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.
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.
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.
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).
2. Confocal and Two-photon Microscopy and Image Analysis
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 1 and 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.
Figure 1. Overview of the experimental procedures. A) 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.
Figure 2. Confocal microscopy setup and representative example for imaging 5FPs-marked HSPC in the bone marrow. A) Spectral (xyλ) 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.
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 µm 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’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. Please click here to view this movie.
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. Please click here to view this movie.
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 “distinct” 8-bits channels. Thus the relative ratio of Cerulean, EGFP, Venus, tdTomato, mCherry within each cell creates a unique “colorprint” 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 “color-print” 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 “colorprint.”
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 “on-the-fly”) complete 3D volumes of great complexity to depths of ~ 30 μm, 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.
The authors have nothing to disclose.
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.
Production of viruses | |||
LeGO-Cer2 | Addgene | 27338 | Expression of Cerulean |
LeGO-G2 | Addgene | 25917 | Expression of eGFP |
LeGO-V2 | Addgene | 27340 | Expression of Venus |
LeGO-T2 | Addgene | 27342 | Expression of tdTomato |
LeGO-C2 | Addgene | 27339 | Expression of mCherry |
Calciumm Phosphate Transfection Kit | Sigma | CAPHOS-1KT | |
Tissue culture dish | BD Falcon | 353003 | |
IMDM | Gibco, Life Technology | 12440-053 | |
Fetal Bovine Serum Heat Inactivated | Sigma | F4135-500ML | |
Pen Strep Glutamine | Gibco, Life Technology | 10378-016 | |
Centrifuge Tubes | Beckman Coulter | 326823 | |
SW28 Ultracentrifuge | Beckman Coulter | L60 | |
Millex Syringe Driven Filter Unite (0.22um) | Millipore | SLGS033SS | |
Mouse cell collection, purification, transduction and transplantation | |||
ACK lysing buffer | Quality Biologicals Inc. | 118-156-101 | |
Lineage Cell Depletion Kit (mouse) | Miltenyi Biotec Inc. USA | 130-090-858 | |
LS columns + tubes | Miltenyi Biotec Inc. USA | 130-041-306 | |
Pre-Separation Filters (30um) | Miltenyi Biotec Inc. USA | 130-041-407 | |
StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500mL) | StemCell Technologies Inc | 9650 | |
murine IL-11 CF | R & D Systems Inc | 418-ML-025/CF | |
recombinant murine SCF | RDI Division of Fitzgerald Industries Intl | RDI-2503 | |
recombinant murine IL-3 | R & D Systems Inc | 403-ML-050 | |
FLT-3 Ligand | Miltenyi Biotec Inc. USA | 130-096-480 | |
12-well plates, Costar | Corning Inc. | 3527 | |
Retronectin | Takara Bio Inc | T100A/B | |
Protamine Sulfate | Sigma | P-4020 | |
Confocal and two-photon microscopy | |||
DMEM | Lonza | 12-614F | |
1M Hepes | Cellgro, Mediatech Inc. | 25-060-CI | |
Glass bottom culture dish P35G-0-20-C | MatTek Corporation | P35G-0-20-C | |
Glass bottom culture dish P35G-0-14-C | MatTek Corporation | P35G-0-14-C | |
Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well | Thermo Scientific | 155383 | |
Leica TCS SP5 AOBS five channels confocal and multi-photon microscope | Leica Microsystems | ||
Chameleon Vision II -TiSaph laser range 680-1080nm | Coherent | ||
Chameleon Compact OPO laser range 1030-1350nm | Coherent | ||
HCX-IRAPO-L 25x/0.95 NA water dipping objective (WD=2.5 mm) | Leica Microsystems | ||
HC-PLAPO-CS 20x/0.70 NA dry objective (WD=0.6 mm) | Leica Microsystems | ||
HC-PL-IRAPO 40x/1.1NA water immersion objective (WD=0.6 mm) | Leica Microsystems | ||
Imaris software version 7.6 | Bitplane |