Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells.

Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.

Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic ß-Cells.

Combinatorial gene and cell therapy as a means of generating surrogate ß-cells has been investigated for the treatment of type 1 diabetes (T1D) for a number of years with varying success. One of the limitations of current cell therapies for T1D is the inability to generate sufficient quantities of functional transplantable insulin-producing cells. Due to their impressive immunomodulatory properties, in addition to their ease of expansion and genetic modification ex vivo, mesenchymal stem cells (MSCs) are an attractive alternative source of adult stem cells for regenerative medicine. To overcome the aforementioned limitation of current therapies, we assessed the utility of ex vivo expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate ß-cells. CD45-/Ly6+ murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, Firefly luciferase (Luc2). The persistence of a subcutaneous (s.c.) transplant of Luc2-expressing MSCs was assessed in immune-competent (NOD) (n = 4) and immune-deficient (NOD/Scid) (n = 4) animal models of diabetes. Luc2-expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/Scid mice. Ex vivo expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin (INS-FUR) and murine NeuroD1 and Pdx1. This was followed by the characterization of pancreatic transdifferentiation via reverse transcriptase polymerase chain reaction (RT-PCR) and static and glucose-stimulated insulin secretion (GSIS). INS-FUR-expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/Scid mice (n = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did not reverse diabetes. The data suggest that ex vivo expanded MSCs lose their multipotent differentiation potential and may be more useful as gene therapy targets prior to expansion.

CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

Due to their ease of isolation, differentiation capabilities, and immunomodulatory properties, the therapeutic potential of mesenchymal stem cells (MSCs) has been assessed in numerous pre-clinical and clinical settings. Currently, whole pancreas or islet transplantation is the only cure for people with type 1 diabetes (T1D) and, due to the autoimmune nature of the disease, MSCs have been utilised either natively or transdifferentiated into insulin-producing cells (IPCs) as an alternative treatment. However, the initial success in pre-clinical animal models has not translated into successful clinical outcomes. Thus, this review will summarise the current state of MSC-derived therapies for the treatment of T1D in both the pre-clinical and clinical setting, in particular their use as an immunomodulatory therapy and targets for the generation of IPCs via gene modification. In this review, we highlight the limitations of current clinical trials of MSCs for the treatment of T1D, and suggest the novel clustered regularly interspaced short palindromic repeat (CRISPR) gene-editing technology and improved clinical trial design as strategies to translate pre-clinical success to the clinical setting.