Strategies to improve the immunosuppressive properties of human mesenchymal stem cells

MSCs vary tremendously in terms of phenotypic and functional characteristics such
as their proliferation capacity, expression of several cell surface markers, and secretion
of cytokines 7]–10]. Interestingly, although MSCs have been continuously adapted in many laboratories,
their heterogeneity is considered to be due mainly to the use of non-standardized
culture protocols, including the starting material, culture media, levels of sera/cytokines/oxygen,
number of passages, and cell density 7], 42], 43]. In this regard, Ho and colleagues 43] classified MSC heterogeneity as follows: (1) cellular heterogeneity of the initial
population, (2) varied expansion capacity of specific subsets of cells and of the
final population, and (3) long-term biological function of MSCs. In particular, ex
vivo expansion of MSCs is used to develop and maintain MSCs for cell therapy, and
the methods used to expand and characterize MSCs are critical for their preparation.
Moreover, MSCs express a wide variety of cytokines, chemokines, and growth factors
that are important for cell migration, homing, and immunomodulation, following reconstitution
of damaged tissues 44]–48]. Based on their functional effects, differences in the secretion of these molecules
by MSCs may be critical for the outcomes of cell therapies. In this regard, it is
important to identify the best subpopulation of cells, to determine how the cells
are expanded and characterized ex vivo, and to determine when the cells should be
used clinically.

Numerous attempts have been made to develop more specific procedures for the isolation
and preparation of appropriate subsets of MSCs from a heterogeneous cell population
7], 11], 43]. The protocol most commonly used in preclinical and clinical studies to isolate MSCs
from various tissues is centrifugation over a density gradient followed by ex vivo
expansion, which removes hematopoietic cell contamination. With this method, cell
recovery from each tissue is variable among operators, and technical expertise is
required to consistently obtain MSCs with a high efficiency. In addition, numerous
putative human MSC surface markers (i.e., CD49a 49], CD73 3], CD105 50], CD106 51], CD271 52], MSC antigen-1 53], Stro-1 54], and stage-specific embryonic antigen-4 55]) have been identified thus far. These markers are used alone or in combination to
enrich homogeneous MSCs and to avoid cellular contamination. Unfortunately, many of
these markers are widely expressed in stromal cells and lack specificity, contributing
to the significant heterogeneity among MSCs derived in a single isolation 56].

MSC culture variables include medium formulation, culture surface substrate, cell
seeding density, physiochemical environment, and subculture protocols. In particular,
the development of well-formulated culture media for the isolation and expansion of
MSCs is imperative; however, this is as an extremely difficult process because of
the high complexity of media formulations. In this regard, the disclosed medium formulations
for MSCs (e.g., those reported in 57]–60]) are best positioned to be further developed by the many investigators interested
in the therapeutic applications of MSCs. Unlike some cell types, MSCs can survive
in hypoxic environments for several days by upregulating survival pathways 61], 62] and increasing cellular metabolism 63]. Cell numbers are also increased when cells proliferate under low oxygen tension
64], 65]. Differentiation into different mesenchymal lineages can be enhanced by culture under
some hypoxic conditions 66], 67]; however, the effects seem to depend on various variables such as the exact oxygen
tension, time in culture, and use of hypoxic preculture. Moreover, hypoxic conditions
enhance the paracrine role of MSCs by altering cytokine and growth factor release
68]–70] and play an import role in mobilizing MSCs and recruiting them to sites of injury
69], 71], 72]. Thus, hypoxic preconditioning of MSCs prior to implantation and associated hypoxic
conditioned medium can improve cell survival in vivo, which has significant effects
on the long-term effectiveness of MSC therapy. However, protocols to prepare and characterize
MSCs have not been standardized. If the heterogeneity of MSCs cannot be minimized,
it might take a long time to produce satisfactory clinical results.

In recent years, preparing cell therapy products using MSCs often required complex
procedures, such as multiple cell-selection steps, ex vivo expansion, cell activation
(e.g., priming or licensing), encapsulation, and genetic modifications 73], 74]. These complex procedures reflect the increasing sophistication of cell therapies
and their production methods but have also occurred in response to the potential risks
and increasingly rigorous regulatory requirements for these novel cell therapies.
In fact, among the methods described above, ex vivo expansion and cell activation
may have only minimal regulatory issues in terms of their clinical application because
ex vivo expansion is a general method used in cell culture and cell activation is
a simple method in which cells are merely primed with cytokines such as IFN-?, which
are commercially available and approved by the US Food and Drug Administration for
the treatment of several diseases 72], 75], 76]. However, there are many concerns regarding the use of engineered or modified cell
therapy products for clinical applications, and appropriate solutions need to be developed
in the near future.