Assessment of Short-Term Engraftment Potential of Ex Vivo Expanded Hematopoietic Stem Cells Using Normal Fetal Mouse in Utero Transplantation Model

Abstract

Objective: Ex vivo expansion is a promising strategy to overcome the low number of human umbilical cord blood hematopoietic stem cells (hUCB-HSCs). Although based on the obtained results in unnatural physiological condition of irradiated genetically immune-deficient mouse models, there has always been concern that the expanded cells have less engraftment potential. The purpose of this study was to investigate effect of common ex vivo expansion method on engraftment potential of hUCB-mononuclear cells (MNCs), using normal fetal mouse, as a model with more similarity to human physiological conditions.

Materials and methods: In this experimental study, briefly, isolated hUCB-MNCs were cultured in common expansion medium containing stem cell factor, Flt3 ligand and thrombopoietin. The unexpanded and expanded cells were transplanted to the fetal mice on gestational days of 11.5-13.5. After administration of human hematopoiesis growth factors (hHGFs), presence of human CD45⁺ cells, in the peripheral blood of recipients, was assessed at various time points after transplantation.

Results: The expanded MNCs showed 32-fold increase in the expression of CD34⁺38^- phenotype and about 3-fold higher clonogenic potential as compared to the uncultured cells. Four weeks after transplantation, 73% (19/26) of expanded-cell recipients and 35% (7/20) of unexpanded-cell recipients were found to be successfully engrafted with human CD45⁺ cells. The engraftment level of expanded MNCs was significantly (1.8-fold) higher than unexpanded cells. After hHGFs administration, the level was increased to 3.2, 3.8 and 2.6-fold at respectively 8, 12, and 16 weeks of post transplantation. The increased expression of CXCR4 protein in expanded MNCs is a likely explanation for the present findings.

Conclusion: The presented data showed that expanded MNCs compared to unexpended cells are capable of more rapid and higher short-term engraftment in normal fetal mouse. It could also be suggested that in utero transplantation (IUT) of normal fetal mice could be an appropriate substitute for NOD/SCID mice in xenotransplantation studies.

Keywords: Chimerism; Cord Blood Stem Cell Transplantation; Hematopoietic Stem Cells.

Fig.1

In utero transplantation of hUCB-MNCs using handmade glass micropipettes. A. Hair removal and sterilization of the surgical site, B. The uterine horns were exteriorized, C. 2-3×106 non-cultured hUCB-MNC or their entire progeny, following 10 days expansion, were injected intraperitoneally to each recipient, and D. The uterine horns were returned to the abdominal cavity and the incision was closed. hUCB-MNCs; Human umbilical cord blood-mononuclear cells.

Fig.2

Characterization of human donor cells. A. Representative dot plots of hUCB-MNCs before and after expansion, B. Number of the total nuclear cells was significantly increased after expansion for 10 days in STF medium (n=9, **; P<0.01), C. Percentage of CD34⁺ and CD34⁺ CD38^- cells in hUCB-MNCs at day 0 and after 10 days of expansion in STF medium, D. Number of CD34⁺ and CD34+CD38- cells were significantly increased after 10 days expansion in STF medium (n=9, *; P<0.05), and E. CFU number in 2000 cells of day 0 uncultured hUCB-MNCs and the progeny of an equivalent number of expanded hUCB-MNCs (n=3, ** P<0.01, ***; P<0.001). Fold expansion was calculated by dividing the absolute output number of the expanded cells expressing a specific phenotype after 10 days of culture by the respective number on day 0. hUCB-MNCs; Human umbilical cord blood-mononuclear cells, CFU; Colony forming unit, STF; SCF+TPO+FLT3L, BFU; Burst forming unit, GM; Granulocyte-macrophage, and GEMM; Granulocyte erythrocyte macrophage monocyte.

Fig.3

CXCR4 overexpression and increased in vitro homing potential of the expanded cells. A. Representative flow-cytometer analysis of CXCR4 expression in different cells. Filled curves indicate isotype control and unfilled curves indicate labeled cells and B. Percentage of the STFexpanded hUBC-MNCs moved through the transwell in response to SDF- 1 versus uncultured cells (day 0) (**; P<0.01, n=5). STF; SCF+TPO+FLT3L, SDF-1; Stromal cell derived factor-1, and hUCB-MNCs; Human umbilical cord blood-mononuclear cells.

Fig.4

Outcome of in utero surgery. A. Number of the pregnant mice and their embryos used through the whole experiment and B. Comparison of survival rates within expanded cell-recipients, unexpanded cell-recipients and sham. MNC; Mononuclear cells.

Fig.5

Short-term in vivo homing of hUCB-MNCs. A. Analysis of PKH26 fluorescence of 20000 MNCs by flow-cytometer, before (unfilled curve) and after staining with PKH26 (gray filled curve) and B. Identification of PKH-stained hUCB-MNCs, 48 hours after infusion. Prepared frozen sections from the spleen and liver of transplanted fetuses were screened for the presence of PKH-bright cells (red: PKH26, blue: DAPI). hUCB-MNCs; Human umbilical cord blood-mononuclear cells.

Fig.6

The ex vivo expanded hUCB-MNCs have higher engraftment potential than unexpanded cells. A. Representative flow-cytometer analysis for human cell engraftment in peripheral blood of the expanded- and unexpanded-cell recipients. Peripheral blood of normal mouse was employed as negative control, B. Mean human engraftment levels in peripheral blood of NMRI mice fetal transplanted with expanded or unexpanded hUCBMNCs. Mice with ≥0.2% human cells were considered chimeric, C. Representative flow-cytometer analysis for human cell engraftment in peripheral blood of the expanded- and unexpanded-cell recipients, and D. Identification of human CD45⁺ cells in the bone marrow of recipient mice. Bone marrow smears of 4-months-old transplanted mice were screened for the expression of human nuclear antigen (arrows; As mentioned they are human CD45⁺ cells). hUCB-MNCs; Human umbilical cord blood-mononuclear cells.