Endothelial Differentiation of Embryonic Stem Cells

Alicia A. Blancas1, Nicholas E. Lauer2, Kara E. McCloskey2

1 Graduate Program in Quantitative and Systems Biology, University of California at Merced, Merced, California, 2 School of Engineering, University of California at Merced, Merced, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.5
DOI:  10.1002/9780470151808.sc01f05s6
Online Posting Date:  September, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Vascular progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications, including cell‐based therapies and tissue engineering. Here, we describe methods for isolating purified proliferating populations of vascular endothelial cells from mouse embryonic stem cells (mESC) using Flk‐1 positive sorted cells, VEGF supplementation, and a rigorous manual selection technique required for endothelial cell purification and expansion. Using this in vitro derivation procedure, it is possible to obtain millions of cells at various stages of differentiation, with the potential for up to 25 population doublings. Curr. Protoc. Stem Cell Biol. 6:1F.5.1‐1F.5.19. © 2008 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; endothelial cells; endothelial progenitor cells; vascular progenitor cells; Flk‐1; VEGF

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Endothelial Cell Differentiation from Mouse ESC
  • Support Protocol 1: Freezing Cultured Cells
  • Support Protocol 2: Thawing Cultured Cells
  • Support Protocol 3: Mitotic Inactivation of Fibroblast Feeder
  • Support Protocol 4: Preparation of Dissecting Pipets
  • Support Protocol 5: Preparing a Mouth Aspirator
  • Alternate Protocol 1: EC Differentiation from Mouse ESC Culture Under Serum‐Free Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Endothelial Cell Differentiation from Mouse ESC

  Materials
  • ES‐D3 or ES‐R1 cells (American Type Culture Collection, cat. no. CRL‐1934 or SCRC‐1036)
  • Mouse ESC medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS), calcium‐ and magnesium‐free (Invitrogen, cat. no. 14190‐144)
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • ESC‐to‐EC differentiating medium (see recipe)
  • Gelatin (for subculturing of cells)
  • Cell dissociation solution (Sigma, cat. no. C‐5914)
  • Fetal bovine serum (FBS), heat inactivated (Cellgro, cat. no. 35‐001‐CV)
  • BSA buffer solution (see recipe)
  • Normal donkey serum (Research Diagnostics, cat. no. RDI‐NSDNKY)
  • Rabbit anti–mouse Flk‐1 (Alpha Diagnostic International, cat. no. FLK11‐A)
  • Donkey anti–rabbit phycoerythrin (PE)‐conjugated (Research Diagnostics, cat. no. RDI‐711116152)
  • Recombinant human vascular endothelial growth factor (VEGF 165; R&D Systems, cat. no. 293‐VE)
  • EC medium (see recipe)
  • Collagen IV (Becton‐Dickinson; cat. no. 354233) or collagen I (Becton‐Dickinson; cat. no. 354236) or fibronectin (Sigma; cat. no. F‐1141) or gelatin (Sigma; cat. no. G‐1890) for coating flasks for expansion
  • Gelatin (Sigma, cat. no. G‐1890)
  • Fibroblast feeder cell–coated 35‐mm dishes ( protocol 4)
  • 15‐ml centrifuge tubes (VWR, cat. no. 21008‐103)
  • Benchtop centrifuge
  • Biocoat collagen IV 35‐mm culture dishes (Becton‐Dickinson, cat. no. 354459)
  • Cell scraper, optional
  • Vortex
  • 5‐ml round‐bottomed polystyrene FACS tube
  • Fluorescent‐activated cell sorter (FACS)
  • 25‐, 75‐, and 175‐cm2 flasks
  • Inverted microscope (for general viewing of cells)
  • Stereomicroscope
  • Additional reagents and equipment for thawing ES‐D3 cells ( protocol 3), performing a viable cell count (unit 1.3), preparing dissecting pipets ( protocol 5), and preparing a mouth aspirator ( protocol 6)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Freezing Cultured Cells

  Materials
  • Cultures to be frozen
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free
  • Appropriate medium for cells containing serum
  • Freezing medium (see recipe)
  • 35‐mm tissue culture dishes
  • Phase contrast microscope
  • Nunc cryovials (VWR, cat. no. 66021‐986)
  • Cryo 1°C freezing containers (Research Products International, cat. no. 5100‐0001)
  • −70° or −80°C freezer
  • Liquid nitrogen storage tank

Support Protocol 2: Thawing Cultured Cells

  Materials
  • Frozen stocks of cells ( protocol 2)
  • Appropriate cell medium
  • 37°C water bath
  • Laminar flow cabinet
  • 15‐ml centrifuge tube

Support Protocol 3: Mitotic Inactivation of Fibroblast Feeder

  Materials
  • Feeder cells to be inactivated: mouse fibroblasts or STO cells (ATCC, cat. no. CRL‐1503)
  • Embryonic fibroblast feeder cell medium (see recipe)
  • Mitomycin C solution (see recipe)
  • Phosphate‐buffered saline (PBS), with calcium and magnesium
  • Phosphate‐buffered saline (PBS), calcium‐ and magnesium free
  • Trypsin/EDTA
  • 175‐cm2 tissue culture flasks (with 0.2‐mm vent cap; Corning, cat. no. 431080)
  • 37°C incubator
  • 15‐ml centrifuge tubes
  • 35‐mm dish
  • Additional reagents and equipment for counting cells (unit 1.3)

Support Protocol 4: Preparation of Dissecting Pipets

  Materials
  • Glass Pasteur pipets (9 in.; VWR, cat. no. 53283‐915)
  • Bunsen burner

Support Protocol 5: Preparing a Mouth Aspirator

  Materials
  • 1000‐µl micropipet tip
  • Aspirator assembly with rubber tubing (Sigma, cat. no. A5177)
  • 0.2‐µm syringe filter (Pall, cat. no. 4192)
  • Dissecting pipet ( protocol 5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adelman, C.A., Chattopadhyay, S., and Bieker, J.J. 2002. The BMP/BMPR/Smad pathway directs expression of the erythroid‐specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum‐free media. Development 129:539‐549.
   Aranguren, X.L., Luttun, A., Clavel, C., Moreno, C., Abizanda, G., Barajas, M.A., Pelacho, B., Uriz, M., Arana, M., Echavarri, A., Soriano, M., Andreu, E.J., Merino, J., Garcia‐Verdugo, J.M., Verfaillie, C.M., and Prosper, E. 2007. In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells. Blood 109:2634‐2642.
   Choi, K, Kennedy, M., Kazarov, A., Papadimitriou, J.C., and Keller, G. 1998. A common precursor for hematopoietic and endothelial cells. Development 125:725‐32.
   Dzau, V.J., Gnecchi, M., Pachori, A.S., Morello, F., and Melo, L.G. 2005. Therapeutic potential of endothelial progenitor cells in cardiovascular diseases. Hypertension 46:7‐18.
   Ferreira, L.S., Gerecht, S., Shieh, H.F., Watson, N., Rupnick, M.A., Dallabrida, S.M., Vunjak‐Novakovic, G., and Langer, R. 2007. Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo. Circ. Res. 101:286‐294.
   Griese, D.P., Ehsan, A., Melo, L.G., Kong, D., Zhang, L., Mann, M.J., Pratt, R.E., Mulligan, R.C., and Dzau, V.J. 2003. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: Implications for cell‐based vascular therapy. Circulation 108:2710‐2715.
   Hirashima, M., Kataoka, H., Nishikawa, S., and Matsuyoshi, N. 1999. Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis. Blood 93:1253‐1263.
   Johansson, B.M. and Wiles, M.V. 1995. Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell Biol. 15:141‐151.
   Kalka, C., Masuda, H., Takahashi, T., Kalka‐Moll, W.M., Silver, M., Kearney, M., Li, T., Isner, J.M., and Asahara, T. 2000. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. U.S.A. 97:3422‐3427.
   Kaushal, S., Amiel, G.E., Guleserian, K.J., Shapira, O.M., Perry, T., Sutherland, F.W., Rabkin, E., Moran, A.M., Schoen, F.J., Atala, A., Soker, S., Bischoff, J., and Mayer, J.E. Jr. 2001. Functional small‐diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat. Med. 7:1035‐1040.
   Kawamoto, A., Gwon, H.C., Iwaguro, H., Yamaguchi, J.I., Uchida, S., Masuda, H., Silver, M., Ma, H., Kearney, M., Isner, J.M., and Asahara, T. 2001. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103:634‐637.
   Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N.M., and Itescu, S. 2001. Neovascularization of ischemic myocardium by human bone‐marrow‐derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7:430‐436.
   Levenberg, S., Golub, J.S., Amit, M., Itskovitz‐Eldor, J., and Langer, R. 2002. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 99:4391‐4396.
   McCloskey, K.E., Lyons, I., Rao, R.R., Stice, S.L., and Nerem, R.M. 2003. Purified and proliferating endothelial cells derived and expanded in vitro from embryonic stem cells. Endothelium 10:329‐336.
   McCloskey, K.E., Gilroy, M.E., and Nerem, R.M. 2005. Use of embryonic stem cell‐derived endothelial cells as a cell source to generate vessel structures in vitro. Tissue Eng. 11:497‐505.
   Ng, E.S., Azzola, L., Sourris, K., Robb, L., Stanley, E.G., and Elefanty, A.G. 2005. The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4‐induced ventral mesoderm patterning in differentiating ES cells. Development 132:873‐884.
   Nishikawa, S.I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., and Kodama, H. 1998. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE‐cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747‐1757.
   Nishikawa, S.I., Hirashima, M., Nishikawa, S., and Ogawa, M. 2001a. Cell biology of vascular endothelial cells. Ann. N.Y. Acad. Sci. 947:35‐40.
   Nishikawa, S.I., Hirashima, M., Nishikawa, S., and Ogawa, M. 2001b. Cell Biology of Vascular Endothelial Cells. Ann. N.Y. Acad. Sci. 947:35.
   Nishikawa, S.I., Jakt, L.M., and Era, T. 2007. Opinion: Embryonic stem‐cell culture as a tool for developmental cell biology. Nat. Rev. Molec. Cell Biol. 8:502‐507.
   Risau, W., Sariola, H., Zerwes, H.G., Sasse, J., Ekblom, P., Kemler, R., and Doetschman, T. 1988. Vasculogenesis and angiogenesis in embryonic‐stem‐cell‐derived embryoid bodies. Development 102:471‐478.
   Soker, S., Machado, M., and Atala, A. 2000. Systems for therapeutic angiogenesis in tissue engineering. World J. Urol. 18:10‐18.
   Tanaka, N., Takeuchi, T., Neri, Q.V., Sills, E.S., and Palermo, G.D. 2006. Laser‐assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: Applications and preliminary results in a murine model. J. Translat. Med. 4:20.
   Vittet, D., Prandini, M.H., Berthier, R., Schweitzer, A., Martin‐Sisteron, H., Uzan, G., and Dejana, E. 1996. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88:3424‐3431.
   Wang, R., Clark, R., and Bautch, V.L. 1992. Embryonic stem cell‐derived cystic embryoid bodies form vascular channels: An in vitro model of blood vessel development. Development 114:303‐316.
   Wang, Z.Z., Au, P., Chen, T., Shao, Y., Daheron, L.M., Bai, H., Arzigian, M., Fukumura, D., Jain, R.K., and Scadden, D.T. 2007. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat. Biotechnol. 25:317‐318.
   Wiles, M.V. and Johansson, B.M. 1999. Embryonic stem cell development in a chemically defined medium. Exp. Cell Res. 247:241‐248.
   Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., and Nakao, K. 2000. Flk1‐positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408:92‐96.
   Yamashita, J.K. 2007. Differentiation of arterial, venous, and lymphatic endothelial cells from vascular progenitors. Trends Cardiovasc. Med. 17:59‐63.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library