Corrigendum: Consistent long-term therapeutic efficacy of human umbilical cord matrix-derived mesenchymal stromal cells after myocardial infarction despite individual differences and transient engraftment.

[This corrects the article DOI: 10.3389/fcell.2021.624601.].

Pharmacological effects of ex vivo mesenchymal stem cell immunotherapy in patients with acute kidney injury and underlying systemic inflammation.

Mesenchymal stem cells (MSCs) have natural immunoregulatory functions that have been explored for medicinal use as a cell therapy with limited success. A phase Ib study was conducted to evaluate the safety and immunoregulatory mechanism of action of MSCs using a novel ex vivo product (SBI-101) to preserve cell activity in patients with severe acute kidney injury. Pharmacological data demonstrated MSC-secreted factor activity that was associated with anti-inflammatory signatures in the molecular and cellular profiling of patient blood. Systems biology analysis captured multicompartment effects consistent with immune reprogramming and kidney tissue repair. Although the study was not powered for clinical efficacy, these results are supportive of the therapeutic hypothesis, namely, that treatment with SBI-101 elicits an immunotherapeutic response that triggers an accelerated phenotypic switch from tissue injury to tissue repair. Ex vivo administration of MSCs, with increased power of testing, is a potential new biological delivery paradigm that assures sustained MSC activity and immunomodulation.

Consistent Long-Term Therapeutic Efficacy of Human Umbilical Cord Matrix-Derived Mesenchymal Stromal Cells After Myocardial Infarction Despite Individual Differences and Transient Engraftment.

Human mesenchymal stem cells gather special interest as a universal and feasible add-on therapy for myocardial infarction (MI). In particular, human umbilical cord matrix-derived mesenchymal stromal cells (UCM-MSC) are advantageous since can be easily obtained and display high expansion potential. Using isolation protocols compliant with cell therapy, we previously showed UCM-MSC preserved cardiac function and attenuated remodeling 2 weeks after MI. In this study, UCM-MSC from two umbilical cords, UC-A and UC-B, were transplanted in a murine MI model to investigate consistency and durability of the therapeutic benefits. Both cellular products improved cardiac function and limited adverse cardiac remodeling 12 weeks post-ischemic injury, supporting sustained and long-term beneficial therapeutic effect. Donor associated variability was found in the modulation of cardiac remodeling and activation of the Akt-mTOR-GSK3ß survival pathway. In vitro, the two cell products displayed similar ability to induce the formation of vessel-like structures and comparable transcriptome in normoxia and hypoxia, apart from UCM-MSCs proliferation and expression differences in a small subset of genes associated with MHC Class I. These findings support that UCM-MSC are strong candidates to assist the treatment of MI whilst calling for the discussion on methodologies to characterize and select best performing UCM-MSC before clinical application.

Mesenchymal stromal cell delivery via an ex vivo bioreactor preclinical test system attenuates clot formation for intravascular application.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.

Author Correction: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells.

Bone marrow mesenchymal stromal cells (MSCs) have been studied for decades as potent immunomodulators. Clinically, they have shown some promise but with limited success. Here, we report the ability of a scalable hollow fiber bioreactor to effectively maintain ideal MSC function as a single population while also being able to impart an immunoregulatory effect when cultured in tandem with an inflamed lymphocyte population. MSCs were seeded on the extraluminal side of hollow fibers within a bioreactor where they indirectly interact with immune cells flowing within the lumen of the fibers. MSCs showed a stable and predictable metabolite and secreted factor profile during several days of perfusion culture. Exposure of bioreactor-seeded MSCs to inflammatory stimuli reproducibly switched MSC secreted factor profiles and altered microvesicle composition. Furthermore, circulating, activated human peripheral blood mononuclear cells (PBMCs) were suppressed by MSC bioreactor culture confirmed by a durable change in their immunophenotype and function. This platform was useful to study a model of immobilized MSCs and circulating immune cells and showed that monocytes play an important role in MSC driven immunomodulation. This coculture technology can have broad implications for use in studying MSC-immune interactions under flow conditions as well as in the generation of ex vivo derived immune cellular therapeutics.

Convergence of Cell Pharmacology and Drug Delivery.

Cellular therapy is enabling new approaches to tackle significant unmet needs in areas such as regenerative medicine and immunotherapy. The pharmacology of cell therapeutics becomes of critical importance to assure that these new drugs work reproducibly and effectively. Cell pharmacology can benefit from adapting principles of classical molecular drug pharmacokinetics (PK) and pharmacodynamics (PD) to quantitatively understand rate-limiting constraints of cell fate after administration. Future innovations focused on improvements in drug delivery using a PK/PD perspective can aid in designing a cell therapeutic product to overcome any pharmacological barriers for a given disease application. Herein, we present a perspective on the development of an ex vivo mesenchymal stromal therapeutic using a PK/PD framework and also present examples of general cell engineering techniques that implicitly influence the PK/PD curve by genetically modifying cells to regulate their in vivo duration, biodistribution, and activity. Stem Cells Translational Medicine 2019;8:874&879.

The Secretome Derived From 3D-Cultured Umbilical Cord Tissue MSCs Counteracts Manifestations Typifying Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder whose treatment is mostly restricted to pain and symptom management and to the delay of joint destruction. Mesenchymal stem/stromal cells from the umbilical cord tissue (UC-MSCs) have previously been proven to be immunomodulatory and more efficient than bone marrow-derived MSCs in causing remission of local and systemic arthritic manifestations in vivo. Given the paracrine nature of UC-MSC activity, their application as active substances can be replaced by their secretome, thus avoiding allogeneic rejection and safety issues related to unwanted grafting. In this work, we aimed at demonstrating the viability of applying the 3D-primed UC-MSC secretome for the amelioration of arthritic signs. A proteomic analysis was performed to both, media conditioned by UC-MSC monolayer (CM2D) and 3D cultures (CM3D). The analysis of relevant trophic factors confirmed secretome profiles with very significant differences in terms of therapeutic potential. Whereas, CM3D was characterised by a prevailing expression of anti-inflammatory cytokines such as IL-10 and LIF, along with trophic factors involved in different mechanisms leading to tissue regeneration, such as PDGF-BB, FGF-2, I-309, SCF, and GM-CSF; CM2D presented relatively higher levels of IL-6, MCP-1, and IL-21, with recognised pro-inflammatory roles in joint disease and pleiotropic effects in the progression of rheumatoid arthritis (RA). Accordingly, different motogenic effects over mouse chondrocytes and distinct capacities of inducing glycosaminoglycan synthesis in vitro were observed between CM3D and CM2D. Finally, the evaluation of arthritic manifestations in vivo, using an adjuvant-induced model for arthritis (AIA), suggested a significantly higher therapeutic potential of CM3D over CM2D and even UC-MSCs. Histological analysis confirmed a faster remission of local and systemic arthritic manifestations of CM3D-treated animals. Overall, the results show that the use of UC-MSC CM3D is a viable and better strategy than direct UC-MSC administration for counteracting AIA-related signs. This strategy represents a novel MSC-based but nonetheless cell-free treatment for arthritic conditions such as those characterising RA.

Biomanufacturing for clinically advanced cell therapies.

The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

BACKGROUND AIMS: The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®. METHODS: Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. RESULTS: Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. DISCUSSION: This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia.

BACKGROUND: Mesenchymal stem cells derived from human umbilical cord tissue, termed UCX®, have the potential to promote a full range of events leading to tissue regeneration and homeostasis. The main goal of this work was to investigate UCX® action in experimentally induced hindlimb ischemia (HLI). METHODS: UCX®, obtained by using a proprietary technology developed by ECBio (Amadora, Portugal), were delivered via intramuscular injection to C57BL/6 females after unilateral HLI induction. Perfusion recovery, capillary and collateral density increase were evaluated by laser doppler, CD31 immunohistochemistry and diaphonisation, respectively. The activation state of endothelial cells (ECs) was analysed after EC isolation by laser capture microdissection microscopy followed by RNA extraction, cDNA synthesis and quantitative RT-PCR analysis. The UCX®-conditioned medium was analysed on Gallios flow cytometer. The capacity of UCX® in promoting tubulogenesis and EC migration was assessed by matrigel tubule formation and wound-healing assay, respectively. RESULTS: We demonstrated that UCX® enhance angiogenesis in vitro via a paracrine effect. Importantly, after HLI induction, UCX® improve blood perfusion by stimulating angiogenesis and arteriogenesis. This is achieved through a new mechanism in which durable and simultaneous upregulation of transforming growth factor ß2, angiopoietin 2, fibroblast growth factor 2, and hepatocyte growth factor, in endothelial cells is induced by UCX®. CONCLUSIONS: In conclusion, our data demonstrate that UCX® improve the angiogenic potency of endothelial cells in the murine ischemic limb suggesting the potential of UCX® as a new therapeutic tool for critical limb ischemia.

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

INTRODUCTION: The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. METHODS: A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. RESULTS: UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor ß1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles. CONCLUSIONS: This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

The Human Umbilical Cord Tissue-Derived MSC Population UCX(®) Promotes Early Motogenic Effects on Keratinocytes and Fibroblasts and G-CSF-Mediated Mobilization of BM-MSCs When Transplanted In Vivo.

Mesenchymal stromal cells (MSCs) play an important role in tissue regeneration mainly through the secretion of trophic factors that enhance the repair of damaged tissues. The main goal of this work was to study the paracrine mechanisms by which an umbilical cord tissue-derived MSC population (UCX(®)) promotes the migration capacity of human dermal fibroblasts and keratinocytes, which is highly relevant for skin regeneration. Furthermore, the differences between paracrine activities of MSCs from the umbilical cord tissue and the bone marrow (BM-MSCs) were also evaluated. In vitro scratch assays revealed that conditioned media (CM) obtained from both growing and stationary-phase UCX(®) cultures induced human dermal fibroblast (HDF) and keratinocyte (HaCaT) migration. These assays showed that the motogenic activity of UCX(®) CM to HaCaTs was significantly higher than to HDFs, in opposition to the effect seen with CM produced by BM-MSCs that preferentially induced HDF migration. Accordingly, a comparative quantification of key factors with vital importance in the consecutive stages of wound healing revealed very different secretome profiles between UCX(®) and BM-MSCs. The relatively higher UCX(®) expression of EGF, FGF-2, and KGF strongly supports early induction of keratinocyte migration and function, whereas the UCX(®)-specific expression of G-CSF suggested additional roles in mobilization of healing-related cells including CD34(-)/CD45(-) precursors (MSCs) known to be involved in tissue regeneration. Accordingly, in vitro chemotaxis assays and an in vivo transplantation model for chemoattraction confirmed that UCX(®) are chemotactic to CD34(-)/CD45(-) BM-MSCs via a cell-specific mobilization mechanism mediated by G-CSF. Overall, the results strongly suggest different paracrine activities between MSCs derived from different tissue sources, revealing the potential of UCX(®) to extend the regenerative capacity of the organism by complementing the role of endogenous BM-MSCs.

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BACKGROUND: ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. METHODS: UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. RESULTS: UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations. CONCLUSION: The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Spontaneous bacterial keratitis in CD36 knockout mice.

PURPOSE: CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis. METHODS: The corneas of CD36(-/-), TSP1(-/-), TLR2(-/-), and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis. The epithelial tight junctions and mucin layer were assessed via LC-biotin and Rose Bengal staining, respectively. Bacterial quantification was performed on corneal buttons and GFP-expressing Staphylococcus aureus was used to study bacterial binding. RESULTS: CD36(-/-) mice develop spontaneous corneal defects that increased in frequency and severity with age. The mild corneal defects were characterized by a disruption in epithelial tight junctions and the mucin layer, an infiltrate of macrophages, and increased bacterial binding. Bacterial quantification revealed high levels of Staphylococcus xylosus in the corneas of CD36(-/-) mice with severe defects, but not in wild-type controls. CONCLUSIONS: CD36(-/-) mice develop spontaneous bacterial keratitis independent of TLR2 and TSP1. The authors conclude that CD36 is a critical component of the corneal epithelial barrier, and in the absence of CD36 the barrier breaks down, allowing bacteria to bind to the corneal epithelium and resulting in spontaneous keratitis. This is the first report of spontaneous bacterial keratitis in mice.

Activation marker expression and apoptotic susceptibility of T-cell clones derived from CD34(+), young and SENIEUR donors.

T-cell clones (TCC) derived from human peripheral blood lymphocytes of a young control, a healthy elderly (SENIEUR) donor, or from CD34(+) hematopoietic progenitor cells were utilised in this study to examine how in vivo and in vitro ageing affects T-cell apoptotic capability. The role of CD25, CD28 and the intracellular proteins, FLICE-inhibitory protein (FLIP), receptor-interacting protein (RIP) and caspase 3 were investigated. We observed an age-related decline in the expression of the IL-2 receptor alpha chain CD25, and absence of the co-stimulatory receptor CD28 on three of the four TCC studied. In young donor- and CD34 cell-derived TCC, but not in SENIEUR donor-derived TCC, we observed an age-related increase in susceptibility of the cells to mFas-L-induced apoptosis, which correlated with the age-related decrease of CD25 expression. Expression levels of full-length RIP and FLIP did not show any correlation to apoptotic susceptibility. However, expression levels of the cleaved form of RIP were greatly reduced in the SENIEUR donor-derived TCC, which together with a trend towards increased caspase 3 activity, could indicate an age-related alteration in utilisation of different apoptotic signalling pathways.

P-glycoprotein decreases with T cell maturation but is not responsible for resistance to CD95-induced apoptosis.

P-glycoprotein (Pgp) is a membrane transporter responsible for resistance to chemotherapy in cancer cells. Its presence in T cells is very well documented, but its function in the immune system is still poorly understood. Recent findings suggest that Pgp may be involved in regulating programmed cell death by inhibiting caspase 8 and caspase 3. Utilising antigenically-activated T cells and the physiologically relevant apoptotic ligand, membrane CD95-L, we have previously reported that while T cells are generally resistant to CD95-induced death at early stages of activation, their susceptibility to apoptosis increases with successive activation and clonal expansion. In this study we investigated whether changes in apoptotic susceptibility were related to T cell Pgp function. Results showed that Pgp expression and function in T cells decreases with maturation, with CD8 cells having the highest Pgp function. However, although Pgp function inversely correlated with caspase 3 activity, no difference was observed between apoptotic susceptible CD25- cells and resistant CD25+ cells. In addition sorting of cells with high and low Pgp function showed no correlation with apoptotic capability. Therefore, whilst Pgp modulates caspase activity, it is not responsible for resistance to apoptosis of early activated T cells nor the increased susceptibility observed at the later stages of maturation in antigenically activated cells.

Caspase involvement in RIP-associated CD95-induced T cell apoptosis.

CD95-induced apoptosis is an important regulatory mechanism in T cells and this complex signalling pathway is now thought to include the protein kinase RIP. Although, RIP is best known for its role in TNF signalling and NF-kappaB activation, it contains a death domain and it is capable of causing apoptosis upon cleavage. In the present study, the role of RIP in CD95-induced apoptosis and its inter-relationship with the caspase cascade was investigated. Studies were performed on both a RIP-/- T cell line and peripheral T lymphocytes, where RIP was degraded through the addition of geldanamycin. Apoptosis was induced by membrane CD95-L, thought to be the most physiological relevant form of CD95-L. Results showed that RIP-/- cells had a decreased susceptibility to death, thus confirming a role for RIP in CD95-induced apoptosis. Furthermore, it was confirmed that RIP is cleaved upon CD95-L stimulation, a process that can be inhibited by Z-VAD. However, only partial inhibition in peripheral T lymphocytes by Z-VAD was observed, suggesting a potential caspase-independent processing of RIP. Studies performed on the activity of effector caspase 3 and on the initiator caspases 2, 8, and 9 revealed that, in the absence of RIP, the activity of these caspases decreases, indicating that RIP-associated apoptosis is caspase-dependent. Hence, these studies support a caspase-related role for RIP in CD95-induced T apoptosis.

Age associated decline in CD25 and CD28 expression correlate with an increased susceptibility to CD95 mediated apoptosis in T cells.

Immunosenescence is believed to contribute to increase susceptibility to infectious diseases and cancer in the elderly, and is caused mainly by changes in the T cell compartment. Longitudinal studies were undertaken to examine T cell surface receptor expression and apoptotic susceptibility using Staphylococcal enterotoxin B (SEB) activated human T cells as an in vitro model of an ageing T cell culture. An intracellular stain Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to assess the number of population divisions (PD) occurring in the ageing T cell culture. One major biomarker of aged T cells is a decrease in expression of CD28 and since this is an essential co-stimulatory molecule, its decreasing expression with age could compromise their activation and apoptotic capacity. Activation of T cells resulted in initial up-regulation of CD25, CD95 and CD28, although expression of CD25 and CD28 subsequently decreased with increasing PD. CD4 and CD8 T cells expressed similar CD25 profiles although CD28 expression was unique in each subset. CD4+ cells expressed the highest CD28 levels, and showed a gradual decline in expression with increasing PD, whereas CD8+ cells were low CD28 expressers, but did not appear to lose their expression as they aged. To determine T cell susceptibility to apoptosis via CD95/CD95-L interactions with increasing age, cells were challenged with CD95-L transfected CHO cells at various PD. Increased death was observed as they aged, which correlated with the decreased expression of activation markers CD25 and CD28.