Isolation of Mesenchymal Stem Cells from Human Cord Blood

Anita Laitinen1, Jarmo Laine1

1 Finnish Red Cross Blood Service, Helsinki
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.3
DOI:  10.1002/9780470151808.sc02a03s1
Online Posting Date:  June, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Cord blood is a rich source of stem cells especially for hematopoietic stem cells. Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. However, these fibroblast‐like multipotent progenitor cells are rather rare in cord blood. Many different methods have been used for their culture. This unit describes one method to obtain MSCs from cord blood and another method to differentiate these cells into osteoblasts, which is one of the lineages that mesenchymal stem cells are capable of differentiating into. The starting material for the protocol is cord blood–derived mononuclear cells. As cord blood contains a great number of erythroid precursors, the glycophorin A–positive cells are depleted using magnetic cell separation to reduce their presence in MSC culture. Osteoblast differentiation and a method to demonstrate the result of the differentiation are also described in this unit. Curr. Protoc. Stem Cell Biol. 1:2A.3.1‐2A.3.7. © 2007 by John Wiley & Sons, Inc.

Keywords: cord blood; mesenchymal stem cells; glycophorin A; osteoblasts; von Kossa staining

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

Table of Contents

  • Basic Protocol 1: Isolation of Mesenchymal Stem Cells from Human Cord Blood Mononuclear Cells
  • Basic Protocol 2: Differentiation of Mesenchymal Stem Cells to Osteoblasts
  • Support Protocol 1: Von Kossa Staining for Osteoblasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Mesenchymal Stem Cells from Human Cord Blood Mononuclear Cells

  Materials
  • Mononuclear cells from fresh human cord blood (unit 2.1)
  • Labeling buffer (see recipe), degassed
  • Glycophorin A MicroBeads (Miltenyi Biotec)
  • MSC culture medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% (w/v) trypsin/1 mM EDTA solution
  • Freezing medium: 50% MSC culture medium/40% (v/v) fetal bovine serum/10% (v/v) dimethyl sulfoxide (DMSO), chilled
  • 10‐ml and 50‐ml centrifuge tubes
  • Centrifuge (preferably swinging‐bucket rotor)
  • MACS LD columns (Miltenyi Biotech)
  • Magnetic cell separator (MidiMACS, Miltenyi Biotec)
  • Fibronectin‐coated 6‐well plates (see recipe)
  • Cryovials
  • Cell‐freezing container
  • Liquid nitrogen
  • Additional reagents and equipment for preparing mononuclear cell suspension (unit 2.1) and cell counting (Phelan, )

Basic Protocol 2: Differentiation of Mesenchymal Stem Cells to Osteoblasts

  Materials
  • Mesenchymal stem cells (MSCs; protocol 1)
  • MSC culture medium (see recipe)
  • Differentiation medium (see recipe)
  • 24‐ or 6‐ well tissue culture plates
  • Additional reagents and equipment for preparation of mesenchymal stem cell suspension ( protocol 1) and von Kossa staining ( protocol 3)

Support Protocol 1: Von Kossa Staining for Osteoblasts

  Materials
  • Differentiated cells ( protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% (w/v) paraformaldehyde (pH 7.2; see recipe)
  • Deionized water
  • 1% (w/v) silver nitrate
  • 2.5% (w/v) sodium thiosulfate
  • Mirror or aluminum foil
  • UV light source or 60‐W lamp
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bieback, K., Kern, S., Kluter, H., and Eichler, H. 2004. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625‐634.
   Friedenstein, A.J., Gorskaja, J.F., and Kulagina, N.N. 1976. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol. 4:267‐274.
   Jaiswal, N., Haynesworth, S.E., Caplan, A.I., and Bruder, S.P. 1997. Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro. J. Cell Biochem. 64:295‐312.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A3F.1‐A.3F.18.
   Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284:143‐147.
   Reyes, M., Lund, T., Lenvik, T., Aguiar, D., Koodie, L., and Verfaillie, C.M. 2001. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615‐2625.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library