Injection of Blastocysts from Immunocompetent Mice with Human HPC Having High ALDH Activity Results in Chimeric Animals with Low-Level Human Contribution in Hematopoietic and Non-Hematopoietic Tissues.
Authors: Giver Cynthia R. , Foster Sandra J. , Martin William D. , Balber Andrew E. , Waller Ned 
Published: October 2019

Abstract
The high cytosolic aldehyde dehydrogenase (ALDH) activity in hematopoietic progenitor cell (HPC) populations allows flow sorting of ALDH bright (ALDHbr), side scatter low cell populations highly enriched for human cells with long-term hematopoietic repopulating capacity. We are determining whether human ALDHbr populations and their CD34+ and CD34neg subsets sorted from umbilical cord blood (UCB) or adult G-CSF-mobilized peripheral blood (MPB) can engraft murine embryos and generate chimeric mice with partial human contribution. For each cell population, 50 – 75 sorted cells were injected into 3.5-day C57BL/6 blastocysts that were then transferred to surrogate mothers. We harvested 4 – 10 viable embryos/group at day 12.5 – 15.5; the ratio of viable embryos recovered/blastocysts transferred was not significantly different among the groups. Because cells in ALDHbr populations express surface markers characteristic of several stem cell types, and because some HPC may contribute to development in other tissues, we analyzed human cell content in both hematopoeitic and non-hematopoeitic tissues of the resulting animals. Human DNA was detected in dissected embryonic tissues by real-time PCR analysis of the repetitive α-satellite DNA on human chromosome 17. The detection limit for human content was 0.00003% (1 human cell / 3.3x106 mouse cells). The frequencies of positive tissues were similar among embryos produced using ALDHbr UCB cells (n=10), ALDHbrCD34+ UCB cells (n=6), and ALDHbrCD34neg MPB cells (n=10): 10–20% for brain and placenta, 20 – 50% for liver, 30 – 80% for lung and yolk sac (p=0.0001 compared to brain and placenta), and 60 – 100% for heart (p<0.0001). Among the positive samples, the average human content was lowest in brain tissue (0.00008%, 1/1.3x106) and highest in heart tissue (0.0015%, 1/6.7 x104). In embryos derived from blastocysts injected with ALDHbrCD34+ MPB cells, 2/4 brain, heart, and placenta samples, 3/4 liver, and all (4/4) lung and yolk sac samples were chimeric. Again, the brain and placenta had lower human content than other tissues. One embryo in the ALDHbrCD34+ MPB group had human content of 0.01% (1/1x104) in heart, 0.03% (1/3.3x103) in yolk sac and 0.2% (1/500) in liver. Long-term chimerism was assayed in 10-week-old pups from blastocysts transplanted with ALDHbrCD34+ or ALDHbrCD34neg MPB cells (5 mice each). In recipients of ALDHbrCD34+ cells, human DNA was detected in 1 lung, 1 spleen, and 3 bone marrow samples. In the ALDHbr CD34− recipients, 1 lung, 1 brain, 2 liver, and 3 bone marrow samples were positive. The human contribution in positive tissues from 10-week pups was less than 0.0005%. No human signal was detected in the heart of any of these pups. In conclusion, the results demonstrate that both ALDHbrCD34+ and ALDHbrCD34neg human cells are capable of expansion in embryos of an immunocompetent mouse strain after blastocyst injection, and suggest that a subset of primitive stem cells reside in the ALDHbrCD34neg population. The reduced human content in 10-week old pup tissues suggests that human cells have a proliferative disadvantage or are cleared following development of the mouse immune system. Additional studies of the distribution and fates of CD34+ and CD34neg human ALDHbr cells in fetal and post-natal murine tissues following blastocyst injection are underway.