Name: clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture
Uploaded: 2018-05-08T13:00:00
Description: Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

We would like to thank Andrew Berger, Stacey Dozono, and Brian Raden in the Fred Hutchinson Flow Cytometry Core for assistance with FACS. This work was supported by the National Institutes of Health NHLBI UO1 grant #HL100395, Ancillary Collaborative Grant #HL099997, and NIDDK grant #RC2DK114777. Brandon Hadland is supported by the Alex&#8217;s Lemonade Stand Foundation and Hyundai Hope on Wheels Foundation.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Fred Hutchinson Cancer Research Center.
1. Preparation of AGM-EC Monolayers for Co-culture
<ol>
	<li>24 h prior to the AGM dissection and sort, make&#160; AGM-EC culture media and filter sterilize.</li>
	<li>Add 0.1% gelatin in water (100 &#181;L/well) to each well of a 96-well plate and incubate at room temperature for 15 min. Aspirate the gelatin and leave the plate in a tissue c...

<ol>
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C., Hiatt, K., Mukherjee, P. <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+repopulating+hematopoietic+stem+cells+are+present+in+the+murine+yolk+sac+at+day+9.0+postcoitus.">In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.</a> Proc Natl Acad Sci U S A. 94 (13), 6776-6780 (1997).</li><li>Arora, N., 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=Effect+of+developmental+stage+of+HSC+and+recipient+on+transplant+outcomes.">Effect of developmental stage of HSC and recipient on transplant outcomes.</a> Dev Cell. 29 (5), 621-628 (2014).</li><li>Butler, J. M., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascular+niche+for+studying+stem+cell+homeostasis.">Generation of a vascular niche for studying stem cell homeostasis.</a> Methods Mol Biol. 904, 221-233 (2012).</li><li>Butler, J. 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=Endothelial+cells+are+essential+for+the+self-renewal+and+repopulation+of+Notch-dependent+hematopoietic+stem+cells.">Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.</a> Cell Stem Cell. 6 (3), 251-264 (2010).</li><li>Kobayashi, 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=Angiocrine+factors+from+Akt-activated+endothelial+cells+balance+self-renewal+and+differentiation+of+haematopoietic+stem+cells.">Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.</a> Nat Cell Biol. 12 (11), 1046-1056 (2010).</li><li>Hadland, B. 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=Endothelium+and+NOTCH+specify+and+amplify+aorta-gonad-mesonephros-derived+hematopoietic+stem+cells.">Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.</a> J Clin Invest. 125 (5), 2032-2045 (2015).</li><li>Hadland, B. 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=A+Common+Origin+for+B-1a+and+B-2+Lymphocytes+in+Clonal+Pre-+Hematopoietic+Stem+Cells.">A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.</a> Stem Cell Reports. 8 (6), 1563-1572 (2017).</li><li>Wilson, N. 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=Combined+Single-Cell+Functional+and+Gene+Expression+Analysis+Resolves+Heterogeneity+within+Stem+Cell+Populations.">Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.</a> Cell Stem Cell. 16 (6), 712-724 (2015).</li><li>Schulte, R., 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=Index+sorting+resolves+heterogeneous+murine+hematopoietic+stem+cell+populations.">Index sorting resolves heterogeneous murine hematopoietic stem cell populations.</a> Exp Hematol. 43 (9), 803-811 (2015).</li><li>Morgan, K., Kharas, M., Dzierzak, E., Gilliland, 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=Isolation+of+early+hematopoietic+stem+cells+from+murine+yolk+sac+and+AGM.">Isolation of early hematopoietic stem cells from murine yolk sac and AGM.</a> J Vis Exp. (16), (2008).</li><li>deBruijn, M. 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=Hematopoietic+stem+cells+localize+to+the+endothelial+cell+layer+in+the+midgestation+mouse+aorta.">Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.</a> Immunity. 16 (5), 673-683 (2002).</li><li>Lorsbach, R. B., 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=Role+of+RUNX1+in+adult+hematopoiesis:+analysis+of+RUNX1-IRES-GFP+knock-in+mice+reveals+differential+lineage+expression.">Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression.</a> Blood. 103 (7), 2522-2529 (2004).</li><li>Holmes, R., Z&#250;&#241;iga-Pfl&#252;cker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009 (2), pdb.prot5156 (2009).</li><li>Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+wave+of+B+lymphopoiesis+develops+independently+of+stem+cells+in+the+murine+embryo.">The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.</a> Ann N Y Acad Sci. 1362, 16-22 (2015).</li><li>Kobayashi, 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=Functional+B-1+progenitor+cells+are+present+in+the+hematopoietic+stem+cell-deficient+embryo+and+depend+on+Cbf&#946;+for+their+development.">Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbf&#946; for their development.</a> Proc Natl Acad Sci U S A. 111 (33), 12151-12156 (2014).</li><li>Yoshimoto, 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=Autonomous+murine+T-cell+progenitor+production+in+the+extra-embryonic+yolk+sac+before+HSC+emergence.">Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.</a> Blood. 119 (24), 5706-5714 (2012).</li><li>Inlay, 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=Identification+of+multipotent+progenitors+that+emerge+prior+to+hematopoietic+stem+cells+in+embryonic+development.">Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.</a> Stem Cell Reports. 2 (4), 457-472 (2014).</li><li>Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell-independent+hematopoiesis:+emergence+of+erythroid,+megakaryocyte,+and+myeloid+potential+in+the+mammalian+embryo.">Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo.</a> FEBS Lett. 590 (22), 3965-3974 (2016).</li></ol>

The study of HSC genesis during embryonic development necessitates means to detect HSC potential in hemogenic precursors yet lacking the competence to provide long-term multilineage hematopoietic reconstitution in transplanted adult recipients. In this protocol, we present a clonal assay of embryonic hemogenic precursors by stromal co-culture on vascular niche ECs from the AGM, which supports the maturation of precursors to HSC, with subsequent functional analysis in transplantation assays. Incorporation of index sorting...

Clonal studies have revealed heterogeneity in the long-term engraftment properties of adult HSCs, providing new insight into HSC subtypes and changes in HSC behavior during aging1. However, similar studies of embryonic HSCs and their precursors have been more challenging. During early embryonic development, HSCs arise from a population of precursors known as hemogenic endothelium in a transient process referred to as the endothelial to hematopoietic transition2. The first HSC, defined by their ability to provide robust, long-term multilineage engraftment following transplantation into conditioned adult recipients, are not detected until after embryonic day 10.5 (E10.5) in the murine embryo, at very low frequency3. During their development, precursors to HSC (pre-HSC) arising from hemogenic endothelium must undergo maturation prior to acquiring the properties of adult HSC which allow for efficient engraftment in transplantation assays4,5,6. Obscuring the study of rare HSC origin, a multitude of hematopoietic progenitors with erythroid, myeloid, and lymphoid potential are already detected prior to the emergence of HSC from pre-HSC7,8. Thus, distinguishing pre-HSC from other hematopoietic progenitors requires methods to clonally isolate cells and provide them with the signals sufficient for their maturation to HSC, to detect their engraftment properties in transplantation assays.
A number of approaches have been described which allow for the detection of pre-HSC by either ex vivo or in vivo maturation to HSC. Ex vivo methods have depended on culture of embryonic tissues, such as the AGM region, where the first HSC are detected in development9. Building on these methods, protocols which incorporate the dissociation, sorting, and re-aggregation of AGM tissues have permitted the characterization of sorted populations containing HSC precursors during development from E9.5 to E11.5 in the para-aortic splanchnopleura (P-Sp)/AGM regions4,5,10; however, these approaches are not amenable to high-throughput analysis of precursors at the single cell level required for clonal analysis. Similarly, in vivo maturation by transplantation into newborn mice, where the microenvironment is presumed to be more suitable for the support of earlier stages of HSC precursors, has also enabled studies of sorted populations from the yolk sac and AGM/P-Sp (P-Sp is the precursor region to the AGM) with characteristics of pre-HSC, but these methods also fail to provide a robust platform for single cell analysis11,12.
Studies from Rafii et al. demonstrated that Akt-activated endothelial cell (EC) stroma can provide a niche substrate for the support of adult HSC self-renewal in vitro13,14,15. We recently determined that Akt-activated EC derived from the AGM region (AGM-EC) provides a suitable in vitro niche for the maturation of hemogenic precursors, isolated as early as E9 in development, to adult-engrafting HSC, as well as the subsequent self-renewal of generated HSC16. Given that this system employs a simple 2-dimensional co-culture, it is readily adaptable for clonal analysis of the HSC potential of individually isolated hemogenic precursors.
We have recently reported an approach to assay the HSC potential of clonal hemogenic precursors by combining index sorting of individual hemogenic precursors from murine embryos with AGM-EC co-culture and subsequent functional analysis in transplantation assays17. Index sorting is a mode of fluorescence-activated cell sorting (FACS) that records (indexes) all phenotypic parameters (i.e., forward scatter (FSC-A), side scatter (SSC-A), fluorescence parameters) of each individually sorted cell such that these features can be retrospectively correlated to subsequent functional analysis following sorting. FACS software records both phenotypic information for each cell and the position/well of the 96-well plate into which it was placed. This technique has previously been elegantly used to identify heterogeneity in adult HSC, determine phenotypic parameters that further enrich for the long-term engrafting subset of HSC, and correlate phenotypic parameters of HSC with transcriptional properties at the single cell level18,19. Here, we provide detailed methodology of this approach that enables identification of unique phenotypic parameters and lineage contributions of pre-HSC during early stages of embryonic development.

<table>
	<tbody>
		<tr>
			<td>AGM-EC culture media </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for culture of endothelial cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td>Use to dissociate AGM-EC monolayers</td>
		</tr>
		<tr>
			<td>Gelatin 0.1% in water</td>
			<td>StemCell Technologies</td>
			<td>7903</td>
			<td>Use to adhere AGM-EC monolayers to plastic</td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Use to co-culture AGM-EC monolayers and index sorted clones</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Use for dissection and FACS staining</td>
		</tr>
		<tr>
			<td>500 ml filter bottles</td>
			<td>Fisher Scientific</td>
			<td>9741202</td>
			<td>Use to sterile filter Endothelial media</td>
		</tr>
		<tr>
			<td>AGM-EC culture media (500 mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td>400 mls</td>
		</tr>
		<tr>
			<td>Hyclone Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH30088.03</td>
			<td>100 mls</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>5 mls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td>50 mg dissolved in 10 mls media</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>StemCell Technologies</td>
			<td>7100</td>
			<td>5 mls</td>
		</tr>
		<tr>
			<td>Endothelial Mitogen (ECGS)</td>
			<td>Alfa Aesar</td>
			<td>J64516 (BT-203)</td>
			<td>Add 10 mls media to dissolve and add</td>
		</tr>
		<tr>
			<td>Materials for AGM index sort</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (0.25%)</td>
			<td>StemCell Technologies</td>
			<td>7902</td>
			<td>Use for dissociation of embryonic tissues</td>
		</tr>
		<tr>
			<td>3ml syringe</td>
			<td>BD Biosciences</td>
			<td>309657</td>
			<td>Use to sterile filter antibody solutions for FACS</td>
		</tr>
		<tr>
			<td>0.22 &#181;M syringe-driven filter</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td>Use to sterile filter antibody solutions for FACS</td>
		</tr>
		<tr>
			<td>5 ml polystyrene tube with cell-strainer cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>Use to remove cell clumps prior to FACS</td>
		</tr>
		<tr>
			<td>DAPI (prepared as 1 mg/ml stock in H2O)</td>
			<td>Millipore</td>
			<td>268298</td>
			<td>Use to exclude dead cells from sort</td>
		</tr>
		<tr>
			<td>anti-mouse CD16/CD32 (FcR block)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>Use to block non-specific staining to Fc receptors</td>
		</tr>
		<tr>
			<td>Antibodies for AGM index sort</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD144 PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-1441-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control, PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4301-81</td>
			<td>Isotype control for CD144 PE-Cyanine7</td>
		</tr>
		<tr>
			<td>Anti-mouse CD201 (EPCR) PerCP-eFluor710</td>
			<td>eBioscience</td>
			<td>46-2012-80</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PerCP-eFlour710</td>
			<td>eBioscience</td>
			<td>46-4031-80</td>
			<td>Isotype control for CD201 PerCP-eFluor710</td>
		</tr>
		<tr>
			<td>Anti-mouse CD41 PE</td>
			<td>BD Biosciences</td>
			<td>558040</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control, PE</td>
			<td>BD Biosciences</td>
			<td>553925</td>
			<td>Isotype control for CD41 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 FITC</td>
			<td>eBioscience</td>
			<td>11-0451-85</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, FITC</td>
			<td>eBioscience</td>
			<td>11-4031-81</td>
			<td>Isotype control for CD45 FITC</td>
		</tr>
		<tr>
			<td>AGM-serum free media (10mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vivo 20</td>
			<td>Lonza</td>
			<td>04-448Q</td>
			<td>10 mls</td>
		</tr>
		<tr>
			<td>recombinant murine stem cell factor (SCF)</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant human FLT3 Ligand (FLT3L)</td>
			<td>Peprotech</td>
			<td>300-19</td>
			<td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant human thrombopoietin (TPO)</td>
			<td>Peprotech</td>
			<td>300-18</td>
			<td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant murine interleukin-3 (IL3)</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td>
		</tr>
		<tr>
			<td>Materials for Staining co-cultured cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, V-bottom</td>
			<td>Corning</td>
			<td>3894</td>
			<td>Use for FACS analysis</td>
		</tr>
		<tr>
			<td colspan="3">Antibodies for analysis of Index sorted clones co-cultured with endothelial cells</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PerCP-Cyanine5.5</td>
			<td>eBioscience</td>
			<td>45-0451-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PE-Cyanine5.5</td>
			<td>eBioscience</td>
			<td>35-4031-80</td>
			<td>Isotype control for CD45 PerCP-Cyanine5.5</td>
		</tr>
		<tr>
			<td>Anti-mouse CD201 (EPCR) PE</td>
			<td>eBioscience</td>
			<td>12-2012-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PE</td>
			<td>eBioscience</td>
			<td>12-4031-81</td>
			<td>Isotype control for CD201 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6A/E (Sca-1) APC</td>
			<td>eBioscience</td>
			<td>17-5981-83</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC</td>
			<td>eBioscience</td>
			<td>17-4321-81</td>
			<td>Isotype control for Ly-6A/E APC</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 FITC</td>
			<td>eBioscience</td>
			<td>11-4801-81</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, FITC</td>
			<td>eBioscience</td>
			<td>11-4321-41</td>
			<td>Isotype control for F4/80 FITC</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G/C (Gr1) FITC</td>
			<td>BD Biosciences</td>
			<td>553127</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, FITC</td>
			<td>BD Biosciences</td>
			<td>556923</td>
			<td>Isotype control for Ly-6G/C and CD3 FITC</td>
		</tr>
		<tr>
			<td>Materials for tail vein injection for transplant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 ml insulin syringes with 29G 1/2&#34; needles</td>
			<td>BD Biosciences</td>
			<td>309306</td>
			<td>Use for tail vein injection for transplantation</td>
		</tr>
		<tr>
			<td>Antibodies for Peripheral Blood analysis following translant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G/C (Gr1) PerCP</td>
			<td>Biolegend</td>
			<td>108426</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PerCP</td>
			<td>Biolegend</td>
			<td>400629</td>
			<td>Isotype control for Ly-6G/C PerCP</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 PE</td>
			<td>eBioscience</td>
			<td>12-4801-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, PE</td>
			<td>eBioscience</td>
			<td>12-4321-80</td>
			<td>Isotype control for F4/80 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 FITC</td>
			<td>BD Biosciences</td>
			<td>555274</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 APC</td>
			<td>BD Biosciences</td>
			<td>550992</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC</td>
			<td>BD Biosciences</td>
			<td>553932</td>
			<td>Isotype control for CD19 APC</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.1 (A20) PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0453-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4321-81</td>
			<td>Isotype control for CD45.1 PE-Cyanine7</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.2 (104) APC-eFluor780</td>
			<td>eBioscience</td>
			<td>47-0454-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC-eFluor780</td>
			<td>eBioscience</td>
			<td>47-4321-80</td>
			<td>Isotype control for CD45.2 APC-eFluor780</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria II with DIVA software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II with plate reader</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>Fisher Scientific</td>
			<td>S17040</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Multi-channel pipette</td>
			<td>Fisher Scientific</td>
			<td>14-559-417</td>
			<td>For dispensing cells</td>
		</tr>
		<tr>
			<td>FACS analysis software</td>
			<td>FlowJo/BD Biosciences</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
	</tbody>
</table>

clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Figure 1A shows a schematic of the experimental design. Once P-Sp/AGM tissues are dissected, pooled, and dissociated in collagenase, they are stained with antibodies to VE-Cadherin and EPCR for index sorting. Pre-HSC are enriched in cells sorted at VE-Cadherin+EPCRhigh (Figure 1B). Other fluorochrome-conjugated antibodies can be included to retrospectively analyze additional phenotypic parameters, which are ...

Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

The overall goal of this method is to identify the phenotypic properties and engraftment potential of clonal Hematopoietic Stem Cell or HSC precursors during murine embryonic development. This method can help answer key questions in the field of developmental hematopoiesis about the unique lineage contributions of clonal HSC precursors during embryonic development. The main advantage of this technique is that hemogenic precursors are analyzed at the single cell level.This allows identification of the unique phenotypic properties that distinguish rare HSC precursors. Begin by adding Aorta-Gonad-Mesonephros, AGM, tissues dissected from embryos between 9.5 and 11.5 days post coitum to a 15 milliliter conical tube containing 10 milliliters of PBS supplemented with 10%FBS on ice. When the tissues have settled to the bottom of the tube, aspirate the supernatant and immediately add one milliliter of 0.25%collagenase to the samples.After 25 minutes in a 37 degree Celsius water bath, stop the reaction with one milliliter of PBS plus FBS and use a one milliliter pipette tip to triturate the tissues 20 to 30 times until a single cell suspension is obtained. Then dilute the cells with eight more milliliters of PBS plus FBS and collect the cells by centrifugation. Resuspend the pellet in 500 microliters of blocking buffer for five minutes on ice.Then add 500 microliters of the antibody cocktail of interest. After a 15 to 20-minute incubation on ice, wash the cells in eight milliliters of PBS plus FBS and resuspend the pellet in one milliliter of fresh PBS plus FBS. Filter the cells through a 35 micrometer cell strainer into a five milliliter tube, washing the strainer with an additional three milliliters of PBS plus FBS to collect all of the cells and centrifuge the cells again.Then resuspend the pellet in 500 microliters of PBS plus FBS and place the cells on ice. To set the gates for sorting in single cell and index sorting mode, load a small aliquot of cells onto the flow cytometer and set the acquisition to the lowest flow rate. Open a forward scatter area versus side scatter area plot and set a gate to include cells of varying size.Open a forward scatter area versus forward scatter width plot and a side scatter area versus side scatter width plot and set a gate in each plot to exclude the doublets. Set a forward scatter area versus DAPI plot to gate the live cells. Then create an anti-vascular endothelial Cadherin versus anti-endothelial protein C receptor plot and gate the vascular endothelial Cadherin positive and high endothelial protein C receptor expressing cells.To index sort the aorta-gonad-mesonephros cells, place a 96-well plate precultured with AGM endothelial cells in AGM serum-free medium plus cytokines into the plate block of the flow cytometer and load the experimental sample. Begin the sample acquisition and confirm that the index sorting single cell mode is selected to sort a single cell into each well of the 96-well plate. When one cell has been sorted into each well of the plate, place the plate into a tissue culture incubator set to 37 degrees Celsius and 5%CO2 for up to seven days.At the end of the co-culture period, hematopoietic colonies of various sizes and morphologies can be visualized using an inverted microscope at a 100X magnification. To analyze the clones by flow cytometry, use a multichannel pipette to dissociate the hematopoietic cells from the endothelial layers without detaching the endothelial cells. Then transfer 100 microliters of cells into individual wells of a new 96-well v-bottom plate and return the original 96-well plate containing the remaining 100 microliters of cells to the tissue culture incubator.Pellet the cells in the v-bottom plate by centrifugation and remove the supernatant by inverting and flicking the plate in a single rapid motion. Resuspend the pellets in each well with 50 microliters of blocking buffer at four degrees Celsius. After five minutes, add the antibody cocktail of interest to each well for a 20-minute incubation at four degrees Celsius.At the end of the incubation, wash each well with 200 microliters of PBS plus FBS, flicking the plate at the end of the centrifugation to discard the supernatant. Resuspend the pellets in 50 microliters of PBS plus FBS and analyze the cells according to standard flow analysis protocols to screen for colonies that contain hematopoietic stem cell potential. Selecting the wells that contain cells with hematopoietic stem cell phenotype by flow analysis, use a multichannel pipette to resuspend the remaining 100 microliters of cells from the original co-culture and add five times 10 to the fourth rescue bone marrow cells in 100 microliters of PBS plus 2%FBS to each well.After adoptive transfer of each clone into individual lethally irradiated congenic recipient animals, collect peripheral blood samples from the engrafted animals at regular intervals post transplant. To identify the clones with long-term engraftment, analyze the samples by flow cytometry according to standard flow analysis protocols. The phenotypic properties of each clone can then be correlated with its engraftment properties.Pre-hematopoietic stem cells can be enriched by vascular endothelial Cadherin positivity and a high level of endothelial protein C receptor expression. Staining with other fluorochrome conjugated antibodies allows the retrospective analysis of additional phenotypic parameters that can be recorded for each cell during index sorting. Following index sorting into aorta-gonad-mesonephros endothelial cell co-cultures, the individual clones integrate into the aorta-gonad-mesonephros endothelial cell layer before the clones begin to round up and form hematopoietic clusters.After five to seven days of co-culture, colonies of various sizes and morphologies can be detected. Here, four different colonies obtained following co-culture are shown with three colonies demonstrating different proportions of phenotypic hematopoietic stem cells along with other more differentiated cell types and the fourth colony lacking cells with hematopoietic stem cell marker expression. Adoptive transfer of the clone cell progeny allows assessment of the long-term multi-lineage engraftment capability of each clone following donor contribution to the myeloid B and T cell compartments of the peripheral blood of the recipient over time.Although most colonies containing cells with a hematopoietic stem cell phenotype achieved long-term engraftment, transplantation is necessary to confirm a functional multi-lineage engraftment as some clones lose engraftment over time. The functional hematopoietic stem cell potential of each clone can then be correlated back to that clone's original index sorting parameters to identify its precise phenotypic characteristics. Following this procedure, other methods like secondary culture of hematopoietic progeny on stroma supporting B and T cell development in vitro can be performed to answer additional questions about the lymphocyte potentials of individual hemogenic precursors.

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Research

JoVE Journal

Developmental Biology

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&amp;#8217;s Cartilage

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...