Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.
BACKGROUND
Regenerative stem cell therapy (SCT) is currently being tested in clinical trials. The ideal type and source of cells have not yet been defined. Lineage (Lin) depletion is an experimental procedure capable of enriching all recently recognized SC types with regenerative potency. This study was performed to define a practicable monoclonal antibody (MoAb) cocktail for Lin depletion and to test whether clinical-scale Lin depletion is possible.


METHODS
MoAbs (CD2/14/15/19/41/56/glycophorin A) were selected to mark seven mature hematopoietic lineages. Lin7-negative (Lin7NEG) cells were analyzed in peripheral blood (PB, n = 9), mobilized PB (MPB, n = 5), umbilical cord blood (UCB, n = 5), and marrow aspirates (BM, n = 4) by flow cytometry. Preclinical Lin depletion was tested with leukapheresis products from PB following good manufacturing practice (GMP) principles.


RESULTS
Lin7NEG cells comprised 0.23 +/- 0.04, 0.27 +/- 0.03, 0.53 +/- 0.07, and 0.49 +/- 0.03 percent of PB, MPB, UCB, and BM, respectively. Basophils, CD34+, and dendritic cells constituted the major Lin7NEG subpopulations (84 +/- 2, 90 +/- 3, 40 +/- 3, and 80 +/- 3% in PB, MPB, UCB, and BM, respectively). Minor populations included CD7- and CD45- cells. Preclinical CD2/14/15/19/56 (Lin5) depletion after automated red blood cell and platelet reduction resulted in up to a 16.7-fold enrichment of CD34+ and CD34-/Lin5NEG cells.


CONCLUSIONS
A seven-MoAb cocktail is sufficient to label more than 99 percent of nucleated cells in PB, MPB, UCB, and BM. Preclinical Lin depletion can be performed under GMP conditions from PB apheresis procedures.
Strunk
Dirk
D
Department of Internal Medicine, Division of Hematology, Medical University, Graz, Austria. dirk.strunk@klinikum-graz.at
Rohde
Eva
E
Lanzer
Gerhard
G
Linkesch
Werner
W
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015415:Biomarkers; D019070:Cell Lineage / Q000276:immunology*; D002469:Cell Separation / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D007937:Leukapheresis / Q000379:methods; D008297:Male; D008565:Membrane Proteins / Q000276:immunology; D008875:Middle Aged
D000911:Antibodies, Monoclonal; D015415:Biomarkers; C119588:LIN-7 protein, mammalian; D008565:Membrane Proteins