Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence.

作者: Bertoncello I , Hodgson Gs , Bradley Tr

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摘要: A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells preferentially home to the marrow of lethally irradiated mice be enriched and separated from majority spleen colony-forming are assayed 13 days after transplantation (CFU-S13). First, bone centrifuged in a discontinuous bovine serum albumin gradient. Low-density (1.060-1.068 g/cm3) harvested labeled with supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled analyzed using fluorescence-activated sorter, sorted on basis relative Rh123 fluorescence within predetermined forward versus 90 degrees red light scatter window has been optimized for recovery enrichment repopulating ability (MRA). Cells MRA were characterized by relatively low could fraction fluoresced more intensely contained CFU-S13 but MRA. The ratios low-fluorescent:high-fluorescent 20:1 0.5:1, respectively, resultant ratio MRA:CFU-S13 was 556:1 low-fluorescent fraction, 48:1 high-fluorescent fraction. platelet cofractionate whereas erythroid remain associated CFU-S13. High-proliferative-potential macrophage form colonies agar presence combined stimulus pregnant mouse uterus extract plus human-spleen-conditioned medium present both fractions. Between 20% 30% recovered approximately 1% total nucleated starting preparation, enrichments 25- 30-fold have achieved permitting reconstitution host as few 400 donor cells.

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