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Background Aims: Cell-based therapies (CBTs) provide opportunities to treat rare and high-burden diseases. Manufacturing development of these innovative products is said to be complex and costly. However, little research is available providing insight into resource use and cost drivers. Therefore, this study aimed to assess the feasibility of estimating the cost of manufacturing development of two cell-based therapy case studies using a CBT cost framework specifically designed for small-scale cell-based therapies., Methods: A retrospective costing study was conducted in which the cost of developing an adoptive immunotherapy of Epstein-Barr virus-specific cytotoxic T lymphocytes (CTLs) and a pluripotent stem cell (PSC) master cell bank was estimated. Manufacturing development was defined as products advancing from technology readiness level 3 to 6. The study was conducted in a Scottish facility. Development steps were recreated via developer focus groups. Data were collected from facility administrative and financial records and developer interviews., Results: Application of the manufacturing cost framework to retrospectively estimate the manufacturing design cost of two case studies in one Scottish facility appeared feasible. Manufacturing development cost was estimated at £1,201,016 for CTLs and £494,456 for PSCs. Most costs were accrued in the facility domain (56% and 51%), followed by personnel (20% and 32%), materials (19% and 15%) and equipment (4% and 2%)., Conclusions: Based on this study, it seems feasible to retrospectively estimate resources consumed in manufacturing development of cell-based therapies. This fosters inclusion of cost in the formulation and dissemination of best practices to facilitate early and sustainable patient access and inform future cost-conscious manufacturing design decisions., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs)., Methods: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs., Results: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions., Conclusions: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies., Methods: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€)., Results: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices., Conclusions: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17- to 188-fold relative to nucleated tissues and 14- to 26-fold relative to samples digested with RNAse R to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched on average 12.7 times in platelets relative to nucleated tissues and identify 3162 genes significantly enriched for circRNAs, including some where all RNAseq reads appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in cultured megakaryocytes, and demonstrate that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost >90% of their progenitor mRNAs and that translation in platelets occurs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts previously classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artifacts., (© 2016 by The American Society of Hematology.)

The creation of red blood cells for the blood transfusion markets represents a highly innovative application of regenerative medicine with a medium term (5-10 year) prospect for first clinical studies. This article describes a case study analysis of a project to derive red blood cells from human embryonic stem cells, including the systemic challenges arising from (i) the selection of appropriate and viable regulatory protocols and (ii) technological constraints related to stem cell manufacture and scale up to clinical Good Manufacturing Practice (GMP) standard. The method used for case study analysis (Analysis of Life Science Innovation Systems (ALSIS)) is also innovative, demonstrating a new approach to social and natural science collaboration to foresight product development pathways. Issues arising along the development pathway include cell manufacture and scale-up challenges, affected by regulatory demands emerging from the innovation ecosystem (preclinical testing and clinical trials). Our discussion reflects on the efforts being made by regulators to adapt the current pharmaceuticals-based regulatory model to an allogeneic regenerative medicine product and the broader lessons from this case study for successful innovation and translation of regenerative medicine therapies, including the role of methodological and regulatory innovation in future development in the field., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34(+) CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 microM. CML CD34(+) cells were still able to expand over 72 hours with 5 microM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSE(max)) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

Imatinib mesylate (IM) therapy for chronic myeloid leukemia (CML) has transformed the treatment of this disease. However, the vast majority of patients, despite major responses, still harbor Philadelphia chromosome-positive (Ph(+)) cells. We have described a population of primitive Ph(+) cells that are insensitive to IM and may be a source of IM resistance. Cell line studies have suggested that the drug transporter ABCG2 may be a mediator of IM resistance, however there is considerable debate about whether IM is an ABCG2 substrate or inhibitor. We demonstrate here that primitive CML CD34(+) cells aberrantly overexpress functional ABCG2 but that cotreatment with IM and an ABCG2 inhibitor does not potentiate the effect of IM. We definitively show that IM is an inhibitor of, but not a substrate for, ABCG2 and that, therefore, ABCG2 does not modulate intracellular concentrations of IM in this clinically relevant cell population.

Dasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P = .031). In addition, CrKL phosphorylation was higher in the primitive CD34(+)CD38(-) than in the total CD34(+) population (P = .002). In total CD34(+) CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34(+)CD38(-) CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs.

Chronic myeloid leukemia (CML) is sustained by a rare population of primitive, quiescent, BCR-ABL+ cells and represents an excellent example of a malignancy in which tumor-initiating cells represent the key to disease eradication. CML is also the first malignancy for which targeted therapy has replaced conventional chemotherapy. Within a vast excess of proliferating progenitor cells that express breakpoint cluster region-abelson (BCR-ABL) and are exquisitely sensitive to the tyrosine kinase inhibitor imatinib mesylate (IM) resides a small population of quiescent leukemic cells that, despite higher levels of BCR-ABL transcripts, exhibits innate insensitivity to IM. These cells remain after IM therapy, even when apparently complete responses are achieved, and they probably explain molecular disease persistence. Although it can be argued that patients may survive for many years with low levels of leukemia still present, it is possible to achieve disease clearance at the molecular level following an allogeneic stem cell transplantation. The emergence of drug resistance with IM monotherapy also argues in favor of complete disease eradication that we believe should remain the ultimate therapeutic goal in CML. New approaches to the elimination of these primitive CML cells may thus be crucial to the development of curative strategies.

Induction of growth arrest and differentiation of some cancer cells by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], and its potent analogs, is well characterized. However, aggressive cancer cell lines are often either insensitive to the antiproliferative effects of 1alpha,25(OH)(2)D(3) or require toxic concentrations to recapitulate them which has, to-date, precluded its use in anticancer therapy. Therefore we are interested in mechanisms by which 1alpha,25(OH)(2)D(3) signaling has become deregulated in malignant cells in order to identify novel therapeutic targets. We observed previously that 1alpha,25(OH)(2)D(3) and its metabolites, generated via the C-24 oxidation pathway, drive simultaneous differentiation and hyper-proliferation within the same cell population. Thus we have proposed that metabolism of 1alpha,25(OH)(2)D(3) via the C-24 oxidation pathway represents a novel-signaling pathway, which integrates proliferation with differentiation. In the current study we examined further the role of this pathway and demonstrated that these effects are not restricted to leukemic cells but are observed also in both normal myeloid progenitors and breast cancer cell lines. Intriguingly, stable transfection of MCF-7 breast cancer cells with antisense vitamin D(3) receptor (VDR) reduced antiproliferative sensitivity to 1alpha,25(OH)(2)D(3) but significantly enhanced growth stimulation, which, in turn, was blocked by inhibiting metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway with ketoconazole. Taken together, these studies indicate that metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway gives rise to ligands with different biologic effects. We propose that this mechanism may allow the co-ordination of population expansion and cell maturation during differentiation. Cancer cells appear to corrupt this process during malignant transformation, by only responding to the pro-proliferative signals, thereby deriving a clonal advantage.

Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.