Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms

Abstract

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.

Figure 1

Donor-derived osteopoietic engraftment after bone marrow cell infusions. (A) Donor osteopoietic cell engraftment, expressed as a percentage of donor bone cells in the metaphysis and epiphysis, when ex vivo–expanded murine MSCs were infused after varying doses of TBI. (B) Donor osteopoietic cell engraftment when NABMC or MSCs were infused after 1125 cGy TBI. All data are mean ± SEM.

Figure 2

Clinical outcome of children with OI after bone marrow MNC infusion. The mean growth velocity (centimeters per month) of patients is shown during the 6 months before MNC infusion (□), the first 3-month interval (), and the second 3-month interval (▤) after MNC infusion.

Figure 3

Photomicrographs of bone from oim/oim mice after cell infusions. Mice were infused with either NABMC or MSCs from wild-type mice and then the bones were immunostained with a polyclonal antibody which recognizes only the proα2 polypeptide (not proα1) and visualized with NOVARed. The positive control (top left panel) is normal mouse bone which demonstrates staining of the trabecular bone but not the growth-plate cartilage on the right side of the section. The negative control (top right panel) is oim/oim mouse bone, which does not express proα2 peptide. Oim/oim mice infused with NABMC (bottom left panel) show red stain (proα2 expression) in the trabecular bone but not the articular cartilage on the left side of the section. Oim/oim mice infused with MSCs (bottom right panel) lack any red staining indicating the lack of detectable proα2. Original magnification, ×200.

Figure 4

The effect of MSCs on chondrocyte proliferation. (A) GFP staining of the growth plate after GFP-positive MSC infusion. Immunostaining for GFP expression of the growth plate from a GFP-transgenic mouse (positive), a mouse after saline infusion (PBS, negative), or a mouse after GFP-transgenic MSC infusion. Original magnification, ×200. (B) Chondrocyte proliferation was analyzed by MTT Cell Proliferation assay after 3-day coculture with MSCs or chondrocytes or control medium on the transwell plates (n = 3). (C) Sera were collected from MSC-injected and PBS-injected mice 2 days and 7 days after the injection. Chondrocyte proliferation assay was performed at day 0, day 2, day 4, and day 6 after culture with the sera, MSC-conditioned medium, and control medium (P < .001, n = 6). The depicted data are a representative experiment. In total, 38 groups of mice (222 experimental, 154 controls) confirmed these findings. (D) Sera from mice injected with control medium, MSC-conditioned medium, or MSCs were applied into chondrocyte culture. Chondrocytes were cultured for 6 days in serum-supplemented medium followed by the proliferation assay measured as fluorescence intensity (*P < .05, n = 9). All data are mean ± SEM.

Figure 5

The effect of MSCs in vivo. (A) PCNA immunohistochemical staining was performed on the sections of MSC- and PBS-injected mice, and the percentage of PCNA-positive cells in the growth plate was determined. 3,3′-diaminobenzidine signals were pseudocolored red. A representative picture of each group is shown. Original magnification, ×200 (P < .001, n = 12). (B left panel) Representative photographs of lumbar vertebrae of 8-week-old oim/oim mice, 4 weeks after PBS or MSC infusion, or PBS-injected oim/oim mice at 8 weeks old. (Right panel) Lumbar vertebral length of each group (P < .001, n = 4). (C left panel) Photographs of representative 8-week-old oim/oim mice 4 weeks after PBS or MSC infusion. (Right panel) The 8-week-old: 4-week-old body-weight ratio of oim/oim after PBS or MSC infusion (P = .003, PBS group, n = 7; MSC group, n = 10). All data are mean ± SEM.

Figure 6

The effect of NABMCs on chondrocyte proliferation. Chondrocytes were cultured in medium supplemented with sera from mice injected with MSCs, NABMCs, or PBS. After 6 days of culture, the proliferation assay was performed (*P < .05, **P < .001, n = 9). Data are mean ± SEM.