The purification of CD34+ cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors

Authors

  • PIERRE CHARBORD,

    1. 0 Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, and 1 Service de Gynécologie Obstétrique, Centre Hospitalier, Besançon, France
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  • ISOBEL NEWTON,

    1. 0 Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, and 1 Service de Gynécologie Obstétrique, Centre Hospitalier, Besançon, France
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  • LAURENT VOILLAT,

    1. 0 Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, and 1 Service de Gynécologie Obstétrique, Centre Hospitalier, Besançon, France
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  • JEAN PATRICK SCHAAL,

    1. 0 Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, and 1 Service de Gynécologie Obstétrique, Centre Hospitalier, Besançon, France
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  • PATRICK HERVE

    1. 0 Laboratoire d'Etude de l'Hématopoièse, Etablissement de Transfusion Sanguine, Besançon, and 1 Service de Gynécologie Obstétrique, Centre Hospitalier, Besançon, France
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Dr P. Charbord Directeur de Recherches INSERM, Establissement de Transfusion Sanguine, BP 1937, 1 boulevard Fleming, 25020 Besançon Cédex, France.

Abstract

This study was performed to assess the methods which gave maximal recovery of purified CD34/45+ cells from a cord blood specimen and optimal growth of progenitors cultured from the purified cells. Cord blood samples were separated using Percoll gradients (either one (1.080) or two successive (1.080 and 1.068) gradient(s)) and commercially available devices for CD34+ cell isolation (affinity columns as manufactured by CellPro Inc. or immunomagnetic separation procedure as devised by Baxter Inc.). ‘CellPro’ or ‘Baxter’ techniques gave similar results in terms of nucleated, CD34/45+ and progenitor cell concentration; however, the yield of CD34/45+ cells in the CD34+ enriched fraction was significantly higher when using the ‘CellPro’ technique. We also found significantly higher numbers of CD34/45+ cells in the CD34+ enriched final fraction when using only one, 1.080, Percoll density gradient in the first separation step. Using one density separation step followed by the ‘CellPro’ technique, we obtained an average of 3×106 purified CD34/45+ cells from samples containing 8.5×108 nucleated cells. Granulomonocytic progenitors (CFU-GM) and mixed progenitors (CFU-GEMM) cells from light-density and purified CD34/45+ cell fractions were evaluated. We found that 20–30% of the light-density cells and the purified CD34/45+ cells, yielded a granulomonocytic colony in serum free medium in the presence of interleukins 3 and 6, erythropoietin, granulomonocytic and granulocytic colony-stimulating factors and stem cell factor. The addition of tumour-necrosis factor α to the cocktail significantly improved the growth of CFU-GEMM allowing 10% of the purified CD34/45+ cells to yield a mixed colony, which confirms the role of this cytokine on CD34+ cells from cord blood. This study provides an improved method for the recovery of CD34/45+ purified cells and their colony formation. These methods may serve as a basis for studies on CD34/45+ cell amplification and gene transfer.

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