An in vivo model of double-unit cord blood transplantation that correlates with clinical engraftment

Abstract

Double-unit cord blood transplantation (DCBT) appears to enhance engraftment despite sustained hematopoiesis usually being derived from a single unit. To investigate DCBT biology, in vitro and murine models were established using cells from 39 patient grafts. Mononuclear cells (MNCs) and CD34(+) cells from each unit alone and in DCB combination were assessed for colony-forming cell and cobblestone area-forming cell potential, and multilineage engraftment in NOD/SCID/IL2R-γ(null) mice. In DCB assays, the contribution of each unit was measured by quantitative short tandem repeat region analysis. There was no correlation between colony-forming cell (n = 10) or cobblestone area-forming cell (n = 9) numbers and clinical engraftment, and both units contributed to DCB cocultures. In MNC transplantations in NOD/SCID/IL2R-γ(null) mice, each unit engrafted alone, but MNC DCBT demonstrated single-unit dominance that correlated with clinical engraftment in 18 of 21 cases (86%, P < .001). In contrast, unit dominance and clinical correlation were lost with CD34(+) DCBT (n = 11). However, add-back of CD34(-) to CD34(+) cells (n = 20) restored single-unit dominance with the dominant unit correlating not with clinical engraftment but also with the origin of the CD34(-) cells in all experiments. Thus, unit dominance is an in vivo phenomenon probably associated with a graft-versus-graft immune interaction mediated by CD34(-) cells.

Figure 1

Methodology of in vitro assays. MNCs or CD34⁺ cells from each unit alone and in DCB combination were plated in methylcellulose and on MS5 stroma for CFC and CAFC assays, respectively. CFC assay was also performed after overnight incubation with thrombopoietin (TPO), kit ligand (KL), and Fms-like tyrosine kinase 3 ligand (Flt3L). CFC and CAFC were harvested from DCB cocultures for DNA extraction and quantitative PCR of short tandem repeats to establish donor origin. (Bottom left photograph) Composite of 4 images taken from the CFC experiments. Photomicrograph taken with Nikon Eclipse Ti-S microscope; objectives: 4× for CFC scoring, 10× and 20× for CAFC scoring. A Spot camera (Diagnostic Instruments) with Spot Version 4.0.2 software was used.

Figure 2

Methodology of murine experiments. NSG mice were sublethally irradiated and transplanted by tail vein injection.

Figure 3

Representative flow cytometric analysis of human cell engraftment of an NSG mouse transplanted with human double-unit CB and gated on live cells. (A) Fluorescence-activated cell sorter analysis of the murine BM, spleen, and thymus after staining with PE-conjugated anti–human CD45 antibodies. (Bottom panel) Percentage of human CD45⁺ cells (51.8% in BM, 32.5% in spleen, and 7.1% in thymus). Analysis of murine BM revealed that the majority (71%) of human CD45⁺ cells were CD19⁺ (B lymphocytes), 6% CD33⁺ (myeloid cells), 3% CD34⁺, and less than 1% CD56⁺ (NK cells; data not shown). Staining for monocytes, erythroid cells, and megakaryocytes was not performed. (B) Human T-cell engraftment in the murine thymus. Of the human CD45⁺ cells, the majority were CD4⁺/CD8⁺.