Q2. What are the common stimuli used to trigger immune response in the presence of MSCs?
For T cells, common stimuli are phytohaemagglutinin (PHA), a polyclonal stimulus leading to robust activation (20), and monoclonal antibodies against CD2, CD3 and CD28, which induce a more “physiologic” responder cell activation (43).
Q3. What is the way to study the differentiation of memory B cells to plasma cells?
Cytofluorimetricevaluation of CD38/CD138 upregulation and parallel downregulation of CD20 seems to be a good approach to study the differentiation of memory B cells to plasma cells.
Q4. What is the role of MSCs in the development of monocytes?
MSCs can also affect differentiation of monocytes to macrophages and dendritic cells,as well as their maturation, migration and functions.
Q5. What type of cells can be used to activate immune effector cells?
Both unselected peripheral blood mononuclear cells (PBMCs) or purified immune effector cells can be used, but the latter usually provide more reproducible results because of the lack of confounding third-party cells (monocytes in particular).
Q6. What is the main issue when evaluating the activity of MSCs in preclinical models?
Two main issues should be considered when investigating the activity of MSCs in preclinical models: whether to measure their immunosuppressive potency or elucidate the mechanisms underlying the therapeutic activities.
Q7. What is the role of iNOS in the inhibition of lymphocytes?
1.4. MSC cellular biochemistryActivation of IDO and iNOS is a pivotal mechanism in lymphocyte inhibition with MSCs, but species-specific differences exist.
Q8. What is the purpose of this working proposal paper?
The sincere hope of this working proposal paper is to contribute to the general discussion in the MSC scientific world, thus leading soon to shared guidelines and common protocols for the immunological characterization of MSCs for clinical use, in order to achieve comparable and unambiguous results on MSC efficacy in human diseases (65).
Q9. What is the reason for the difficulty of injection of MSCs in mice?
Intravenous injection of MSCs in mice is notoriously difficult because of the extremely high incidence of lethal pulmonary embolism, even with subtherapeutic doses.
Q10. What is the ratio of MSCs to T cells?
A ratio of MSCs to T cells of 1:5-10 is generally suitable to obtain a measurable effect (36,44), but a ratio of 1:1 or 1:5 would be preferable when MSCs are co-cultured with B cells (6,36,40,44) or natural killer cells (25,36,44,48,49).
Q11. What is the expectation that robust potency assays be developed and implemented as part of cell?
There is also an expectation that robust potency assays be developed and implemented as part of cell manufacturing, which may subsequently be associated with clinical effect and serve as a “gold standard” for inter-study analysis.
Q12. What is the way to study the immune function of MSCs in vitro?
In the human setting, a large number of MSC:immune effector cell ratios has been tested to assess the best coculture conditions to unravel immune regulatory effects, thus showing that the modulation of immune functions in vitro requires the presence of adequate numbers of MSCs.
Q13. What is the method to monitor the function of MSCs?
The potential advantage of the latter method is that cell preparation is devoid of cells that might release factors influencing MSC and neutrophil functions regardless of their reciprocal interaction (57); consequently, high-purification procedure would be preferable to obtain more reproducible results.
Q14. What is the way to assess the effects of MSCs on neutrophils?
Different stimuli (e.g., lipopolysaccharide, poly(I:C), phorbol esters), ratios of neutrophils to MSCs (from 1,000:1 to 10:1, in direct contact or in Transwell® conditions) and functional assays (e.g., CD16 and CD11b expression as surrogate markers of neutrophil viability and activation, respectively; ELISA for cytokine detection; superoxide anion release for respiratory burst quantification) may be used to assess the effects of MSC–neutrophil interactions (56,57).
Q15. What is the way to study the effects of MSCs on macrophages?
MSC effects on macrophages should be carefully characterized, as macrophages play a major role in many diseases, i.e. myocardial infarction, stroke and sepsis, for which MSCs have been suggested as possible therapeutic strategy.