Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BACKGROUND: According to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens. METHODS: As this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction. RESULTS: Our results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3-22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3-73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1ß, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-ß1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-ß1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-ß1. However, treatment of MSCs with TGF-ß1 for 24 h completely abrogated constitutive expression of IDO. CONCLUSIONS: Irrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products.

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

Manufacturing and validation of Good Manufacturing Practice-compliant regulatory dendritic cells for infusion into organ transplant recipients.

BACKGROUND AIMS: Regulatory (or "tolerogenic") dendritic cells (DCregs) are a highly promising, innovative cell therapy for the induction or restoration of antigen-specific tolerance in immune-mediated inflammatory disorders. These conditions include organ allograft rejection, graft-versus-host disease following bone marrow transplantation and various autoimmune disorders. DCregs generated for adoptive transfer have potential to reduce patients' dependence on non-specific immunosuppressive drugs that can induce serious side effects and enhance the risk of infection and certain types of cancer. Here, our aim was to provide a detailed account of our experience manufacturing and validating comparatively large numbers of Good Manufacturing Practice-grade DCregs for systemic (intravenous) infusion into 28 organ (liver) transplant recipients and to discuss factors that influence the satisfaction of release criteria and attainment of target cell numbers. RESULTS: DCregs were generated in granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 from elutriated monocyte fractions isolated from non-mobilized leukapheresis products of consenting healthy adult prospective liver transplant donors. Vitamin D3 was added on day 0 and 4 and IL-10 on day 4 during the 7-day culture period. Release and post-release criteria included cell viability, purity, phenotype, sterility and functional assessment. The overall conversion rate of monocytes to DCregs was 28 ± 8.2%, with 94 ± 5.1% product viability. The mean cell surface T-cell co-inhibitory to co-stimulatory molecule (programmed death ligand-1:CD86) mean fluorescence intensity ratio was 3.9 ± 2.2, and the mean ratio of anti-inflammatory:pro-inflammatory cytokine product (IL-10:IL-12p70) secreted upon CD40 ligation was 60 ± 63 (median = 40). The mean total number of DCregs generated from a single leukapheresis product (n = 25 donors) and from two leukapheresis products (n = 3 donors) was 489 ± 223 × 106 (n = 28). The mean total number of DCregs infused was 5.9 ± 2.8 × 106 per kg body weight. DCreg numbers within a target cell range of 2.5-10 × 106/kg were achieved for 25 of 27 (92.6%) of products generated. CONCLUSIONS: High-purity DCregs meeting a range of quality criteria were readily generated from circulating blood monocytes under Good Manufacturing Practice conditions to meet target cell numbers for infusion into prospective organ transplant recipients.

Advanced Technologies for Potency Assay Measurement.

Crucial for their application, cell products need to be well-characterized in the cell manufacturing facilities and conform to regulatory approval criteria before infusion into the patients. Mesenchymal Stromal Cells (MSCs) are the leading cell therapy candidate in clinical trials worldwide. Early phase clinical trials have demonstrated that MSCs display an excellent safety profile and are well tolerated. However, MSCs have also exhibited contradictory efficacy in later-phase clinical trials with reasons for this discrepancy including poorly understood mechanism of MSC therapeutic action. With likelihood that a number of attributes are involved in MSC derived clinical benefit, an assay that measures a single quality of may not adequately reflect potency, thus a combination of bioassays and analytical methods, collectively called "assay matrix" are favoured for defining the potency of MSC more adequately. This chapter highlights advanced technologies and targets that can achieve quantitative measurement for a range of MSC attributes, including immunological, genomic, secretome, phosphorylation, morphological, biomaterial, angiogenic and metabolic assays.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting. METHODS: We collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions. RESULTS: The survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product. CONCLUSIONS: Survey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.

Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation.

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Joint EURADOS-EANM initiative for an advanced computational framework for the assessment of external dose rates from nuclear medicine patients.

BACKGROUND: In order to ensure adequate radiation protection of critical groups such as staff, caregivers and the general public coming into proximity of nuclear medicine (NM) patients, it is necessary to consider the impact of the radiation emitted by the patients during their stay at the hospital or after leaving the hospital. Current risk assessments are based on ambient dose rate measurements in a single position at a specified distance from the patient and carried out at several time points after administration of the radiopharmaceutical to estimate the whole-body retention. The limitations of such an approach are addressed in this study by developing and validating a more advanced computational dosimetry approach using Monte Carlo (MC) simulations in combination with flexible and realistic computational phantoms and time activity distribution curves from reference biokinetic models. RESULTS: Measurements of the ambient dose rate equivalent H*(10) at 1 m from the NM patient have been successfully compared against MC simulations with 5 different codes using the ICRP adult reference computational voxel phantoms, for typical clinical procedures with 99mTc-HDP/MDP, 18FDG and Na131I. All measurement data fall in the 95% confidence intervals, determined for the average simulated results. Moreover, the different MC codes (MCNP-X, PHITS, GATE, GEANT4, TRIPOLI-4®) have been compared for a more realistic scenario where the effective dose rate E of an exposed individual was determined in positions facing and aside the patient model at 30 cm, 50 cm and 100 cm. The variation between codes was lower than 8% for all the radiopharmaceuticals at 1 m, and varied from 5 to 16% for the face-to face and side-by-side configuration at 30 cm and 50 cm. A sensitivity study on the influence of patient model morphology demonstrated that the relative standard deviation of H*(10) at 1 m for the range of included patient models remained under 16% for time points up to 120 min post administration. CONCLUSIONS: The validated computational approach will be further used for the evaluation of effective dose rates per unit administered activity for a variety of close-contact configurations and a range of radiopharmaceuticals as part of risk assessment studies. Together with the choice of appropriate dose constraints this would facilitate the setting of release criteria and patient restrictions.

Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies.

Mesenchymal stromal cells (MSCs) have gained immense attention over the past two decades due to their multipotent differentiation potential and pro-regenerative and immunomodulatory cytokine secretory profiles. Their ability to modulate the host immune system and promote tolerance has prompted several allogeneic and autologous hMSC-based clinical trials for the treatment of graft-versus-host disease and several other immune-induced disorders. However, clinical success beyond safety is still controversial and highly variable, with inconclusive therapeutic benefits and little mechanistic explanation. This clinical variability has been broadly attributed to inconsistent MSC sourcing, phenotypic characterization, variable potency, and non-standard isolation protocols, leading to functional heterogeneity among administered MSCs. Homogeneous MSC populations are proposed to yield more predictable, reliable biological responses and clinically meaningful properties relevant to cell-based therapies. Limited comparisons of heterogeneous MSCs with homogenous MSCs are reported. This review addresses this gap in the literature with a critical analysis of strategies aimed at decreasing MSC heterogeneity concerning their reported immunomodulatory profiles. STATEMENT OF SIGNIFICANCE: This review collates, summarizes, and critically analyzes published strategies that seek to improve homogeneity in immunomodulatory functioning MSC populations intended as cell therapies to treat immune-based disorders, such as graft-vs-host-disease. No such review for MSC therapies, immunomodulatory profiles and cell heterogeneity analysis is published. Since MSCs represent the most clinically studied experimental cell therapy platform globally for which there remains no US domestic marketing approval, insights into MSC challenges in therapeutic product development are imperative to providing solutions for immunomodulatory variabilities.

[The Nationwide Survey on Avoidance of Targeted Radionuclide Therapy in Patients with Limited Activities of Daily Living and Those Undergoing Hemodialysis].

Targeted radionuclide therapy with high-dose radioisotopes should be performed in isolation rooms. Patients can be released only after radioactivity remaining in their bodies becomes less than the limits determined by the release criteria in order to secure public protection. Patients are asked to stay in isolation rooms for a few days. Physicians often face difficulties to carry out therapy in patients with limited activities of daily living and those undergoing hemodialysis, and have to avoid therapy in such cases. The Japanese Society of Nuclear Medicine conducted a nationwide survey in order to find out the actual situation. The survey results should reflect future improvement of therapeutic environment in collaborating with related societies and administrative bodies.

Principal Criteria for Evaluating the Quality, Safety and Efficacy of hMSC-Based Products in Clinical Practice: Current Approaches and Challenges.

Human Mesenchymal Stem Cells (hMSCs) play an important role as new therapeutic alternatives in advanced therapies and regenerative medicine thanks to their regenerative and immunomodulatory properties, and ability to migrate to the exact area of injury. These properties have made hMSCs one of the more promising cellular active substances at present, particularly in terms of the development of new and innovative hMSC-based products. Currently, numerous clinical trials are being conducted to evaluate the therapeutic activity of hMSC-based products on specific targets. Given the rapidly growing number of hMSC clinical trials in recent years and the complexity of these products due to their cellular component characteristics and medicinal product status, there is a greater need to define more stringent, specific, and harmonized requirements to characterize the quality of the hMSCs and enhance the analysis of their safety and efficacy in final products to be administered to patients. These requirements should be implemented throughout the manufacturing process to guarantee the function and integrity of hMSCs and to ensure that the hMSC-based final product consistently meets its specifications across batches. This paper describes the principal phases involved in the design of the manufacturing process and updates the specific technical requirements needed to address the appropriate clinical use of hMSC-based products. The challenges and limitations to evaluating the safety, efficacy, and quality of hMSCs have been also reviewed and discussed.

Flow cytometric assays for identity, safety and potency of cellular therapies.

Cellular therapeutics are a fast-growing, highly innovative area of medicine. This field encompasses well-established immune therapies for infection and cancer, as well as newer cell therapies aimed at regenerating diseased tissue. Flow cytometry is arguably the most important tool in the development of advanced cellular therapeutics and plays a role in many aspects of manufacturing. Quality control of raw materials, assessing rates of cellular growth during complex in vitro culture processes, differentiation status, as well as final product characterization, viability, and product stability are all essential data to be collected and documented. This requires robust and validated flow cytometric analysis. In this review, we discuss flow cytometer set up; design and use of assays to maintain quality control in cell processing, and the building of a robust set of release criteria. The use of flow cytometric assays as surrogates of functional assays to allow comparison and release of cellular products for clinical use are reviewed with reference to monocyte-derived and T cell products.

Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine.

Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age-related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone-forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical-grade production to achieve predictably favorable treatment outcomes for stem cell therapy. Stem Cells Translational Medicine 2017;6:2173-2185.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

BACKGROUND: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). METHODS: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. RESULTS: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. CONCLUSIONS: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. CLINICAL TRIALS: Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

Regulatory Considerations for NK Cells Used in Human Immunotherapy Applications.

Translating cellular therapy from the laboratory to the clinic is a complicated process that involves scale-up of procedures to generate clinically relevant cell numbers, adaptation to reagents and equipment that are qualified for human use, establishing parameters of safety for reagents and equipment that are not already qualified for human use, codifying these processes into standards of practice and rules of conduct, and obtaining approval from regulatory bodies based on those codified standards and rules. As the laws and regulations that apply to cellular therapy will vary by time and geography, this chapter reviews some common key principles for the manufacturing of NK cells for human use that will need to be considered within the constraints of local policies and regulations.