Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation.

BACKGROUND: Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. METHODS: An in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. RESULTS: IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. CONCLUSIONS: We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP.

BACKGROUND: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. METHODS: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. RESULTS: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. CONCLUSION: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

Neurotoxicity is mediated by a variety of modes-of-actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC-derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM)) of maturation-supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA-grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC-derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity.

Development of the Concept for Stem Cell-Based Developmental Neurotoxicity Evaluation.

Human brain development consists of a series of complex spatiotemporal processes that if disturbed by chemical exposure causes irreversible impairments of the nervous system. To evaluate a chemical disturbance in an alternative assay, the concept evolved that the complex procedure of brain development can be disassembled into several neurodevelopmental endpoints which can be represented by a combination of different alternative assays. In this review article, we provide a scientific rationale for the neurodevelopmental endpoints that are currently chosen to establish assays with human stem/and progenitor cells. Assays covering these major neurodevelopmental endpoints are thought to assemble as building blocks of a DNT testing battery.

Current Availability of Stem Cell-Based In Vitro Methods for Developmental Neurotoxicity (DNT) Testing.

There is evidence that chemical exposure during development can cause irreversible impairments of the human developing nervous system. Therefore, testing compounds for their developmentally neurotoxic potential has high priority for different stakeholders: academia, industry, and regulatory bodies. Due to the resource-intensity of current developmental neurotoxicity (DNT) in vivo guidelines, alternative methods that are scientifically valid and have a high predictivity for humans are especially desired by regulators. Here, we review availability of stem-/progenitor cell-based in vitro methods for DNT evaluation that is based on the concept of neurodevelopmental process assessment. These test methods are assembled into a DNT in vitro testing battery. Gaps in this testing battery addressing research needs are also pointed out.

Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro.

Developmental neurotoxicity (DNT) testing performed in rats is resource-intensive (costs, time, animals) and bears the issue of species extrapolation. Thus, reliable alternative human-based approaches are needed for predicting neurodevelopmental toxicity. Human induced pluripotent stem cells (hiPSCs) represent a basis for an alternative method possibly being part of an alternative DNT testing strategy. Here, we compared two hiPSC neural induction protocols resulting in 3D neurospheres: one using noggin and one cultivating cells in neural induction medium (NIM protocol). Performance of Nestin+/SOX2+ hiPSC-derived neural progenitor cells (NPCs) was compared to primary human NPCs. Generally, primary hNPCs first differentiate into Nestin+ and/or GFAP+ radial glia-like cells, while the hiPSC-derived NPCs (hiPSC-NPC) first differentiate into ßIII-Tubulin+ neurons suggesting an earlier developmental stage of hiPSC-NPC. In the 'Neurosphere Assay', NIM generated hiPSC-NPC produced neurons with higher performance than with the noggin protocol. After long-term differentiation, hiPSC-NPC form neuronal networks, which become electrically active on microelectrode arrays after 85days. Finally, methylmercury chloride inhibits hiPSC-NPC and hNPC migration with similar potencies. hiPSC-NPCs-derived neurospheres seem to be useful for DNT evaluation representing early neural development in vitro. More system characterization by compound testing is needed to gain higher confidence in this method.