Engraftment of Cultured Human Hematopoietic Cells in Sheep

Abstract

In an effort to expand human hematopoietic progenitors and stem cells in vitro, we cultured human CD34+c-kitlow bone marrow cells in suspension in the presence of KIT ligand, FLK2/FLT3 ligand, interleukin-6 (IL-6), and erythropoietin with or without IL-3 and tested their engrafting capabilities by injecting them into sheep fetuses. As markers for engraftment, we analyzed CD45+ cells and karyotypes of the colonies grown in methylcellulose culture. In three separate experiments, day-60 engraftment in the bone marrow was seen with both fresh cells and cells cultured in the presence or absence of IL-3. When fetuses were allowed to be born and analyzed for CD45+cells, no long-term engraftment was seen with cultured cells. We then pooled the CD45+ cells of the fetal samples and transplanted them into secondary recipient fetuses. Day-60 engraftment in the secondary recipients was again noted when transplantation in the primary recipients was initiated with fresh cells. There were 3 cases in which cultured cells showed signs of engraftment in the secondary recipients, but the remaining 24 cases showed no signs of engraftment. These data documented that suspension culture for 2 weeks of enriched adult human bone marrow cells can maintain short-term (2 months) engrafting cells, but may not maintain longer term engrafting cells. This sheep/human xenograft model may serve as an excellent method for the evaluation of the engraftment potential of in vitro-expanded cells.

Murine Bone Marrow Cells Cultured Ex Vivo in the Presence of Multiple Cytokine Combinations Lose Radioprotective and Long-Term Engraftment Potential

“…The addition of IL-11 to cytokine combination (i) and then IL-3 to cytokine combination (ii) would provide evidence if single cytokines influenced survival rates and engraftment levels in the transplanted animals. Previous reports indicated that the addition of IL-3 to cell cultures prior to transplantation had a detrimental effect on long-term engraftment in the sheep (31) as well as in the NOD-SCID mouse (21,32). Although our results with cytokine combinations containing IL-3 were not positive, the results obtained by transplanting cells exposed to IL-3 were not demonstrably worse.…”

Asymmetric Cell Divisions of Human Hematopoietic Stem and Progenitor Cells Meet Endosomes

“…And although it was found that HSC can be maintained in close contact to certain stroma cells, [15][16][17][18][19] stroma-free culture conditions regularly fail to even support long-term ex vivo maintenance of primitive hematopoietic cells. [20][21][22] Therefore, the surrounding environment and thus a special combination of extrinsic factors are required to keep HSC in a primitive state. These observations are compatible with the model of the HSC niche that was already hypothesized by Raymond Schofield in 1978.…”

Stem Cell Transplantation in the Fetus

“…141,151,152 Despite the genetic differences between sheep and humans, maintenance of human HSCs in the sheep was demonstrated by harvesting human cells from primary recipients and successfully transplanting them into secondary sheep. 142,[153][154][155] These studies demonstrate that IUT can result in hematopoietic chimerism, even across widely disparate species barriers.…”

“…Furthermore, liver engraftment was observed in some nonhuman primates transplanted with fetal hepatocytes. 155,180,181 For the purpose of producing a specific liver gene product, hepatocytes might not be the only cells that can be transplanted. Takahashi et al 182 used amniotic epithelial cells that were transfected with adenoviral vector and transplanted into fetal rat liver to show that other cell types can be used to deliver gene products to a fetus.…”