three-color 2P imaging use Ti-Sa tuned at 860 nm simultaneously with the OPO laser (tuned at 1,130 nm for tdTomato; or 1,140 nm mCherry) (Movie 2).
7. Reconstruction and Analysis of 3D Volumes and 4D time sequences
   1. 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 described^3,4 (Movie 1) step-by-step example:
      1. In Imaris, Open the z-stack image; Select “Surpass” in order to display the 3D volume visualization.
      2. Use “navigate” to turn the volume around 360°.
      3. Select “Animation” 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).
   2. 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 described^3,4. 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.
   3. 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” colorprint” 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.