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Mesenchymal Stem Cells (MSCs)
Mesenchymal stem cells (MSCs) are multipotent stem cells that have been
isolated from a variety of anatomical locations including the bone
marrow, peripheral blood, and placenta. MSCs are sometimes referred to
as multipotent adult progenitor cells (MAPCs). While the
standard test to confirm their multipotency is differentiation of the
cells into osteoblasts, adipocytes, and chondrocytes, it is clear that
under the influence of appropriate signals
these MAPCs have been found to be able to evolve into
cells with ectodermal, endodermal and mesodermal characteristics. It
may be that MSCs in adult tissues are reservoirs of reparative cells,
ready to mobilize and differentiate in response to wound signals or
disease conditions. However, recent evidence suggests their efficacy may
be related to their secretion of cytokines or other potent immune
modulators.
A
significant amount of information has been obtained in recent years
surrounding the expansion of MSCs, control of their differentiation and
their cytokine production. While most of this work has been performed on
MSCs that have been derived from autologous bone-marrow, Pluristem has
focused on using MSCs derived from the placenta and used allogeneically.
Pluristem’s Expansion of MSCs
Traditionally MSCs have been plated and enriched using standard cell
culture techniques. Cells are usually cultured in basal medium in the
presence of fetal bovine serum (FBS) with the addition of growth factor
supplements such as fibroblast growth factor-2
(FGF-2), leukemia inhibitory factor (LIF) and epidermal growth factor (EGF).
Pluristem’s proprietary position in the expansion of placental-derived
MSCs surrounds the growth of these cells in a three dimensional (3D)
bioreactor (on a non-woven fibrous matrix), termed PluriX™. In the
PluriX™ system, MSCs home onto the polystyrene rungs of the matrix and
expand to as much as 6.5x106 cells/gr. of matrix without the
use of growth factors or other supplements. Pluristem has termed these
placental-derived, 3D expanded MSCs as PLacental eXpanded
(PLX) cells.
Figure 1: Photomicrographs of Pluristem’s PLX cells expanding within the
PluriX™ Bioreactor
The major steps involved in the production of PLX cells include
(a) the receiving, recovery and processing of the MSCs from the
disease-free placenta of a full term delivery (b) certifying the
placenta for use as 2D-Cell-Stock (2DCS) (c) inoculating the 2DCS into
the PluriX™ 3D bioreactor and (d) harvesting, filing and the freezing of
the PLX cells in liquid nitrogen.
The Cytological, Immuno-phenotypic and Cytokine Expression
Characteristics of Pluristem’s PLX Cells
Scientists at Pluristem’s research center in Haifa, Israel have
demonstrated that PLX cells show an almost identical surface
profile related to the expression of mesenchymal markers and the absence
of hematopoietic, dendritic and endothelial markers. Typical MSC surface
markers, such as CD105, CD73, CD90 and CD29, are highly expressed by
PLX cells. Markers for hematopoietic (CD45, CD34), endothelial
(CD31) or dendritic cells (costimulatory molecules CD80 [B7-1], CD86
[B-7-2]) could only be located at extremely low levels in some cells.
The surface markers of these adherent PLX cells, compared to bone
marrow-derived MSCs, strongly indicate a MSC-like phenotype.
Additionally, it has been shown by both Pluristem as well as independent
researchers that PLX cells possess immunomodulatory
characteristics that suggest these cells are not only immuno-privileged
but also immunosuppressive. For example, when compared to MSCs derived from the bone marrow, PLX
cells have been demonstrated to prevent the proliferation of
pro-inflammatory cells, down regulate pro-inflammatory cytokines and
enhance the production of anti-inflammatory cytokines.
Figure 2: The Cytological and Immunomodulation Properties of Pluristem’s
PLX cells
PLX cells seem to escape the immune system,
and this makes them potentially useful for various transplantation
purposes.
PLX cells
express intermediate levels of HLA major histocompatibility complex (MHC)
class I molecules and do not express HLA class II antigens on the cell
surface.
It has been shown in the literature that after the differentiation of MSCs into bone,
cartilage, or adipose tissue, both adult and fetal MSCs continued to
express HLA class I, but not class II.
The
way in which
PLX cells
suppress T-cell activation and modulate the immune response has not been
completely resolved. However, several mechanisms have been proposed and
MSCs have been shown to have a variety of significant effects. Immune
suppression seems to be mediated by soluble factors produced by
PLX
cells.
It is unlikely that the factors are constitutively secreted by
the cells,
because cell-free MSC culture supernatants fail to suppress
alloreactivity, whereas supernatants from MSC/lymphocyte cocultures are
suppressive.
When
PLX cells were present in mixed lymphocyte cultures (MLC) T-or to
lymphocytes that were stimulated by mitogens, cell proliferation was
modulated in a dose dependent manner. The suppression of allogeneic
T-cell proliferation, as well as the T cell response to mitogens, was
clearly observed by the addition of PLX cells and suggesting
these cells are immunosuppressive.
Pluristem believes important immunomodulation differences such as those
noted above could potentially allow the PLX cell to play a major
role in the prevention or treatment of cellular transplantation
reactions such as Graft-versus-Host Disease (GvHD). Additionally, the
Company believes the uniqueness of the PLX cell could be the
basis for a cell proprietary only to Pluristem.
PLX-PAD - Pluristem’s First Product for Critical Limb Ischemia
Peripheral vascular disease of the lower extremities comprises a
clinical spectrum that goes from asymptomatic patients to patients with
chronic critical limb ischemia (CLI) that might result in amputation and
limb loss. Critical limb ischemia is a persistent and relentless problem
that severely impairs the patient’s functional status and quality of
life, and is associated with an increased cardiovascular mortality and
morbidity. It can present acutely (i.e. distal embolization, external
compression, acute thrombosis, etc.) or, in the majority of cases, as
chronic CLI.
In
the vast majority of cases chronic CLI is related to advanced
atherosclerotic disease, which, in turn is often seen in diabetic
patients. Chronic CLI secondary to atherosclerosis develops when
arterial stenosis reaches a critical point in which the blood flow
supplied to the distal extremity is insufficient to provide the basal
tissue oxygen demand. When the basal tissue oxygen demand cannot be met
by the peripheral vascular system, ischemic injury occurs in the tissues
with the lowest blood supply and necrosis results leading to tissue
destruction, the appearance of ulceration, gangrene, and rest-pain.
Industry thought leaders have recently concluded that an estimated two
million people in the U.S. have CLI. Reflecting the ageing population,
this number is projected to grow to almost 2.8 million by 2020. However,
if the prevalence of diabetes continues to increase, there could be over
3.5 million cases of CLI by 2020.
Under a collaborative research agreement with the
Berlin-Brandenburg Center for Regenerative Therapy (BCRT) animal studies
were conducted in mice on CLI at Charite, partner to the BCRT and
Europe’s largest university research hospital. The femoral artery was
ligated on one side of cohort and control mice. One million PLX-PAD
cells were injected
intramuscularly five hours post
ligation with the control group receiving saline. Blood flows on the
legs and hips from both sides were measured using a non-contact laser
Doppler. A significant increase in perfusion was seen in those limbs
treated with PLX-PAD versus the control group. These animals were
then sacrificed and histological analysis of the PLX-PAD treated
limbs showed evidence of neo-vascularity versus the control group (fig.
4).
Figure 4: Histological Evidence of Neo-vascularity with PLX-PAD
(arrows point to endothelial capillary cells)
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