1) being run through a magnetic separator column. The

1) Techniques
for isolation and propagation, and characteristics of Mesenchymal Stem Cells
from various sources

Isolation
Techniques of Mesenchymal stem cells from  derived from Bone Marrow aspirate and
umbilical cord:

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 Earlier MSC
were isolated from Bone marrow and it is frequently used source for MSC. Bone
marrow has been used for both autologous and allogenic purpose. Bone marrow
derived MSCs have some disadvantage as it involves painful collection  procedure. Recently several kinds of MSCs
have been isolated successfully from various sources such as umbilical cord,
peripheral blood,adipocyte and placenta. Umbilical cord derived MSC shows
similar gene expression profile to that of Embryonic stem cell and it has
faster self renewing capacity than BM derived MSC.  Bone marrow and Umbilical card derived MSCs  have both advantage and disadvantage for  each clinical application and it is
significant to know about it.

Techniques involved in BM-MSCs:

a)Magnetic-activated
cell sorting:

  The desired 
MSCs cell expressing a specific antigen binds to an antibody-bound
magnetic particle before being run through a magnetic separator column. The
MSCs will retain on the column.

b)Fluorescence-activated
cells sorting :The MSCs containing sample which have earlier been tagged
earlier  with a fluorescent marker, is
introduced into the system where the cells are hit by a laser. After observing
the  resulting fluorescence and scatter
data by a computer, with the help of charged plates the cells of interest are
finally isolated.

c)Microfluidics:

 The separation system requires  placing the sample on the chip, and using
different flow rates, based on their ability to pass through channels of
different size the cells could be isolated accordingly.

Techniques involved in
UC-MSCs:

a) The
explant method:The UC compartment is fragmented and seeded onto tissue culture
dishes after the process cell and tissue are filtered.

b) The
enzymatic digestion method: Wharton’s jelly is exposed into enzymatic solution
followed by enzymatic digestion process resulting in cell detachment.

Propagation of MSC: The
following step after isolation is the expansion of MSCs. During
expansion use of  growth factors will
increase the time and enhance the yield of the cells. MSCs can be
expanded enormously within a short span of time. This rapid proliferation could
result in an expansion of a thousand-fold in two to three weeks time. In vitro
growth of MSCs is characterized by three phases (i) starting lag phase, followed
by (ii) a rapid expansion (log phase)and ends with (iii) a stationary phase.
Proliferation
of MSCs is influenced by a number of cytokines and growth factors.

characteristics of MSC:
The
various characteristics  linked  with MSC can be explained by differences in
isolation methods, culture condition and tissue origin. Analysis such as Flow
cytometry analysis, BrdU cell proliferation assay, Immunocytochemistry,
Immunofluorescence, Colony forming unit and RT-PCR are performed to
characterized MSCs based on their expression markers.

 Immunophenotype :The initial source
for primary mesenchymal stem cells were immunophenotyped for widely used
MSC-markers, such as CD29, CD44 and Sca-1.The specific genotype and proteonomic
profiles analysis of multipotential MSCs  has been carried out to elucidate the
characterization of MSCs.

2)Ability of Mesenchymal stem
cells to differentiate into various lineages both in vitro and in vivo:

 Mesodermal
tri-lineage differentiation: MSCs
P3 were first  cultured under adipogenic,
osteogenic and chondrogenic conditions and compared with cells grown under
control condition to determine capability of MSCs P3 to differentiate into
mesodermal lineages .After staining process, around 80% cells were found to be
differentiated into adipocytes. Likewise, MSCs P3 were cultured in osteocyte
induction medium for 16-18 days and checked for expression of alkaline
phosphatase, a marker for osteocytes. The control cells either varied  in morphology or showed alkaline phosphatase
activity, while retractation of differentiated cells takes place, with the
passage of time and stained majorly positive for action of alkaline
phosphatase. In addition, there were several concentric layers of extracellular
depositions found around the MSC-differentiated osteocytes. About 80% cells
were observed to be alkaline phosphatase positive.

 To examine the chondrocyte differentiation
potential, MSCs-pellets were incubated individually in chondrocyte-induction
medium in CO2 incubator at 37°C.In chondrogenic induction medium the pellet
size and density increases by three weeks time during incubation period, and
the pellet is found to be  compact and
bigger in size when compared to the control pellet. For chondrogenic evaluations,
these pellets (control and differentiation sets) were washed, fixed,
paraffin-embedded and cut into 5 ?m to 7 ?m thin portions. These portions were
stained with safranin O, an extracellular matrix staining dye. The control
pellet derived portion indicates lighter staining and also less
compression.Contrarily, the pellet-derived portions of chondrogenic induction
were very compact and stained darkly by safranin O staining. Moreover, dark
stained nodule and compact of chondrocytes representing the cartilage-like
tissue was observed.

The in
vitro neuronal differentiation of bone marrow-derived MSCs is attained  using different combinations of neurogenic
inducers like retinoic acid, b-FGF and, ?- ME.Additionaly, RA plays significant
role in neuronal development including anterior-posterior patterning in the
nerve cord,creation of tubus medullaris, and cortical and motor neuronal
regeneration.

Simultaneous
and different  stages of osteogenic
differentiation: During osteogenic differentiation, both morphological and
cellular heterogeneities are retained and preserved, which could be monitored
till the end of the differentiation process. After haematoxylin and alkaline
phosphatase double-staining process, MSCs in the control found to be well
stained only by haematoxylin, while the differentiated cells (osteocytes)
indicates strong staining for both haematoxylin and alkaline phosphatase.
Resultant heterogeneity at the cellular morphology, differential intercellular
and intracellular alkaline phosphatase activities is noted. Following induction
by osteocyte differentiation medium, MSCs attained round, oval and star-shaped
morphologies.Cellular heterogeneity was found to be clear  in terms of the alkaline phosphatase
activities among the differentiated cells owing to differential colour
intensity.Moreover, these alkaline phosphatase positive cells showed various
morphologies, including star-shaped, rounded and oval, and some globular shape,
evoking the morphological heterogeneity between the differentiated osteocytes.
Cells at last stages of osteogenic differentiation shows completely oval/round
shape morphology, becomes non-adherent, and starts secreting significant
quantity of extracellular matrix .The Extracellular matrix deposition with
definite  pattern was also discovered
significantly.

 

3)Therapeutic
mechanism of action of mesenchymal stem cell:

The
hypoimmunogenicity of MSCs supports their therapeutic concern in a diversity of
diseases related to alloreactive immunity or 
autoimmunity. The
immunosuppressive nature of MSCs has great significance in the field of allogeneic
transplantations. MSCs are immunoprivileged and it is  used to minimize the severity of
graft-versus-host disease.MSCs induce the inhibition of mixed lymphocyte
cultures and T cells, regardless of the MHC, may have clinical significance.

 Respective studies have shown
that MSCs contribute to tissue repair and 
this cells have significant impact in the regeneration of various
tissues of the organism. In an experimental model of Duchenne muscular dystrophy
in mice, human MSCs isolated from the synovial membrane helps in the
regeneration of skeletal muscle tissue. Similarly, the transplantation of stem
cells from human exfoliated deciduous teeth (SHEDs) with hydroxyapatite and
tricalcium phosphate was able to restore the bone defects created in the
calvaria of mice with significant bone formation.

Osteogenesis
imperfecta (OI) is a genetic due to 
deficiency in the type I collagen production. It is the major structural
protein in bone, Deficiency leads to bone fragility and growth deficiency. The
use of purified bone marrow MSCs and allogeneic bone marrow transplantation
results in significant increase in body length and in the bone mineralization.
These studies shows that the use of MSCs to treat severe OI may be a promising
treatment for this condition. The
recognized immunomodulatory characteristics of MSCs is used to develop novel
therapies to treat autoimmune diseases. In a murine model of experimental
arthritis treatment, the use of human adipose-derived MSCs significantly
reduced disease prevalence. This therapeutic strategy minimized the production
of different inflammatory cytokines and chemokines, decreased antigen-specific
Th1/Th17 cell expansion and induced the production of interleukin-10 in lymph
nodes and joints.

In
addition, the production of antigen-specific Tregs which is capable of
suppressing self-reactive T effector responses was observed. In association to
autoimmune type I diabetes, the administration of allogeneic murine MSCs
delayed the onset of disease in pre-diabetic non-obese diabetic (NOD) mice,
thereby promoting a shift toward Th2 immune response. Likewise, murine MSCs
were capable of preventing beta cell destruction in NOD mice through the
induction of Tregs. MSC transplantation has a significant therapeutic
effects in the systemic lupus erythematosus mouse model by reconstructing the osteoblastic
niche and restoring immune homeostasis. In the research for more effective and
safe therapies to treat systemic lupus erythematosus (SLE), Sun et al. used an
infusion of allogeneic bone marrow MSCs (1 x 106 cells per kg) in four patients
with acute disease and nephritis caused by SLE and significant results were
observed, such as the retrieval of the levels of Tregs (CD4+Foxp3+) and a
significant improvement in renal function.

Overall,
the current data about therapeutic mechanism of MSCs shows that the MSCs
constitutes a promising alternative strategy for treating various
immune-mediated diseases. MSCs
are promising tools for the treatment of diverse conditions as they have a
multitude of bioactive molecules which helps in tissue reformation at the sites
of injury.

 

 

4)Ability of MSC to
escape host allogenic responses :

 The
use of allogeneic MSC from both in vitro and in vivo studies shows that  MSCs  avoid normal alloresponses. T cell recognition by the
recipient of alloantigen is the major confine to solid organ graft survival .
Direct recognition, involving recognition by recipient CD8+ or CD4+ T cells of
donor MHC class I and class II molecules and Indirect mechanisms involving
recognition of peptides from the allogeneic tissue are two important mechanism
which mediates the potent rejection response. The peptides molecules produced
from alloantigen by antigen presenting cells represents it to native T cells on
self-MHC molecules. Yet there are some exclusions to these allorejection
processes; the fetal allograft escapes rejection by the mother through a series
of actions. Likewise tissue which has specific lymphatic drainage is found to
be less prone to allorejection. Although tumor cell is not allogeneic in many
events is said to be  both self altered
and immunogenic but it frequently involve in active modulation of  the 
immune reactions to escape the immune surveillance. Thus mechanisms of
tumor evasion of the immune system may renders tolerance of  allogeneic MSCs by the mismatched host.

 The controversy that MSC avoid
allogeneic responses has also emerge from a 
in vitro experiments, which normally involves the  co-culture or mixed lymphocyte reactions
(MLR).confirmation from these studies depicts that employing mismatched MSC
does not  stimulates a proliferative T
cell response in allogeneic MLR, therefore indicating an immunosuppressive role
for MSC. Le Blanc et al, showed that MSC failed to evoke  proliferation of allogeneic lymphocytes. In
addition, they also exhibited that even after 
IFN-? stimulation the MSCs stayed immunosuppressive, studies from
Krampera et al supported these findings, they demonstrated that murine MSCs
lack MHC class II and suppressed  T cell
proliferation.

 MSC seem to escape allogeneic
rejection by a) being hypoimmunogenic; b) regulating T cell phenotype and c)
creating an immunosuppressive local environment. These three mechanisms are
inter-connected and  involves cell  dependent and independent interactions.

The
absence of co-stimulatory molecules shows that any residual involvement of the
T cell receptor on Th cells will result in lack of immune response to a
specific antigen and leads to tolerance instead of  allogeneic responses. Although this is a
assuring work, based largely on in vitro studies, it cannot fully explicate the
evasion mechanism of alloreactivity showed by MSC. Experiments which involved
allogeneic co-cultures or MLR have showed that both cell-cell interaction  and action by soluble factors imparts  to the immunomodulatory role of MSC.

MSC avoidance
of alloreactivity  show analogue to tumor
evasion: Escape from
immune surveillance is considered to be a primary characteristics of malignant
disease in humans. The immune effector response is sub-optimal because tumors
creates complex strategies to escape immune excision. These strategies may
provide hints to how MSC promote tolerogenic mechanisms during allogeneic
engraftment. Regulation of tumor antigen expression, particularly MHC class I
and II is a specifically usual component of tumor immune evasion.