Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds.

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.

Post-transplant cyclophosphamide and bortezomib inhibit dendritic cell maturation and function and alter their IκB and NFκB.

Impairing dendritic cell (DC) function to prevent graft versus host disease (GvHD) is an appealing concept. DC antigen presentation is NF-κB pathway-dependent and bortezomib might therefore play a role in preventing alloreactivity. We obtained DC from the blood of patients enrolled in a phase I study using post-transplant cyclophosphamide and bortezomib for prevention of GvHD. Control samples were obtained from patients receiving standard GvHD prevention regimen. Pre-treatment samples were also collected from enrolled patients. DC isolated on days +1, +4, and +7 showed progressive decrease in the expression of maturation markers in comparison to control. In a DC-CD4+ mixed lymphocyte reaction (MLR) where DC isolated from the recipient blood before graft infusion were the stimulator cells, T cell proliferation measured by bromodeoxyuridine (BrdU) integration was decreased in samples obtained on days +14 and +21 in comparison to control group. Finally, measured by real-time PCR, the expression of IκB progressively increased while the expression of NF-κB decreased in DC on days +1, +4, and +7, in comparison to pre-treatment paired controls. We conclude that our data further justify exploring the role of bortezomib in GvHD prevention and propose a novel mechanism of action of bortezomib in DC.

Effect of novel proteasome and immunoproteasome inhibitors on dendritic cell maturation, function, and expression of IκB and NFκB.

Dendritic cells (DC) play a central role in the pathophysiology of graft versus host disease (GvHD). Their antigen presenting capacity is nuclear factor κB- (NF-κB) dependent. Consequently, DC have emerged as a potential target for the prevention of GvHD and clinical trials with bortezomib are underway. We explored the activity of novel proteasome and immunoproteasome inhibitors on healthy volunteer peripheral blood DC. After incubation with the drug or drug combination, DC were stimulated with lipopolysaccharide, stained for maturation surface markers and then analyzed by flow cytometry. We found that the different molecule(s) inhibited DC maturation marker expression to variable degrees, with the constitutive proteasome-selective agent being the least active. In a DC and allogeneic CD4+ mixed lymphocyte reaction, DC incubation with the studied proteasome and immunoproteasome inhibitor(s), impeded T cell proliferation as measured by BrDU incorporation. Finally, we found that DC incubation with the drug(s) enhanced IκB expression and that oprozomib inhibited NF-κB expression. We concluded that based on its activity and oral bioavailability, oprozomib merits further investigation in an animal GvHD prevention model. We also suggest that altering IκB and NF-κB expressions may, in DC, represent a new mechanism of action of proteasome and immunoproteasome inhibitors.