This work was supported by the Bone Disease Program of Texas Award, the Caroline Wiess Law Fund Award, and the NIAMS of the National Institutes of Health under award numbers R01 AR072018, R21 AG064345, R01 CA221946 to D.P.&#160; We thank M.E. Dickinson and T.J. Vadakkan in the BCM Optical Imaging and Vital Microscopy Core&#160;and the&#160;BCM Advanced Technology Cores&#160;with funding from NIH (AI036211,&#160;CA125123, and DK056338).
.

All mice were maintained in pathogen-free conditions, and all procedures were approved by Baylor College of Medicine&#8217;s Institutional Animal Care and Use Committee (IACUC).
1. Mouse preparation
<ol>
	<li>Cross Mx1-Cre17 and Rosa26-loxP-stop-loxP-tdTomato (Tom)18 mice (purchased from the Jackson Laboratory) with &#945;SMA-GFP mice (provided here by Drs. Ivo Kalajzic and Henry Kronenberg) to generate Mx1-C...

<ol>
	<li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+mechanisms+and+interventions.">Fracture healing: mechanisms and interventions.</a> Nature Reviews Rheumatology. 11 (1), 45-54 (2015).</li><li>Murao, H., Yamamoto, K., Matsuda, S., Akiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periosteal+cells+are+a+major+source+of+soft+callus+in+bone+fracture.">Periosteal cells are a major source of soft callus in bone fracture.</a> Journal of Bone and Mineral Metabolism. 31 (4), 390-398 (2013).</li><li>Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+cell+fate+decisions+within+periosteum+and+bone+marrow+during+bone+regeneration.">Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration.</a> Journal of Bone and Mineral Research. 24 (2), 274-282 (2009).</li><li>Roberts, S. J., van Gastel, N., Carmeliet, G., Luyten, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+the+periosteum+for+skeletal+regeneration:+the+stem+cell+that+lies+beneath.">Uncovering the periosteum for skeletal regeneration: the stem cell that lies beneath.</a> Bone. 70, 10-18 (2015).</li><li>Duchamp de Lageneste, 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=Periosteum+contains+skeletal+stem+cells+with+high+bone+regenerative+potential+controlled+by+Periostin.">Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.</a> Nature Communications. 9 (1), 773 (2018).</li><li>Olivos-Meza, 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=Pretreatment+of+periosteum+with+TGF-beta1+in+situ+enhances+the+quality+of+osteochondral+tissue+regenerated+from+transplanted+periosteal+grafts+in+adult+rabbits.">Pretreatment of periosteum with TGF-beta1 in situ enhances the quality of osteochondral tissue regenerated from transplanted periosteal grafts in adult rabbits.</a> Osteoarthritis Cartilage. 18 (9), 1183-1191 (2010).</li><li>Debnath, 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=Discovery+of+a+periosteal+stem+cell+mediating+intramembranous+bone+formation.">Discovery of a periosteal stem cell mediating intramembranous bone formation.</a> Nature. 562 (7725), 133-139 (2018).</li><li>Ransom, R. 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=Axin2-expressing+cells+execute+regeneration+after+skeletal+injury.">Axin2-expressing cells execute regeneration after skeletal injury.</a> Scientific Reports. 6, 36524 (2016).</li><li>Ouyang, Z., 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=Prx1+and+3.2kb+Col1a1+promoters+target+distinct+bone+cell+populations+in+transgenic+mice.">Prx1 and 3.2kb Col1a1 promoters target distinct bone cell populations in transgenic mice.</a> Bone. 58, 136-145 (2013).</li><li>Wilk, K., 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=Postnatal+Calvarial+Skeletal+Stem+Cells+Expressing+PRX1+Reside+Exclusively+in+the+Calvarial+Sutures+and+Are+Required+for+Bone+Regeneration.">Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration.</a> Stem Cell Reports. 8 (4), 933-946 (2017).</li><li>Ortinau, L. 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=Identification+of+Functionally+Distinct+Mx1+alphaSMA++Periosteal+Skeletal+Stem+Cells.">Identification of Functionally Distinct Mx1+alphaSMA+ Periosteal Skeletal Stem Cells.</a> Cell Stem Cell. 25 (6), 784-796 (2019).</li><li>Deveza, L., Ortinau, L., Lei, K., Park, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+gene+expression+identifies+distinct+molecular+signatures+of+bone+marrow-+and+periosteal-skeletal+stem/progenitor+cells.">Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells.</a> PLoS One. 13 (1), 0190909 (2018).</li><li>Schindeler, A., McDonald, M. M., Bokko, P., Little, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+remodeling+during+fracture+repair:+The+cellular+picture.">Bone remodeling during fracture repair: The cellular picture.</a> Seminars in Cell and Developmental Biology. 19 (5), 459-466 (2008).</li><li>Shi, Y., 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=Gli1+identifies+osteogenic+progenitors+for+bone+formation+and+fracture+repair.">Gli1 identifies osteogenic progenitors for bone formation and fracture repair.</a> Cell Stem Cell. 15 (2), 782-796 (2017).</li><li>Zhou, B. O., Yue, R., Murphy, M. M., Peyer, J. G., 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=Leptin-receptor-expressing+mesenchymal+stromal+cells+represent+the+main+source+of+bone+formed+by+adult+bone+marrow.">Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.</a> Cell Stem Cell. 15 (2), 154-168 (2014).</li><li>Grcevic, D., 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=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30 (2), 187-196 (2012).</li><li>Kuhn, R., Schwenk, F., Aguet, M., Rajewsky, K. <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+targeting+in+mice.">Inducible gene targeting in mice.</a> Science. 269 (5229), 1427-1429 (1995).</li><li>Srinivas, 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=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1, 4 (2001).</li><li>Park, D., 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=Endogenous+bone+marrow+MSCs+are+dynamic,+fate-restricted+participants+in+bone+maintenance+and+regeneration.">Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.</a> Cell Stem Cell. 10 (3), 259-272 (2012).</li><li>Nitzsche, F., 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=Concise+Review:+MSC+Adhesion+Cascade-Insights+into+Homing+and+Transendothelial+Migration.">Concise Review: MSC Adhesion Cascade-Insights into Homing and Transendothelial Migration.</a> Stem Cells. 35 (6), 1446-1460 (2017).</li><li>Adler, J., Pagakis, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+image+distortions+due+to+temperature-related+microscope+stage+drift.">Reducing image distortions due to temperature-related microscope stage drift.</a> Journal of Microscopy. 210, 131-137 (2003).</li><li>Roberts, S. J., Ke, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anabolic+Strategies+to+Augment+Bone+Fracture+Healing.">Anabolic Strategies to Augment Bone Fracture Healing.</a> Current Osteoporosis Reports. 16 (3), 289-298 (2018).</li></ol>

L.C.O. and D.P. disclose a pending patent entitled &#8220;Periosteal Skeletal Stem Cells in Bone Repair&#8221; (BAYM.P0264US.P1). The remaining authors declare no competing interests.

During bone healing, periosteal cells are the main source of newly differentiated chondrocytes and osteoblasts within an injury callus3. Similar to bone marrow, osteogenic/chondrogenic SSCs have also been identified in the periosteum4,5,6. Assessing endogenous P-SSC functional characteristics is technically challenging. Often, migratory dynamics of SSCs are assessed in vitro, making interpretation of thei...

Fracture repair is a dynamic, multicellular process that is distinct from embryonic skeletal development and remodeling. During this process, the induction of injury signals from damaged tissue is followed by the rapid recruitment, proliferation, and subsequent differentiation of skeletal stem/progenitor cells, which are all critical for the overall stability and fixation of fractures1. In particular, early stages of fracture healing require soft callus formation, which is mainly attributed to periosteal resident cells2. When bones are injured, a subset of periosteal cells rapidly respond and contribute to newly differentiated cartilage intermediates and osteoblasts within the callus3, implicating the presence of a distinct skeletal stem/progenitor cell population within the periosteum. Therefore, the identification and functional characterization of the periosteum-resident skeletal stem cells (SSCs) constitute a promising therapeutic approach for degenerative bone diseases and bone defects4.&#160;
SSCs are thought to reside in multiple tissue locations, including the bone marrow. Similar to bone marrow, osteogenic/chondrogenic SSCs have also been identified in the periosteum4,5,6.&#160; These periosteal SSCs (P-SSCs) can be labeled with early mesenchymal lineage markers (i.e., Prx1-Cre5, Ctsk-Cre7, and Axin2-CreER8) during fetal bone development4,7,8,9,10. A significant limitation of these single genetic lineage tracing models is that there is substantial heterogeneity within labeled cell populations. Furthermore, they cannot distinguish labeled SSCs from their progeny in vivo. To address this limitation, we recently developed a dual reporter mouse (Mx1-Cre+Rosa26-Tomato+&#945;SMA-GFP+) to distinctly visualize P-SSCs from bone marrow SSCs (BM-SSCs)11. With this model, it has been determined that P-SSCs are marked by Mx1+&#945;SMA+ dual labeling, whereas more differentiated Mx1+ cells reside in the bone marrow, endosteal and periosteal surfaces, and cortical and trabecular bones11,12.&#160;
Multiple cytokines and growth factors are known to regulate bone remodeling and repair and have been tested for the enhancement of skeletal repair in critical segment defects1,13. However, due to the cellular complexity of injury models generated through disruption of the bone compartment&#39;s physical barrier, the direct effects of these molecules on endogenous P-SSC migration and activation during healing are unclear. The functional characteristics and migratory dynamics of SSCs are often assessed in vitro by performing a transwell or scratch assay, in combination with cytokines or growth factors known to induce migration in other cell populations. Thus, interpretation of results from these in vitro experiments for application in their corresponding in vivo systems is challenging. Currently, in vivo assessment of skeletal stem/progenitor cell migration is not typically observed in real-time; rather, it is measured at fixed timepoints post-fracture5,7,14,15,16.&#160;&#160;
A limitation of this method is that migration is not assessed at a single-cell level; rather, it is measured via changes in cell populations. Due to recent advances in live animal intravital microscopy and generation of additional reporter mice, in vivo tracking of individual cells is now possible. Using live animal intravital microscopy, we observed the distinct migration of P-SSCs from the calvaria suture niche to a bone injury within 24&#8211;48 h post-injury in Mx1/Tomato/&#945;SMA-GFP mice.&#160;
CCL5/CCR5 was recently identified as a regulatory mechanism influencing the recruitment and activation P-SSCs during the early injury response. Interestingly, there was no detection of substantial P-SSC migration in response to an injury in real-time. However, treatment of an injury with CCL5 produces a robust, directionally distinct migration of P-SSCs, which can be captured in real-time. Therefore, the objective of this protocol is to provide a detailed methodology for recording the in vivo migration of P-SSCs in real-time after treatment with CCL5.

<table border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="719">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">&#189;&#8221; optical post</td>
			<td style="width:175px;">ThorLabs</td>
			<td style="width:135px;">TR2</td>
			<td style="width:247px;">For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">1 mL syringe</td>
			<td style="width:175px;">BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">27G needle</td>
			<td style="width:175px;">BD PercisionGlide</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">29G insulin syringe</td>
			<td style="width:175px;">McKesson</td>
			<td>102-SN05C905P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">50 mL conicol tube</td>
			<td style="width:175px;">Falcon</td>
			<td>352098</td>
			<td>For mouse restraint</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Adjustable angle plate</td>
			<td style="width:175px;">Renishaw</td>
			<td>R-PCA-5023-50-20</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Alcohol wipes</td>
			<td style="width:175px;">Coviden</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">betadine surgical scrub</td>
			<td style="width:175px;">Henry Schen</td>
			<td>67618-151-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Buprenorphine SR-LAB</td>
			<td style="width:175px;">ZooPharm</td>
			<td></td>
			<td>1mg/mL Sustained Release</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Combo III</td>
			<td style="width:175px;">Obtained from staff veterinarian</td>
			<td>N/A</td>
			<td style="width:247px;">37.6 mg/mL Ketamine; 1.9 mg/mL Xylazine; 0.37 mg/mL Acepronazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Coverslip</td>
			<td style="width:175px;">Fisher</td>
			<td>12-545-87</td>
			<td>24 x 40 premium superslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Fine tip forcepts</td>
			<td style="width:175px;">FST</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Ketamine</td>
			<td style="width:175px;">KetaVed</td>
			<td>50989-161-06</td>
			<td style="width:247px;">100 mg/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Leica TCS SP8MP with DM6000CFS</td>
			<td style="width:175px;">Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Matrigel</td>
			<td style="width:175px;">R &#38; D Systems</td>
			<td>344500101</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:164px;">Medical tape</td>
			<td style="width:175px;">McKesson</td>
			<td>100199</td>
			<td>3&#34; x 10 yds (7.6 cm x 9.1 m)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Methocellulose</td>
			<td style="width:175px;">Electron Microscopy Sciences</td>
			<td>19560</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Microdissection scissors</td>
			<td style="width:175px;">FST</td>
			<td>1456-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Motorized stage</td>
			<td style="width:175px;">Anaheim automation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Needle holder</td>
			<td style="width:175px;">FST</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Nonabsorbable sutures</td>
			<td style="width:175px;">McKesson</td>
			<td>S913BX</td>
			<td style="width:247px;">monofilament nylon 5-0 nonabsorbable sutures with attached C-1 reverse cutting needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Opthalmic ointment</td>
			<td style="width:175px;">Rugby</td>
			<td>0536-1086-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">RANTES</td>
			<td style="width:175px;">Biolegend</td>
			<td>594202</td>
			<td>10 &#181;g/50 &#181;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Right-angle clamp for &#189;&#8221; post, 3/16&#8221; Hex</td>
			<td style="width:175px;">ThorLabs</td>
			<td>RA90</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Spring-loaded 3/16&#8221; Hex-locking &#188;&#8221; thumbscrew</td>
			<td style="width:175px;">ThorLabs</td>
			<td>TS25H</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Sterile cotton swabs</td>
			<td style="width:175px;">Henry Schen</td>
			<td>100-9249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Sterile DPBS (1x)</td>
			<td style="width:175px;">Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:164px;">Sterile drapes</td>
			<td style="width:175px;">McKessen</td>
			<td>25-517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Surgical gloves</td>
			<td style="width:175px;">McKessen</td>
			<td>3158VA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Triple antibiotic ointment</td>
			<td style="width:175px;">Taro Pharmaceuticals U.s.a., Inc.</td>
			<td>51672-2120-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Vacutainer blood collection set</td>
			<td style="width:175px;">BD</td>
			<td style="width:135px;">REF 367298</td>
			<td style="width:247px;">25G butterfly needle infusion set with 12&#34; tubing</td>
		</tr>
	</tbody>
</table>

real-time imaging of ccl5-induced migration of periosteal skeletal stem cells in mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

Skeletal progenitors have been proposed to have migratory or circulatory potential20. Recently, Mx1-Cre+Rosa26-Tomato+&#945;SMA-GFP+ (Mx1/Tomato/&#945;SMA-GFP) reporter mice were generated, in which P-SSCs are marked by Mx1+&#945;SMA+ dual labeling (Figure 2A,B). Substantial migration of Mx1+&#945;SMA+...

Watch this Scientific Journal Video about Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice at JoVE.com

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

The standardized protocol can be used to assess endogenous cell migration and can be applied to several cell types as well as skeletal phenotypes. The advantage of this technique is that it allows in vivo cell migration to be tracked for individual cells rather than a population of cells in response to injury and/or cytokine treatment. Before beginning the procedure, confirm a lack of response to toe pinch in the anesthetized mouse and apply ointment to the animal's eyes.Next, make a transverse, less than one centimeter incision from immediately medial to the right ear toward the left ear and make a second incision from the initial incision, approximately two to three millimeters past the right eye toward the nose. Use forceps to separate the skin from the periosteum and use scissors to gently cut through to connective tissue to separate the skin from the calvaria periosteum. Using a sterile cotton swab, apply ophthalmic ointment to the top of the skin flap and gently fold the flap over the left eye.The intersection of the sagittal and coronal sutures should be clearly exposed. Flush the open surface with sterile PBS to clean the area of any blood and residual hair and place the tip of a bevel one to two needle widths toward the nose from the coronal suture and one to two needle widths to the right of the sagittal suture. Using a very small amount of downward pressure, carefully rotate the syringe clockwise one time and counterclockwise several times.Then use a 27 gauge needle to widen the microfracture to approximately 40 micrometers. If bone fragments remain, flush the microfracture with PBS. If bone fragments still remain, use a 29 gauge needle to gently scoop the fragments out.For CCL5 treatment, use a 100 nanogram per milliliter stock CCL5 solution and basement membrane matrix to obtain a 10 nanogram per milliliter working chemoattractant solution. To treat the injury, load a 220 microliter pipette with five microliters of the solution and apply two microliters of the chemokine to the suture. Then allow the matrix to solidify and gently cover the calvaria with the skin flap to ensure that the tissue does not become dehydrated during the one hour chemokine treatment.For intravital imaging of periosteal stem cell migration in real time, before moving the mouse to the microscope, apply gentle pressure to the back of the mouse's head to check the visual plane of the calvaria. If the plane is not level, gently rotate the mouse restraint to the left or the right to adjust the position. Next, cover the entire calvaria in skin flap with sterile 2%methylcellulose in water and place the mouse on the XYZ axis motorized microscope stage.Using the epifluorescent light, align the mouse so that it is facing the microscope and that the intersection of the coronal and sagittal sutures is the centered reference point of the stage. Then place a glass cover on the imaging area and double check the alignment. For time-lapse imaging of the migration into and out of the suture, select a low magnification water immersion lens to scan the calvaria.Acquire a reference image of the sagittal and coronal suture intersection and using the SHG and fluorescent signals from the cells, locate each injury site and record the XYZ coordinates as well as the distances from the sagittal and coronal sutures. Select a location on either the coronal or sagittal sutures for long-term imaging and obtain a detailed ZStack image of this area from reference during imaging. Use the time-lapse software settings to record an image every one minute for at least one hour, comparing the current field of view with the initial field of view in the time between snapshots.If the fields of view are different, use the ZStack image to determine in which direction the field of view needs to be adjusted. Recently, Mx1 tomato alpha-SMA GFP reporter mice were generated in which periosteal skeletal stem cells are marked by Mx1 positive alpha-SMA positive dual labeling. Little to no Mx1 positive alpha-SMA positive periosteal skeletal stem cell migration is observed over a one hour period of imaging, 24 hours after suture placement.The CCL5 chemokine treatment of a calvaria defect 24 hours post injury induces directionally distinct migration away from the coronal suture and toward the injury site. In this representative analysis, within one hour of imaging, one of the Mx1 positive alpha-SMA positive periosteal skeletal stem cells migrated approximately 50 micrometers toward the injury. The most important aspect of this protocol to remember is to limit the CCL5 treatment to the injury site in order to stimulate cell-specific migration.Once this procedure is complete, localized treatment can be continued for one week. A micro CT can be used to assess calvaria injury healing and mineral deposition.

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Research

JoVE Journal

Developmental Biology

2.2K ビュー.  Baylor College of Medicine. The standardized protocol can be used to assess endogenous cell migration and can be applied to several cell types as well as skeletal phenotypes. The advantage of this technique is that it allows in vivo cell migration to be tracked for individual cells rather than a population of cells in response to injury and/or cytokine treatment. Before beginning the procedure, confirm a lack of response to toe pinch in the anesthetized mouse and apply ointment to the animal's eyes.Next, make a t...