Improved Culture Expansion of Human Mesenchymal Stem Cells (MSCs) Using Fibroblastic Growth Factor-2 for the Treatment of Graft Versus Host Disease (GVHD).

Abstract

Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of diseases. However, graft-versus-host disease (GVHD) remains a significant obstacle to the successful outcome of this procedure. We have demonstrated in Phase I clinical trials that co-transplantation of culture expanded human MSCs during allogeneic HSC transplantation can facilitate engraftment without increasing the risk of GVHD. MSC have been shown to have immunomodulatory activity, and decrease T-cell interferon (IFN)-γ production. More recently, clinical studies have suggested that MSC infusion can also reduce the severity of GVHD. Based on these data, we have initiated a Phase I clinical trial (CWRU 3Y03) using allogeneic same-sibling donor MSC infusion of 1–6 × 106 culture expanded MSCs per kg for the treatment of acute or chronic GVHD of clinical grade II – IV after sibling donor HSC transplant. One of the main hurdles to overcome in MSC infusion protocols is the expansion of single donor MSCs to achieve the prescribed cell dose in a specified time frame. Here we report that the addition of recombinant human FGF-2 to the culture medium expedites and enhances MSC expansion capacity in normal adult donors cultured under clinically relevant conditions. In pre-clinical studies, MSCs from 13 normal donors (median age 28.5; range 21 – 40) were cultured in standard growth medium with or without FGF-2. The cells were expanded for up to 8 passages. MSCs expanded in the presence of FGF-2 exhibited shorter population doubling times and achieved a greater number of population doublings (31 ± 3) than those expanded in standard conditions (23 ± 2). These FGF-stimulated MSCs exhibited the same phenotype and immunomodulatory potential as MSCs grown in conventional medium. For each patient enrolled on CWRU 3Y03, donor MSCs were harvested, culture expanded and cryopreserved until indicated. Twenty-three culture expansions were attempted from 21 different donors (18 without FGF; 3 with FGF; median donor age 52, range 38 – 67). From the cultures grown in the absence of FGF, 11 donors were expanded to an infusion dose of 0.5 – 2.4 × 106 cells/kg (based on patient weight) with a mean of 161 ± 54 × 106 MSCs at harvest and a median cell expansion time of 41 days (range 23 – 66). Nine cultures failed to reach a minimum cell dose of 0.5 × 106 cells/kg during an 8-week culture period. The three MSC cultures grown in the presence of FGF -2 (R&D systems) were successful and reached infusion doses of 1.65 – 2.4 × 106 cells/kg. In these 3 cultures, the mean number of MSCs was 135 × 106 and the median day to harvest was 28 (range 27 – 41). The immunomodulatory potential of these three MSC preparations was tested in vitro in an IFN-γ EliSpot assay in which they inhibited IFN-γ production by 87.6 ± 5.4%. Cells from one donor that failed to expand in media without FGF-2 reached 171 × 106 (2.8 × 106/kg) MSCs in 27 days when MSCs from a second marrow harvest from the same donor were cultured in medium containing FGF-2, suggesting that FGF-2 supplementation may rescue a culture that may be otherwise unable to expand. Thus, FGF-2 can facilitate MSC culture expansion for clinical use while retaining their immunomodulatory function.