CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties

Abstract

Background In vitro proliferative and differentiation potential of mesenchymal stromal cells generated from CD271(+) bone marrow mononuclear cells (CD271-mesenchymal stromal cells) has been demonstrated in several earlier and recent reports. In the present study we focused, in addition to proliferative and differentiation potential, on in vitro and in vivo immunosuppressive and lymphohematopoietic engraftment-promoting potential of these mesenchymal stromal cells compared to bone marrow-derived mesenchymal stromal cells generated by plastic adherence (plastic adherence-mesenchymal stromal cells).

Design and methods: We set up a series of experimental protocols in order to determine the phenotype of CD271-mesenchymal stromal cells, and their clonogenic, proliferative, differentiation and immunosuppressive potential. The potential of CD271-mesenchymal stromal cells to improve the engraftment of CD133(+) hematopoietic stem cells at co-transplantation was evaluated in immunodeficient NOD/SCID-IL2Rgamma(null) mice.

Results: In vitro studies demonstrated that CD271-mesenchymal stromal cells differentiate along adipogenic, osteogenic and chondrogenic lineages (trilineage potential), produce significantly higher levels of cytokines than plastic adherence-mesenchymal stromal cells, and significantly inhibit the proliferation of allogeneic T-lymphocytes in mixed lymphocyte reaction assays. Elevated levels of prostaglandin E(2), but not nitric monoxide, mediated the majority of this immunosuppressive effect. In vivo studies showed that CD271-mesenchymal stromal cells promoted significantly greater lymphoid engraftment than did plastic adherence-mesenchymal stromal cells when co-transplanted with CD133(+) hematopoietic stem cells at a ratio of 8:1 in immunodeficient NOD/SCID-IL2Rgamma(null) mice. They induced a 10.4-fold increase in the number of T cells, a 2.5-fold increase in the number of NK cells, and a 3.6-fold increase in the number of B cells, indicating a major qualitative difference between these two mesenchymal stromal cell populations. Conclusions Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multipotent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. The co-transplantation of such cells together with hematopoietic stem cells in patients with hematologic malignancies may prove valuable in the prevention of impaired/delayed T-cell recovery and graft-versus-host disease.

Figure 1.

Phenotype of ex vivo expanded CD271- MSC and their in vitro differentiation potential. (A) CD271-MSC express high levels of all typical MSC markers such as CD73, CD90, CD146, CD166 and CD105. They also express HLA- class I antigens but do not express HLA-DR, CD45, CD14, CD184, or hematopoietic stem cells markers (CD34 and CD133). (B) Differentiation potential of CD271-MSC and PA-MSC (C). Like PA-MSC (Ci-iii) these MSC differentiate into adipocytes (Bi), osteoblasts (Bii) and chondrocytes (Biii). Accumulation of intracellular lipid vacuoles was shown by oil red-O staining for 18 days in culture with NH AdipoDiff medium, while osteoblast differentiation was detected by alkaline phosphatase activity after 10 days in NH OsteoDiff medium. Cartilage matrix deposition along with chondrocytes in lacunae was demonstrated by metachromatic toluidine blue staining after 24 days in NH ChondroDiff medium. Magnification for microphotographs of MSC and osteoblasts was 20x; the magnification for adipocytes was 200x and for chondrocytes 400x.

Figure 2.

The effect of CD271-MSC and PA-MSC on proliferation of mitogen-activated lymphocytes and allogeneic reaction. The figure shows the inhibitory effect of the CD271-MSC population (A) and PA-MSC population at passage 4 at the ratio 1:1 (B). In each experiment, 100,000 peripheral blood lymphocytes were stimulated in triplicate with phytohemagglutin (PHA), concanavalin A (con-A), pokeweed mitogen (PWM), interleukin- 2 (IL-2) and staphylococcal enterotoxin B (SEB) in the presence or absence (control group) of lethally irradiated mesenchymal stromal cells. The values represent the mean of the triplicate experiments ± standard error of mean (SEM) (n=5). Statistically significant differences between groups were assessed with a Student’s ttest. P values of less than 0.05 were considered to be statistically significant: *P<0.02; **P<0.003; P<0.0001. Inhibition of allogeneic reaction through CD271- MSC (C) and PA-MSC (D). PB-MNC of two unrelated donors were cultured for 5 days either alone (control group) or mixed with lethally irradiated third-party MSC at the ratio of 1:1 (10⁵ PB-MNC: 10⁵ MSC). For the prostaglandine E₂ (PGE₂) inhibition experiments, the MSC were cultured with MNC in the presence or absence of the PGE₂ inhibitor, indomethacin (IM, final concentration: 5 μM). Proliferation levels of PB-MNC were determined on day 6 by means of the BrdU assay. The significance of inhibition of proliferation of allogeneic cells by MSC and abrogation of this effect by indomethacin was assessed with a Student’s t test. P values of less than 0.05 were considered to be statistically significant: **P<0.005; P<0.0001. The experiment was performed with four unrelated MSC-PBMC donor pairs. The inhibitory effect of CD271-MSC or PA-MSC correlated very well with the elevated levels of PGE₂ in the supernatants of mixed lymphocyte reactions containing MNC and CD271-MSC or MNC and PA-MSC (E).

Figure 3.

Hematopoietic engraftment of human CD133⁺ cells in bone marrow of NOD/SCID IL-2Rγnull mice. (A) Experimental design of the in vivo studies. Five groups of NOD/SCID IL-2Rγnull mice (5 mice each) were sublethaly irradiated and on the next day transplanted with 100,000 human CD133⁺ cells only (control group), 100,000 CD133⁺ cells and 100,000 human GFP-transduced CD271- MSC (ratio 1:1) and the other group with 100,000 CD133⁺ cells and 700,000 human GFP-transfected CD271-MSC (ratio 1:8). At the same ratios 100,000 CD133⁺ cells were transplanted with GFP-transduced PA-MSC. The overall human cell engraftment in the bone marrow (as measured by anti-human CD45 immunostaining) 14 weeks post-transplantation is shown in the right panel. The top right panel shows the graph for co-transplantation of CD133⁺ cells with CD271-MSC; the bottom right panel shows co-transplantation of CD133⁺ cells with PA-MSC. (B) A representative dot plot of the group transplanted with 100,000 mobilized peripheral blood CD133⁺ cells only, the group co-transplantated with PA-MSC at the ratio 1:8 (C) and the group co-transplanted with CD271-MSC at the ratio 1:8 (D). (E) Multilineage differentiation of CD133⁺ without or with co-transplantation of CD271-MSC or PA-MSC, respectively. The percentage of positive cells for each lineage marker was determined by gating on human CD45⁺ cells as shown in panels B, C and D. Differences in the engraftment level of each cell population between the groups were tested by Student’s t-test, A P value of less than 0.05 was considered to be statistically significant. (F) Representative flow cytometric data on the engraftment of human T, NK and NKT cells in the group transplanted with 10⁵ CD133⁺ cells and 7x10⁵ CD271-MSC (1:8 ratio).

Figure 4.

Distribution of human cells in the tissues of NOD/SCID mice 14 weeks after transplantation. (A) Real-time PCR amplification of human albumin gene in tissue samples of mice co-transplanted with human CD133⁺ cells and CD271-MSC at a ratio of 1:1. (B) Real-time PCR amplification of human albumin gene in tissue samples of mice co-transplanted with human CD133⁺ cells and CD271-MSC at a ratio 1:8. Standard curve achieved from serial dilutions of human DNA in mouse DNA (10⁻¹ to 10⁻⁴) is depicted with dashed lines. (C) Organ distribution of human cells in mouse tissues. Fourteen weeks after transplantation genomic DNA was extracted and the presence of human-specific DNA in the organs of transplanted mice was confirmed by real-time polymerase chain reaction (RQ-PCR) for human albumin gene. The values represent calculated mean percentages of human DNA ± SD of four experiments. Significant differences were observed in the brain of groups co-transplanted with HSC and MSC at the 1:1 ratio as compared with the content of human DNA in the brain of groups co-transplanted with HSC and MSC at a 1:8 ratio (*P<0.04 when HSC were co-transplanted with CD271-MSC and *P<0.01 when HSC were co-transplanted with PA-MSC). In contrast, when HSC were co-transplanted with MSC at a 1:8 ratio a significantly higher amount of human DNA was observed in the lungs as compared to the content of human DNA in the same organ of mice co-transplanted with HSC and MSC at a 1:1 ratio (*P<0.01 when HSC were co-transplanted with CD271-MSC and *P<0.04 when HSC were co-transplanted with PA-MSC) (D) and (E) A representative immunostaining of GFP-transduced CD271-MSC in the tissue sections with phycoerythrin (PE)-conjugated mouse anti-human HLA-class I and Alexa 488-conjugated anti-GFP antibody. Localization of MSC-derived cells in the lungs (D) of mice co-transplanted with 7x10⁵ CD271-MSC and 10⁵ CD133⁺ HSC and brain (E) when CD271-MSC were co-transplanted with CD133⁺ HSC at a ratio of 1:1 (magnification 600x).