An improved panning technique for the selection of CD34+ human bone marrow hematopoietic cells with high recovery of early progenitors.
The human hematopoietic pluripotent repopulating "stem cell" is thought to be present within a minor subpopulation of CD34+ cells. This has not been definitively shown, although the more primitive CD34+ cell subset contains precursors for all lymphoid and nonlymphoid cell lineages. When purifying CD34+ cells, it is important to recover these early progenitors, which are more strongly immunoadsorbent to the separation devices. Using a commercialized panning system (AIS CELLector flasks), we observed that a high degree of purity requires a thorough washing procedure so that cells not binding or weakly binding to CD34 antibodies are removed. High recoveries can be obtained if the adherent cells are then efficiently detached by a 2-hour incubation in culture medium without added cytokines. In this way, we can routinely obtain 93.5 +/- 3.4% purity of CD34+ cells with a 74% yield of the multipotent colony-forming units (CFU-GEMM). Complete recovery of the putative "stem cell," or at least the early progenitor cell compartment (CD34+ CD38low/- CD34+ Thy-1+ cells), is also obtained. More than 30% of these cells can generate day-14 colonies in vitro. Comparable results were obtained when the separation was scaled up for clinical application using appropriate large-scale devices. The various incubation times of the procedure can be easily adjusted to the work schedule. This renders the procedure easy to handle, efficient, safe, and, because the cells can be observed under light microscopy, easy to control.
Cardoso
A A
AA
Laboratoire de Biologie Cellulaire et Moleculaire des Facteurs de Croissance, CNRS UPR 272, IRSC, Villejuif, France.
Watt
S M
SM
Batard
P
P
Li
M L
ML
Hatzfeld
A
A
Genevier
H
H
Hatzfeld
J
J
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation / Q000032:analysis; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D004921:Erythropoietin / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000032:analysis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D007163:Immunosorbent Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D016184:Receptors, Colony-Stimulating Factor / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D018800:Thy-1 Antigens / Q000032:analysis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D003115:Colony-Stimulating Factors; D006684:HLA-DR Antigens; D007377:Interleukin-3; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D004921:Erythropoietin; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1