In vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells

Abstract

Summary. This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non-obese diabetic/severe combined immunode®cient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34 + cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL-3), IL-6 and granulocyte colony-stimulating factor, all at 10 ng/ml in serum-replete conditions. Transplantation of mice with fresh CB containing 3´10 4 CD34 + cells and 1±2 SCID repopulating cells (SRC) resulted in a median of 7á4% (0á4%-76á8%) human engraftment. When mice received the expanded product of 1±2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two-to fourfold.Expanded SRC produced long-term culture-initiating cells, clonogenic cells and CD34 + cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0á8%, range 0á0±24á0%) compared with 1±2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we con®rmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two-to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.

Identification of the CD33‐related Siglec receptor, Siglec‐5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias

“…Cord blood or bone marrow mononuclear cells (MNC) were prepared from 40–50 ml of CB or 5 ml bone marrow using lymphoprep ( d = 1·077 g/ml, Nycomed, Birmingham, UK) density gradient separation followed by treatment with ammonium chloride lysing solution for 15 min at 4°C to remove red cells. CD34 + cells were prepared as previously described (Denning‐Kendall et al , 2002) using the immunomagnetic MiniMACS column system with the CD34 + stem cell isolation kit (Miltenyi Biotec, Bisley, UK) as specified by the manufacturer. These preparations contained >80% CD34 + cells by flow cytometry analysis.…”

“…Siglec-5 is upregulated later than CD33 during in vitro differentiation of CD34 + cells To further investigate when Siglec-5 might be switched on during myelopoiesis, CB purified CD34 + cells were cultured with a combination of cytokines (SCF, IL-3, IL-6, G-CSF, Flt-3 and TPO) previously shown to induce both expansion and maturation of myeloid progenitors from CD34 + stem cells (Denning-Kendall et al, 2002).…”

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34+CD133+CXCR4low fraction

“…In this way, we obtained unequivocal proof that SRC activity was enhanced after the ex vivo expansion culture. To quantify these data, we performed a limiting dilution assay 31 on the CD34 + CXCR low CD133 + population at the beginning and after culture (see further text for the CD133 issue), the results of which showed an ~4.2-fold expansion of SRCs after 7 days with respect to day 0 ( Figure 3A-C ). Furthermore, the mean individual SRC proliferative capacity was ~4-fold higher after 7 days of expansion culture compared to the capacity at day 0 ( Figure 3D ).…”

Ex vivo expansion of CD34+ and T and NK cells from umbilical cord blood for leukemic BALB/C nude mouse transplantation

“…Ex vivo expansion of human UCB stem and progenitor cells becomes a critical issue in UCB clinical transplantation and immunotherapy. A small number of groups reported that certain combination of cytokines could preserve the activity of transplanted cells [13,22,23]. Guenechea et al [7] found that these cells retained their capacity to support long-term repopulation with delayed engraftment as compared to fresh cells, whereas Piacibello et al [24] reported that CD34 + CB cells, expanded for up to 10 weeks, maintained their in vivo repopulating potential.…”