The authors would like to acknowledge the Baker Foundation and the Royal Children's Foundation 'Women in Science Fellowship' support of Zerina Lokmic. Andrew G. Elefanty and Edouard G. Stanley are NHMRC Senior Research Fellows. Work in their laboratories was supported by NHMRC and Stem Cells Australia.

Ethics statement: Ethical approval for collection of lymphatic malformation and foreskin tissues was obtained from the Human Research Ethics Committees at the Royal Children&#8217;s Hospital, Melbourne, Australia. Signed consent was received from patients&#8217; parents prior to surgery. Tissue samples were collected from patients diagnosed with LMs undergoing surgical procedures as part of their clinical management and patients undergoing elective circumcision. All experiments were performed in accordance with guideline...

<ol>
	<li>Baluk, P., 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=Functionally+specialized+junctions+between+endothelial+cells+of+lymphatic+vessels.">Functionally specialized junctions between endothelial cells of lymphatic vessels.</a> J Exp Med. 204 (10), 2349-2362 (2007).</li><li>Yao, L. C., Baluk, P., Srinivasan, R. S., Oliver, G., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+button-like+junctions+in+the+endothelium+of+airway+lymphatics+in+development+and+inflammation.">Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.</a> Am J Pathol. 180 (6), 2561-2575 (2012).</li><li>Zawieja, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+physiology+of+lymphatics.">Contractile physiology of lymphatics.</a> Lymphat Res Biol. 7 (2), 87-96 (2009).</li><li>Witte, M. 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=Structure+function+relationships+in+the+lymphatic+system+and+implications+for+cancer+biology.">Structure function relationships in the lymphatic system and implications for cancer biology.</a> Cancer Metastasis Rev. 25 (2), 159-184 (2006).</li><li>Andrade, M., Jacomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+human+lymphatic+system.">Anatomy of the human lymphatic system.</a> Cancer Treat Res. 135, 55-77 (2007).</li><li>Kumar, V., Abbas, A. K., Aster, J. C., Ch Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cotran Pathologic Basis of Disease. 43-78. 2, (2009).</li><li>Shayan, R., Achen, M. G., Stacker, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+cancer+metastasis:+bridging+the+gaps.">Lymphatic vessels in cancer metastasis: bridging the gaps.</a> Carcinogenesis. 27 (9), 1729-1738 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas and. , 1059-1076 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas. 14, 562-594 (2013).</li><li>Bellini, C., Hennekam, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+disorders+of+primary+malfunctioning+of+the+lymphatic+system.">Clinical disorders of primary malfunctioning of the lymphatic system.</a> Adv Anat Embryol Cell Biol. 214, 187-204 (2014).</li><li>Schook, C. 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=Primary+lymphedema:+clinical+features+and+management+in+138+pediatric+patients.">Primary lymphedema: clinical features and management in 138 pediatric patients.</a> Plast Reconstr Surg. 127 (6), 2419-2431 (2011).</li><li>Brouillard, P., Boon, L., Vikkula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+lymphatic+anomalies.">Genetics of lymphatic anomalies.</a> J Clin Invest. 124 (3), 898-904 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=for+the+Study+of+Vascular+Anomalies+(ISSVA)+classification.">for the Study of Vascular Anomalies (ISSVA) classification.</a> Available from: http://www.issva.org/classification.com. , (2014).</li><li>Croteau, S. E., 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=Kaposiform+lymphangiomatosis:+a+distinct+aggressive+lymphatic+anomaly.">Kaposiform lymphangiomatosis: a distinct aggressive lymphatic anomaly.</a> J Pediatr. 164 (2), 383-388 (2014).</li><li>Lokmic, 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=Isolation+of+human+lymphatic+malformation+endothelial+cells,+their+in+vitro+characterization+and+in+vivo+survival+in+a+mouse+xenograft+model.">Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model.</a> Angiogenesis. 17 (1), 1-15 (2014).</li><li>Smith, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+malformations.">Lymphatic malformations.</a> Lymphat Res Biol. 2 (1), 25-31 (2004).</li><li>Chen, E. 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=Similar+histologic+features+and+immunohistochemical+staining+in+microcystic+and+macrocystic+lymphatic+malformations.">Similar histologic features and immunohistochemical staining in microcystic and macrocystic lymphatic malformations.</a> Lymphat Res Biol. 7 (2), 75-80 (2009).</li><li>Norgall, 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=Elevated+expression+of+VEGFR-3+in+lymphatic+endothelial+cells+from+lymphangiomas.">Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.</a> BMC cancer. 7 (2), 75-80 (2007).</li><li>Garzon, M. C., Huang, J. T., Enjolras, O., Frieden, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+malformations.">Vascular malformations.</a> Part I. J Am Acad Dermatol. 56 (3), 353-370 (2007).</li><li>Osborn, A., Dickie, P., Adams, D., Dickie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 20th International workshop on Vascular Anomalies. ISSVA. , (2014).</li><li>Baluk, P., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+microscopic+imaging+of+lymphangiogenesis+and+angiogenesis.">Markers for microscopic imaging of lymphangiogenesis and angiogenesis.</a> Ann N Y Acad Sci. 1131, 1-12 (2008).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+microcirculation.+Tissue+engineering.">Engineering the microcirculation. Tissue engineering.</a> Part B, Reviews. 14 (1), 87-103 (2008).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+and+stereological+assessment+of+blood+and+lymphatic+vessels.">Visualisation and stereological assessment of blood and lymphatic vessels.</a> Histol Histopathol. 26 (6), 781-796 (2011).</li><li>Hagerling, 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=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> EMBO J. 32 (5), 629-644 (2013).</li><li>Pollmann, C., Hagerling, R., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+lymphatic+vessel+development,+growth,+and+function.">Visualization of lymphatic vessel development, growth, and function.</a> Adv Anat Embryol Cell Biol. 214, 167-186 (2014).</li><li>Breiteneder-Geleff, 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li><li>Kaipainen, 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=Expression+of+the+fms-like+tyrosine+kinase+4+gene+becomes+restricted+to+lymphatic+endothelium+during+development.">Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.</a> Proc Natl Acad Sci U S A. 92 (8), 3566-3570 (1995).</li><li>Schacht, V., 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=T1alpha/podoplanin+deficiency+disrupts+normal+lymphatic+vasculature+formation+and+causes+lymphedema.">T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.</a> EMBO J. 22 (14), 3546-3556 (2003).</li><li>Kriehuber, E., 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=Isolation+and+characterization+of+dermal+lymphatic+and+blood+endothelial+cells+reveal+stable+and+functionally+specialized+cell+lineages.">Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.</a> J Exp Med. 194 (6), 797-808 (2001).</li><li>Florez-Vargas, 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=Comparative+analysis+of+D2-40+and+LYVE-1+immunostaiing+in+lymphatic+malformations.">Comparative analysis of D2-40 and LYVE-1 immunostaiing in lymphatic malformations.</a> Lymphology. 41 (3), 103-110 (2008).</li><li>Makinen, 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=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes Dev. 19 (3), 397-410 (2005).</li><li>Connell, F. 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=Analysis+of+coding+regions+of+VEGFR3+and+VEGFRC+in+Milroy+disese+and+other+primary+lymphoedemas.">Analysis of coding regions of VEGFR3 and VEGFRC in Milroy disese and other primary lymphoedemas.</a> Hum Genet. 124 (6), 625-631 (2009).</li></ol>

LECs play an important role in maintaining fluid homeostasis, immune response to foreign antigens and absorption and transport of some nutrients. LEC homeostasis can be affected by disease processes such as bacterial infections and tumor metastasis, but LECs can also develop somatic mutations that result in formation of dysfunctional lymphatic vessels and significant morbidity for affected patients. To gain more understanding of lymphatic malformation etiology through in vivo implantation and ex vivo ex...

A major function of the lymphatic vascular system is to absorb lymph, an excess interstitial fluid containing lipids, proteins and cellular components, and conduct it to the blood venous system. A network of lymphatic capillaries directs lymph to the lymph nodes where it is screened for presence of foreign antigens, an important process in immune surveillance and deployment of white blood cells to neutralize foreign antigens.
The uni-directional lymphatic system starts in tissues with the initial lymphatic capillary, a unique structure with a discontinuous single layer of thin-walled flat endothelial cells with specialized cell junctions that permit lymph entry1,2. These capillaries are attached to the neighboring connective tissue matrix via anchoring filaments to prevent vessel collapse in presence of increased interstitial pressure3. The initial lymphatic capillaries empty into collecting lymphatic capillaries that coalesce into larger lymphatic vessels or veins. In comparison to initial lymphatic capillary vessels, collecting lymphatic vessels have thicker vessel walls, paired lymphatic valves and are encased by a discontinuous basement membrane in which a few smooth muscle cells are embedded4. Coordinated opening and closure of lymphatic valves and contraction of smooth muscle cells facilitates flow of lymph3. In humans, the lymphatic veins from various regions of the body join to form lymphatic trunks which merge to form two lymphatic ducts: the thoracic duct and the right lymphatic duct. The thoracic duct drains lymph from the left side of the body and from the right side below the chest while the right lymphatic duct drains lymph from the right arm and right side of the head, neck, and thorax. Both ducts conduct lymph into the subclavian veins in the neck5.
Disorders of the lymphatic system are broadly grouped into acquired and congenital (Table 1). Examples of acquired conditions are lymphangitis and secondary lymphedema. Lymphangitis is an inflammation of a lymphatic vessel due to bacterial infection. The affected lymphatics dilate and fill with exudate containing polymorphonuclear cells. In skin, these lymphatics are visible as red, painful subcutaneous streaks often accompanied by enlargement of the associated draining lymph node (lymphadenitis)6. Secondary lymphedema arises as a consequence of damage or obstruction to the lymphatic vessel or lymph node obstruction. This leads to chronic progressive swelling due to accumulation of lymph distal to the damage or obstruction. In developed countries, secondary lymphedema is most commonly associated with malignancy where metastasizing tumors obstruct lymphatic vessels or regional lymph nodes, or as a consequence of anti-cancer therapy following surgical removal of lymph nodes, post-irradiation fibrosis and post-inflammatory thrombosis and scarring7. In other parts of the world, secondary lymphedema may be secondary to lymphatic obstruction caused by parasitic worms such as Wuchereria bancrofti6.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Disorders of Lymphatic Vascular System</td>
		</tr>
		<tr>
			<td>Acquired</td>
			<td colspan="3">Congenital</td>
		</tr>
		<tr>
			<td rowspan="3">Lymphadenitis 
			Secondary lymphedema</td>
			<td>Primary Lymphedema10</td>
			<td>Sporadic Lymphatic Malformations13</td>
			<td>Lymphatic Malformations Associated with Syndromes13</td>
		</tr>
		<tr>
			<td rowspan="2">e.g. 
			Milroy Syndrome 
			Meige Syndrome</td>
			<td>Simple: 
			Lymphatic malformations</td>
			<td rowspan="2">e.g. 
			Klippel-Tranaunay Syndrome 
			Parks Weber Syndrome 
			Sturge-Weber Syndrome</td>
		</tr>
		<tr>
			<td>Combined: 
			Capillary-lymphatic malformations 
			Capillary-lymphatic-venous malformation 
			Capillary-lymphatic-arteriovenous malformation 
			Capillary-lymphatic venous-arteriovenous malformation</td>
		</tr>
	</tbody>
</table>
Table 1. Overview of the disorders of lymphatic vascular system.
Congenital disorders of the lymphatic system include primary (idiopathic) lymphedema thought to be caused by genetic mutations, lymphangiectasia and anomalies of the lymphatic system8,9. Primary lymphedema can be sporadic presumably caused by de novo mutations, or inherited. Lymphatic disorders can also be isolated or comprise part of a more generalized syndrome10. In the pediatric population, 97% of lymphedema is sporadic with abnormalities in lymphatic vessel structure that impair regional lymph drainage11. Milroy disease is an example of primary lymphedema caused by mutation in the VEGFR-3 gene evident at birth or soon after12. Although mostly familial condition, the Milroy disease can also be identified in infants without family history of Milroy disease32. The severity of any lymphedema is dependent on the amount of lymph production and ability to transport lymph back to venous circulation6.
Based on clinical presentation and in situ endothelial cell proliferation, anomalies of the lymphatic system are classified as lymphatic tumors or lymphatic malformations13. Kaposiform lymphangiomatosis is an example of an LEC tumor14. Lymphatic malformations are thought to arise during embryonic development and grow in proportion to the child15,16. They rarely regress but can remain asymptomatic until trauma or infection precipitates rapid growth leading to clinical complications. The orderly structure of lymphatic network and conduction of lymph from the tissue to venous circulation described above is perturbed in lymphatic malformations which consist of localized collections of abnormal cystic structures filled with lymphatic fluid. While there is no clinical or experimental evidence that these cystic vessels are connected to the lymphatic circulation or that they contain functional lymphatic valves, their lymphatic identity is confirmed by expression of range of lymphatic cell markers such as PODOPLANIN, CD31, Lymphatic Vessel Endothelial Receptor 1 (LYVE-1), Prospero homeobox protein 1 (PROX-1) and VEGFR-315,17,18. These cystic structures can be either small (microcystic) or large (macrocystic), but most lymphatic malformations contain both microcystic and macrocystic components (Figure 1)16. Following surgery, injection sclerotherapy and/or radiofrequency ablation the lymphatic malformations often reoccur.
<img alt="Figure 1" src="/files/ftp_upload/52691/52691fig1highres.jpg" width="700" /> 
Figure 1. Morphology of human lymphatic vessels and lymphatic malformations. Normal human lymphatic (A) and lymphatic malformation vessels (B and C) labelled with antibody to PODOPLANIN (brown label, arrow). Human lymphatic malformation vessels are characterized by marked dilation and considerable variation in lumen size. These localized abnormal cystic structures can be either small (microcystic, *) (B) or large (macrocystic, #) (C). Most lymphatic malformations contain both microcystic and macrocystic components. <a href="https://www.jove.com/files/ftp_upload/52691/52691fig1highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>
Some investigators have suggested that lymphatic malformations represent a developmental disorder of lymphatic vasculature in which the LECs do not have abnormal growth potential but instead have failed to connect to the normal circulation19. However, we have found that the LM LECs proliferate faster and are more resistant to apoptosis than foreskin LECs15 suggesting that there is a primary defect in the LM LECs. When LM LECs are implanted in a mouse xenograft model, they form structures reminiscent of lymphatic malformations15. This supports a hypothesis that lymphatic malformations may be caused by one or more somatic mutations arising in LM LECs during fetal development. Indeed, recent reports have identified one such mutation in the p110&#945; catalytic subunit of Phosphoinositide-3-Kinase (PIK3CA) gene20.
Given the advances in DNA sequencing technology, relevant mutations could be more readily identified in isolated LM LECs, guiding future studies of these conditions. The isolation of viable LECs would facilitate comparisons between abnormal and normal LECs in assays such as migration, proliferation, tube forming ability and survival in response to reduced nutrient availability or pro-apoptotic agents15. Isolated LECs would further enable us to perform cell-specific gene expression and proteomic studies, to delineate new LEC subpopulations and discover novel pharmacological agents suitable for clinical management of lymphatic malformations.
We have previously published a LEC isolation method based on magnetic bead separation of LECs from neonatal foreskin and lymphatic malformations15. We reported a strategy of separating normal and diseased LECs from vascular endothelial cells based on the absence of CD34 expression, followed by subjecting CD34Neg cell fraction to positive selection for CD31. However, this method was hampered by the presence of residual non-endothelial cells. This was independent of removing epidermis prior to subsequent connective tissue digestion. These contaminants generally proliferated more rapidly and thus eventually overgrew the endothelial cell cultures despite subsequent attempts to repeat LEC isolation. Indeed, an initial contamination of non-endothelial cells as low as 2% to 5% was sufficient to overwhelm the LEC population15. This prompted us to explore fluorescently activated cell sorting method as an option to improve LEC cell yield and purity. In addition, we used multi-parameter sorting to enhance the specificity of the LEC populations, adding VEGFR-3 and PODOPLANIN to the selection markers to identify CD34LowCD31PosVEGFR-3PosPODOPLANINPos LECs.
The rationale for selecting these markers was based on the reports that while LECs and blood vascular endothelial cells have many cell surface markers in common such as CD31, LECs show phenotypic variation in their expression of CD34, PODOPLANIN and VEGFR-3 cell surface marker when compared to blood vascular endothelial cells21-23. CD31 is a 130 kDa transmembrane glycoprotein also known as platelet endothelial cell adhesion molecule 1 (PECAM-1). It is considered to be a pan-endothelial cell marker since it is expressed on all types of blood and lymphatic vessels21,24,25. CD34 is 110-kDa transmembrane glycoprotein present on most hematopoietic progenitor and stem cells, vascular endothelial cells and some lymphatic vessels26.
VEGFR-3, the receptor for vascular endothelial growth factors C and D, is initially present on the developing veins in the mouse embryo, but following lymphatic specification regulated by the transcription factors SRY-related HMG-box (SOX)-18, chicken ovalbumin upstream promoter transcription factor 2 (COUPTF-II) and PROX-1, VEGFR-3 venous expression is lost and it becomes restricted to embryonic LECs25,27. PODOPLANIN, a 38 kDa membrane mucoprotein, is first noted on lymphatic vessels at approximately embryonic day 11 (~E11.0) of mouse embryonic development 28 and whilst it is strongly expressed by microvascular lymphatic vessels, PODOPLANIN expression by macrocystic lymphatic endothelium in lymphatic malformations is more variable15. Flow cytometry experiments suggest that at least some CD34HighCD31Pos endothelial cells express the lymphatic marker PODOPLANIN29. Although systematic evaluation of LYVE-1 and PODOPLANIN staining in human lymphatic malformations showed that both are effective at staining lymphatic malformation endothelium30, in normal tissues, LYVE-1 was reported to be strongly present in the initial lymphatic capillary endothelium but reduced and even absent in the collecting lymphatic endothelium31. As our aim is to isolate both the initial and collecting lymphatic endothelial cells we have opted not to use LYVE-1 as part of our cell selection strategy. Finally, the decision to employ these markers was also based on the availability of antibodies that are used diagnostically for labelling lymphatic vessels for microscopic imaging, a feature that would permit correlation between flow cytometry and immunofluorescent studies.
This article will describe the tissue digestion method, cell staining and FACS settings required for successful isolation of CD34LowCD31PosVEGFR-3PosPODOPLANINPos LECs as well as CD34HighCD31PosVEGFR-3PosPODOPLANINPos endothelial cells from foreskin and lymphatic malformation tissue.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies (Gibco)</td>
			<td>11965-092</td>
			<td>Used to collect tissue samples from the operating theatre.</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R3-IGF-1 and ascorbic acid.</td>
		</tr>
		<tr>
			<td>VEGF-C</td>
			<td>R&#38;D Systems</td>
			<td>2179-VC-025</td>
			<td>Complete endothelial cell media is supplemented with 50 ng/mL VEGF-C</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution (100x)</td>
			<td>Life Technologies (Gibco)</td>
			<td>15240-062</td>
			<td>This solution contains 10,000U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml of Fungizone antimycotic. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4 degrees and maintained sterile. Use 7 mL to coat 150 cm2 flask.</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell dissociation reagent</td>
			<td>Life Technologies (Gibco)</td>
			<td>A11105-01</td>
			<td>StemPro&#174;Accutase&#174; is used at 0.05 ml per cm2 to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.</td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue dye</td>
			<td>Life Technologies (Gibco)</td>
			<td>15250-061</td>
			<td>Used as a viability stain.</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche Applied Biosciences</td>
			<td>4942078001</td>
			<td>Used at 0.04%</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Worthington Lab</td>
			<td>4176</td>
			<td>Used at 0.25%</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche Applied Biosciences</td>
			<td>11284932001</td>
			<td>Used at 0.01%</td>
		</tr>
		<tr>
			<td>70 mm nylon cell strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated VEGFR-3 clone 9D9F9, clone WM59</td>
			<td>BioLegend</td>
			<td>356204</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated CD34, clone 581</td>
			<td>BioLegend</td>
			<td>343516</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti human CD31, clone WM59</td>
			<td>BioLegend</td>
			<td>303116,</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated rat anti human PODOPLANIN, clone NC-80.</td>
			<td>BioLegend</td>
			<td>337006</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>PE-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400112</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400126</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400120</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758</td>
			<td>BioLegend</td>
			<td>400525</td>
			<td>Used at 1:200 dilution</td>
		</tr>
	</tbody>
</table>

isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Following initial tissue digestion, after 24 hours in culture of unfractionated samples, distinct endothelial cell colonies can be observed (Figure 2A) together with fibroblast-like cells and smooth muscle cells. Following sorting and after 24 hours in cell culture, the CD34LowCD31PosVEGFR-3PosPodoplaninPos cells attach and show typical cobblestone morphology (Figure 2B and C). Using the FACS method described above, we are able...

Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

isolation-human-lymphatic-endothelial-cells-multi-parameter

Research

JoVE Journal

Medicine

14.2K Aufrufe.  The Royal Children&#146;s Hospital.  Das Ziel des Protokolls ist es, lymphatische Endothelzellen menschlicher Lymphmissbildung zystenartige Gefäße und Vorhaut mittels Fluoreszenz-aktivierter Zellsortierung (FACS) isoliert werden. Anschließende Zellkultur und Expansion dieser Zellen ermöglicht eine neue Stufe der experimentellen Raffinesse für genetische, Proteomik, funktionale und Zelldifferenzierung Studien. 