Identification of individual CD34+lin− daughter cells that retain multilineage differentiation potential after one division

Abstract

There is a need to determine whether culture conditions may exist which favor solely proliferative self-renewal of hematopoietic stem cells as opposed to proliferation with differentiation. A key to answering this is the ability to detect self-renewal in vitro. We have previously reported that TPO and FLT-3L can interact to promote doubling and phenotypic self-renewal of single cord blood CD34+Thy 1+lin− (lin−) cells and this cell fate/phenotype decision can be detected s early as the first cell division. To determine if individual daughter cells that remained lin− after 1 division still retained lymphoid/myeloid differentiation potential, single cell micromanipulation of doublets was performed. CB lin− cells were plated in Terasaki plates containing FLT3L+TPO. Each well was monitored daily for evidence of cell division. When a division was detected the cell doublet was stained for phenotype assessment and daughter cells that remained lin− were separated by micromanipulation and placed in different wells on AFT024 stroma with IL-7, SCF, FLT-3L. After 28 days, wells in which the single cell had proliferated were stained with FITC and PE-conjugated antibodies against lymphoid (CD2/3, CD16, CD19, CD56) and myeloid (CD13, CD15, CD33) specific markers to identify which cells of the doublet had multi-lineage differentiation potential. In preliminary experiments, with 783 single cells, 52% of the cells that divided, over 7 days, remained lin− (405 doublets). Of the 810 total cells, 73.5% of those were successfully separated and transferred to stroma. Staining, revealed that 27 doublets (90%) had at least 1 daughter cell that generated progeny with both L/M markers, and that 23% of the doublets separated had both daughters capable of generating L/M positive progeny. This assay may be useful to improve in vitro stem cell expansion conditions by identifying which single cells with retained phenotype can generate progeny with multilineage potential.