A risk-based approach for cell line development, manufacturing and characterization of genetically engineered, induced pluripotent stem cell-derived allogeneic cell therapies.

Advances in cellular reprogramming and gene-editing approaches have opened up the potential for a new class of ex vivo cell therapies based on genetically engineered, induced pluripotent stem cell (iPSC)-derived allogeneic cells. While these new therapies share some similarities with their primary cell-derived autologous and allogeneic cell therapy predecessors, key differences exist in the processes used for generating genetically engineered, iPSC-derived allogeneic therapies. Specifically, in iPSC-derived allogeneic therapies, donor selection and gene-editing are performed once over the lifetime of the product as opposed to as part of the manufacturing of each product batch. The introduction of a well-characterized, fully modified, clonally derived master cell bank reduces risks that have been inherent to primary-cell derived autologous and allogeneic therapies. Current regulatory guidance, which was largely developed based on the learnings gained from earlier generation therapies, leaves open questions around considerations for donor eligibility, starting materials and critical components, cell banking and genetic stability. Here, a risk-based approach is proposed to address these considerations, while regulatory guidance continues to evolve.

Production, safety and efficacy of iPSC-derived mesenchymal stromal cells in acute steroid-resistant graft versus host disease: a phase I, multicenter, open-label, dose-escalation study.

The therapeutic potential of donor-derived mesenchymal stromal cells (MSCs) has been investigated in diverse diseases1, including steroid-resistant acute graft versus host disease (SR-aGvHD)2. However, conventional manufacturing approaches are hampered by challenges with scalability and interdonor variability, and clinical trials have shown inconsistent outcomes3,4. Induced pluripotent stem cells (iPSCs) have the potential to overcome these challenges, due to their capacity for multilineage differentiation and indefinite proliferation5,6. Nonetheless, human clinical trials of iPSC-derived cells have not previously been completed. CYP-001 (iPSC-derived MSCs) is produced using an optimized, good manufacturing practice (GMP)-compliant manufacturing process. We conducted a phase 1, open-label clinical trial (no. NCT02923375) in subjects with SR-aGvHD. Sixteen subjects were screened and sequentially assigned to cohort A or cohort B (n = 8 per group). One subject in cohort B withdrew before receiving CYP-001 and was excluded from analysis. All other subjects received intravenous infusions of CYP-001 on days 0 and 7, at a dose level of either 1 × 106 cells per kg body weight, to a maximum of 1 × 108 cells per infusion (cohort A), or 2 × 106 cells per kg body weight, to a maximum dose of 2 × 108 cells per infusion (cohort B). The primary objective was to assess the safety and tolerability of CYP-001, while the secondary objectives were to evaluate efficacy based on the proportion of participants who showed a complete response (CR), overall response (OR) and overall survival (OS) by days 28/100. CYP-001 was safe and well tolerated. No serious adverse events were assessed as related to CYP-001. OR, CR and OS rates by day 100 were 86.7, 53.3 and 86.7%, respectively. The therapeutic application of iPSC-derived MSCs may now be explored in diverse inflammatory and immune-mediated diseases.

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.

Feasibility, Safety, and Tolerance of Mesenchymal Stem Cell Therapy for Obstructive Chronic Lung Allograft Dysfunction.

Feasibility, tolerance, and safety of intravenous infusions of allogeneic mesenchymal stem cell (MSC) therapy in lung transplant recipients with bronchiolitis obliterans syndrome (BOS) are not well established. MSCs were manufactured, cryopreserved, transported to our facility, thawed, and infused into nine recipients with moderate BOS (average drop in forced expiratory volume in 1 second was 56.8% ± 3.2% from post-transplant peak) who were refractory to standard therapy and not candidates for retransplant. Cells were viable and sterile prior to infusion. Patients received a single infusion of either 1 (n = 3), 2 (n = 3), or 4 (n = 3) million MSCs per kg. Patients were medically evaluated before; during; and at 24 hours, 1 week, and 1 month after infusion for evidence of infusion-related adverse events and tolerance of therapy. Vital signs, pulmonary function test results, Borg Dyspnea Index, and routine laboratory data were recorded. Vital signs and O2 saturation did not significantly change during or up to 2 hours after MSC infusion. There were no significant changes in gas exchange variables, pulmonary function test results, or laboratory values at 1, 7, and 30 days postinfusion compared with preinfusion values. Infusion of MSCs in patients with BOS was feasible, safe, and well tolerated and did not produce any significant adverse changes in clinical, functional, or laboratory variables during or up to 30 days after infusion. Manufacturing, transport, and administration of intravenous, allogeneic bone marrow-derived MSCs in doses from 1 to 4 million MSCs per kg is safe in lung transplant recipients with BOS. Stem Cells Translational Medicine 2018;7:161-167.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

Biodistribution and Clearance of Human Mesenchymal Stem Cells by Quantitative Three-Dimensional Cryo-Imaging After Intravenous Infusion in a Rat Lung Injury Model.

: Cell tracking is a critical component of the safety and efficacy evaluation of therapeutic cell products. To date, cell-tracking modalities have been hampered by poor resolution, low sensitivity, and inability to track cells beyond the shortterm. Three-dimensional (3D) cryo-imaging coregisters fluorescent and bright-field microcopy images and allows for single-cell quantification within a 3D organ volume. We hypothesized that 3D cryo-imaging could be used to measure cell biodistribution and clearance after intravenous infusion in a rat lung injury model compared with normal rats. A bleomycin lung injury model was established in Sprague-Dawley rats (n = 12). Human mesenchymal stem cells (hMSCs) labeled with QTracker655 were infused via jugular vein. After 2, 4, or 8 days, a second dose of hMSCs labeled with QTracker605 was infused, and animals were euthanized after 60, 120, or 240 minutes. Lungs, liver, spleen, heart, kidney, testis, and intestine were cryopreserved, followed by 3D cryo-imaging of each organ. At 60 minutes, 82% ± 9.7% of cells were detected; detection decreased to 60% ± 17% and 66% ± 22% at 120 and 240 minutes, respectively. At day 2, 0.06% of cells were detected, and this level remained constant at days 4 and 8 postinfusion. At 60, 120, and 240 minutes, 99.7% of detected cells were found in the liver, lungs, and spleen, with cells primarily retained in the liver. This is the first study using 3D cryo-imaging to track hMSCs in a rat lung injury model. hMSCs were retained primarily in the liver, with fewer detected in lungs and spleen. SIGNIFICANCE: Effective bench-to-bedside clinical translation of cellular therapies requires careful understanding of cell fate through tracking. Tracking cells is important to measure cell retention so that delivery methods and cell dose can be optimized and so that biodistribution and clearance can be defined to better understand potential off-target toxicity and redosing strategies. This article demonstrates, for the first time, the use of three-dimensional cryo-imaging for single-cell quantitative tracking of intravenous infused clinical-grade mesenchymal stem cells in a clinically relevant model of lung injury. The important information learned in this study will help guide future clinical and translational stem cell therapies for lung injuries.

Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion.

BACKGROUND AIMS: In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo-generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. METHODS: Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. RESULTS: The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. DISCUSSION: Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.

Intravenous Followed by X-ray Fused with MRI-Guided Transendocardial Mesenchymal Stem Cell Injection Improves Contractility Reserve in a Swine Model of Myocardial Infarction.

The aim of this study is to determine the effects of early intravenous (IV) infusion later followed by transendocardial (TE) injection of allogeneic mesenchymal stem cells (MSCs) following myocardial infarction (MI). Twenty-four swine underwent balloon occlusion reperfusion MI and were randomized into 4 groups: IV MSC (or placebo) infusion (post-MI day 2) and TE MSC (or placebo) injection targeting the infarct border with 2D X-ray fluoroscopy fused to 3D magnetic resonance (XFM) co-registration (post-MI day 14). Continuous ECG recording, MRI, and invasive pressure-volume analyses were performed. IV MSC plus TE MSC treated group was superior to other groups for contractility reserve (p = 0.02) and freedom from VT (p = 0.03) but had more lymphocytic foci localized to the peri-infarct region (p = 0.002). No differences were observed in post-MI remodeling parameters. IV followed by XFM targeted TE MSC therapy improves contractility reserve and suppresses VT but does not affect post-MI remodeling and may cause an immune response.

A reproducible immunopotency assay to measure mesenchymal stromal cell-mediated T-cell suppression.

BACKGROUND AIMS: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression. METHODS: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. RESULTS: Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen-DR expression. CONCLUSIONS: We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy.

Bilateral administration of autologous CD133+ cells in ambulatory patients with refractory critical limb ischemia: lessons learned from a pilot randomized, double-blind, placebo-controlled trial.

BACKGROUND AIMS: CD133+ cells confer angiogenic potential and may be beneficial for the treatment of critical limb ischemia (CLI). However, patient selection, blinding methods and end points for clinical trials are challenging. We hypothesized that bilateral intramuscular administration of cytokine-mobilized CD133+ cells in ambulatory patients with refractory CLI would be feasible and safe. METHODS: In this double-blind, randomized sham-controlled trial, subjects received subcutaneous injections of granulocyte colony-stimulating factor (10 µg/kg per day) for 5 days, followed by leukapheresis, and intramuscular administration of 50-400 million sorted CD133+ cells delivered into both legs. Control subjects received normal saline injections, sham leukapheresis and intramuscular injection of placebo buffered solution. Subjects were followed for 1 year. An aliquot of CD133+ cells was collected from each subject to test for genes associated with cell senescence. RESULTS: Seventy subjects were screened, of whom 10 were eligible. Subject enrollment was suspended because of a high rate of mobilization failure in subjects randomly assigned to treatment. Of 10 subjects enrolled (7 randomly assigned to treatment, 3 randomly assigned to control), there were no differences in serious adverse events at 12 months, and blinding was preserved. There were non-significant trends toward improved amputation-free survival, 6-minute walk distance, walking impairment questionnaire and quality of life in subjects randomly assigned to treatment. Successful CD133+ mobilizers expressed fewer senescence-associated genes compared with poor mobilizers. CONCLUSIONS: Bilateral administration of autologous CD133+ cells in ambulatory CLI subjects was safe, and blinding was preserved. However, poor mobilization efficiency combined with high CD133+ senescence suggests futility in this approach.

Induced pluripotent stem cells for post-myocardial infarction repair: remarkable opportunities and challenges.

Coronary artery disease with associated myocardial infarction continues to be a major cause of death and morbidity around the world, despite significant advances in therapy. Patients who have large myocardial infarctions are at highest risk for progressive heart failure and death, and cell-based therapies offer new hope for these patients. A recently discovered cell source for cardiac repair has emerged as a result of a breakthrough reprogramming somatic cells to induced pluripotent stem cells (iPSCs). The iPSCs can proliferate indefinitely in culture and can differentiate into cardiac lineages, including cardiomyocytes, smooth muscle cells, endothelial cells, and cardiac progenitors. Thus, large quantities of desired cell products can be generated without being limited by cellular senescence. The iPSCs can be obtained from patients to allow autologous therapy or, alternatively, banks of human leukocyte antigen diverse iPSCs are possible for allogeneic therapy. Preclinical animal studies using a variety of cell preparations generated from iPSCs have shown evidence of cardiac repair. Methodology for the production of clinical grade products from human iPSCs is in place. Ongoing studies for the safety of various iPSC preparations with regard to the risk of tumor formation, immune rejection, induction of arrhythmias, and formation of stable cardiac grafts are needed as the field advances toward the first-in-man trials of iPSCs after myocardial infarction.

Induced pluripotent stem cells as custom therapeutics for retinal repair: progress and rationale.

Human pluripotent stem cells have made a remarkable impact on science, technology and medicine by providing a potentially unlimited source of human cells for basic research and clinical applications. In recent years, knowledge gained from the study of human embryonic stem cells and mammalian somatic cell reprogramming has led to the routine production of human induced pluripotent stem cells (hiPSCs) in laboratories worldwide. hiPSCs show promise for use in transplantation, high throughput drug screening, "disease-in-a-dish" modeling, disease gene discovery, and gene therapy testing. This review will focus on the first application, beginning with a discussion of methods for producing retinal lineage cells that are lost in inherited and acquired forms of retinal degenerative disease. The selection of appropriate hiPSC-derived donor cell type(s) for transplantation will be discussed, as will the caveats and prerequisite steps to formulating a clinical Good Manufacturing Practice (cGMP) product for clinical trials.

Banking human induced pluripotent stem cells: lessons learned from embryonic stem cells?

The generation of human embryonic stem cell banking networks has ensured that well-characterized and quality controlled stem cell lines are broadly accessible to researchers worldwide. Here, we provide recommendations for engaging these established networks in efforts to build similar resources for the distribution and collection of induced pluripotent stem cells.

Cell therapy for lung diseases. Report from an NIH-NHLBI workshop, November 13-14, 2012.

The National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health convened the Cell Therapy for Lung Disease Working Group on November 13-14, 2012, to review and formulate recommendations for future research directions. The workshop brought together investigators studying basic mechanisms and the roles of cell therapy in preclinical models of lung injury and pulmonary vascular disease, with clinical trial experts in cell therapy for cardiovascular diseases and experts from the NHLBI Production Assistance for Cell Therapy program. The purpose of the workshop was to discuss the current status of basic investigations in lung cell therapy, to identify some of the scientific gaps in current knowledge regarding the potential roles and mechanisms of cell therapy in the treatment of lung diseases, and to develop recommendations to the NHLBI and the research community on scientific priorities and practical steps that would lead to first-in-human trials of lung cell therapy.

A call for standardized naming and reporting of human ESC and iPSC lines.

Human embryonic and induced pluripotent stem cell lines are being generated at a rapid pace and now number in the thousands. We propose a standard nomenclature and suggest the use of a centralized database for all cell line names and a minimum set of information for reporting new derivations.

The International Stem Cell Banking Initiative (ISCBI): raising standards to bank on.

The International Stem Cell Banking Initiative (ISCBI) aims to create a global network of stem cell banks to facilitate best practice in stem cell research and clinical cell delivery, primary objectives of national and local governments worldwide and stem cell organizations such the International Stem Cell Forum and the International Society of Stem Cell Research. This paper is a brief overview of ISCBI, its primary activities, potential network participants, and the challenges for harmonizing stem cell banking on a global level.