Name: combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches
Uploaded: 2017-03-21T13:00:00
Description: Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Imaging was carried out in the Indiana Center for Biological Microscopy at Indiana University, directed by Dr. Ken Dunn. The stereotaxic device is a prototype designed and made by Mark Soonpaa, Wells Center for Pediatric Research. This work was supported by NIH/R01DK097837-09 (NC), NIH/R01HL068256-05 (NC), NIH/NIDDK1U54DK106846-01 (NC), the MPN research Foundation (NC) and the CTSI Collaborative project IUSM/Notre Dame (NC).

All procedures involving the use of animals were performed with authorization of the Animal Care and Use Committee of Indiana University School of Medicine. Ensure to adhere to the legislation on animal experimentation of the country where the work is performed.
1. Preparation of Mx1CreRBPJ-/- Recipient Mice
<ol>
	<li>Cross Mx1-Cre+ mice with RBPJlox/lox mice10 to obtain Mx1-Cre positive RBPJlox/lox mic...

<ol>
	<li>Carlesso, N., Cardoso, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+regulatory+niches+and+their+role+in+normal+and+malignant+hematopoiesis.">Stem cell regulatory niches and their role in normal and malignant hematopoiesis.</a> Curr Opin Hematol. 17 (4), 281-286 (2010).</li><li>Lo Celso, C., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124 (PT 21), 3529-3535 (2011).</li><li>Calvi, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425 (6960), 836-841 (2003).</li><li>Mendez-Ferrer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li><li>Naveiras, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-marrow+adipocytes+as+negative+regulators+of+the+haematopoietic+microenvironment.">Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.</a> Nature. 460 (7252), 259-263 (2009).</li><li>Scheiermann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+nerves+govern+circadian+leukocyte+recruitment+to+tissues.">Adrenergic nerves govern circadian leukocyte recruitment to tissues.</a> Immunity. 37 (2), 290-301 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 716-726 (2000).</li><li>Han, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+knockout+of+transcription+factor+recombination+signal+binding+protein-J+reveals+its+essential+role+in+T+versus+B+lineage+decision.">Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision.</a> Int Immunol. 14 (6), 637-645 (2002).</li><li>Lo Celso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457 (7225), 92-96 (2009).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch-dependent+repression+of+miR-155+in+the+bone+marrow+niche+regulates+hematopoiesis+in+an+NF-kappaB-dependent+manner.">Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner.</a> Cell Stem Cell. 15 (1), 51-65 (2014).</li><li>Miyamoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+or+lymphoid+promiscuity+as+a+critical+step+in+hematopoietic+lineage+commitment.">Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment.</a> Dev Cell. 3 (1), 137-147 (2002).</li><li>Luc, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down+Regulation+of+Mpl+marks+the+transition+to+lymphoid-primed+multipotent+progenitors+with+gradual+loss+of+granulocyte-monocyte+potential.">Down Regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.</a> Blood. 111 (7), 3424-3434 (2008).</li><li>Suzuki, M., Moriguchi, T., Ohneda, K., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+the+Gata1+gene+hematopoietic+enhancer+to+erythroid+differentiation.">Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.</a> Mol Cell Biol. 29 (5), 1163-1175 (2009).</li><li>Essers, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458 (7240), 904-908 (2009).</li><li>Pietras, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-entry+into+quiescence+protects+hematopoietic+stem+cells+from+the+killing+effect+of+chronic+exposure+to+type+I+interferons.">Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.</a> J Exp Med. 211 (2), 245-262 (2014).</li><li>Scott, M. 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This protocol describes an experimental design optimized to study the kinetics of hematopoietic cells engraftment by Intravital Florescent Microscopy. In this study, the expansion of myeloid progenitor cells in a WT BM or in a Notch signaling defective BM was tracked in the bone calvarium by following Lys-GFP positive myeloid cells after BMT into RBPJWT or RBPJKO recipients. This approach is proposed as a model that can be applied to address similar questions, for example: i) to determine the expansion and localization i...

Intravital multiphoton fluorescence microscopy (IVFM) is a powerful imaging technique that allows for the high-resolution, real-time imaging of tissues with depth up to 1mm, depending on the tissue. When applied to the mouse calvarium, it permits observing the behavior of the hematopoietic cells within the BM in a non-invasive manner up to 60-100 &#956;m11. This approach is used here to determine the kinetics of engraftment of normal myeloid progenitors in the BM of RBPJ knock-out mice lacking canonical Notch signaling.
Recent work from our group demonstrated that defective canonical Notch signaling in the BM microenvironment leads to a myeloproliferative-like disease12. Loss of Notch signaling was obtained by conditional deletion of the DNA binding domain of RBPJ, the critical transcription factor downstream of canonical Notch signaling, using Mx1-Cre induced recombination10. In this study, the Mx1-Cre/RBPJlox/lox mice model was used. Conditional deletion of the DNA-binding motif of RBPJ results in the loss of signaling from all Notch receptors. In the Mx1-Cre model, Cre expression is driven by the Mx1 promoter activated upon administration of polyI:C resulting in the induction of targeted gene deletion in blood cells as well as in stromal components of multiple organs, including BM, spleen and liver.
Mx1-Cre+/RBPJlox/lox and Mx1-Cre-/RBPJlox/lox mice induced with polyI:C (hereon indicated as RBPJKO and RBPJWT, respectively) were lethally irradiated and transplanted with normal, wild type hematopoietic cells. Starting from week 4 after transplantation, RBPJKO recipients developed significant leukocytosis followed by splenomegaly. Although RBPJKO mice presented increased percentage of myeloid progenitors in the BM at week 8 after transplant and at later time points, analysis of BM at weeks 4 and 6 did not reveal striking differences in their myeloid cell content compared to control RBPJWT recipients. This observation, together with the fact that Mx1-Cre is expressed in different hematopoietic organs, raised the question whether the BM microenvironment had direct impact on the initiation of the myeloproliferative phenotype.
To determine whether the BM was a critical initial site of disease development, IVFM of the mouse calvarium was used in combination with BM transplantation (BMT), the RBPJ knock-out model, and a lineage tracking system. Transgenic mice expressing EGFP under the control of the specific lysozyme promoter (Lys-GFP)9 were used to obtain donor cells that could be visualized during BM imaging after BMT. Lysozyme expression is specific to myeloid cells and Lys-GFP marks cells from the common myeloid progenitor (CMP) to the mature granulocyte13.
IVFM of the BM at different time points demonstrated that Lys-GFP cells homed similarly to the BM of RBPJWT and RBPJKO recipients, but expanded and engrafted faster in the BM of RBPJKO recipients. This difference was dramatic at the earlier time point (week 2) and decreased over time (weeks 4 and 6). However, at these later time points, evaluation of the hematopoietic compartment in the same recipient showed a steady increase in the number of myeloid cells circulating in the PB and localized in the spleen of RBPJKO mice, indicating an increased output of cells from the BM into the circulation. Analysis of Lys-GFP cells localization in the BM of transplanted mice at 6 weeks revealed that myeloid cells were residing further from the vasculature in the RBPJKO microenvironment than in the control.
Collectively, the combination of IVFM with these specific animal models provided insights in the engraftment dynamics of myeloid cells in the RBPJKO BM microenvironment. The experimental design and quantitative approach described here is proposed as a paradigm that can be applied to address similar questions. For example, the use of other cell specific lineage tracking models, such as RAG1-GFP14 or Gata1-GFP15 mice, may allow following the behavior of lymphoid or erythroid progenitors, respectively, in the BM.

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			<td>Other non-absorbable suture may be used</td>
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			<td height="21" style="height:21px;width:216px;">WillCo- glass bottom dish</td>
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			<td height="21" style="height:21px;width:216px;">Stereotaxic stage insert&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Olympus XLUMPLFL 20xW, NA 0.95 objective&#160;</td>
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			<td>3/4 D Image Visualization and Analysis software</td>
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combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Cohorts of 2 RBPJKO and 2 RBPJWT recipients were imaged in an individual imaging session at different time points: 24 h and 2, 4 and 6 weeks after transplantation of BM Lys-GFP cells (workflow is illustrated in Figure 1A).
In each mouse, images were acquired from 6 standard regions of the BM calvarium, identified by their position in relation to the bifurcation of the central vein (Figure 2A, <s...

Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

The overall goal of this procedure is to visualize and measure the in vivo engraftment of transplanted blood cells to recipient bone marrow niches. The main advantage of this technique is that it allows visualization of cellular processes like homing, expansion, and localization to different bone marrow niches of donor hematopoietic cells following transplantation. Used in combination with the engineered animal models, this approach can help in defining molecular pathways critical for hematopoietic cell regeneration.Unlike histology or other imaging techniques, intravital microscopy allows capturing cell-to-cell interactions in a live animal on a cellular level and in real time. We decided to use this method to determine whether the bone marrow microenvironment was the initial site of the development of the myeloproliferative disease caused by loss of Notch signaling. Challenges for individuals new to this technique include minimizing breathing artifact, optimally positioning and stabilizing the head and avoiding image saturation.One to two hours before the transplant, spray a lysed EGFP mouse with 70%ethanol. Next, use surgical scissors to make an incision in the skin of each leg around the ankle and surgical forceps to remove the skin and fur together to expose the clean muscle tissue. Remove as much muscle from the legs as possible.Then cut the bones at the knee and ankle joints and use gauze sponges to remove any remaining muscle tissue from the femurs and tibias. Rinse the cleaned bones in a six-well plate containing DMEM supplemented with 10%FBS. Then transfer all four hind leg bones into 10 milliliters of cold EDTA in PBS into a mortar and crush the bones with a pestle.Triturate the bone marrow cells to obtain a single-cell suspension and strain the slurry through a 70 micron filter into a 15 milliliter centrifuge tube. Rinse the filter with two to three milliliters of PBS. Then centrifuge the cells and resuspend the pellet in 10 milliliters of fresh DMEM plus 10%FBS.For the injection, centrifuge the cell suspension then count and adjust the concentration of cells to 1.5 times 10 to the seventh cells per milliliter in IMDM without serum. Place the cells on ice. Five to six hours after the second dose of radiation, inject three times 10 to the sixth bone marrow cells into the tail vein of each radiated recipient animal.Add the appropriate time point post-transplantation. Place the anesthetized recipient mouse onto a 37 degrees Celsius heating pad and confirm a lack of response to toe pinch. Apply ointment to the animal's eyes and administer 100 microliters of the appropriate fluorescent marker via the tail vein.Next, use small electric clippers to remove the hair from the dorsal surface of the animal's head, followed by the application of a depilatory cream. After five minutes, remove the cream with gauze sponges and rinse off any remaining solution with saline. Disinfect the scalp with a 70%ethanol soaked cotton swab.Then use fine forceps and scissors to make a 10 to 20 millimeter midline skin incision on the scalp to expose the underlying dorsal skull surface. Using 5-0 surgical silk, place two stay sutures on each side of the incision creating a flap to expose the calvarium for imaging. Apply a droplet of oil on the exposed skull.With the mouse in the supine position, submerge the exposed scalp in a glass bottom dish filled with microscope oil. Then place the animal on a multiphoton imaging stage with the calvarium above the objective and cover the animal with a 37 degrees Celsius heating pad. It is essential that the head remain stabilized in the correct position during the multiphoton image acquisition.Use a stereotactic device or secure the mouse with tape as appropriate. Using an inverted confocal system modified for multiphoton imaging, tune the two photon laser to 830 nanometers. Next, open the appropriate image acquisition software.Under the acquisition settings menu, confirm that the one directional scanning mode is selected and set the scanning speed to four microseconds per pixel, the frame rate to 512 by 512 pixels and the zoom to 1.5. Select the 20X watt in a 0.95 objective. Open the dye list window and select two photon.Then select the RDM690 mirror in the microscope controller window. In the light path and dice window, select the laser unit two box to check if the two photon laser shutter is open. Select EPI Lamp and choose the B/G EPI filter cube.Then focus the objective on the specimen to visualize the vascular flow in the calvarium bone marrow niche, using the bifurcation of the central vein and the coronary suture as reference points. Using the non-descanned mode, select three external detectors and image the specimen at a scan rate of four microseconds per pixel with no averaging and a constant laser power and detector gain to utilize the full dynamic range of the detector with a minimal saturation. After collecting a series of sections through the depth of the tissue, open the appropriate dedicated 3 to 4D image quantitation and visualization software.Interactively visualize the Z stacks using the appropriate 3D rendering algorithm according to the manufacturer's instructions. Then use the spot object segmentation module to segment the GFP cells and apply stack arithmetic processing to eliminate the false positive GFP cells in the green and red channels. Finally, use the surface segmentation module to reform the segmentation of the vasculature and bone surface, applying X tension distance of spot to surface algorithms to calculate the distances of the cells to any of the distal surfaces as necessary.Here, Z stacks collected from each of the six standard regions of the bone marrow calvarium were rendered as 3D maximal intensity projection, 3D shadow projection including the bone component, and 3D segmented images, allowing quantitation of the cells and the measurement of their distances from the bone and vessels. In this experiment, in vivo imaging of myeloid cells in the Notch-defective bone marrow microenvironment revealed that donor lysed GFP cells have a similar homing efficiency in the wild type and knockout recipients at 24 hours after transplant while they exhibit different engraftment at later time points. Analysis of the dynamics of myeloid progenitor expansion showed that adaptively bone marrow transplanted cells expanded more rapidly and in greater numbers in knockout recipients at two weeks after transplant.The donor bone marrow cells in wild type recipients expanded later but in two-fold higher numbers than the donor cells and knockout recipients at four weeks, a time where donor cells in the knockout bone marrow are decreased because it's going rapidly into circulation. And that by six weeks, the differences in the number of donor cells in the bone marrow calvarium level out. Further, 3D segmentation analysis of the lower middle region of knockout and wild type calvaria at six weeks post-bone marrow transplant indicates that the lysed GFP cells localized at the same distance from the bone in both types of recipient animals, but interestingly, they reside more distantly from the vasculature in the knockout mice.Once mastered, the intravital bone marrow imaging can be completed in one hour and 30 minutes per mouse if performed properly. While using this approach, it's important to remember to sample multiple areas of the bone marrow of the calvarium per mouse and to analyze at least three mice per experiment to address any intra and inter sample variations. After watching this video, you should have a good understanding of how to combine bone marrow transplantation and the intravital imaging to determine the kinetics of the engraftment of specific blood subpopulation to the bone marrow microenvironment.

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Developmental Biology

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

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 ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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 ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...