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10.5 Multi-dynamic Properties of EnSCs
10.5.1 Clonogenecity
The clonogenic potential of EnSCs was first reported by Chan et al. [ 1 ]. Epithelial
and stromal cells were separated into single cell suspensions and cultured at a clon-
ing density of 300–500 cells/cm^2 in different culture conditions such as in the pres-
ence of serum containing medium or serum-free medium supplemented with
different growth factors. Cloning efficiencies of epithelial and stromal cells were
reported to be 0.22% and 1.25% respectively. Thirty-seven percentage of epithelial
colonies were small, with large, loosely arranged cells whereas 1 in 60 of stromal
colonies were large colonies, comprising small, densely packed cells. Cloning of
MSCs obtained from endometrial stromal cells described by other groups were dif-
ferent from the technique followed by Chan et al. wherein pure cultures of endome-
trial stromal cells were subjected to serial dilution and single cell cloning [ 1 ]. In
other reports, passage 5 stromal cells were serially diluted in 96 well culture plates
in order to obtain a cell count of one cell/well. Dividing clonal populations obtained
from single cell cultures of stromal cells were further cultured as regenerating
MSCs [ 16 , 17 ]. There was no variation in clonogenecity of EnSCs isolated from
epithelial and stromal cells along the different stages of the menstrual cycle, i.e.,
from proliferative to secretory stage or between active, cycling and inactive endo-
metrium [ 1 ].
10.5.2 Immunogenicity
Immunosuppression is one of the hallmark features of MSCs [ 34 , 35 ] and the
immuno-modulatory properties of MSCs isolated from different tissues were
reported in several experimental settings [ 6 , 10 , 36 , 37 ]. This cardinal ability of
MSCs helps them in curbing many immune disorders [ 34 , 37 , 38 ]. Several studies
have shown that MSCs in culture can mediate suppression of T-cell proliferation
[ 34 ]. Immunosuppressive property of MSCs could be validated in vitro using PBMC
proliferation assays [ 5 ]. Briefly, mitogen activated PBMCs are co-cultured with
MSCs at a ratio of 10:1 for 48 h in multi-well culture plates. MTT assay is per-
formed following incubation and percent change in cell proliferation is validated.
Percent change in suppression of PBMC proliferation is calculated using the for-
mula: % change = ({(mean OD of triplicate wells of PBMC + PHA) − (mean OD
of triplicate wells of PBMC + MSC + PHA)}/(mean OD of triplicate wells of
PBMC + PHA)) × 100. Immunosuppressive property of endometrial stromal cell
derived MSCs has been validated in a recent study from our laboratory [ 17 ]. In vitro
co-culture experiments involving healthy human endometrial MSCs (eutopic
MSCs) resulted in approximately 50% reduction in proliferation of mitogen acti-
vated PBMCs [ 17 ].
K.G. Aghila Rani and T. Madan