We thank the staff in the core facilities at the Francis Crick Institute (Biological Research facility, In Vivo Imaging and Experimental Histopathology) and Yolanda Saavedra-Torres and Mercedes Sanchez-Garzon, the vets at Crick and LRI respectively, for their valuable help.&#160;We are grateful to Dr. W. Grey for critically reading the manuscript. D.P. was supported by a non-clinical research fellowship from the EHA. This work was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001045), the UK Medical Research Council (FC001045), and the Welcome Trust (FC001045).

All animal experiments were performed under PPL 70/8904, approved by the U.K. Home Office and in accordance with Cancer Research UK guidelines. The use of human umbilical cord blood (UCB) and primary human acute myeloid leukemia (AML) samples was approved by the East London Ethical Committee after receiving consent and was carried out in accordance with the Declaration of Helsinki.
1. Bioengineering Collagen-based Scaffolds with Human Hematopoietic and Stromal Cells
NOTE: The entire protocol should be performed in sterile conditions and with sterile material. Cell culture medium 1 corresponds to hMSC medium (MEM-&#945;, P/S, and 10% hMSC-FBS); cell culture medium 2 corresponds to EC medium (M199, 20% FBS, P/S, 10mM HEPES, 50 &#181;g/mL heparin, 2 mM glutamine and 50 &#956;g/mL ECGS) and cell culture medium 3 corresponds to hematopoietic cell medium (H5100 and P/S).
<ol>
	<li>Prepare umbilical cord blood mononuclear cells (CB-MNCs) or primary AML (bone marrow or peripheral blood MNCs) using a Ficoll-Paque density gradient, according to well-established protocols53.
	<ol>
		<li>For AML, deplete the T cells using OKT.3 antibody. Incubate 4 &#181;g of OKT.3 per 1 x 106 AML MNCs for 30 min at room temperature before washing the cells in PBS. If required, cryopreserve the MNCs in FBS with 10% DMSO at 2 x 108 cells per mL.</li>
	</ol>
	</li>
	<li>Prepare hMSC (culture medium 1) and EC (culture medium 2) cell cultures. Plate hMSC cells (see the Table of Materials) on regular cell culture flasks in hMSC medium. Plate ECs (see the Table of Materials) on collagen 1-coated surfaces in EC medium.</li>
	<li>At 85-90% cell confluence remove the medium, wash twice with PBS, and add trypsin-EDTA solution (20 &#181;L per cm2).
	<ol>
		<li>After 5 min, check that cells are detached. Recover the cells by diluting 1:3 with cell culture medium. Centrifuge at 300 x g for 5 min and re-suspend in the corresponding medium for each cell type and count the cells using a Neubauer chamber.</li>
		<li>Use cells at 2 x 106 - 107 cells per mL. If both cell types need to be used together, mix the hMSC and EC suspensions in a 1:1 ratio.</li>
	</ol>
	</li>
	<li>Transfer the cell suspension to an insulin syringe.</li>
	<li>Using a scalpel, cut the sterilized gelatin sponge (e.g., gelfoam) initial scaffold (20 mm x 60 mm x 7 mm) into 24 pieces of similar size (6.6 mm x 7.5 mm x 7 mm; Figure 1A and B).</li>
	<li>Reconstitute gelatin scaffolds by immersion in PBS (5 min).</li>
	<li>One by one, gently put the scaffolds on a sterile tissue to remove excess PBS. Transfer the scaffolds to one well of a 24-well plate (ultra-low attachment surface) and use the syringe to inject 50 &#181;L containing 105 - 106 cells (hMSC alone or in combination with hEC; Figure 1C).</li>
	<li>Repeat step 1.7 with each scaffold until all required scaffolds are seeded with stromal cells.</li>
	<li>Incubate for 1 h inside a cell culture incubator (37 &#176;C and CO2 5%).</li>
	<li>Fill each well with 3 mL of cell culture medium 1 (Figure 1D) and return the scaffolds to a cell culture incubator (37 &#176;C and CO2 5%) for overnight incubation. Use cell culture medium 2 if ECs are used in the scaffolds.</li>
	<li>Thaw CB-MNCs and isolate CD34+ cells from them following an appropriate CD34+ section kit protocol. Suspend the selected CD34+ cells in medium 3 and count them in a Neubauer chamber. 
	NOTE: Here, cells were used at a concentration of 2 x 106 cells per mL.
	<ol>
		<li>If AML patient-derived primary samples are used, thaw the cells, dilute them 1:10 in FBS, centrifuge them for 5 min at 300 x g, resuspend the cells in PBS-2% FBS, add anti-human CD3 antibody (2 - 4 &#181;g per 106 cells), and incubate at room temperature for 30 min. Centrifuge for 5 min at 300 x g and resuspend in hematopoietic cell medium supplemented with cytokines (20 ng/mL granulocyte-colony stimulating factor (G-CSF), 20 ng/mL IL-3, and 20 ng/mL thrombopoietin (TPO)).</li>
	</ol>
	</li>
	<li>Repeat steps 1.7 - 1.9 but in this case seed 1 x 105 human hematopoietic cells instead of stromal components, as above.
	<ol>
		<li>Fill the well with 3 mL of cell culture medium 3 (Figure 1D) and return the scaffolds to a cell culture incubator (37 &#176;C and CO2 5%) for overnight incubation. Use a 1:1 mix of cell culture media 2 and 3 If ECs are used in the scaffolds.</li>
	</ol>
	</li>
	<li>For only recombinant human bone morphogenetic protein-2 (rhBMP-2) carrier scaffolds, gently put the scaffolds one by one on a sterile tissue to remove excess PBS. Transfer the scaffolds to one well of a u-bottom, 96-well plate (Figure 1E) and add 5 &#181;L of rhBMP-2, thoroughly covering the scaffold.
	<ol>
		<li>Add 20 &#181;L of thrombin followed by 20 &#181;L of fibrinogen, thoroughly covering the scaffold each time (Figure 1F). Repeat the procedure for each scaffold until all required scaffolds are treated and then incubate for 5 - 10 min at 37 &#176;C. Check if coagulation has successfully formed.</li>
	</ol>
	</li>
</ol>
2. Surgical Implantation of Bioengineered Scaffolds
NOTE: Here, either male or female, 6- to 12-week-old NSG mice were used. As the animals are immunodeficient, all procedures should be done in sterile conditions. Steps 2.10 - 2.13 are related to survival strategies and post-surgical care.
<ol>
	<li>60 - 120 min before surgery, subcutaneously administer pain medication (carprofen, 5 mg/kg bodyweight/mouse).</li>
	<li>Induce anesthesia in a chamber with 0.5% isoflurane and 2 L/min O2. &#160;Mice should be monitored continuously during the procedure. Transfer the animal to the surgical area in prone position in order to have easy access to the back. Keep the animal under anesthesia using a nose cone supplying 1.5% isoflurane and 2 L/min O2. Keep the mouse under anesthesia during the surgical procedure and frequently check the animal state.</li>
	<li>While it is under anesthesia, use ophthalmic gel on the eyes of the mouse to prevent dryness, and keep the mouse at 37 &#176;C.</li>
	<li>Shave the surgical area on the back of the mouse using an electric trimmer. To sterilize the skin, dip a cotton tip in diluted clorexidine (diluted 1:10 in PBS) and use this tip to clean the skin surface. Repeat this procedure twice.</li>
	<li>Using sterile forceps and a scalpel (or scissors), make a 0.5- to 0.7-cm anterior-to-posterior full incision of the skin. Use the forceps inserted under the subcutaneous tissue to make a pocket.</li>
	<li>Insert the scaffold subcutaneously, making sure that it is placed deep within the pocket (Figure 1G and H). Close the incision with surgical glue (Figure 1I and J).</li>
	<li>To treat post-surgical pain, subcutaneously administer buprenorphine (0.1 mg/kg&#160;bodyweight).</li>
	<li>During recovery, place the animal on its side in a pre-warmed cage and monitor recovery until normal behavior is observed.</li>
	<li>Dilute pain medication in water (carprofen, 0.1 mg/mL of water) and provide it to animals as drinking water for 4 days after surgery.</li>
	<li>Check the animal and the wound frequently during the 48-h post-surgery period for possible adverse effects.</li>
</ol>
3. Mouse Treatments, Euthanasia, and Sample Retrieval for Imaging
NOTE: Analysis of the scaffolds is performed between 8 and 24 weeks post-implantation.
<ol>
	<li>60 min before imaging, keep the implanted mouse warm in a heating box at 37 &#176;C and intravenously administer 100 &#181;L of human immunoglobulin to block unspecific sites.</li>
	<li>30 min before imaging, intravenously administer 10 &#181;g (per mouse) of specific antibodies to label the cells of interest.</li>
	<li>5 min before imaging, intravenously administer 15 &#181;L (diluted in 100 &#181;L of PBS) of 655-nm fluorophore-labeled, vessel-pooling agent (655-VPA) to visualize vascular structures.</li>
	<li>Euthanize the mouse via cervical dislocation.</li>
	<li>Using sharp scissors, make a longitudinal skin incision on the back of the mouse, near the original implantation site.</li>
	<li>With the help of tweezers and scissors, carefully separate the skin from the subcutaneous pocket where the scaffold has been implanted.</li>
	<li>Hold the scaffold with tweezers and gently explant it from the skin by cutting the residual membrane and tissue surrounding the scaffold using scissors. See examples of scaffolds to be recovered in Figure 2.</li>
	<li>Secure the scaffold with fast-acting adhesive glue to an imaging plate (a 35 mm x 10 mm Petri dish) and fill with saline solution (PBS) at room temperature.</li>
	<li>For BMP scaffolds, before filling the plate with PBS, use a surgical microdrill to thin the bone surface under a microsurgical microscope; this allows fluorophore visualization and high-resolution image capture. Use either the 1.2- or 1.6-mm burrs, depending upon the size of the scaffold. 
	NOTE: The user will realize how much to drill depending upon the thickness of the bone. In general, as the BMP scaffolds are vascularized, the bone will slightly change color and become more red when approaching the correct thickness for imaging.</li>
	<li>Insert the plate onto the stage of the confocal microscope.</li>
</ol>
4. Live-imaging Using Two-photon Microscopy
NOTE: When using non-descanned detectors (NDD), always use the NDD slider for imaging to direct the fluorescence to the NDD. The microscope configuration is provided in Figure 3.
<ol>
	<li>Switch on the microscope and the computer, start the software by clicking &#34;Start System,&#34; and go to the &#34;Acquisition&#34; mode.</li>
	<li>Tick the &#34;Show manual tools&#34; box. In the &#34;Laser&#34; menu switch &#34;on&#34; the two-photon laser and allow it to warm up and stabilize.</li>
	<li>In the &#34;Imaging Setup&#34; menu, simultaneously activate &#34;Channel Mode&#34; and &#34;Switch track every Frame.&#34; In the &#34;Light Path&#34; menu, select &#34;Non Descanned&#39;&#34; and &#34;Main Beam Splitter MBS 760+.&#34; Tick to activate the four NDDs and set the configuration as illustrated in Figure 3. 
	NOTE: With this configuration, the collagen signal from bone structures (second harmonic generation, SHG) is collected at 380 - 485 nm, FITC-hCD31+ human endothelial cells and AF488- hCD45+ human hematopoietic cells at 500-550 nm, and 655-VPA at 640 - 690 nm.</li>
	<li>In the &#34;Channels&#34; menu, set the &#34;laser wavelength&#34; to 890 nm and the power to 50%. Set the &#34;Gain (Master)&#34; to 500-600, the &#34;Digital Offset&#34; to 0, and the &#34;Digital Gain&#34; to 15 for each channel. Adjust these values once the acquisition has started.</li>
	<li>In &#34;Acquisition Mode,&#34; set up the required parameters to obtain high-resolution images without damaging the tissue and bleaching the fluorophores. Set &#34;Scan Mode&#34; to &#34;Frame,&#34; &#34;Frame Size&#34; to &#34;x512 y512,&#34; Line Step&#34; to 1, &#34;Speed&#34; to 9, &#34;Averaging number&#34; to 8, &#34;Bit Depth&#34; to &#34;8 Bit,&#34; &#34;Mode&#34; to &#34;line,&#34; &#34;Direction&#34; to &#34;bidirectional,&#34; and &#34;Method&#34; to &#34;mean.&#34;
	<ol>
		<li>Set the &#34;Zoom&#34; to 1 for the initial scan of the image, and increase it if required to focus on particular areas.</li>
	</ol>
	</li>
	<li>Place the plate containing the scaffold on the microscope stage under the 20X, 1.0 NA water-immersion lens and lower the lens until it touches the saline solution. Set the focus of the lens on the scaffold using the microscope eyepieces, using a lamp as the light source.</li>
	<li>Activate the &#34;Z-stack&#34; menu, select the &#34;First/Last&#34; function, and set the required interval between two sub-sequential slices (e.g., 2-&#181;m Z-stack). Keep the intervals constant within the Z-stack.</li>
	<li>Select &#34;Live&#34; to image a live scan of the sample and adjust the &#34;Digital Gain&#34; and &#34;Digital Offset&#34; for optimal exposure. To visualize multiple channels at the same time, select the &#34;Split&#34; function.</li>
	<li>In the &#34;Stage&#34; menu, while in &#34;Live&#34; mode, scan the image and &#34;Mark&#34; regions of interest (ROI), such as the location of human hematopoietic cells and vascular structures. When the scan of the sample is complete, move to the first ROI to start imaging.</li>
	<li>In &#34;Live&#34; mode, set the top and bottom of the 3D Z-stack surrounding the area of interest using the &#34;Set first&#34; and &#34;Set last&#34; functions. Once finished, set the center, &#34;C.&#34; Start the acquisition of the ROI with the &#34;start experiment&#34; button. See examples of images in Figure 4, Figure 5, and Figure 6.</li>
	<li>Once acquisition is complete, save the image in the designated folder. Move to the next ROI and repeat step 4.10. Once the experiment is complete, remove the imaging plate from the microscope, carefully detach the sample from the plate, and clean any residual glue.</li>
	<li>Prepare the sample for the next analysis technique.</li>
</ol>
5. Sample Processing for Histology and Immunostaining
NOTE: Samples are processed according to the protocol described in the JoVE general laboratory techniques54 describing sample fixation, embedding, and sectioning processes. Bone-forming samples should be treated for 7 days in an EDTA-based decalcifying agent between the fixation and embedding processes. The blocking/permeabilization solution is 10 mM PBS pH 7.4 buffer with 1% Triton X-100, 1% bovine serum albumin (BSA), and 10% normal goat serum (NGS).
<ol>
	<li>Put the slices in xylene for 10 min), xylene for 5 min, 100% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and H2O for 5 min.</li>
	<li>Transfer the slices to a citrate-based antigen unmasking working solution.</li>
	<li>Boil the slices for 15 min and allow them to cool to room temperature.</li>
	<li>Wash the slices in 10 mM PBS pH 7.4 solution with 1% Triton X-100 (5 min, 3 times).</li>
	<li>Transfer the samples to the blocking/permeabilization solution and incubate for 30 min.</li>
	<li>Add primary antibody diluted in blocking/permeabilization solution and incubate overnight at 4 &#176;C.</li>
	<li>Wash the slices in 10 mM PBS pH 7.4 solution with 1% Triton X-100 (5 min, 3 times).</li>
	<li>Add secondary antibody diluted in blocking/permeabilization solution for 1 h in the dark at room temperature.</li>
	<li>Wash with H2O (5 min, 3 times).</li>
	<li>To reduce background fluorescence, immerse the slices in Sudan Black working solution for 10 min, in the dark and at room temperature.</li>
	<li>Wash the slices in H2O (5 min, 3 times).</li>
	<li>Mount the slices using fluorescent mounting medium with DAPI (0.5 &#181;g/mL).</li>
	<li>Store the slices at 4 &#176;C and check that the mounting medium is dry before conducting fluorescent imaging. See example images in Figure 7.</li>
</ol>

The authors have nothing to disclose.

Significance with Respect to Existing Methods:
In this protocol, we described a method to generate different humanized microenvironments in mice and to visualize their architecture via two-photon microscopy and histology. The representative data provided shows the feasibility of the approach, using different stromal cells to engineer humanized tissues. The protocol has specific applications to the study of human hematopoietic cells and bone marrow niche-derived cells in normal and pathological conditions. These applications include the study of clonal evolution, drug screening, and crosstalk between human HSCs and stromal components. In the emerging field of tissue engineering, several alternative approaches have been proposed. Approaches of note include the development of 3D humanized BM structures in vitro55,56,57,58,59,60,61,62,63 and the orthotopic graft of humanized BM scaffolds in mice64. Our approach has the advantage of combining both the complexity of the in vivo system with the easy anatomical accessibility of the humanized tissue graft.
Modifications and Troubleshooting:
A source of variability in this protocol can be found in the selection of cells used to seed the scaffolds. In our work, we used BM-derived hMSCs. However, mesenchymal cells can be obtained from several tissues, which may show distinctive properties depending upon the origin. Therefore, the use of hMSCs derived from different organs can be considered. However, their ability to form bone tissue in vivo should be tested prior to use in this protocol.This protocol uses a commercially available human endothelial cell source (i.e., E4ORF1-transduced HUVEC). Recently, the use of organ-specific endothelial cells for different purposes has been reported65,66. Furthermore, the use of primary hECs derived from the BM could represent an interesting improvement to the protocol. Therefore, the use of different sources of endothelial cells may produce different in vivo outcomes.
We used NSG immunocompromised recipient mice to favor the implantation of humanized scaffolds and to avoid tissue rejection. We do not exclude the possibility of using this protocol to engineer ectopic bone marrow tissues in other mouse strains. Indeed, rhBMP-2 can induce bone formation in different mammalian models47,48,49,50,52. However, differences in cell viability and long-term transplantation are likely to be observed using different strains/models.
The timing of scaffold recovery can also be flexible, depending upon the final purpose of the experiment. In the presented protocol, we recover samples at 8 - 12 weeks after implantation to assess long-term hematopoietic engraftment. To study early steps of the human BM niche formation (e.g., osteochondral tissue formation47 or vascular development), different time points can be chosen.
The live-imaging technique we described in this protocol is indicated for short-term imaging of explants. The use of an equilibrated chamber for maintaining physiological temperature, oxygen tension, and CO2 concentration should be considered in cases of long-term imaging, such as to study motility behaviors.
Critical Steps within the Protocol:
Among the challenges related to the protocol, we would highlight the technical skills required for some steps. Mesenchymal and endothelial cells should be used at low cell passage numbers; otherwise, they will not be able to support human hematopoietic cell engraftment in vivo or to participate in de novo vasculature and bone formation in vivo. We recommend the use of hMSCs and hECs at passages 1 - 5. Scaffold preparation and cell-seeding steps require basic cell-culture skills and knowledge of the properties of the specific cells used in the procedure. The surgery protocol is quite straightforward but requires some practice. Maintenance of an aseptic environment to avoid contamination of the implanted scaffolds in immunodeficient mice is crucial to ensure the success of the experiment. Sample explant and live imaging require surgical practice (especially for the use of the microdrill) and knowledge of the microscope system. Finally, sample processing and histology require basic knowledge of the techniques to be used.
Limitations of the Technique:
The approach we describe allows for the visualization of live human hematopoietic cells seeding a humanized bone marrow microenvironment, with human endothelial cells forming vascular structures and mesenchymal cells forming bone/bone marrow space. As the tissue is formed in vivo, the final engineered scaffold will still be chimeric (human and murine). This issue should be taken into account, as the chimeric tissue may not fully mimic human bone marrow complexity and environment.
The scaffolds implanted have a limited size (we tried a maximum of 6.6 x 7.5 x 7 mm), and therefore, they are able to host a limited number of cells for xenotransplantation. The absolute number of recovered cells will also be limited; thus, the number of implanted scaffolds should be calculated as a function of the number of cells required for the experiment.
The imaging application we described is particularly useful for observing large areas of live tissue at depths of 150-200 &#181;m from the surface without disrupting the architecture and damaging the cells. Therefore, it does not allow for the visualization of the whole scaffold. If a complete scan of the tissue is required, standard immunofluorescence approaches would be more appropriate.
Future Applications:
The future direction of this bioengineered model would be to increase the complexity of the human components in the tissue. The knowledge and characterization of the human BM niche has progressed in recent years67, and the described protocol could be an interesting platform to study the function of these new cellular components and soluble factors, as well as their role in supporting normal/malignant HSCs.
Furthermore, the imaging technique provides the potential for intravital imaging of the scaffolds in longitudinal studies, which would require technical improvements in imaging the scaffolds in live, anesthetized mice, with post-surgery recovery. This approach would require additional steps and is currently under investigation in the laboratory.

Cell fate decisions observed in stem cell compartments are tightly regulated by both intrinsic and extrinsic factors. In particular, it is now widely recognized that the BM microenvironment plays a fundamental role in controlling the switch in HSCs from a quiescent to an active state, as well as in their self-renewal or differentiation fate decision1. Moreover, recent findings indicate that hematological malignancies affect the function of the BM microenvironment, pointing to the existence of active crosstalk between the two compartments2,3,4,5,6. Despite recent advances, many key questions remain about how the activity of specific BM-niche components contribute to HSC behavior and malignant transformation.
The BM microenvironment is a highly heterogeneous and complex mixture of many different cell types, each with specialized functions. The abundant endothelial (EC) and vascular component regulate nutrient and metabolite turnover, the ingress and egress of different cells to and from the BM, and several HSC functions7,8. Mesenchymal stromal cells (MSCs), a heterogeneous population of undifferentiated stem cells and progenitors committed through three different lineages (i.e., osteogenic, chondrogenic, an adipogenic), are another fundamental component of the BM niche. These MSCs localize both in central areas of the BM and in the proximity to the endosteal region. They may be associated with vascular structures and are implicated in the regulation of HSC function9,10,11,12,13,14,15.
Many reports suggest that HSCs reside in various defined sites within the marrow and that their function might depend upon their precise localization. Most of the present knowledge regarding HSCs and their interaction with the BM microenvironment derives from murine studies1. The use of xenograft models has extended this knowledge to human normal and malignant HSCs, engrafting within the murine BM of immunodeficient mice16,17,18,19,20. Although this represents a valid model, it still presents many challenges, such as the need to condition the recipient mouse in most cases to allow for human HSC homing and engraftment or the cross-species barrier and its poorly understood influence on cell-cell interactions and functions.
The use of neutralizing antibodies and genetically modified mice, along with xenotransplantation, has been instrumental in highlighting the complex dialog that human HSCs establish with their microenvironments. The introduction and development of intravital two-photon confocal microscopy has moved these studies a step forward, allowing for the direct, high-resolution, and dynamic imaging of the bone marrow19,20,21,22 and provides a powerful tool for the functional characterization of the BM microenvironment and its role in regulating HSC function. In order to circumvent some of the problems arising in classical xenotransplantation models, the concept of engineering a humanized BM structure has been brought to the fore. Merging biomaterials and cell-implantation concepts, reports have shown the feasibility of mimicking the human bone marrow microenvironment in heterotopic regions23,24,25,26,27. This opens the possibility of using bioengineering in mouse models to study human normal and malignant hematopoiesis28,29,30,31,32,33,34,35,36,37,38,39, tumorigenesis, and metastasis40,41,42,43,44.
Based on previous experience in bone tissue engineering and in vivo imaging19,22,45,46,47,48,49,50,51,52, we describe a protocol to bioengineer and live-image organotypic human BM tissues. These structures originate from the implantation of human BM-derived stromal cells into collagen-based scaffolds subcutaneously grafted in immunodeficient mice. In a previous report, we demonstrated that human MSCs ensure the formation of a human microenvironment adequate for the engraftment of human hematopoietic cells45. Furthermore, here we describe how the co-implantation of other human BM cellular components, such as human endothelial cells (hEC), and/or cytokines important for bone formation (e.g., hBMP2), cooperate with hMSCs to generate different humanized microenvironments, which can be live-imaged in situ.

<table border="0" cellpadding="0" cellspacing="0" width="1378">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Ficoll-Paque</td>
			<td style="width:277px;">Ge Healthcare</td>
			<td style="width:229px;">17-1440-03</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Cd34 positive selection Kit</td>
			<td style="width:277px;">Stemcell</td>
			<td style="width:229px;">18056</td>
			<td style="width:480px;">Store a 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Magnet</td>
			<td style="width:277px;">Stemcell</td>
			<td style="width:229px;">18000</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Anti-human CD3 antibody, clone OKT-3</td>
			<td style="width:277px;">Bioxcell</td>
			<td style="width:229px;">BE0001-2</td>
			<td style="width:480px;">Store at 4&#176;C. Used for T cell depletion in primary AML samples.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">Cytokines (IL3, G-CSF, and TPO)</td>
			<td style="width:277px;">PeproTech</td>
			<td style="width:229px;">200-03, 300-23 and 300-18</td>
			<td style="width:480px;">For doing the stock: Dilute each one of the cytokines in 100&#956;L of water and mix them. Add 200&#956;L, do alicuots of 45&#956;L and Store at -20&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">DMSO</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">D4540</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:392px;">FBS</td>
			<td style="width:277px;">Life technologies</td>
			<td style="width:229px;">10270106</td>
			<td style="width:480px;">Heat-inactivate at 56&#176;C during 30 minutes, do aliquots and freeze down. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">MEM-&#945;</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">32571-028</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Myelocult H5100</td>
			<td style="width:277px;">corning</td>
			<td style="width:229px;">5100</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">199</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">41150-020</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">hMSC-FBS, Heat-inactivated</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">12662-029</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">P/S</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">P0781</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">ECGS</td>
			<td style="width:277px;">Millipore</td>
			<td style="width:229px;">02-102</td>
			<td style="width:480px;">Dilute in culture media and use 0.22 mm filter.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:392px;">HEPES</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">S1558-50ML</td>
			<td style="width:480px;">Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Heparin</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">H3149</td>
			<td style="width:480px;">Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Glutamine</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">25030</td>
			<td style="width:480px;">Store at -20&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Collagen 1 coated cell culture plate</td>
			<td style="width:277px;">corning</td>
			<td style="width:229px;">354505</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Trypsin-EDTA solution</td>
			<td style="width:277px;">Thermo-Fisher</td>
			<td style="width:229px;">25200056</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:392px;">hMSC</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:229px;">PT-2501</td>
			<td style="width:480px;">Alternatively, hMSCs we also kindly provided by Dr. Dosquet (University Paris Diderot, Paris) from human bone marrow obtained during orthopaedic surgery under ethical approval 10-038 from IRB00006477.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">VeraVec HUVEC endothelial cells</td>
			<td style="width:277px;">Angiocrine bioscience</td>
			<td style="width:229px;">hVera101</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:392px;">Human hematopoietic cells</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Umbilical Cord blood or primary Acute Myeloid Leukemia (AML) samples were obtained from the Royal London Hospital (London, UK) after informed consent and protocol of use was approved by the East London Ethical Committee and carried out in accordance with the Declaration of Helsinki.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:392px;">Gelfoam, Size 12-7mm</td>
			<td style="width:277px;">Pfizer</td>
			<td style="width:229px;">00009-0315-08</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">PBS</td>
			<td style="width:277px;">Thermo-Fisher</td>
			<td style="width:229px;">10010023</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Surgical material</td>
			<td style="width:277px;">Multiple</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Sterile forceps, tweezers and sharp scissors.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Sterile tissues</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Heat-sterilize paper tissues.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">1 ml Syringe with needle of 25G</td>
			<td style="width:277px;">Terumo</td>
			<td style="width:229px;">SS+01H25161</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">Ultra Low Attachment Multiple Well Plates</td>
			<td style="width:277px;">Corning</td>
			<td style="width:229px;">3473 or 3471</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">BMP2</td>
			<td style="width:277px;">Noricum</td>
			<td style="width:229px;">rhBMP-2</td>
			<td style="width:480px;">Dilute at 5 mg/ml in acetic acid 50 mM and store at 4 &#176;C.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Thrombin</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">T9010</td>
			<td style="width:480px;">Dilute in CaCl2 2%, 500 mL per vial, and store at 4 &#176;C.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:392px;">Fibrinogen</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">F3879</td>
			<td style="width:480px;">Dilute at 4 mg/100 mL in PBS. Store at -20C. Warm before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:392px;">Tissue-culture dishes 35 mm x 10 mm</td>
			<td style="width:277px;">Falcon</td>
			<td style="width:229px;">353001</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:392px;">U-Botton 96 well plate</td>
			<td style="width:277px;">Falcon</td>
			<td style="width:229px;">353077</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">NSG mice</td>
			<td style="width:277px;">The Jackson Laboratory</td>
			<td style="width:229px;">5557</td>
			<td style="width:480px;">NSG mice were a kind gift from Dr Leonard Shultz (The Jackson Laboratory).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Chlorhexidine</td>
			<td style="width:277px;">G9</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Dilute 1:10 before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Carprofen</td>
			<td style="width:277px;">Pfizer</td>
			<td style="width:229px;">Rimadyl</td>
			<td style="width:480px;">5 mg/g of mouse</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Buprenorphine</td>
			<td style="width:277px;">Alstoe</td>
			<td style="width:229px;">Vetergesic</td>
			<td style="width:480px;">0.1 mg/g of mouse body weight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Isoflurane</td>
			<td style="width:277px;">Abbott</td>
			<td style="width:229px;">B506</td>
			<td style="width:480px;">Induction of anaesthesia 2%, maintenance 1%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Trimmer</td>
			<td style="width:277px;">Wella</td>
			<td style="width:229px;">Contura HS61</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Surgical glue</td>
			<td style="width:277px;">3M</td>
			<td style="width:229px;">Vetbond</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">carbomer (polyacrylic acid)&#160; as Ophthalmic gel</td>
			<td style="width:277px;">Novartis</td>
			<td style="width:229px;">Viscotears Liquid Gel</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">Human Normal Immunoglobulin</td>
			<td style="width:277px;">Gammaplex</td>
			<td style="width:229px;">10g vial</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before infusion of specific antibody.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">NT-QTracker</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">Q21021MP</td>
			<td style="width:480px;">Vessel-pooling agent. Administrate 15 ml/mouse intravenously 5 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">AF488-hCD45</td>
			<td style="width:277px;">Biolegend</td>
			<td style="width:229px;">304017</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">FITC-hCD31</td>
			<td style="width:277px;">BD Pharmigen</td>
			<td style="width:229px;">555445</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Super glue</td>
			<td style="width:277px;">Loctite&#160;</td>
			<td style="width:229px;">Super Glue</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Micro-Drill Kit</td>
			<td style="width:277px;">IDEAL - Fisher Scientific</td>
			<td style="width:229px;">NC9010016</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgical microscope</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">No specific brand/company is adviced.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">LSM 710 NLO</td>
			<td style="width:277px;">Zeiss</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Upright confocal microscope with motorized stage, two-photon laser and 20x 1.0 NA water immersion lens. Alternatively, a microscope with similar specifications could be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">MaiTai &#8220;High Performance&#8221; fully automated 1-box 517 mode-locked Ti:Sapphire laser with DeepSee dispersion compensation</td>
			<td style="width:277px;">&#160;Spectra-Physics</td>
			<td style="width:229px;"></td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Formalin solution, neutral buffered, 10%&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">HT501128</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Ethanol</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">32294</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Osteosoft</td>
			<td style="width:277px;">Millipore</td>
			<td style="width:229px;">1.01728.1000</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Polysine slices</td>
			<td style="width:277px;">Thermo scientific</td>
			<td style="width:229px;">J2800AMNZ</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Antigen unmasking solution</td>
			<td style="width:277px;">Vector</td>
			<td style="width:229px;">H-3300</td>
			<td style="width:480px;">Store at 4 &#176;C. Dilute 1:100 in H2O for working solution.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Triton 100x</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">T9284</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Bovine serum albumin (BSA)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">A96470</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Normal Goat serum (NGS)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">G9023</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Endomucin Antibody</td>
			<td style="width:277px;">Santa Cruz</td>
			<td style="width:229px;">sc-65495</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hVimentin antibody</td>
			<td style="width:277px;">Santa Cruz</td>
			<td style="width:229px;">sc-6260</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hCD31 antibody</td>
			<td style="width:277px;">DAKO</td>
			<td style="width:229px;">M0823</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hCD45 antibody</td>
			<td style="width:277px;">DAKO</td>
			<td style="width:229px;">M0701</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">ADRP (Perilipin2)</td>
			<td style="width:277px;">antibodies-online</td>
			<td style="width:229px;">ABIN283918</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:392px;">Goat anti mouse secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11029 or A11005</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:392px;">Goat anti Rabbit secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11037 or A11008</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Goat anti Rat secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11007 or A21247</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">Sudan Black</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">S2380</td>
			<td style="width:480px;">Prepare a stock of 1% sudan black in ethanol 70%. Store at RT. Prepare working solution of 0.1% sudan black in ethanol 70% and filter using filter-paper before use.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">DAPI</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">D8417</td>
			<td style="width:480px;">Prepare stock in H20 at 100 mg/mg. Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Fluorescent mounting media</td>
			<td style="width:277px;">Dako</td>
			<td style="width:229px;">S3023</td>
			<td style="width:480px;">Add DAPI before use (1:400 from DAPI stock).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Cover glass</td>
			<td style="width:277px;">VWR</td>
			<td style="width:229px;">631-0147</td>
			<td style="width:480px;"></td>
		</tr>
	</tbody>
</table>

bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

In Figure 1, representative images of the scaffold cell seeding and implantation processes are shown. In Figure 1C, note that cells are injected directly into the scaffold. In Figure 1G, note that an incision is made in the back of the mouse, where the subcutaneous pocket is created and the scaffold is implanted. Figure 2 shows the gross morphology of different scaffolds implanted in NSG mice and retrieved after 8 weeks. Note the slight vascularization in hMSC seeded scaffolds (Figure 2A). The co-seeding of human ECs with hMSCs in the scaffold allows for the formation of more relevant vasculature in scaffolds (Figure 2B). Finally, the presence of rhBMP-2 induces bone formation. The retrieved scaffolds are bigger in this case, and they are constituted by bone-resembling hard tissue.
Figure 3 shows the channel configuration setup on the microscope for live-imaging with NDD (details in the figure legend). Figure 4 and Video 1 show human hematopoietic cells in hMSC-coated scaffolds. Scaffolds were explanted 8 weeks post-implantation and after the intravenous inoculation of AF488-hCD45 antibody and 655-VPA. This procedure allows for the visualization of implanted human hematopoietic cells and the vascular structure by two-photon confocal microscopy. In this case, the images show blood vessels (655-VPA) in scaffolds and the long-term engraftment of human hematopoietic cells (AF488-hCD45) in the scaffold parenchyma. Figure 5 and Video 2 correspond to human scaffolds seeded with hECs and hMSCs. 8 weeks after surgery, scaffolds were explanted after the intravenous inoculation of FITC-hCD31 antibody and 655-VPA, and images were acquired with a two-photon confocal microscope, as mentioned before. Images show the participation of hECs in vessel formation in the scaffold, resulting in a murine-human chimeric vasculature.
Figure 6A shows representative data of the approach used to stimulate bone formation in MSC scaffolds. Similar to previous figures, 8 weeks after implantation, the intravenous inoculation of 655-VPA was performed, scaffolds were retrieved, and images were acquired with two-photon confocal microscopy. rhBMP-2-stimulated scaffolds induce the formation of bone tissue, which could be visualized due to the SHG (cyan color in the images) provided by the calcium in the bone. The provided images also show the formation of cavities and vascularized endosteal tissue, which highly resemble the BM endosteal tissue. In Figure 6B and Video 3, hECs were co-implanted with hMSCs. Scaffolds were retrieved after the intravenous inoculation of FITC-hCD31 antibody and 655-VPA, and two-photon confocal microscopy images show the participation of hECs in the neovascularization in a bone-forming scaffold.
Figure 7 shows representative images of histology, a procedure performed to corroborate previously described results. Immunofluorescent images show mouse vasculature, hECs, hMSCs, and long-term engrafted human hematopoietic cells in the scaffold structures. Scaffolds retrieved from mice were fixed and used for immunofluorescence. In the rhBMP-2 carrier bone-forming scaffolds (Figure 7D-F), note the morphology of the tissue, resembling mature bone marrow with adipose tissue. In this bone-forming scaffold, we show that hMSCs are fibroblasts, which would indicate that they contribute to newly formed tissue as stromal cells. We also show human adipocyte marker expression, which would indicate that hMSCs also contribute to adipose tissue formation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55914/55914fig1.jpg" /> 
Figure 1. Representative Images of the Cell-seeding and Implantation Processes. A) Initial scaffold and its cutting method using a scalpel. B) 24 pieces obtained from the initial scaffold. C) Scaffold cell-seeding method using a syringe. D) Cell-seeded scaffolds with culture medium, ready to be implanted. E-F) Specific steps for bone-forming scaffolds: E) scaffold being transferred to a 96-well, u-bottom plate and F) representative image of the method used to add rhBMP2, thrombin, and fibrinogen to the scaffold. G-J) Surgical implantation procedure under general anesthesia: G) wound created in the skin and scaffold implantation, H) implantation method, and I-J) wound closing procedure using surgical glue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55914/55914fig2.jpg" /> 
Figure 2. Different Scaffolds Retrieved from Mice. Representative images of MSC scaffolds (left), MSC+EC scaffolds (middle), and MSC+EC+BMP scaffolds (right). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55914/55914fig3.jpg" /> 
Figure 3. Channel Configuration. The microscope filter setup is shown. A) There are four NDD detector modules: in the first module, there are two filter cubes; the second and third modules have one filter cube each; and the last module has no cube (not used). The first photomultiplier tube (PMT) is for the far-red channel (640 - 690 nm), reflecting the lower wavelengths; the following ones are 380 - 485 nm, 500 - 550 nm, and 555 - 625 nm (the order is always from the lower to higher wavelengths). B) The fluorophore emissions detectable with the above configurations (color coded). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55914/55914fig4.jpg" /> 
Figure 4. MSC Scaffolds Allow for the Formation of a Niche for Human Hematopoietic Cells. A) and B) 3D reconstructions of Z-stacks taken post-explant, after intravenous inoculation with AF488-hCD45 (green), to label human hematopoietic cells, and 655-VPA (magenta)m to label vasculature. Scale bars represent 20 &#181;m in A and B (upper panels) and 5 &#181;m in B (lower panels). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/55914/55914video1.jpg" /> 
Video 1. MSC Scaffolds Allow for the Formation of a Niche for Human Hematopoietic Cells. 3D reconstruction of human hematopoietic cells (AF488-hCD45) associated with the vasculature (655-VPA) in the MSC scaffold (collagen structures: SHG, cyan). Each stack measures 140 x 140 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914video1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55914/55914fig5.jpg" /> 
Figure 5. Human ECs Participate in the Formation of Humanized Vessels in MSC Scaffolds. 3D reconstruction of vasculature (655-VPA) lined by ECs of human origin (FITC-hCD31) in MSC+EC scaffolds. Scale bars represent 20 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 2" src="/files/ftp_upload/55914/55914video2.jpg" /> 
Video 2. Human ECs Participate in the Formation of Humanized Vessels in MSC Scaffolds. 3D reconstruction of human ECs (FITC-hCD31) lining the vasculature (655-VPA) in MSC+EC scaffolds. Each stack measures 240 x 240 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914video2.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55914/55914fig6.jpg" /> 
Figure 6. rhBMP-2 Carrier Scaffolds have Bone Surfaces and Humanized Vasculature Similar to the Bone Marrow. A) 3D reconstruction of MSC+BMP scaffolds showing the formation of bone structures (SHG-cyan) and vasculature (655-VPA). Scale bars represent 100 &#181;m (left), 70 &#181;m (middle), and 50 &#181;m (right). B) 3D reconstruction of MSC+EC+BMP scaffolds showing humanized vessels (655-VPA) lined with human ECs (FITC-hCD31). Scale bars represent 50 &#181;m (left) and 30 &#181;m (middle and right). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 3" src="/files/ftp_upload/55914/55914video3.jpg" /> 
Video 3. rhBMP-2 Carrier Scaffolds have Bone Surfaces and Humanized Vasculature Similar to the Bone Marrow. 3D reconstruction of MSC+VERA+BMP scaffold (bone: SHG; vessels: 655-VPA; human ECs: FITC-hCD31). Each stack measures 600 x 600 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914video3.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55914/55914fig7.jpg" /> 
Figure 7. Representative Images of Immunofluorescence Performed on Fixed Scaffolds. A-F) Immunofluorescence studies performed to locate human cells implanted in scaffolds. A-C) Scaffolds implanted with hECs, hMSCs, and hHSCs. D-F) Bone-forming scaffolds implanted with hECs, hMSCs, and hHSCs. The color channels are as follows: A-D) human EC (hCD31) and mouse vascular structure (endomucin, ENDOM), B-E) hMSCs (hVimentin, hVIM) and mouse vascular cells (endomucin, ENDOM), C) human hematopoietic cells (hCD45) and mouse vasculature (endomucin, ENDOM), and F) human adipose differentiation-related protein (hADRP) and mouse vasculature (endomucin, ENDOM). Scale bars represent 10 &#181;m (A and C), 20 &#181;m (B and E), and 40 (D and F) &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55914/55914fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging at JoVE.com

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

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Developmental Biology

11.6K Views.  The Francis Crick Institute. A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Video: Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...