Evaluating teratoma formation risk of pluripotent stem cell-derived cell therapy products: a consensus recommendation from the Health and Environmental Sciences Institute's International Cell Therapy Committee.

Aim& methods: The Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee launched an international, multisite study to evaluate the sensitivity and reproducibility of the highly efficient culture (HEC) assay, an in vitro assay to detect residual undifferentiated human pluripotent stem cells (hPSCs) in cell therapy products. Results: All facilities detected colonies of human induced pluripotent stem cells (hiPSCs) when five hiPSCs were spiked into 1 million hiPSC-derived cardiomyocytes. Spiking with a trace amount of hiPSCs revealed that repeatability accounts for the majority of reproducibility while the true positive rate was high. Conclusion: The results indicate that the HEC assay is highly sensitive and robust and can be generally applicable for tumorigenicity evaluation of hPSC-derived cell therapy products.

To the Editor: The molecular mechanisms and the control of smooth muscle cell (SMC) differentiation have been extensively investigated because of its therapeutic potential.1 To date, different cell types have been used to study SMC differentiation, including a variety of mouse embryonic stem cells,2 adult stem cells,3,4 and others.5 Because several fundamental differences exist between mouse and human embryonic development,6 lack of a good model system to study human SMC differentiation has hampered the progress of translating SMC knowledge to novel clinical therapies. Human embryonic stem (hES) cells provide a valuable source of cells for studying human cell differentiation and developing therapeutic potentials in regenerative medicine. Since the initial report describing the derivation of hES cells,7 a variety of studies have established in vitro differentiation strategies to several lineages. Recently, it has been demonstrated that vascular progenitors derived from hES cells could be differentiated into endothelial cells and SMCs by endothelial …

Human pluripotent stem cells are considered to be ideal cell sources for regenerative medicine, but their clinical and industrial application is hindered by their tumorigenic potential. Previously we have identified a pluripotent stem cell-specific lectin rBC2LCN recognizing podocalyxin as a cell surface ligand. More recently, podocalyxin was found to be a soluble ligand of rBC2LCN that is secreted specifically from human pluripotent stem cells into cell culture media. Taking advantage of this phenomenon, we have previously developed a sandwich assay targeting the soluble podocalyxin using rBC2LCN as a capturing probe and another lectin rABA as an overlay probe to detect human pluripotent stem cells residing in cell therapy products derived from human pluripotent stem cells. A drawback to this, however, was that cell culture media containing fetal bovine serum was found to cause a substantial background signal to the sandwich assay. To reduce the background and increase the sensitivity, we screened different overlay probes to detect the soluble podocalyxin. Among them, an anti-keratan sulfate monoclonal antibody called R-10G showed the highest sensitivity and provided a low background signal to fetal bovine serum. The established sandwich assay using rBC2LCN and R-10G was proved to be powerful, which allowed the high-sensitive detection of human induced pluripotent stem cells residing among clinical-grade cardiomyocytes and neural stem cells, both derived from human induced pluripotent stem cells. The developed method has a possibility to be a standard technology to detect human induced pluripotent stem cells resided in various types of cell therapy products.

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.

A brief overview of cell therapy and its product

Promise of Human Induced Pluripotent Stem Cells in Skin Regeneration and Investigation

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

Recognizing the challenges faced by researchers and clinicians working in the field of cellular therapy, the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, established the Production Assistance for Cellular Therapies (PACT) program in 2003 and expanded it in 2010. The PACT program provides both clinical product manufacturing support that furthers the mission of NHLBI in the areas of cardiac, lung, and blood diseases and broad support of translational development across all disease areas to serve the entire cell therapy community. The program also provides access to expertise in project management, regulatory affairs, and quality assurance and control. Education initiatives include webinars, cell processing facility-hosted workshops, national workshops, and active participation and leadership within the cell therapy community through collaboration with other cell therapy organizations and academia. So far, over 650 PACT-manufactured cell therapy products have been administered in 32 clinical trials for a range of illnesses and diseases such as acute myocardial infarction, sickle cell disease, and graft-versus-host disease.

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Collaborative science in action: A 20 year perspective from the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee

12 - Biomarker Qualification – Past, Present and Future: Initiating a Cross-Sector Toxicogenomics Biomarker Initiative – Health and Environmental Sciences Institute (HESI) Committee on Application of Genomics to Mechanism-Based Risk Assessment

Administration of a new drug candidate in a first-in-human (FIH) clinical trial is a particularly challenging phase in drug development and is especially true for immunomodulators, which are a diverse and complex class of drugs with a broad range of mechanisms of action and associated safety risks. Risk is generally greater for immunostimulators, in which safety concerns are associated with acute toxicity, compared to immunosuppressors, where the risks are related to chronic effects. Current methodologies for FIH dose selection for immunostimulators are focused primarily on identifying the minimum anticipated biological effect level (MABEL), which has often resulted in sub-therapeutic doses, leading to long and costly escalation phases. The Health and Environmental Sciences Institute (HESI) - Immuno-Safety Technical Committee (ITC) organized a project to address this issue through two complementary approaches: (i) an industry survey on FIH dose selection strategies and (ii) detailed case studies for immunomodulators in oncology and non-oncology indications. Key messages from the industry survey responses highlighted a preference toward more dynamic PK/PD approaches as in vitro assays are seemingly not representative of true physiological conditions for immunomodulators. These principles are highlighted in case studies. To address the above themes, we have proposed a revised decision tree, which expands on the guidance by the IQ MABEL Working Group (Leach et al. 2021). This approach facilitates a more refined recommendation of FIH dose selection for immunomodulators, allowing for a nuanced consideration of their mechanisms of action (MOAs) and the associated risk-to-benefit ratio, among other factors.

Cell therapy as a new medical technology in the 21st century has become the consensus of medical community in the world. The US Food and Drug Administration divides cell therapy products into stem cell-derived cell therapy products and mature/ functionally differentiated cell-derived cell therapy products. Cell therapy community in China has not clearly defined these two kinds of cell therapy products. One serious issue is that cell therapy community in China misuses the identification standards of mesenchymal stromal cell established by International Society of Cellular Therapy as the identification standard of mesenchymal stem cell, which has led to wrong policy, regulation and administration of cell therapy from the relevant government ministries, and hinder the development of cell therapy in China. In terms of the long history of clinical cell therapy of neruorestoration, published papers, number of treated patients, treatment outcome and impact in professional field, China is currently in the forefront of the world in the clinical cell therapy of neurorestoration. Only the academic community of cell therapy defines the clear concept of both stem cell therapy products and mature/ functionally differentiated cell therapy products and determines their boundaries, and related government ministries may develop laws and regulations with a clear boundary between two kinds of cell therapy products, which can promote clinical cell therapy innovation in China. Key words: Stem cells; Standards; Mesenchymal stromal cell; Neurorestoration

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

SummaryInduced pluripotent stem cells (iPSCs) are valuable in disease modeling because of their potential to expand and differentiate into virtually any cell type and recapitulate key aspects of human biology. Functional genomics are genome-wide studies that aim to discover genotype-phenotype relationships, thereby revealing the impact of human genetic diversity on normal and pathophysiology. In this review, we make the case that human iPSCs (hiPSCs) are a powerful tool for functional genomics, since they provide an in vitro platform for the study of population genetics. We describe cutting-edge tools and strategies now available to researchers, including multi-omics technologies, advances in hiPSC culture techniques, and innovations in drug development. Functional genomics approaches based on hiPSCs hold great promise for advancing drug discovery, disease etiology, and the impact of genetic variation on human biology.