Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors.

BACKGROUND: Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS: The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION: This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

Isolation and culture of rhesus adipose-derived stem cells.

Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Rhesus monkeys are physiologically and phylogenetically similar to humans, and they and their cells are valuable for biomedical research and evaluation of preclinical therapies. Here, we describe methods for the isolation, culture, and differentiation of rhesus adipose-derived stem cells (rASCs).

Estimates of heritability for reproductive traits in captive rhesus macaque females.

Records from a colony of captive Indian rhesus macaques (Macaca mulatta) were used to estimate heritability for a number of reproductive traits. Records were based on a total of 7,816 births by 1,901 females from 1979 to 2007. Heritability was estimated with a linear animal model using a multiple trait derivative free REML set of programs. Because no male parents were identified, the numerator relationship matrix contained female kinships established over six generations. Reproductive traits included female age at the birth of the first, second and last infant, age at death, inter-birth intervals, number of infants born per female and infant survival. Heritability for each trait was estimated as the ratio of the additive genetic variance to phenotypic variance adjusted for significant fixed effects. Estimates of heritability for early reproduction ranged from 0.000+/-0.072 for birth interval after the first reproduction to 0.171+/-0.062 for age of female at the first infant. Higher estimates of heritability were found for female longevity [0.325+/-0.143] and for productivity of deceased females born before 1991 [0.221+/-0.138]. Heritability for infant survival ranged from 0.061+/-0.018 for survival from 30 days to 1 year to 0.290+/-0.050 for survival from birth to 30 days when adjusted to an underlying normal distribution. Eight of the 13 estimates of heritability for reproductive traits in this study were different from zero [P<0.05]. Generally, heritability estimates reported in this study for reproductive traits of captive rhesus macaque females are similar to those reported in the literature for free-ranging rhesus macaque females and for similar reproductive traits of other species. These estimates of heritability for reproductive traits appear to be among the first for a relatively large colony of captive rhesus macaque females.

Differentiation of nonhuman primate embryonic stem cells along neural lineages.

The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14-16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), beta-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and beta-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, beta-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen beta-tublinIII (73+/-4.7%) and some expressed intermediate neuron antigen MAP2 (18+/-7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%+/-1.2%), GFAP (4.93+/-1.9%), S100 (7+/-3.5%) and O4 (0.27+/-0.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.

Large animal models of neurological disorders for gene therapy.

he development of therapeutic interventions for genetic disorders and diseases that affect the central nervous system (CNS) has proven challenging. There has been significant progress in the development of gene therapy strategies in murine models of human disease, but gene therapy outcomes in these models do not always translate to the human setting. Therefore, large animal models are crucial to the development of diagnostics, treatments, and eventual cures for debilitating neurological disorders. This review focuses on the description of large animal models of neurological diseases such as lysosomal storage diseases, Parkinsons disease, Huntingtons disease, and neuroAIDS. The review also describes the contributions of these models to progress in gene therapy research.

Postthaw survival of rhesus macaque sperm: variation in the response of individual males to different freezing protocols.

This study was designed to assess the effects of various cryopreservation protocols on the postthaw survival of rhesus macaque sperm. Multiple ejaculates of five mature males were collected and frozen by each of four different methods. Cryopreservation significantly reduced motility in all samples, regardless of the method, and the response of different ejaculates of the same male to each method proved to be consistent. The freezing method was a significant factor in determining the immediate postthaw motility, although this effect disappeared after 4 hr. Moreover, there was a significant interaction between individual males and freezing method suggesting that developing a single freezing protocol that is universally suitable may be difficult.

Adipose-derived stem cells: isolation, expansion and differentiation.

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

Age as a determinant of reproductive success among captive female rhesus macaques (Macaca mulatta).

A retrospective analysis was performed on fertility outcomes among a colony of captive Indian rhesus monkeys. The analysis covered over 30 years and was based on 1443 females with a total of 11,453 pregnancies. Various determinants of fertility were assessed including birth rates, pregnancy loss, infant survival, interbirth intervals, and interval from last birth to death. Binary variables were analyzed with generalized linear models with random intercepts, while linear mixed models were used for analysis of continuous variables. Age of the dam was a significant factor in determining whether a pregnancy resulted in a birth and whether an infant survived the first 30 days with primiparous or older mothers being less likely to produce an infant surviving to that age. In contrast, sex proved to be the only significant factor in determining whether an infant lived to 1 year, with females being more likely to survive. The interval between births proved to be affected primarily by dam age, while the late death of an infant depressed the likelihood of an extended time interval between her last birth and her death. Overall, these results demonstrate that maternal age contributes significantly to a decline in fertility and older females can live relatively long periods following birth of their last infant.