Nonirradiated NOD,B6.SCID Il2rγ−/− KitW41/W41 (NBSGW) Mice Support Multilineage Engraftment of Human Hematopoietic Cells

In this study, we demonstrate a newly derived mouse model that supports engraftment of human hematopoietic stem cells (HSCs) in the absence of irradiation. We cross the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) strain with the C57BL/6J-KitW-41J/J (C57BL/6.KitW41) strain and engraft, without irradiation, the resulting NBSGW strain with human cord blood CD34+ cells. At 12-weeks postengraftment in NBSGW mice, we observe human cell chimerism in marrow (97% ± 0.4%), peripheral blood (61% ± 2%), and spleen (94% ± 2%) at levels observed with irradiation in NSG mice. We also detected a significant number of glycophorin-A-positive expressing cells in the developing NBSGW marrow. Further, the observed levels of human hematopoietic chimerism mimic those reported for both irradiated NSG and NSG-transgenic strains. This mouse model permits HSC engraftment while avoiding the complicating hematopoietic, gastrointestinal, and neurological side effects associated with irradiation and allows investigators without access to radiation to pursue engraftment studies with human HSCs

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

Background: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. Methodology: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit + hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. Conclusions/Significance: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind t

Characterization of Kit-129 binding and ligand-blocking activity.

<p>(a) Kit-129 has high specificity for BJAB c-kit cells. Shown are mean fluorescence values of Alexa-647-labeled Kit-129 aptamer tested in serial dilutions from 0 to 100 nM by flow cytometry. (b) Alexa-647-labeled Kit-129 binds to mouse mast cell line 11P0-1 (which naturally expresses c-kit) as well as BJAB c-kit cells, while a scrambled version of this aptamer (Kit-129SC) does not. (c) Kit-129 blocks SCF binding to c-kit expressed by BJAB cells. Serial dilutions of Kit-129 or Kit-129SC were added to either cell line followed by addition of biotin-labeled SCF and streptavidin-Alexa-647 conjugate. SCF binding was measured by flow cytometry.</p

Characterization of c-kit aptamer binding by flow cytometry.

<p>(a) Mean fluorescence values from duplicate binding experiments with 10 c-kit aptamers tested at 100 nM with BJAB c-kit (blue) or BJAB parental (red) cells. FACS analysis with 50 nM Kit-129 tested on (b) BJAB c-kit cells and <b>(c)</b> BJAB parental cells.</p

Top 20 c-kit aptamer sequences isolated via STACS (boldface indicates aptamers chosen for further analysis).

<p>Top 20 c-kit aptamer sequences isolated via STACS (boldface indicates aptamers chosen for further analysis).</p

Kit-129 competes with ACK2 in binding to BJAB c-kit cells.

<p>Buffer or Kit-129 aptamer and phycoerythrin (PE)-labeled anti-c-kit antibody ACK2 were added to BJAB-c-kit cells and incubated for 1 hour on ice. ACK2-PE binding was measured by flow cytometry.</p

Identification of c-kit expressing bone marrow cells.

<p>Double labeling of mouse bone marrow cells with anti-CD45 antibody and (a) Kit-129 aptamer or (b) rabbit anti-c-kit antibody 2B8.</p