技术资料

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients,eliminate virus infections,then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs),they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs,but there was little apparent expansion of these cells in patients. In that study,VSTs were gene-modified on day 19 of culture and we hypothesized that by this time,sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism,we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV),Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates,so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively,and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2,TNF-α,IFN-γ,MIP-1α,MIP-1β and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype,they should expand and persist in vivo,simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR). View Publication

Following haematopoietic stem cell transplantation,monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood,bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood,a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid,inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit,Accumol,Calgary,AB,Canada) (P = 0.59). Of the 303 samples tested by flow cytometry,290 (95.7%) exceeded 90% purity,and 215 (70.95%) were over 99% pure. There were some outlying samples,showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols,allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature,assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results. View Publication