Reconstruction of hematopoietic inductive microenvironment after transplantation of VCAM-1-modified human umbilical cord blood stromal cells

Abstract

The hematopoietic inductive microenvironment (HIM) is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood-derived stromal cells (HUCBSCs) and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34(+) cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1)-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were "homed in" to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.

Figure 1. Dynamic observation of the growth of Dexter-cultured HUCBSCs.

1-A: Dexter-cultured HUCBSCs formed adherent cells on day 3. 1-B: Dexter-cultured HUCBSCs formed smaller stromal cell colonies on day 14. 1-C: There were larger and more stromal cell colonies in Dexter-cultured HUCBSCs on day 21. 1-D: Stromal cells became confluent in Dexter-cultured HUCBSCs on day 28. (inverted microscope, ×100). 1-E: Fibroblast-like (thick arrow) and “small-round” cells (thin), (Wright's staining, inverted microscope×1000). 1-F: Macrophage-like cells, (inverted microscope ×1000).

Figure 2. HUCBSCs transfected with the Ad-VCAM-1-EGFP adenovirus plasmid.

2-A: HUCBSCs before VCAM-1 transfection. 2-B: HUCBSCs after VCAM-1 transfection. 2-C: Detection of VCAM-1 expression before and after transfection using RT-PCR, M: DNA marker; 1,2,3: before transfection; 3,4,5: after transfection. 2-D: The weak expression of VACM-1 in HUCBSCs before transfection. 2-E: The increased expression of VCAM-1 in HUCBSCs after transfection. 2-F: Immunofluorescence detection of VCAM-1 of HUCBSCs after transfection.

Figure 3. The effect of HUCBSCs on the in vitro proliferation of cord blood CD34⁺ cells.

3-A: Culture of CFU-GM from human cord blood CD34⁺ cells. 3-B: Culture of BFU-E from human cord blood CD34⁺ cells. 3-C: Culture of CFU-Meg from human cord blood CD34⁺ cells. 3-D: Comparison of numbers of colonies in the three groups. * compared with control group, P<0.01; ** compared with control and HUCBSCs groups, p<0.01.

Figure 4. Dynamic observation of the nude mouse hemogram, bone marrow stromal CFU-F count, and bone marrow pathological slices after different doses of irradiation.

4-A: WBC counts. 4-B: RBC counts. 4-C: Hemoglobin counts. 4-D: PLT counts. 4-E: CFU-F counts in bone marrow stromal cells, * compared with control, 3.5 and 5 Gy groups, P<0.01. 4-F: Nude mouse bone marrow pathological slices after 6.5 Gy irradiation. 4-G: Nude mouse bone marrow pathological slices after 8 Gy irradiation.

Figure 5. Expression of bone marrow stromal cell VCAM-1 in nude mice after HUCBSCs transplantation.

5-A: control group +14 d; 5-B: control group +21 d; 5-C: HUCBSCs group +14 d; 5-D: HUCBSCs group +21 d; 5-E: VCAM-1-HUCBSCs-1 group +14 d; 5-F: VCAM-1-HUCBSCs-1 group +21 d; 5-G: VCAM-1-HUCBSCs-2 group +14 d; 5-H: VCAM-1-HUCBSCs-2 group +21 d (immunohistochemistry under inverted microscope×400); 5-I: flow cytometry for the expression of VCAM-1, * compared with control group, P<0.05, * *compared with control group, P<0.01,^Δ compared with HUCBSCs group P<0.05.

Figure 6. Dynamic changes in peripheral WBC and PLT counts in nude mice after transplantation.

6-A: Dynamic changes in WBC counts after transplantation. 6-B: Dynamic changes in PLT counts after transplantation.

Figure 7. Dynamic changes in the myelogram in nude mice after HUCBSCs transplantation after 6.5 Gy irradiation.

7-A: The degree of bone marrow hyperplasia in control nude mice on day +1 after transplantation. 7-B: Myelogram of control nude mice on day +14. 7-C: Myelogram of control nude mice on day +21. 7-D: Myelogram of nude mice in the HUCBSC group on day +14. 7-E: Myelogram of nude mice in the HUCBSC group on day +21. 7-F: Myelogram of nude mice in the VCAM-1-HUCBSC-1 group on day +14. 7-G: Myelogram of nude mice in the VCAM-1-HUCBSC-2 group on day +7. 7-H: Myelogram of nude mice in the VCAM-1-HUCBSC-1 group on day +21. 7-I: Counting of bone marrow nucleated cells in nude mice after HUCBSCs transplantation, * compared with control group, P<0.01; ^Δ compared with HUCBSCs group P<0.05; ^ΔΔ compared with HUCBSCs group, P<0.01.

Figure 8. Effects of nude mouse bone marrow stromal cells on CFU-F, myeloid, erythroid, and megakaryocytic lineage cells after transplantation of HUCBSCs.

8-A: changes of CFU-F in nude mice at different time points after HUCBSCs transplantation. 8-B: changes of CFU-GM in nude mice at different time points after HUCBSCs transplantation. 8-C: changes of BFU-E in nude mice at different time points after HUCBSCs transplantation. 8-D: changes of CFU-Meg in nude mice at different time points after HUCBSCs transplantation.. * compared with control group, P<0.05;** compared with control group, P<0.01; ^Δ compared with HUCBSCs group P<0.05; ^ΔΔ compared with HUCBSCs group, P<0.01; ^⋆ ccompared with VCAM-1-HUCBSCs-1 group P<0.05; ^⋆⋆compared with VCAM-1-HUCBSCs-1 group P<0.01.

Figure 9. Observation of the pathological slides of nude mouse bone marrow under light microscope on day +21 after transplantation.

9-A: control group; 9-B: HUCBSCs group; 9-C: VCAM-1-HUCBSCs-1 group; 9-D: VCAM-1-HUCBSCs-2 group, (HGF staining, inverted microscope, ×100). Red arrows represent new fibroblast-like cells.

Figure 10. Observation of the fluorescences in the bone marrow pathological slices of the VCAM-1HUCBSC groups.

10-A: VCAM-1-HUCBSC-1 group on day +7 after transplantation; 10-B: VCAM-1-HUCBSC-1 group on day +14 after transplantation; 10-C: VCAM-1-HUCBSC-1 group on day +21 after transplantation; 10-D: VCAM-1-HUCBSC-2 group on day +7 after transplantation; 10-E: VCAM-1-HUCBSC-2 group on day +14 after transplantation; 10-F: VCAM-1-HUCBSC-2 group on day +21 after transplantation (fluorescence microscope ×100).