OBJECTIVES:Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro.MATERIALS AND METHODS:We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro.RESULTS:Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.CONCLUSIONS:This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 x 10(7) cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-target alterations and the biosafety of cells intended for therapeutic applications. Genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 nuclease systems are being investigated as safer and efficient alternatives for site-directed genome modification. Using site-specific integration into the AAVS1 locus of primary human cells as an example, we present an integrated approach to multimodal investigation of off-target alterations and an evaluation of potential genotoxicity induced by ZFN-mediated integration of a therapeutic transgene.

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth‐limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in‐house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 10 6 cells mL −1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 10 6 cells mL −1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96–3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half‐liter stirred‐bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O 2 binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.

Figure 1. Expansion of hPSC in agitated MC cultures result in reduced BPM4-based hematopoietic differentiation as compared to non-agitated static cultures. (A) Schematic showing the entire differentiation process starting from hPSC expansion on MCs (under static/agitation) to differentiation and expansion of hematopoietic precursors and erythroblasts followed by terminal maturation. All subsequent steps were performed under static conditions. (B) Images of hES3 MC aggregates following 7 days of static or 3 and 7 days of agitated culture. Flow cytometry evaluation of (C) pluripotency markers (3 or 7 days post hPSC expansion), (D) T-Bra (48 hrs post differentiation) and KDR (4 days post differentiation) expression in hES-3-MC aggregates initially expanded under static (static hPSC expansion) or agitated (3 or 7 days agitated hPSC expansion) condition during the pluripotent expansion stage. *P<0.05; P<0.001 as compared to static hPSC expansion condition. (E) Real-time RT-PCR data showing mean fold change in expression (relative to undifferentiated hES-3) of early hematopoietic specification markers (CD31, GATA2, GATA1, SCL, RUNX1) from hES-3-MC aggregates differentiated for 4 days. *P<0.05; P<0.001 as compared to static hPSC expansion condition. (F) Images of hematopoietic precursors on day 2 and day 14 post expansion in methylcellulose-based medium. Scale bar =1000 micron. (G) Total counts of hematopoietic precursors (day 14 post expansion in BGM medium following initial seeding of 1 x 10 cells) and erythroblasts (Day 14 post seeding in erythroblast expansion medium) differentiated from hES-3-MC aggregates derived from static and 3 or 7 days agitation cultures (P<0.001 as compared to static hPSC expansion condition). (H) Table summarizing flow cytometry expression (%) of CD235aCD71, total CD235a and fetal hemoglobin (HbF) expressing cells and total yield of viable cells 28 days post differentiation of hES3-MC aggregates. Corresponding P-values for comparison between static and agitation cultures as well as images of erythroblast cell pellets at termination of experiment (day 28) are shown. All data are mean ± SEM, n=3. A

In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.

Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC invivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implantedxFVIII activity secreted/million BMSCs/24hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.

Techniques enabling precise genome modifications enhance the safety of gene-based therapy. DLC1 is a hot spot for phiC31 integrase-mediated transgene integration in vitro and in vivo. Here we show that integration of a coagulation factor VIII transgene into intron 7 of DLC1 supports durable expression of factor VIII in primary human umbilical cord-lining epithelial cells. Oligoclonal cells with factor VIII transgene integrated in DLC1 did not have altered expression of DLC1 or neighbouring genes within a 1-Mb interval. Only 1.9% of all expressed genes were transcriptionally altered; most were downregulated and mapped to cell cycle and DNA repair pathways. DLC1-integrated cells were not tumourigenic in vivo and were normal by high-resolution genomic DNA copy number analysis. Our data identify DLC1 as a locus for durable transgene expression that does not incur features of insertional oncogenesis, thus expanding options for developing ex vivo cell therapy mediated by site-specific integration methods.

Introduction 1.1 Overview of gene therapyProgress in understanding the cellular and molecular bases of human health and disease in recent decades has spawned research in the fields of regenerative medicine and gene therapy.These novel approaches to medical treatments offer new possibilities of mitigating, and even curing, a plethora of medical conditions ranging from rare inherited monogenic disorders, metabolic diseases, infections and even complex disorders such as cancer.In a simplified form, gene therapy can be defined as any procedure aimed at genetically altering or modifying cells or tissues with exogenous genetic materials that encompasses RNA, DNA and even oligonucleotides.These molecules may be directly delivered, in vivo into patients, often with the goal of targeting particular tissues (or organs).Alternatively, patients' cells may be isolated, expanded and modified ex vivo before reimplantation into the same subject (figure 1).Whilst gene therapy appears to be a relatively new concept in the field of biomedicine, the original conceptualization of treating diseases by genetic engineering dates back as early as the 1940s.Avery, MacLeod and McCarthy pioneered the notion and demonstrated that genes could be transferred within nucleic acids (Avery et al., 1944).Early visionary investigators such as Tatum (Tatum, 1966) envisioned " that viruses will be effectively used for man's benefit, in theoretical studies in somatic-cell genetics and possibly in genetic therapy..." And at the end of that same decade, the earliest experimentation of gene delivery in humans was carried out controversially by Rogers and colleagues, who explored the idea of using Shope papilloma virus to treat three patients with arginase deficiency (Wolff & Lederberg, 1994).The decades that followed witnessed tremendous advances in recombinant DNA technology and enabled the first approved human gene therapy clinical trial in 1990 for treating infants with adenosine deaminase deficiency (Blaese et al., 1995).By the turn of the millennium, almost 4000 patients had received gene therapy from more than 500 clinical trials worldwide (Scollay, 2001), albeit with varying and limited successes.Nonetheless, these trials were helpful in highlighting several aspects of gene therapy that demanded improvements and refinements to achieve meaningful therapeutic efficacy and patient safety.

Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by non-viral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated <= 2 transgene(s). Expression of 95.6% of genes was unaltered in modified cells. Only three small regions showed genome copy number changes that did not correlate with altered gene expression or integration sites. Spectral karyotyping revealed rare nonrecurrent occurrence of three different translocations. Integrase-modified cells were not tumorigenic in immunocompromised mice for at least 4 months. Stable integration of a human factor VIII (FVIII) construct conferred durable FVIII secretion in vitro. Xeno-implantation of FVIII-secreting CLECs in immunocompetent hemophilic mice achieved significant phenotypic correction. Pre-evaluated clonal populations of phiC31 integrase-modified CLECs could be useful as bioimplants for monogenic diseases such as hemophilia.

BackgroundCurrent gene- and cell-based therapies have significant limitations which impede widespread clinical application. Taking diabetes mellitus as a paradigm, we have sought to overcome these limitations by ex vivo electrotransfer of a nonviral insulin expression vector into primary hepatocytes followed by immediate autologous reimplantation in a preclinical model of diabetes.Methods and ResultsIn a single 3-hour procedure, hepatocytes were isolated from a surgically resected liver wedge, electroporated with an insulin expression plasmid ex vivo and reimplanted intraparenchymally under ultrasonic guidance into the liver in each of 10 streptozotocin-induced diabetic Yorkshire pigs. The vector was comprised of a bifunctional, glucose-responsive promoter linked to human insulin cDNA. Ambient glucose concentrations appropriately altered human insulin mRNA expression and C-peptide secretion within minutes in vitro and in vivo. Treated swine showed correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities for ≥47 weeks. Metabolic correction correlated significantly with the number of hepatocytes implanted. Importantly, we observed no hypoglycemia even under fasting conditions. Direct intrahepatic implantation of hepatocytes did not alter biochemical indices of liver function or induce abnormal hepatic lobular architecture. About 70% of implanted hepatocytes functionally engrafted, appeared histologically normal, retained vector DNA and expressed human insulin for ≥47 weeks. Based on structural tissue analyses and transcriptome data, we showed that early correction of diabetes attenuated and even prevented pathological changes in the eye, kidney, liver and aorta.ConclusionsWe demonstrate that autologous hepatocytes can be efficiently, simply and safely modified by electroporation of a nonviral vector to express, process and secrete insulin durably. This strategy, which achieved significant and sustained therapeutic efficacy in a large preclinical model without adverse effects, warrants consideration for clinical development especially as it could have broader future applications for the treatment of other acquired and inherited diseases for which systemic reconstitution of a specific protein deficiency is critical.

We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10–20 min of exposure to 25 mM glucose or 60 μM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes (∼8 × 105 viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P⩽0.001) without fasting hypoglycemia and better glucose tolerance (P⩽0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for β-galactosidase activity and with anti-human insulin antibody.

Pt and Pt-SnOx catalysts prepared on gamma-Al2O3 spherical supports via ion-exchange and impregnation have been characterised using techniques that included AAS, XRF, BET, N-2 adsorption, Hg porosimetry, TPR, H-2 chemisorption, XPS, TEM and XRD. Deactivation in hydrogenolysis was more prominent with n-butane than propane. The activities shown by Pt/alumina and Pt/SnOx/alumina during a decreasing temperature sequence are lower than those seen during an earlier heating sequence and this suggests irreversible coking. The pseudo Arrhenius plots were non-linear and also showed some hysteresis. On cooling the S-2 selectivities were decreased, but S-1,S-3 values were increased. Apparently, the effects of coke deposition and presence of SnOx on product selectivity in n-butane hydrogenolysis were identical; terminal C-C bond scission being preferred over the internal C-C bond scission. Thus the Pt/SnOx/alumina catalyst showed lower S-2 (and higher S-1,S-3) than the Pt/alumina sample.

Ion-exchange of Pt(NH3)2+4 produces more highly dispersed platinum on silica than impregnation. For the first time, the kinetics of formation of Pt(NH3)2+4 are shown to be rather slow. The ion-exchange process at high pH involves a decrease in pH, a drop in concentration of platinum in solution, but an increase in silicon solution concentration as the silica undergoes dissolution. The latter process affects the total surface area and probably the dispersion and properties of the final platinum particles on the silica surface.