Mesenchymal Stromal Cells and Immunological Potency Assays

Publiceret Marts 2016

Cellular therapy has received increasingly more attention the past decade. This is due to the possibility of providing treatment for diseases rather than addressing the symptoms of a disease, which is what most medicine is currently doing. Mesenchymal stromal cells (MSCs) are "supportive cells" located next to the blood vessels throughout the tissues in the body. They are unspecialised cells characterised by a number of surface proteins, their plastic adherence, and their ability to divide asymmetrically. Asymmetrical cell division is a cellular division resulting in one cell similar to the parent cell, and one cell which is more differentiated towards a certain tissue lineage.

It is proposed that the main purpose of the MSCs is to function as supportive cells for other tissues in case of injury. Following injury, the MSCs enter the bloodstream and home towards the affected area. If you think of the MSCs as micro-engineers, you are not far from the current opinion in the field. Practicing their function, the MSCs appear in the affected area, sense what is wrong, resolves what is possible, and disappears from the area again when their job is done.

The ability of the MSCs to adapt to their surroundings has been extensively studied in vitro. As an example, when the cells are exposed to lowered oxygen tension they respond by secreting proteins responsible for the sprouting of new blood vessels. A similar specific response is seen when the MSCs are faced with apoptotic cells or an inflammatory environment. The fact that the MSCs can adjust and repair in this manner makes the MSCs a very interesting candidate for the treatment of a great variety of diseases.

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Figure 1. MSCs and their interplay with central immune cells. MSC can affect T lymphocytes, either by direct contact or indirectly. T lymphocyte subsets are represented by Cytotoxic T Lymphocyte (CTL), T helper lymphocytes (TH), and Regulatory T lymphocytes (Treg).

Cellular therapies using MSCs have proven beneficial for patients who were otherwise beyond medical treatment, such as patients with severe heart disease or graft versus host disease following transplants. At Cardiology Stem Cell Centre, patients with severe heart disease are treated with cell therapy. In a placebo controlled double blinded randomized trial, an increase in pump function and decreased scar area was found using MSC therapy (1,2). This cannot be achieved by medical treatment, emphasising the promise of cellular therapy. For the first clinical studies, the patients' own cells were extracted, cultured, and administered into the heart muscle. This requires a lot of resources, and is far from feasible. Therefore, in order to increase the number of patients to be treated, treatment with a standardised cell product from a cell bank is the next step. To make this possible, MSCs will have to be isolated from a donor for treatment of one or more other recipients; in other words, a shift from autologous to allogeneic transplantation is needed.

Allogeneic cell therapy is only possible because MSCs are generally perceived to be immunoevasive. This means that the MSCs do not express the proteins of "the usual suspects", whereas an array of active immunosuppressive paracrine mechanisms can be activated. Hence, they are able to evade recognition by the immune cells of the recipients.

Despite the growing attention and clinical evidence much remains unknown about exact mechanisms of action behind allogeneic use and immunosuppressive capabilities of MSCs. Some can be attributed to expression of membrane receptors and direct cell-to-cell contact. Contact serves as a mean for transmission of signals, and brings MSCs in to close contact with immune cells, which potentiates the effect of secreted soluble factors. Several adhesion molecules have been described as being important for the immunosuppressive potential of MSCs, while a low expression of major histocompatibility complex and co-stimulatory molecules render the initiation of an immune response unlikely.

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Figure 2. Potency assays work flow related to clinical use, The need for potency assays is present at numerous steps of production, from isolation of MSCs from a donor, during and following production, and as part of validation before use for treatment.

Aside from the contact-associated attributes, several secreted factors have been identified as central to modulation of different parts of the immune system. For instance, some factors target innate immune cells, such as macrophages and NK cells, while others preferentially affects the adaptive branch, e.g. T cells or B cells; however, most factors has some influence on both (3).

A key cell type for conveying information from the innate to the adaptive immune system is the dendritic cell, the most potent antigen-presenting cell. This function makes the dendritic cell pivotal for cell-based therapies and as such, efforts have been made to generate dendritic cell assays in vitro. Circulating monocytes can be isolated from the blood and differentiated into a mature phenotype. Upon activation, these cells express a number of maturation markers and cytokines required to stimulate leukocytes. Analysis of markers and cytokines provides quantifiable data, and the addition of MSC in a co-culture setup can be used to document the immunosuppressive capabilities of these (4). In supplement to specific mechanisms of action from the co-cultures, the dendritic cells can be transferred to purified T cells where the resulting proliferation offers functional insights to MSC immunomodulation.

A less refined, yet more multifaceted setup utilizes peripheral blood mononuclear cells (PBMCs) to model a mixed leukocyte reaction. Based on the high alloreactivity of this heterogeneous population, PBMCs from one donor can be challenged by those of another. Measuring the proliferation of leukocytes in general and subtypes in particular provides valuable information of how MSCs can affect even such complex interplays and skewer the "distribution" of cell types towards more regulatory phenotypes (4). From the more elaborate models, it is becoming more evident that inflammatory signals affect MSC, and that the MSC respond in return. This opens for intriguing possibilities to pre-condition MSCs to heighten their potential as active immunosuppressors rather than a one-fits-all or ‘passive' treatment.

At Cardiology Stem Cell Centre, the primary aim of MSC treatment is to regenerate damaged myocardium. Given inflammation is part of normal wound healing and due to the allogeneic approach, elucidation and documentation of regenerative and immunological modes of action of the cell product are essential.

Expansion of cells for clinical use is classified as production of a medicinal product, and as such is bound to comply with rules for pharmaceutical manufacturing as issued by the European Medicines Agency. Critical elements of pharmaceutical production are to characterize the product, identify its biological function and assure quality, reproducibility, and stability during production and storage. At this point above mentioned in vitro assays will come in handy, not just for the sake of scientific curiosity and basic research, but as necessary tools during production.

Regulatory authorities use the term "potency". It essentially means that you should identify which biological function of your cell product is relevant for its clinical purpose and be able to test this function in a quantitative and reproducible manner. This is an extraordinary challenging task working with cell products because cells use multiple and complex mechanisms of action and respond to their environment in equally complex manners. As such, a single assay will never suffice (5).

Identifying immunosuppressive activity of a cell product, whether it is for the sake of allogeneic use or active immunosuppression per se, means that you need to have a thorough understanding of not just your own product, but also disease mechanisms and the inflammatory environment in which the product is meant to make a difference. The use of PBMCs is a useful tool for screening, but establishing assays with purified effector cells important for the particular disease in mention are necessary supplements. First encounter of allogeneic MSCs in a chronic ischemic myocardial environment will activate innate immunity. As such, mentioned dendritic cell assays are meaningful supplements to PBMC screening for the sake of ischemic heart disease.

The goal of a potency assay is to predict clinical efficiency and it should be used as a tool during production to assure that all batches released are equally effective. Predicting immunosuppressive activity of every batch of a cell product means that potency assays should be performed at multiple steps during production, including screening of donor material, in-process controls, and final release. To assure consistency of measures, traditional in vitro assays need translation into standard analytical methods. This means that assays must be feasible, robust, and fully validated - not to mention low-cost. A full validation includes identification of accuracy, precision, specificity, linearity, range and suitability. As such, once you have documented mechanisms of action during pre-clinical work, you most likely still have a lot of quality work ahead of you. As clinical stem cell research disseminates, the regulatory requirements for production of cell products will increase, and there is no doubt that the need for analytical assays for quality control and quality assurance will grow with it.

References

  1. Mathiasen AB, Haack-Sørensen M, Jørgensen E, Kastrup J. Autotransplantation of mesenchymal stromal cells from bone-marrow to heart in patients with severe stable coronary artery disease and refractory angina-final 3-year follow-up. Int J Cardiol. 2013;170:246-251.
  2. Mathiasen AB, Qayyum AA, Jørgensen E, et al. Bone marrow-derived mesenchymal stromal cell treatment in patients with severe ischaemic heart failure: a randomized placebo-controlled trial (MSC-HF trial). Eur Heart J. 2015;36:1744-1753.
  3. Zhao S, Wehner R, Bornhäuser M, Wassmuth R, Bachmann M, Schmitz M. Immunomodulatory Properties of Mesenchymal Stromal Cells and Their Therapeutic Consequences for Immune-Mediated Disorders. September 2010. http://online.liebertpub.com/doi/abs/10.1089/scd.2009.0345. Accessed January 13, 2016.
  4. Follin B, Juhl M, Cohen S, et al. Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel: Phenotypic and immunomodulatory evaluation. Cytotherapy. 2015;17:1104-18.
  5. Galipeau J, Krampera M, Barrett J, et al. International society for cellular therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials. Cytotherapy. 2016;18:151-9.