generation-induced-pluripotent-stem-cells-from-frozen-buffy-coats

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

European Academy Bozen/Bolzano (EURAC)

Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

Baculovirus-mediated gene delivery into Mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression.

Schwann cells, the myelin-forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl-transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase-N stripping of PSA. PSA-positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX-transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co-cultures. Thus, migratory properties of PNS myelin-forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro.

Schwann cells (SCs) are among the most attractive cellular candidates for the development of remyelination therapies for CNS lesions. Yet, their integration in the CNS is inhibited by astrocytes and therefore the use of genetically modified SCs with improved properties is an alternative promising approach. Our strategy for ameliorating the therapeutic potential of SCs has been to alter their adhesive properties by expressing on their surface the polysialylated (PSA) form of the neural cell adhesion molecule NCAM. In the present study, SCs from transgenic GFP-mice were transduced with a retroviral vector encoding sialyl-transferase X (STX), the enzyme responsible for transferring PSA on NCAM. Engineered STX-GFP-SCs with sustained PSA expression were thus generated and were found to have improved ability to associate with astrocytes in vitro. Importantly, when these cells were transplanted in vivo in a mouse model of spinal cord injury they promoted faster and significantly greater functional recovery as compared to using SCs transduced with a control retroviral vector or no cells at all. Morphological analysis indicated that the improved locomotor recovery correlated with earlier and enhanced remyelination by grafted STX-GFP-SCs, increased remyelination by host SCs as well as enhanced differentiation/remyelination by resident oligodendrocyte precursors. Moreover, sprouting of regenerating serotonergic nerve fibres, which are known to be important for locomotion and recovery after injury, was observed into and across the lesion site. These results underline the potential therapeutic benefit of early activation of myelin-forming cells to differentiate and remyelinate severed axons thus restoring functions in CNS trauma and/or demyelinating diseases.

Grafts of Schwann cells engineered to express PSA-NCAM promote functional recovery after spinal cord injury.

To evaluate whether amniotic fluid cells contain a germ-like cell subpopulation.

Deleted in Azoospermia-Like (DAZL) gene-expressing cells in human amniotic fluid: a new source for germ cells research?

Circulating 'free' non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core(173)-eGFP, eGFP-core(191) and eGFP-core(173) can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells - the main target of HCV - but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.

Green fluorescent protein - Tagged HCV non-enveloped capsid like particles: development of a new tool for tracking HCV core uptake.

Functional recovery after spinal cord lesion remains an important goal. A combination of inhibitory molecules and lack of appropriate permissive factors in the lesioned spinal cord results in failure of fiber tract reconnection and function. Experimental transplantation in rodent and primate models of CNS injuries has led to the idea that Schwann cells (SCs) are promising candidates for autologous transplantation to assist myelination of lesions and to deliver therapeutic agents in the CNS. In this study, we used retroviral transduction to genetically modify SCs from transgenic GFP-mice in order to overexpress the cell adhesion molecule L1, a protein promoting neurite outgrowth and implicated in myelination. SCs transduced to express L1 or its chimeric secreted form L1-Fc were mixed and grafted rostrally to the lesion site of adult mice immediately after spinal cord compression injury. Our results indicate that 3 weeks postoperatively, but not thereafter, mice transplanted with L1/L1-Fc-expressing SCs exhibited faster locomotor recovery as compared to animals which received SCs transduced with a control vector or no cells at all. Morphological analysis indicated that the accelerated functional recovery correlated with earlier and enhanced myelination by both grafted and host SCs. Moreover, increased sprouting of serotonergic fibers into and across the lesion site was observed in the L1/L1-Fc group as compared with controls. Our results suggest that transplantation of L1-overexpressing SCs enhances early events in spinal cord repair after injury and may be considered in combinatorial strategies together with other regeneration-promoting molecules.

Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury.

The intrinsic inability of the central nervous system to efficiently repair traumatic injuries renders transplantation of neural stem/precursor cells (NPCs) a promising approach towards repair of brain lesions. In this study, NPCs derived from embryonic day 14.5 mouse cortex were genetically modified via transduction with a lentiviral vector to overexpress the neuronal lineage-specific regulator BM88/Cend1 that coordinates cell cycle exit and differentiation of neuronal precursors. BM88/Cend1-overexpressing NPCs exhibiting enhanced differentiation into neurons in vitro were transplanted in a mouse model of acute cortical injury and analyzed in comparison with control NPCs. Immunohistochemical analysis revealed that a smaller proportion of BM88/Cend1-overexpressing NPCs, as compared with control NPCs, expressed the neural stem cell marker nestin 1 day after transplantation, while the percentage of nestin-positive cells was significantly reduced thereafter in both types of cells, being almost extinct 1 week post-grafting. Both types of cells did not proliferate up to 4 weeks in vivo, thus minimizing the risk of tumorigenesis. In comparison with control NPCs, Cend1-overexpressing NPCs generated more neurons and less glial cells 1 month after transplantation in the lesioned cortex whereas the majority of graft-derived neurons were identified as GABAergic interneurons. Furthermore, transplantation of Cend1-overexpressing NPCs resulted in a marked reduction of astrogliosis around the lesioned area as compared to grafts of control NPCs. Our results suggest that transplantation of Cend1-overexpressing NPCs exerts beneficial effects on tissue regeneration by enhancing the number of generated neurons and restricting the formation of astroglial scar, in a mouse model of cortical brain injury.

Transplantation of embryonic neural stem/precursor cells overexpressing BM88/Cend1 enhances the generation of neuronal cells in the injured mouse cortex.

Strategies to enhance neural stem/precursor cell (NPC) capacity to yield multipotential, proliferative, and migrating pools of cells that can efficiently differentiate into neurons could be crucial for structural repair after neurodegenerative damage. Here, we have generated a lentiviral vector for expression of insulin-like growth factor-I (IGF-1) and investigated the impact of IGF-1 transduction on the properties of cultured NPCs (IGF-1-NPCs). Under proliferative conditions, IGF-1 transduction promoted cell cycle progression via cyclin D1 up-regulation and Akt phosphorylation. Remarkably upon differentiation-inducing conditions, IGF-1-NPCs cease to proliferate and differentiate to a greater extent into neurons with significantly longer neurites, at the expense of astrocytes. Moreover, using live imaging we provide evidence that IGF-1 transduction enhances the motility and tissue penetration of grafted NPCs in cultured cortical slices. These results illustrate the important consequence of IGF-1 transduction in regulating NPC functions and offer a potential strategy to enhance the prospective repair potential of NPCs.

Lentivirus-mediated expression of insulin-like growth factor-I promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons.

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

The inability of the central nervous system (CNS) to efficiently repair damages results in severe functional impairment after trauma or neurodegenerative/demyelinating diseases. Regeneration failure is attributed to inhibitory molecules creating a nonpermissive environment for axonal regrowth, and dictates the necessity for the development of novel therapeutic strategies. An emerging approach for improving regeneration is the use of gene therapy to manipulate cell adhesion molecule expression in experimental animal models of degeneration. Alternatively, cell transplantation to replace lost neurons and the grafting of myelinating cells to repair demyelinating lesions are promising approaches for treating CNS injuries and demyelination. Schwann cells (SCs), oligodendrocyte progenitors, olfactory ensheathing cells and embryonic and neural stem cells have been shown to form myelin after transplantation into the demyelinated CNS. The repair capacity of the peripheral nervous system (PNS) is much higher, but there is still a limit to the amount of nerve loss that can be bridged after injury, and longer nerve gaps call for the use of conduits populated with living cells. In both cases, the interaction of grafted cells with the host environment is of paramount importance for the incorporation and functional integration of these cells and the manipulation of cell adhesion molecules is an attractive approach towards achieving this goal. In this review we summarize data from the recent literature regarding the manipulation of cell adhesion molecule expression towards CNS and PNS repair and discuss the prospects for future therapeutic applications.

Alexandros A. Lavdas

Center for Biomedicine

European Academy Bozen/Bolzano (EURAC)

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Alexandros A. Lavdas

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European Academy Bozen/Bolzano (EURAC)

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Center for Biomedicine