Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction

Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1–8 µg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical

Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch

Hypoglycaemic and anti-hyperglycaemic activity of Tabernanthe iboga aqueous extract in fructose-fed Streptozotocin type 2 diabetic rats

Publisher Copyright: © 2020, Institute of Korean Medicine, Kyung Hee University. This is a post-peer-review, pre-copyedit version of Bading-Taïka, B., Souza, A., Bourobou Bourobou, HP. et al. Hypoglycaemic and anti-hyperglycaemic activity of Tabernanthe iboga aqueous extract in fructose-fed streptozotocin type 2 diabetic rats. ADV TRADIT MED (ADTM) (2020). https://doi.org/10.1007/s13596-020-00484-0Root bark preparations of the Gabonese plant Tabernanthe iboga (T. iboga) has long been used in traditional medicine in Central and West African regions for the management of type 2 diabetes (T2D). This study is the first investigation of in vivo hypoglycaemic activity in healthy rats and anti-hyperglycaemic activity of T. iboga in a 10% fructose-fed (40 mg/kg i.p.) streptozotocin (STZ) injected type 2 diabetic rat model. T. iboga at 50 to 200 mg/kg induced hypoglycaemia activity over 3 h fasted glucose tolerance in healthy Wistar rats and anti-hyperglycaemic effects on non-fasted and fasted blood glucose in fructose-fed STZ T2D rats with no toxicity. Fructose-fed STZ T2D rats developed characteristic type 2 diabetic complications over 6 weeks exhibiting significantly elevated fasting and non-fasting blood glucose, polydipsia, reduced body weight gain and glucose and insulin tolerance compared with STZ alone and normal control rats. T. iboga (50 mg/kg and 200 mg/kg bw) administered p.o. once daily for 4 weeks significantly improved diabetic symptoms of polydipsia, reduced body weight, hyperglycaemia, glucose and insulin tolerance (as AUC) compared with fructose-fed STZ T2D rats. T. iboga aqueous extract (50 mg/kg and 200 mg/kg) also significantly reversed altered actions of marker enzymes of liver including alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, HbA1c and elevated triglycerides in fructose-fed STZ type 2 diabetic rats. Our outcomes show that daily oral provision of T. iboga improves type 2 diabetes complications, superior to glibenclamide, in rat fructose-fed STZ model and offers the potential for safe clinical management of T2D in Gabon.Peer reviewe

Large Animal Models of Diabetes

Safe and reliable large animal diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents but is often critiqued in higher animals due to reduced efficacy, relevant side effects, and inadequate mortality rate. In this chapter, we aim to describe both pharmacological and surgical approaches for reproducible and safe diabetes models in minipigs and primates. In addition, genetically modified pig models for diabetes research are described