Generation, Expansion, and Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived From the Umbilical Cords of Newborns

Richard S. Song1, Jeanne M. Carroll1, Lisette Acevedo2, Dongmei Wu3, Yang Liu3, Evan Y. Snyder4

1 Department of Pediatrics, University of California at San Diego and Rady Children's Hospital, San Diego, California, 2 Moores Cancer Center, University of California at San Diego, La Jolla, California, 3 Sanford‐Burnham Medical Research Institute, La Jolla, California, 4 Sanford Consortium for Regenerative Medicine, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.16
DOI:  10.1002/9780470151808.sc01c16s29
Online Posting Date:  May, 2014
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Abstract

The umbilical cord is tissue that is normally discarded after the delivery of the infant, but it has been shown to be a rich source of stem cells from the cord blood, Wharton's jelly, and umbilical endothelial cells. Patient‐specific human induced pluripotent stem cells (hiPSCs) reprogrammed from patient specific human umbilical vein endothelial cells in the neonatal intensive care unit (NICU) population (specifically, premature neonates) have not been shown in the literature. This unit describes a protocol for the generation and expansion of hiPSCs originating from umbilical cords collected from patients in the NICU. Curr. Protoc. Stem Cell Biol. 29:1C.16.1‐1C.16.13. © 2014 by John Wiley & Sons, Inc.

Keywords: umbilical cord; HUVEC; reprogramming; iPS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Procurement of HUVECs from Umbilical Cord
  • Basic Protocol 2: Reprogramming of HUVECs into hiPSCs
  • Basic Protocol 3: Affirming Pluripotence of the hiPSCs by Demonstrating Multi‐Germ Layer Lineage Differentiation Capability In Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Procurement of HUVECs from Umbilical Cord

  Materials
  • Umbilical cord segments
  • Phosphate‐buffered saline (DPBS, 1×; Corning, cat. no. 21‐031‐CV) or clinical‐grade normal saline (0.9% NaCl)
  • 0.2% collagenase
  • HUVEC medium (see recipe)
  • 3 oz. specimen container with lid (Medical Action Industries, Part no. 4938)
  • Clean pads (e.g., Durasorb Plus Disposable Chux Underpad)
  • Bard‐Parker protected disposable scalpels (BD Medical, cat. no. 372610)
  • 22 G × ½‐in. needles
  • Hemostat
  • 10‐ and 20‐ml syringes
  • 100‐ml sterile beakers
  • 50‐ml conical tubes
  • 37°C incubator
  • Centrifuge
  • Collagen‐coated 6‐well tissue culture (TC) plates

Basic Protocol 2: Reprogramming of HUVECs into hiPSCs

  Materials
  • HUVECs (see protocol 1)
  • HUVEC medium (see recipe)
  • Phosphate‐buffered saline (DPBS, 1×; Corning, cat. no. 21‐031‐CV)
  • Episomal DNA
    • Cocktail #1‐ pCXLE‐Oct4/shP53 (1 µg/µl) Cocktail #2‐ pCXLE‐Sox2/Klf4 (1 µg/µl) Cocktail #3‐ pCXLE‐LMyc/Lin28 (1 µg/µl)
  • Neon kit (Life Technologies, cat. no. MPK10025) containing:
    • Buffer R
  • Irradiated mouse embryonic fibroblasts (MEFs)
  • NutriStem medium (Stemgent)
  • bFGF (see recipe)
  • KOSR medium (see recipe)
  • ROCK inhibitor
  • 4% paraformaldehyde (PFA; Affymetrix, cat. no. 19943)
  • Blocking buffer (see recipe)
  • Primary antibodies including:
    • Mouse anti‐Tra‐1‐60
    • Mouse anti‐SSEA3
    • Rabbit anti‐Sox2
    • Rabbit anti‐Oct4
    • Mouse anti‐Tra‐1‐81
    • Rabbit anti‐Nanog
  • Secondary antibodies including:
    • CyTM3 goat anti‐mouse IgG + IgM antibody
    • FITC goat anti‐rabbit IgG Antibody
  • DAPI
  • 15‐ml conical tubes
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Neon electroporation system
  • 6‐well tissue culture (TC) plates
  • Dissection microscope
  • 24‐well Matrigel‐coated tissue culture (TC) plates
  • Aluminum foil

Basic Protocol 3: Affirming Pluripotence of the hiPSCs by Demonstrating Multi‐Germ Layer Lineage Differentiation Capability In Vitro

  Materials
  • Matrigel
  • hiPSCs (see protocol 2)
  • NutriStem medium
  • ROCK inhibitor
  • Endoderm induction medium (see recipe)
  • Phosphate‐buffered saline (DPBS, 1×; Corning, cat. no. 21‐031‐CV)
  • 4% paraformaldehyde (PFA; Affymetrix, cat. no. 19943)
  • Blocking buffer (see recipe)
  • Primary antibodies including:
    • Goat anit‐Sox17
    • Goat anti‐FoxA2
    • Rabbit anti‐PAX6
    • Mouse anti‐SMA
  • Secondary antibodies including:
    • Goat anti‐mouse
    • Goat anti‐rabbit
  • DAPI
  • Ectoderm differentiation medium (see recipe)
  • Dispase
  • Embryoid body (EB) differentiation medium (see recipe)
  • 0.1% gelatin (see recipe for gelatinized plates)
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Figures

Videos

Literature Cited

Literature Cited
  Batsali, A.K., Kastrinaki, M.C., Papadaki, H.A., and Pontikoglou, C. 2013. Mesenchymal stem cells derived from Wharton's Jelly of the umbilical cord: Biological properties and emerging clinical applications. Curr. Stem Cell Res. Ther. 8:144‐155.
  Baudin, B., Bruneel, A., Bosselut, N., and Vaubourdolle, M. 2007. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat. Protoc. 2:481‐485.
  Cohen, M.A., Itsykson, P., and Reubinoff, B.E. 2007. Neural differentiation of human ES cells. Curr. Protoc. Cell Biol. 36:23.7.1‐23.7.20.
  Hofmeister, CC, Zhang, J, Knight, KL, Le, P, and Stiff, PJ. 2007. Ex vivo expansion of umbilical cord blood stem cells for transplantation: Growing knowledge from the hematopoietic niche. Bone Marrow Transplant. 39:11‐23.
  Mahabeleshwar, G.H., Somanath, P.R., and Byzova, T.V. 2006. Methods for isolation of endothelial and smooth muscle cells and in vitro proliferation assays. Methods Mol. Med. 129:197‐208.
  Martín de Llano, J.J., Fuertes, G., García‐Vicent, C., Torró, I., Fayos, J.L., and Lurbe, E. 2007. Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels. Transl Res. 149:1‐9.
  Nagano, M., Yamashita, T., Hamada, H., Ohneda, K., Kimura, K., Nakagawa, T., Shibuya, M., Yoshikawa, H., and Ohneda, O. 2007. Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood. Blood 110:151‐160.
  Park, H.J., Zhang, Y., Georgescu, S.P., Johnson, K.L., Kong, D., and Galper, J.B. 2006. Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Rev. 2:93‐102.
  Roura, S., Bagó, J.R., Soler‐Botija, C., Pujal, J.M., Gálvez‐Montón, C., Prat‐Vidal, C., Llucià‐Valldeperas, A., Blanco, J., and Bayes‐Genis, A. 2012. Human umbilical cord blood‐derived mesenchymal stem cells promote vascular growth in vivo. PLoS One 7:e49447.
  Simões, I.N., Boura, J.S., dos Santos, F., Andrade, P.Z., Cardoso, C.M., Gimble, J.M., da Silva, C.L., and Cabral, J.M. 2013. Human mesenchymal stem cells from the umbilical cord matrix: Successful isolation and ex vivo expansion using serum‐/xeno‐free culture media. Biotechnol. J. 8:448‐458.
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