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.
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Method Article
In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy
In This Article
Erratum Notice
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Summary
Abstract
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.
Introduction
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 fluo....
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Protocol
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....
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Results
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
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Discussion
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 cel.......
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Disclosures
The authors declare no competing financial interests.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 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 (0.22 µm) | 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 (30 µm) | Miltenyi Biotec Inc. USA | 130-041-407 | |
| StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500 ml) | StemCell Technologies Inc | 9650 | |
| Murine IL-11 CF | R & D Systems Inc | 418-ML-025/CF | 100 µg/ml is 1,000x |
| Recombinant murine SCF | RDI Division of Fitzgerald Industries Intl | RDI-2503 | 100 µg/ml is 2,000x |
| Recombinant murine IL-3 | R & D Systems Inc | 403-ML-050 | 20 µg/ml is 2,000x |
| FLT-3 Ligand | Miltenyi Biotec Inc. USA | 130-096-480 | 100 µg/ml is 1,000x |
| 12-well plates, Costar | Corning Inc. | 3527 | |
| Retronectin | Takara Bio Inc | T100A/B | Resuspend at 50 µg/ml |
| Protamine Sulfate | Sigma | P-4020 | 4 µg/ml is 1,000x |
| Confocal and Two-photon Microscopy | |||
| DMEM | Lonza | 12-614F | |
| 1 M 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 | |
| Coverslips 22 mm No 2 | Thomas Scientific | 6662-Q55 | |
| Leica TCS SP5 AOBS five channels confocal and multi-photon microscope | Leica Microsystems | ||
| Chameleon Vision II -TiSaph laser range 680-1,080 nm | Coherent | ||
| Chameleon Compact OPO laser range 1,030-1,350 nm | 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/1.1 NA water immersion objective (WD=0.6 mm) | Leica Microsystems | ||
| Imaris software version 7.6 | Bitplane |
References
- Lo Celso,, C,, Scadden, D. T. The haematopoietic stem cell niche at a glance. J Cell Sci. 124, 3529-3535 (2011).
- Lo Celso, C., et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature. 457, 92-96 (2009).
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Erratum
Formal Correction: Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy
Posted by JoVE Editors on 1/01/1970. Citeable Link.
A correction was made to In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy. 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 “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.
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 “on-the-fly”) complete 3D volumes of great complexity to depths of ~ 300 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.
