Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy in Vivo

Abstract

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire-Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >10 10 cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous b-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal cells merit further development as autologous cell therapy for diabetes.

Durable engraftment of genetically modified FVIII‐secreting autologous bone marrow stromal cells in the intramedullary microenvironment

“… BMSCs from fresh bone marrow were cultured in a 3:1 (v/v) mixture of low‐glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) as previously described . BMSCs were expanded in culture for 3 weeks after bone marrow aspiration.…”

“…D, Flow cytometry profile of GFP‐positive primary BMSCs of dog #5954 (intramedullary implantation) 24 h after electroporation with a GFP‐expressing plasmid. E, A representative profile of positive and negative markers of mesenchymal stromal cells present in early passage BMSCs in this study analysed by flow cytometry as previously described . Broken lines denote the corresponding isotype control.…”

“…All methods for primary BMSC culture, immunophenotyping and plasmid constructs for ZFN-mediated integration of a FVIII transgene (Figure 1A-C) were as previously described. 4,9,10 ZFN-modified autologous BMSCs were implanted in vivo without genetic selection after taking an aliquot for secreted FVIII activity assay. Parallel electroporation of a GFP plasmid was performed to determine the efficiency of gene transfer in each case (Figure 1D).…”

“…Bone marrow is an inexhaustible source of primary BMSCs which have a great capacity for ex vivo expansion. Naïve BMSCs physiologically secrete endogenous FVIII and can be genetically modified to express transgenic proteins durably in vitro and in vivo . A few studies have shown efficacy of naïve or genetically modified BMSCs implanted in haemophilic animals [reviewed in ].…”

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Progress and Challenges

“…The authors reported that hyperglycemia was normalized within 3-4 days and maintained for several months, which was astonishing since it followed a xenogeneic transplantation without immunoisolation or immunosuppression. Thi Do and associates transfected porcine bone marrow-derived MSCs with the insulin gene using a lentiviral vector [70]. Autologous transplantation of the treated cells in the liver of pigs with STZ-induced diabetes resulted in partial improvement of their hyperglycemia.…”