Mouse mesenchymal stem cells can support human hematopoiesis both in vitro and in vivo: the crucial role of neural cell adhesion molecule

Abstract

Background: We previously established a mesenchymal stem cell line (FMS/PA6-P) from the bone marrow adherent cells of fetal mice. The cell line expresses a higher level of neural cell adhesion molecule and shows greater hematopoiesis-supporting capacity in mice than other murine stromal cell lines.

Design and methods: Since there is 94% homology between human and murine neural cell adhesion molecule, we examined whether FMS/PA6-P cells support human hematopoiesis and whether neural cell adhesion molecules expressed on FMS/PA6-P cells contribute greatly to the human hematopoiesis-supporting ability of the cell line.

Results: When lineage-negative cord blood mononuclear cells were co-cultured on the FMS/PA6-P cells, a significantly greater hematopoietic stem cell-enriched population (CD34(+)CD38(-) cells) was obtained than in the culture without the FMS/PA6-P cells. Moreover, when lineage-negative cord blood mononuclear cells were cultured on FMS/PA6-P cells and transplanted into SCID mice, a significantly larger proportion of human CD45(+) cells and CD34(+)CD38(-) cells were detected in the bone marrow of SCID mice than in the bone marrow of SCID mice that had received lineage-negative cord blood mononuclear cells cultured without FMS/PA6-P cells. Furthermore, we found that direct cell-to-cell contact between the lineage-negative cord blood mononuclear cells and the FMS/PA6-P cells was essential for the maximum expansion of the mononuclear cells. The addition of anti-mouse neural cell adhesion molecule antibody to the culture significantly inhibited their contact and the proliferation of lineage-negative cord blood mononuclear cells.

Conclusions: These findings suggest that neural cell adhesion molecules expressed on FMS/PA6-P cells play a crucial role in the human hematopoiesis-supporting ability of the cell line.

Figure 1.

Human hemopoiesis-supporting capacity of FMS/PA6-P cells in vitro. (A) Morphology of L⁻CBMC and formation of cobblestone colonies in the co-culture of L⁻CBMC with FMS/PA6-P cells. The purified L⁻CBMC were stained with May-Giemsa reagent (left photograph). The L⁻CBMC (7×10³/well) were inoculated to the monolayers of confluent FMS/PA6-P cells (20 Gy-irradiated) and the formation of cobblestone areas was observed on day 2–3 of culture (middle and right photographs). Phase-contrast images (B) Expansion of L⁻CBMC on the monolayer of FMS/PA6-P cells. The L⁻CBMC (7×10³/well) were cultured with or without the FMS/PA6-P cells (20 Gy-irradiated). At 1 and 2 weeks, all cells in the well were collected by trypsin-EDTA treatment and the number of hematopoietic cells was counted. The number of CD34⁺ cells and CD34⁺CD38⁻cells per well was calculated from the hematopoietic cell number per well and the percentages of these populations obtained by flow cytometric analyses. The number of CFU-C per well was also calculated from the hematopoietic cell number per well and the number of CFU-C/10⁴ cells obtained by clonal cell culture assay. Fold Increase = the number of hematopoietic cells per well after culture/the number of hematopoietic cells per well before culture. The fold increases of CD34⁺ cells, CD34⁺CD38⁻ cells and CFU-C counts were also calculated from the number of these populations before and after culture, respectively. Each sample was run in triplicate. Representative data from three independent experiments. **P<0.01; *P<0.05. (C) Multi-lineage differentiation of L⁻CBMC in co-culture with FMS/PA6-P cells. At 2 weeks of culture, non-adherent cells were collected from the co-culture of the L⁻CBMC and FMS/PA6-P cells, and were stained with May-Giemsa reagent.

Figure 2.

Reconstitution of human cells in SCID mice that received culture-expanded L⁻CBMC. (A) Higher engraftment of human cells in SCID mice that received L⁻CBMC co-cultured with FMS/PA6-P cells. The L⁻CBMC were cultured in a 12-well plate with the FMS/PA6-P cells for 2 weeks and all cells in one well, including the FMS/PA6-P cells and hematopoietic cells, were harvested and injected into a single SCID mouse via the intravenous route. All cells in one well, cultured without the FMS/PA6-P cells, were also collected and injected into a single SCID mouse. Eight weeks after the transplantation, BM and peripheral blood cells were collected and the percentages of human CD45⁺ cells, CD34⁺ cells and CD34⁺CD38⁻ cells were measured using FACScan. **P<0.01; *P<0.05. (B) Representative flow cytometric pattern of BM cells from SCID mice that received L⁻CBMC cultured with or without FMS/PA6-P cells. BM cells collected from the SCID mice were double-stained with anti-human CD45 and anti-human CD34 monoclonal antibodies or with anti-human CD38 and anti-human CD34 monoclonal antibodies and analyzed by a FACScan. The values indicate the percentages of the population in whole BM cells. (C) Detection of human DNA in SCID mice that received L⁻CBMC co-cultured with FMS/PA6-P cells. Four of five mice, transplanted with the expanded cells produced in the co-culture of L⁻CBMC with FMS/PA6-P cells, contained human DNA in the BM, whereas no human DNA was detected in the BM of six mice transplanted with the expanded cells produced in culture of L⁻CBMC without FMS/PA6-P cells.

Figure 3.

Higher proliferation and differentiation of L⁻CBMC in contact culture with FMS/PA6-P cells. The monolayer of FMS/PA6-P cells was prepared in a 24-well plate and the L⁻CBMC were loaded into the culture chamber inserts or directly into the FMS/PA6-P cell monolayer. As a negative control, L⁻CBMC were cultured without FMS/PA6-P cells. Two weeks later, all cells in the well were collected by trypsin-EDTA treatment, and the fold increases of total cells, CD34⁺ cells, CD34⁺CD38⁻ cells and CFU-C counts were calculated, as mentioned in Figure 1B. Each sample was run in triplicate. Representative data from three independent experiments. ** P<0.01; *P<0.05, N.S.: not significant.

Figure 4.

Inhibitory effect of anti-NCAM monoclonal antibody on the proliferation of L⁻CBMC. The L⁻CBMC were cultured on the FMS/PA6-P cells in the presence of anti-mouse NCAM monoclonal antibody (5 wells/sample). As a control, the same concentration of isotype control (mouse IgG2a) was added to the culture. Two weeks later, all cells in the well were collected by trypsin-EDTA treatment, and the fold increases of total cells, CD34⁺ cells, CD34⁺CD38⁻ cells and CFU-C counts were calculated, as mentioned in Figure 1B. Mean ± SD of five wells. Representative data from three independent experiments. *P<0.05, N.S.: not significant.