Circulating TGF-β Pathway in Osteogenesis Imperfecta Pediatric Patients Subjected to MSCs-Based Cell Therapy

Abstract

Osteogenesis Imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in COL1A1 or COL1A2, which encode type I collagen. There is no cure for OI, and real concerns exist for current therapeutic approaches, mainly antiresorptive drugs, regarding their effectiveness and security. Safer and effective therapeutic approaches are demanded. Cell therapy with mesenchymal stem cells (MSCs), osteoprogenitors capable of secreting type I collagen, has been tested to treat pediatric OI with encouraging outcomes. Another therapeutic approach currently under clinical development focuses on the inhibition of TGF-β pathway, based on the excessive TGF-β signaling found in the skeleton of severe OI mice models, and the fact that TGF-β neutralizing antibody treatment rescued bone phenotypes in those OI murine models. An increased serum expression of TGF-β superfamily members has been described for a number of bone pathologies, but still it has not been addressed in OI patients. To delve into this unexplored question, in the present study we investigated serum TGF-β signalling pathway in two OI pediatric patients who participated in TERCELOI, a phase I clinical trial based on reiterative infusions of MSCs. We examined not only the expression and bioactivity of circulating TGF-β pathway in TERCELOI patients, but also the effects that MSCs therapy could elicit. Strikingly, basal serum from the most severe patient showed an enhanced expression of several TGF-β superfamily members and increased TGF-β bioactivity, which were modulated after MSCs therapy.

Keywords: TGF-β; cell therapy; mesenchymal stem cells; osteogenesis imperfecta; stem cells.

FIGURE 1

Schematic diagram illustrating TERCELOI clinical trial cellular infusions and the current study of circulating TGF-β expression and bioactivity in patients’ sera collected before (basal serum) and after the five consecutive MSCs infusions. The post-infusions sera included those collected 1 week (1w), 1 month (1 m) and 4 months (4 m) after each cell infusion, as well as the follow-up sera, collected 1 year and 2 years after the last cell infusion.

FIGURE 2

Basal circulating TGF-β superfamily members’ expression and bioactivity in OI patients. (A) Left heatmap shows the fluorescent signal intensities for target proteins expressed in P01 and P02 basal sera samples. Only the spots with a fluorescent intensity cutoff ≥300 above the background were considered. Right heatmap magnified shows the fluorescent intensities of the TGF-β superfamily members present in basal P01 and P02 sera are shown (right heatmap). (B) Graph showing the total number of expressed proteins in P01 and P02 basal sera, grouped according to their fluorescent signal intensity: low (300-2,000), medium (2,000-10,000) and high (>10,000). (C) Left, schematic representation of the HEK-Blue-TGF-β^TM reporter cell line assay. Right, Scatter plot with bars showing TGF-β-induced SEAP in HEK-Blue-TGF-β^TM cells. The TGF-β induced SEAP from patients’ sera samples is shown normalized versus that shown by the negative control (C-; w/o TGF-β). C+ stands for the positive control, cells stimulated with TGF-β (10 ng/ml). The experiments were performed in octuplicates for each condition and repeated two independent times. Data are mean ± standard deviation obtained from the two independent experiments. Each dot represents an independent experiment.

FIGURE 3

Circulating TGF-β superfamily members expression and bioactivity after MSCs therapy in OI patients. (A) Heatmap showing the expression ratio of TGF-β superfamily members after the 1st MSCs infusion in P01 and P02, calculated as the fluorescent signal intensity of target proteins after the MSCs therapy, 1 week (1w), 1 month (1 m) or 4 months (4 m) versus the fluorescent intensity of target proteins from each patient’s basal serum. To be considered, a cutoff ratio of ±1.5 was established. (B) Scatter plot showing TGF-β induced SEAP of HEK-Blue-TGF-β^TM cells exposed to P01 and P02 sera collected after the first MSCs infusion and compared to the results obtained with basal serum for each patient. The TGF-β induced SEAP is shown normalized versus the negative control. (C) Scatter plot showing TGF-β induced SEAP of HEK-Blue-TGF-β^TM cells exposed to the sera from P01 obtained during the 2nd, 3rd, 4th, 5th infusions and follow-up visits, and compared to the results obtained with basal serum. Two independent experiments were performed with P01 and P02 sera, each of one with their respective positive (c+) and negative controls (c-). Within each independent experiment, each condition was performed in octuplicates. Data are mean ± standard deviation obtained from the two independent experiments. Each dot represents an independent experiment.Dashed horizontal lines illustrate the TGF-β bioactivity level of basal P01 and P02 sera.