Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone

Abstract

Treatment with isolated allogeneic mesenchymal cells has the potential to enhance the therapeutic effects of conventional bone marrow transplantation in patients with genetic disorders affecting mesenchymal tissues, including bone, cartilage, and muscle. To demonstrate the feasibility of mesenchymal cell therapy and to gain insight into the transplant biology of these cells, we used gene-marked, donor marrow-derived mesenchymal cells to treat six children who had undergone standard bone marrow transplantation for severe osteogenesis imperfecta. Each child received two infusions of the allogeneic cells. Five of six patients showed engraftment in one or more sites, including bone, skin, and marrow stroma, and had an acceleration of growth velocity during the first 6 mo postinfusion. This improvement ranged from 60% to 94% (median, 70%) of the predicted median values for age- and sex-matched unaffected children, compared with 0% to 40% (median, 20%) over the 6 mo immediately preceding the infusions. There was no clinically significant toxicity except for an urticarial rash in one patient just after the second infusion. Failure to detect engraftment of cells expressing the neomycin phosphotransferase marker gene suggested the potential for immune attack against therapeutic cells expressing a foreign protein. Thus, allogeneic mesenchymal cells offer feasible posttransplantation therapy for osteogenesis imperfecta and likely other disorders originating in mesenchymal precursors.

Figures

Figure 1

Analysis of MSCs before infusion. (A) Flow cytometric analysis of MSCs (from patient 5) to exclude contamination by lymphohematopoietic cells. Anti-CD45 perCP (peridinin-chlorophyll) was used to screen for mature and progenitor white blood cells, anti-CD14-PE (phycoerythrin) for monocytes/macrophages, and anti-CD3 FITC (fluorescein isothiocyanate) for T cells. The percentage of positive cells among the total analyzed is shown. Isotype controls are shown on the left. (B) Induction of osteogenic differentiation with osteoinductive medium as described in the text. The Alizarin Red stain reveals foci of mineral deposition in the induced cells (Right), contrasted with the lack of mineralized foci in noninduced control cells (Left).

Figure 2

PCR analysis of engraftment. DNA isolated from osteoblasts, stromal cells, and skin fibroblasts of each patient, at 4 to 6 wk after the second cell infusion, was evaluated. DNA isolated from human cells transduced with either the G1PLII or LNc8 vector served as a positive control; normal human DNA was the negative control. Analysis of the γ-globin gene controlled for the quality and quantity of DNA. MC and E designate whether the signal represents minimally cultured cells or expanded cells, respectively. The vectors were alternated among the patients to avoid vector bias. ND, Not determined.

Figure 3

T cell response against transduced MSCs. Cytotoxic T lymphocyte-mediated lysis of donor MSCs (patient 4) transduced with either the LNc8 or G1PLII retroviral vector or mock supernatant only. E:T designates the effector-to-target cell ratio. Each bar represents the mean of triplicate determinations.

Figure 4

Antibody response against FBS proteins. ELISA assay measuring antifetal bovine serum antibodies in the sera of patients before (░⃞) and after (■) both MSC infusions. Each bar represents the mean of triplicate determinations. Pos, positive control; Neg, negative control.

Figure 5

Growth stimulation after MSC engraftment. Growth velocity of the patients during the 6 mo immediately before (░⃞) and after (■) the first MSC infusion. The values are percentages of the median growth of unaffected children of the same age and sex (23).