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Method Article
Istvan Blazsek
Istvan Blazsek, Inserm U972, Group Hospitalier Paul Brousse14, avenue Paul-Vaillant Couturier, 94807 Villejuif Cedex FRANCE Villejuif
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Self-renewing organs in adult mammals are composed of numerous tissue-specific functional units, such as intestinal crypts/villi or hair follicles, which all involve stem cell (SC) niches. Analogous tissue units in haematopoietic systems, however, have remained elusive. We design here a step-by-step protocol for in situ mapping, purification, enumeration and structural-functional analysis of the haematopoietic tissue unit, termed haematon. Longitudinal bisection of mouse femur and careful, limited dispersion of bone marrow (BM) parenchyma reveals compact, node-like haematons at discreet capillary loops along the diaphysis and spongy metaphysis. Recovery and fractionation of the whole BM in a bulk cell suspension, haematon units and endosteal layer provides a reproducible tool for quantitative and topographical analysis of putative SC niches in defined tissue compartments. We show examples how to characterize haematons in native state or following long-term culture using laser-scanning confocal microscopy, flow cytometry, clonal bioassays and videomicroscopy.
Keywords
Organogenesis, Tissue units, Blood formation, Haematopoietic stem cell niche, Haematon, Tissue processing
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The authors declare no competing financial interest.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.mov
*Video 1C* *General appearence of the haematon compartment * Nodular haematon particles appear following gentle flushing of the BM shaft through a 200-µl pipette tip. <iframe width="480" height="360" src="http://www.youtube.com/embed/tThOkJR796Y?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 1B* *Recovery and careful dispersion of the whole BM parenchyma. * <iframe width="480" height="360" src="http://www.youtube.com/embed/ACr6KXpzI4o?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 5* *Mixed hepaton-haematon units from the fetal mouse liver* Fetal mouse (E13) liver haematons keep their integrity, attach slightly to the dish and tend to form an interactive network in short-term organotypic culture. (Figure 4Ac|) Snapshot 1 frame/60; play 15 frame/s; length 45 s. SAR-2,-3,-4| <iframe width="480" height="360" src="http://www.youtube.com/embed/IYbwzF9YP3A?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 6* *Haematon units from adult human BM in long-term culture* A cohort of ~100 purified haematons was spreaded in 0.7 ml My-LTC medium in a 25-cm^2^ flask until firm adherence (2-3 days). 5 ml fresh medium was overlaid and haematons filmed through day 10-12 of culture. Note the integrity, spontaneous contractil behavior and myelopoietic activity of two adherent haematons. Phase contrast microscopy, x4 objective.(Figure 4Ae|). Snapshot 1 frame/120, play 15 frame/s; movie length 65 s. SAR-5,-6,-8,-9| <iframe width="480" height="360" src="http://www.youtube.com/embed/jlm17BRym_g?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 4* *Adult mouse BM: Persistent haematopoietic activity of cobbleston-like area at d42 in culture, generated by a single haematon Haematons attach to the plastic dish and give rise typical cobbleston-area forming islands (CAFI). Phase-dark cells under the stroma maintain haematopoietic cell proliferation and myeloid differentiation in My-LTC medium over 6 weeks. CAFI+ haematon switches to B-lymphopoiesis in B-cell growth medium. (Box 1|). Snapshot 1 frame /60 s, play 15 frame/s; length 45 s. SAR-1,-4,-5| <iframe width="480" height="360" src="http://www.youtube.com/embed/aY7Morq5noU?rel=0" frameborder="0" allowfullscreen></iframe>
- supplement0.docx
*Boxes (1-3)* *Processing haematons for structural and functional analysis*
- supplement0.docx
Supplementary Tables Commonly used markers, LSCM and FACS settings
- supplement0.docx
*Supplementary Abstract References* International Congress Presentations
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*Video 1A* *Longitudinal bisection of adult mouse femur.* <iframe width="480" height="360" src="http://www.youtube.com/embed/bpQHyQRzWCg?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 1D* *General aspect of the ground endosteal layer and the bisected, cleaned femoral bones* Recovery of the endosteum fraction using a fine scalpel contains spongy and lamellar bone particles and some residual firmly attached haematons. Real time videos have been taken by a Celestron #4432 handheld digital camera. <iframe width="480" height="360" src="http://www.youtube.com/embed/6LazQrozT7c?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 2* *3D LCSM of a typical haematon* Isolated haematons were fixed (Zn-7, 4 h), washed and embedded in a fine sheet of alginate as shown in Box 2|. A cohort of haematons (20-30 particles) were dehydrated upto 70% ethanol (1 h), rehydrated and then stained for Sca-1(FITC), c-Kit (PE) and DNA (DAPI). The preparation was transferred on the cleaned glass slide in 100 µl Mowiol for 30 min, covered and sealed using nail polish. Samples were viewed by x20 immersion (H~2~O) objective (20x/0.5 Plan Neofluar), serial optical sections taken (58x1 µm z-step) and 3D video animation was generated using Zeiss LSM softwar 3.2 image acquisition systeL. (SAR-7,-8|) <iframe width="480" height="360" src="http://www.youtube.com/embed/GjyWf2N5Q7M?rel=0" frameborder="0" allowfullscreen></iframe>
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*Video 3* *Adult mouse BM: Cell production in long-term organotypic haematon culture, d14* Long-term culture was established with BM haematons maintained at 33°C, as described (Box 1| and Box 2|). A single haematon is shown at d14 using dark-field illumination from a circular halogen source (Schott, KL1500) (x4 objective). Note the structural integrity of haematon involving Alg-PB tracked vascular meshwork (Figure 4Aa|, blue flash) and continuous egress of haematopoietic cell from the haematon. Snapshot 1 frame/120 s, play 15 frame/s; length 45 s. SAR-1,-4,-5| <iframe width="480" height="360" src="http://www.youtube.com/embed/zXR167kLbO8?rel=0" frameborder="0" allowfullscreen></iframe>
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Posted 10 Jan, 2013
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Associated Publications
Dual SP/ALDH Functionalities Refine the Human Hematopoietic Lin − CD34 + CD38 − Stem/Progenitor Cell Compartment
by Olivier Pierre-Louis, Denis Clay, Philippe Brunet de la Grange, +9
Stem Cells (04 January, 2013)
Aminobisphosphonate Stimulates Bone Regeneration and Enforces Consolidation of Titanium Implant into a New Rat Caudal Vertebrae Model
by József Blazsek, Csaba Dobó Nagy, István Blazsek, +4
Pathology & Oncology Research (04 January, 2013)
Method Article
Istvan Blazsek
Istvan Blazsek, Inserm U972, Group Hospitalier Paul Brousse14, avenue Paul-Vaillant Couturier, 94807 Villejuif Cedex FRANCE Villejuif
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 10 Jan, 2013
Community comments: 2
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Self-renewing organs in adult mammals are composed of numerous tissue-specific functional units, such as intestinal crypts/villi or hair follicles, which all involve stem cell (SC) niches. Analogous tissue units in haematopoietic systems, however, have remained elusive. We design here a step-by-step protocol for in situ mapping, purification, enumeration and structural-functional analysis of the haematopoietic tissue unit, termed haematon. Longitudinal bisection of mouse femur and careful, limited dispersion of bone marrow (BM) parenchyma reveals compact, node-like haematons at discreet capillary loops along the diaphysis and spongy metaphysis. Recovery and fractionation of the whole BM in a bulk cell suspension, haematon units and endosteal layer provides a reproducible tool for quantitative and topographical analysis of putative SC niches in defined tissue compartments. We show examples how to characterize haematons in native state or following long-term culture using laser-scanning confocal microscopy, flow cytometry, clonal bioassays and videomicroscopy.
Figure 1
Figure 2
Figure 3
Figure 4
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Associated Publications
Dual SP/ALDH Functionalities Refine the Human Hematopoietic Lin − CD34 + CD38 − Stem/Progenitor Cell Compartment
by Olivier Pierre-Louis, Denis Clay, Philippe Brunet de la Grange, +9
Stem Cells (04 January, 2013)
Aminobisphosphonate Stimulates Bone Regeneration and Enforces Consolidation of Titanium Implant into a New Rat Caudal Vertebrae Model
by József Blazsek, Csaba Dobó Nagy, István Blazsek, +4
Pathology & Oncology Research (04 January, 2013)