Development of a cost efficient platform for the industrial manufacturing of pluripotent stem cell derived products for cell therapy: Cell expansion is the starting point

The development of stem cell-derived allogeneic therapeutics requires manufacturing processes able to generate high-density cultures of pluripotent stem cells (PSCs) to be further differentiated to target somatic cells. The Cell Plasticity platform of The Cell and Gene Therapy Catapult (CGT) is a core program that focuses on the cost efficient development of bioprocesses for the industrial manufacture of PSC-derived products in 2D and 3D culture systems. We started this program by establishing banks of PSCs adapted to defined culture systems and used conventional analytical techniques to characterise the cells to industry standards. Defined media were evaluated for the expansion of induced pluripotent stem cells (iPSC) in adherent culture. Scale-down high-throughput tools along with Design of Experiment methodology have been employed to establish a baseline process for the expansion of PSC as cellular aggregates in stirred-suspension culture and targeting cell yield \u3e 5x106 viable cells/mL. We are currently investigating bioengineering parameters for scale-up and evaluating cell retention devices for the dissociation of PSC aggregates in a closed and automated fashion. In parallel, a framework of analytical assays comprising imaging, flow-cytometry and gene expression is under development for process monitor and control using a proprietary multi-parametric analysis approach

ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model

Abstract Background Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice. Methods Parasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load. Results This study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission. Conclusions These results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration

Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

<div><p>Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of <i>Plasmodium chabaudi</i> in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of <i>P</i>. <i>chabaudi</i> infection and <i>P</i>. <i>yoelii</i> infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated <i>P</i>. <i>chabaudi</i>-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the <i>Il21</i> gene, or B cells with a targeted disruption of the <i>Il21r</i> gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic <i>P</i>. <i>chabaudi</i> infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, <i>Il21</i>, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.</p></div

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-specific antibody levels of a hyper-immune standard plasma defined as 1000 U. In the cases where levels of antibodies were below background, arbitrary values of 2 log lower than the mean value observed in WT C57BL/6 mice were set to be able to perform the statistical test. (D) MSP1₂₁-specific IgG-producing ASC in BM obtained from one femur and one tibia, and (E) MBC per spleen, determined by ELISPOT 32 days post-infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01), or comparing with the data obtained from the WT C57BL/6 group (# #, P<0.01). The Mann Whitney U test was used in the case of IgG subtypes, comparing <i>Il21</i>^<i>-/-</i> vs WT C57BL/6 mice (#, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 3–8 mice per time point.</p

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Additional file 3. Similar levels of tissue parasites were seen for kidney or gut in infections of icam1−/−, cd36−/−, and their respective controls, at days 5, 7 and 9 post-infection

Mice deficient in IL-21 signaling fail to develop immunity to a secondary <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Scheme describing the experimental approach. CQ = chloroquine. (B and C) <i>P</i>. <i>chabaudi</i>-infected mice were treated with chloroquine to eliminate parasitemia as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004715#sec009" target="_blank">materials and methods</a>, and re-infected with 10⁵<i>P</i>. <i>chabaudi-</i>infected rbc (day 0 post-secondary infection). The graphs show the course of secondary <i>P</i>. <i>chabaudi</i> infection in WT C57BL/6 (black circles), <i>Il21</i>^<i>-/-</i> (red circles) and <i>Il21r</i>^<i>-/-</i> (brown circles) mice; course of primary infection in <i>Il21</i>^<i>-/-</i> (gray circles) and <i>Il21r</i>^<i>-/-</i> (gray squares) are overlaid. (D and E) Number of Tfh cells per spleen post-primary and secondary infection, respectively. (F and G) Number of IFN-γ⁺CD4⁺ T cells per spleen post-primary and secondary infection, respectively. Data are representative of two independent experiments and are obtained in groups of 3–10 mice per time point. Statistical significance was obtained using Mann Whitney U test (**, P<0.01) or Kruskal-Wallis test (#, P<0.05). Error bars correspond to mean ± SEM.</p

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-specific antibody levels of a hyper-immune standard plasma defined as 1000 U. In the cases where levels of antibodies were below background, arbitrary values of 2 log lower than the mean value observed in WT C57BL/6 mice were set to be able to perform the statistical test. (D) MSP1₂₁-specific IgG-producing ASC in BM obtained from one femur and one tibia, and (E) MBC per spleen, determined by ELISPOT 32 days post-infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01), or comparing with the data obtained from the WT C57BL/6 group (# #, P<0.01). The Mann Whitney U test was used in the case of IgG subtypes, comparing <i>Il21</i>^<i>-/-</i> vs WT C57BL/6 mice (#, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 3–8 mice per time point.</p

IL-21 is produced during <i>P</i>. <i>chabaudi</i> infection and required to control chronic infection.

<p>(A) IL-21 mRNA in spleen cells of <i>P</i>. <i>chabaudi</i>-infected mice measured by real-time quantitative RT-PCR. Parasitemia (B) and total rbc counts (C) were determined in WT C57BL/6 (closed circles), <i>Il21</i>^<i>-/-</i> (open circles) and <i>Il21r</i>^<i>-/-</i> (open squares) mice. (D) Individual examples of spleens from <i>Il21r</i>^<i>-/-</i> (a) <i>Il21</i>^<i>-/-</i> (b) and WT C57BL/6 (c) mice at day 120 post-infection, and a spleen from an age-matched WT C57BL/6 naïve mouse (d). Bar, 1 cm. (E) Total number of nucleated live splenocytes were determined with a hemocytometer in WT C57BL/6 (black bars), <i>Il21</i>^<i>-/-</i> (open bars) and <i>Il21r</i>^<i>-/-</i> (stripped bars) mice. (F) Numbers of Ter119⁺ and Ter119^– cells in the spleen of WT C57BL/6 (black bars) and <i>Il21r</i>^<i>-/-</i> (striped bars) at day 32 post-infection. Data are representative of two or more independent experiments and are obtained in groups of 5–10 mice per time point. Statistical significance was obtained using Mann Whitney U test or Kruskal-Wallis test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Error bars correspond to mean ± SEM.</p

MOESM1 of ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model

Additional file 1. a) Constructs used to generate P. chabaudi smac mutants: (i) Δsmac; (ii) ΔsmacEFluc. b) PCR verification of insertion into the SMAC locus. Integration of the plasmid was verified with primer set P1/P2 and the loss of the wild type locus is shown using primer set P2/P3. Lanes 1–5 contain samples from parasites transfected with Δ smac (1), ΔsmacEFluc (2), wild-type parasites (3–4), water control (5). c) Integration of the plasmids into chromosome 1. PFG separated chromosomes hybridized with a 3′UTR pbdhfr/ts probe show insertion of the plasmid into chromosome 1: P. chabaudi wild-type DNA (1), ΔsmacEFluc (2–3), The probe also hybridizes to the endogenous P. chabaudi dhfr locus on chromosome 7. d) Relative light emission levels are similar for Δsmac and ΔsmacEFluc parasites

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Additional file 2. PCR primers for verification of integration