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Topic Introduction
Purification and Culture of Central Nervous System Endothelial Cells Lu Zhou,1 Fabien Sohet,2 and Richard Daneman2,3 1
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125; Department of Anatomy, UCSF, San Francisco, California 94143-0452
2
Blood vessels are critical for delivering oxygen and nutrients to all tissues in the body. This is especially important in the central nervous system, which is extremely sensitive to hypoxia and ischemia. Blood vessels are made of two main cell types: endothelial cells and mural cells. Endothelial cells form the walls of the blood vessels that generate a lumen through which blood flows. Mural cells are support cells thought to be involved in vessel contractility, vascular remodeling, and regulation of endothelial permeability. On large vessels, including arteries and veins, mural cells are termed vascular smooth muscle cells. On the small vessels of the capillary bed, they are called pericytes. Here, we provide a brief introduction to the methods for purification of endothelial cells, including an immunopanning method that we developed for isolating these cells from the rodent brain and optic nerve.
ENDOTHELIAL CELLS IN THE CENTRAL NERVOUS SYSTEM
Endothelial cells form the walls of the blood vessels, which are critical for delivering blood to all parts of the body, including the central nervous system (CNS). Because of their location, endothelial cells regulate the interaction between the blood and each tissue, including the delivery of nutrients, regulation of clotting, and initiation of immune surveillance. The properties of endothelial cells vary throughout the body, depending on the specific requirements of the tissues they vascularize and the branch of the vascular tree in which they reside (i.e., the arteries, arterioles, capillaries, venules, or veins). The endothelial cells of the CNS form a physiological structure termed the blood–brain barrier (BBB), which is critical for regulating the interaction between the blood and the nervous tissue. This barrier tightly regulates the neuronal environment by limiting the passive movement of molecules and ions from the blood to the brain and provides the CNS with specific nutrients through selective transport. To accomplish this level of control, CNS endothelial cells differ from endothelial cells in nonneural tissue in that they are held together by tight junctions and possess few transcytotic vesicles; these properties limit the paracellular and transcellular movement of hydrophilic molecules and ions between the blood and the brain (Rubin and Staddon 1999). CNS endothelial cells express a variety of molecular transporters. These transporters deliver specific nutrients down their concentration gradient into the CNS or use ATP to pump out potential toxins up to their concentration gradient (Zlokovic 2008; Hermann and Elali 2012). Although these functions are manifested in the endothelial cells, key transplantation studies have showed that they are not intrinsic to the endothelial cells, but induced by cellular interactions with the neural tissues (Stewart and Wiley 1981). Astrocytes, pericytes, neurons, 3
Correspondence: [email protected]
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Purification and Culture of CNS Endothelial Cells
and neural stem cells have all been implicated in regulating these functions (Janzer and Raff 1987; Rubin et al. 1991b; Weidenfeller et al. 2007). Purification and culture of CNS endothelial cells alone or in coculture with other neural cells allow for examination of the cellular and molecular mechanisms that regulate barrier properties of the endothelial cells. For instance, astrocyte–endothelial cell cocultures have been used to show that close apposition of astrocytes to endothelial cells enhances the electrical resistance of the endothelial cell tight junctions (Beck et al. 1984; Rubin et al. 1991a). Formation of blood vessels (vasculogenesis and angiogenesis) is critical for both the development of tissues and the progression of tumors (Carmeliet 2003), and purification of endothelial cells from many tissues has facilitated studies of the molecular mechanisms of these processes. Endothelial cells have also been implicated in modulating the development and function of tissues throughout the body, and purification and culture of CNS endothelial cells has been used to identify their role in development of the brain. For instance, purified endothelial cells have been shown to regulate astrocyte differentiation, neuronal survival, and neural stem cell self-renewal and neurogenesis (Mi et al. 2001; Shen et al. 2004; Dugas et al. 2008). PURIFICATION OF ENDOTHELIAL CELLS
Several different techniques have been used to purify endothelial cells from the CNS. One method involves homogenization of tissue, followed by isolation of microvessels by centrifugation through dextran or sucrose gradients, and then isolation of endothelial cells by selective adherence to specific substrates. This general procedure has been used to purify endothelial cells from the brains of mice, rats, cows, pigs, and humans. An alternative approach has been to purify the endothelial cells by using magnetic beads coupled to anti-CD31 antibodies (van Beijnum et al. 2008; Springhorn 2011). In many cases, purification has been coupled with culturing the cells in puromycin for a limited period (Perrière et al. 2005). Puromycin is toxic to the cells, but is pumped out by efflux transporters specifically expressed by CNS endothelial cells, so addition of puromycin to cell culture medium kills contaminating nonendothelial cells. Puromycin treatment has proven effective with cells from adult specimens, but the efflux transporters have lower activity at earlier developmental time periods, so it is not efficient to select endothelial cells from embryonic or neonatal rodent brains. We have achieved extremely pure endothelial cell populations by using fluorescence-activated cell sorting (FACS) with a Tie2GFP transgenic mouse line (Daneman et al. 2010). FACS, however, is expensive and time consuming. To avoid the need for FACS, we have developed a method for purifying CNS endothelial cells by immunopanning, as described in Purification of Endothelial Cells from Rodent Brain by Immunopanning (Zhou et al. 2014). This prospective isolation method, based on the binding of cells to a dish coated with anti-CD31 antibodies, produces pure populations of CNS endothelial cells that can be analyzed acutely or cultured. REFERENCES Beck DW, Vinters HV, Hart MN, Cancilla PA. 1984. Glial cells influence polarity of the blood–brain barrier. J Neuropathol Exp Neurol 43: 219– 224. Carmeliet P. 2003. Angiogenesis in health and disease. Nat Med 9: 653–660. Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. 2010. The mouse blood–brain barrier transcriptome: A new resource for understanding the development and function of brain endothelial cells. PLoS ONE 5: e13741. Dugas JC, Mandemakers W, Rogers M, Ibrahim A, Daneman R, Barres BA. 2008. A novel purification method for CNS projection neurons leads to the identification of brain vascular cells as a source of trophic support for corticospinal motor neurons. J Neurosci 28: 8294–8305. Hermann DM, Elali A. 2012. The abluminal endothelial membrane in neurovascular remodeling in health and disease. Sci Signal 5: re4.
Janzer RC, Raff MC. 1987. Astrocytes induce blood–brain barrier properties in endothelial cells. Nature 325: 253–257. Mi H, Haeberle H, Barres BA. 2001. Induction of astrocyte differentiation by endothelial cells. J Neurosci 21: 1538–1547. Perrière N, Demeuse P, Garcia E, Regina A, Debray M, Andreux JP, Couvreur P, Scherrmann JM, Temsamani J, Couraud PO, et al. 2005. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier–specific properties. J Neurochem 93: 279–289. Rubin LL, Staddon JM. 1999. The cell biology of the blood–brain barrier. Annu Rev Neurosci 22: 11–28. Rubin LL, Barbu K, Bard F, Cannon C, Hall DE, Horner H, Janatpour M, Liaw C, Manning K, Morales J, et al. 1991a. Differentiation of brain endothelial cells in cell culture. Ann NY Acad Sci 633: 420–425.
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top070987
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L. Zhou et al. Rubin LL, Hall DE, Porter S, Barbu K, Cannon C, Horner HC, Janatpour M, Liaw CW, Manning K, Morales J. 1991b. A cell culture model of the blood–brain barrier. J Cell Biol 115: 1725–1735. Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S. 2004. Endothelial cells stimulate selfrenewal and expand neurogenesis of neural stem cells. Science 304: 1338–1340. Springhorn JP. 2011. Isolation of human capillary endothelial cells using paramagnetic beads conjugated to anti-PECAM antibodies. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4479. Stewart PA, Wiley MJ. 1981. Developing nervous tissue induces formation of blood–brain barrier characteristics in invading endothelial cells:
46
A study using quail-chick transplantation chimeras. Dev Biol 84: 183– 192. van BeijnumJR, RouschM, Castermans K, van der Linden E, Griffioen AW. 2008. Isolation of endothelial cells from fresh tissues. Nat Protoc 3: 1085–1091. Weidenfeller C, Svendsen CN, Shusta EV. 2007. Differentiating embryonic neural progenitor cells induce blood–brain barrier properties. J Neurochem 101: 555–565. Zhou L, Sohet F, Daneman R. 2014. Purification of endothelial cells from rodent brain by immunopanning. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot074963. Zlokovic BV. 2008. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57: 178–201.
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Topic Introduction
Purification and Culture of Central Nervous System Endothelial Cells Lu Zhou,1 Fabien Sohet,2 and Richard Daneman2,3 1
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125; Department of Anatomy, UCSF, San Francisco, California 94143-0452
2
Blood vessels are critical for delivering oxygen and nutrients to all tissues in the body. This is especially important in the central nervous system, which is extremely sensitive to hypoxia and ischemia. Blood vessels are made of two main cell types: endothelial cells and mural cells. Endothelial cells form the walls of the blood vessels that generate a lumen through which blood flows. Mural cells are support cells thought to be involved in vessel contractility, vascular remodeling, and regulation of endothelial permeability. On large vessels, including arteries and veins, mural cells are termed vascular smooth muscle cells. On the small vessels of the capillary bed, they are called pericytes. Here, we provide a brief introduction to the methods for purification of endothelial cells, including an immunopanning method that we developed for isolating these cells from the rodent brain and optic nerve.
ENDOTHELIAL CELLS IN THE CENTRAL NERVOUS SYSTEM
Endothelial cells form the walls of the blood vessels, which are critical for delivering blood to all parts of the body, including the central nervous system (CNS). Because of their location, endothelial cells regulate the interaction between the blood and each tissue, including the delivery of nutrients, regulation of clotting, and initiation of immune surveillance. The properties of endothelial cells vary throughout the body, depending on the specific requirements of the tissues they vascularize and the branch of the vascular tree in which they reside (i.e., the arteries, arterioles, capillaries, venules, or veins). The endothelial cells of the CNS form a physiological structure termed the blood–brain barrier (BBB), which is critical for regulating the interaction between the blood and the nervous tissue. This barrier tightly regulates the neuronal environment by limiting the passive movement of molecules and ions from the blood to the brain and provides the CNS with specific nutrients through selective transport. To accomplish this level of control, CNS endothelial cells differ from endothelial cells in nonneural tissue in that they are held together by tight junctions and possess few transcytotic vesicles; these properties limit the paracellular and transcellular movement of hydrophilic molecules and ions between the blood and the brain (Rubin and Staddon 1999). CNS endothelial cells express a variety of molecular transporters. These transporters deliver specific nutrients down their concentration gradient into the CNS or use ATP to pump out potential toxins up to their concentration gradient (Zlokovic 2008; Hermann and Elali 2012). Although these functions are manifested in the endothelial cells, key transplantation studies have showed that they are not intrinsic to the endothelial cells, but induced by cellular interactions with the neural tissues (Stewart and Wiley 1981). Astrocytes, pericytes, neurons, 3
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top070987
44
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on March 18, 2015 - Published by Cold Spring Harbor Laboratory Press
Purification and Culture of CNS Endothelial Cells
and neural stem cells have all been implicated in regulating these functions (Janzer and Raff 1987; Rubin et al. 1991b; Weidenfeller et al. 2007). Purification and culture of CNS endothelial cells alone or in coculture with other neural cells allow for examination of the cellular and molecular mechanisms that regulate barrier properties of the endothelial cells. For instance, astrocyte–endothelial cell cocultures have been used to show that close apposition of astrocytes to endothelial cells enhances the electrical resistance of the endothelial cell tight junctions (Beck et al. 1984; Rubin et al. 1991a). Formation of blood vessels (vasculogenesis and angiogenesis) is critical for both the development of tissues and the progression of tumors (Carmeliet 2003), and purification of endothelial cells from many tissues has facilitated studies of the molecular mechanisms of these processes. Endothelial cells have also been implicated in modulating the development and function of tissues throughout the body, and purification and culture of CNS endothelial cells has been used to identify their role in development of the brain. For instance, purified endothelial cells have been shown to regulate astrocyte differentiation, neuronal survival, and neural stem cell self-renewal and neurogenesis (Mi et al. 2001; Shen et al. 2004; Dugas et al. 2008). PURIFICATION OF ENDOTHELIAL CELLS
Several different techniques have been used to purify endothelial cells from the CNS. One method involves homogenization of tissue, followed by isolation of microvessels by centrifugation through dextran or sucrose gradients, and then isolation of endothelial cells by selective adherence to specific substrates. This general procedure has been used to purify endothelial cells from the brains of mice, rats, cows, pigs, and humans. An alternative approach has been to purify the endothelial cells by using magnetic beads coupled to anti-CD31 antibodies (van Beijnum et al. 2008; Springhorn 2011). In many cases, purification has been coupled with culturing the cells in puromycin for a limited period (Perrière et al. 2005). Puromycin is toxic to the cells, but is pumped out by efflux transporters specifically expressed by CNS endothelial cells, so addition of puromycin to cell culture medium kills contaminating nonendothelial cells. Puromycin treatment has proven effective with cells from adult specimens, but the efflux transporters have lower activity at earlier developmental time periods, so it is not efficient to select endothelial cells from embryonic or neonatal rodent brains. We have achieved extremely pure endothelial cell populations by using fluorescence-activated cell sorting (FACS) with a Tie2GFP transgenic mouse line (Daneman et al. 2010). FACS, however, is expensive and time consuming. To avoid the need for FACS, we have developed a method for purifying CNS endothelial cells by immunopanning, as described in Purification of Endothelial Cells from Rodent Brain by Immunopanning (Zhou et al. 2014). This prospective isolation method, based on the binding of cells to a dish coated with anti-CD31 antibodies, produces pure populations of CNS endothelial cells that can be analyzed acutely or cultured. REFERENCES Beck DW, Vinters HV, Hart MN, Cancilla PA. 1984. Glial cells influence polarity of the blood–brain barrier. J Neuropathol Exp Neurol 43: 219– 224. Carmeliet P. 2003. Angiogenesis in health and disease. Nat Med 9: 653–660. Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. 2010. The mouse blood–brain barrier transcriptome: A new resource for understanding the development and function of brain endothelial cells. PLoS ONE 5: e13741. Dugas JC, Mandemakers W, Rogers M, Ibrahim A, Daneman R, Barres BA. 2008. A novel purification method for CNS projection neurons leads to the identification of brain vascular cells as a source of trophic support for corticospinal motor neurons. J Neurosci 28: 8294–8305. Hermann DM, Elali A. 2012. The abluminal endothelial membrane in neurovascular remodeling in health and disease. Sci Signal 5: re4.
Janzer RC, Raff MC. 1987. Astrocytes induce blood–brain barrier properties in endothelial cells. Nature 325: 253–257. Mi H, Haeberle H, Barres BA. 2001. Induction of astrocyte differentiation by endothelial cells. J Neurosci 21: 1538–1547. Perrière N, Demeuse P, Garcia E, Regina A, Debray M, Andreux JP, Couvreur P, Scherrmann JM, Temsamani J, Couraud PO, et al. 2005. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier–specific properties. J Neurochem 93: 279–289. Rubin LL, Staddon JM. 1999. The cell biology of the blood–brain barrier. Annu Rev Neurosci 22: 11–28. Rubin LL, Barbu K, Bard F, Cannon C, Hall DE, Horner H, Janatpour M, Liaw C, Manning K, Morales J, et al. 1991a. Differentiation of brain endothelial cells in cell culture. Ann NY Acad Sci 633: 420–425.
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top070987
45
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on March 18, 2015 - Published by Cold Spring Harbor Laboratory Press
L. Zhou et al. Rubin LL, Hall DE, Porter S, Barbu K, Cannon C, Horner HC, Janatpour M, Liaw CW, Manning K, Morales J. 1991b. A cell culture model of the blood–brain barrier. J Cell Biol 115: 1725–1735. Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S. 2004. Endothelial cells stimulate selfrenewal and expand neurogenesis of neural stem cells. Science 304: 1338–1340. Springhorn JP. 2011. Isolation of human capillary endothelial cells using paramagnetic beads conjugated to anti-PECAM antibodies. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4479. Stewart PA, Wiley MJ. 1981. Developing nervous tissue induces formation of blood–brain barrier characteristics in invading endothelial cells:
46
A study using quail-chick transplantation chimeras. Dev Biol 84: 183– 192. van BeijnumJR, RouschM, Castermans K, van der Linden E, Griffioen AW. 2008. Isolation of endothelial cells from fresh tissues. Nat Protoc 3: 1085–1091. Weidenfeller C, Svendsen CN, Shusta EV. 2007. Differentiating embryonic neural progenitor cells induce blood–brain barrier properties. J Neurochem 101: 555–565. Zhou L, Sohet F, Daneman R. 2014. Purification of endothelial cells from rodent brain by immunopanning. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot074963. Zlokovic BV. 2008. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57: 178–201.
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top070987
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on March 18, 2015 - Published by Cold Spring Harbor Laboratory Press
Purification and Culture of Central Nervous System Endothelial Cells Lu Zhou, Fabien Sohet and Richard Daneman Cold Spring Harb Protoc; doi: 10.1101/pdb.top070987 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Immunoaffinity Purification (37 articles) Immunoseparation (24 articles) Mouse (322 articles) Neural Cell Culture (55 articles) Neuroscience, general (263 articles) Other Laboratory Organisms (59 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
