Selection of CD34⁺ HSC from Pooled Cord Blood Samples Does Not Result in Co-Purification of Potential Alloreactive CD3⁺ T Cells.

Most systems for studying the ex vivo expansion of human UCB-HSC require enrichment of the HSC population, typically by CD34 selection. Because the number of HSC present in a single UCB collection is limited, one approach to increase the total HSC is to pool multiple UCB samples. A potential complicating factor with this method is that alloreactive T lymphocytes present in whole UCB samples would be expected to bind to cells displaying allogeneic HLA, including HSC, and would therefore co-purify with CD34⁺ UCB-HSC during isolation. Such complexes would be excluded from ISHAGE analysis by the side scatter gate, which is set to exclude aggregates. Alloreactive T cells would be expected to contain cytotoxic T lymphocytes [CTL], which could potentially inhibit or completely abrogate HSC expansion.

In order to determine whether CD3⁺ T lymphocytes co-purify with CD34⁺ UCB-HSC in pooled samples, UCB pools were prepared containing 2 to 4 UCB samples. CD34⁺ HSC were isolated by MACS and analyzed by flow cytometry and antibodies to human CD45, CD34 and CD3, with and without ISHAGE gates. Samples of CD34⁺ HSC purified from a single UCB collection were analyzed concurrently to give the background value for co-purification of syngeneic T cells and formation of T:HSC aggregates in the CD34-selected products. We found a limited number of CD3⁺ T cells present in CD34⁺ HSC isolated from single UCB collections [mean = 0.25%, range = 0 – 1.1%]. That value was slightly elevated when pools of UCB were used [mean = 0.44%; range = 0 – 1.9%]. There are few, if any, CD34⁺/CD3⁺ cells that can be detected by either the standard ISHAGE gating or by gating merely on CD45⁺ cells with no side scatter gating [0.02 – 0.04%]. This was true of CD34⁺ HSC isolated from either pooled UCB or single UCB collections.

Based on these results, we conclude that there is not significant co-purification of CD3⁺ T cells with CD34⁺ UCB-HSC, and that any such complexes that form are not found at any greater frequency in UCB pools than in single UCB collections.