The role of placental-derived adherent stromal cell (PLX-PAD) in the treatment of critical limb ischemia.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are spindle-shaped plastic-adherent cells isolated from bone marrow (BM), adipose tissue and other organs, including the placenta. Autologous BM-derived MSC have been studied in animals with experimentally induced critical limb ischemia (CLI) as a model of end-stage peripheral vascular disease. While demonstrating therapeutic benefit, the use of these cells is limited by the need to surgically extract BM and the fear of thrombosis secondary to the use of granulocyte-colony-stimulating factor (G-CSF) to mobilize the cells. METHODS: We studied the use of placental-derived adherent stromal cells (ASC) in a standard limb ischemia model of male Balb/c mice. These placental-derived cells, termed PLX-PAD, share the adherence and marker expression of BM-derived MSC but lack their differentiation potential. PLX-PAD are isolated from human placenta following a Caesarean section and cultured in a bioreactor, termed the PluriX System. The PluriX System provides a three-dimensional (3-D) microenvironment that enables the large-scale growth of these cells. PLX-PAD are stable adhesive cells that can be expanded in vitro without the loss of phenotype and without showing signs of karyotypic changes. RESULTS: The intramuscular (i.m.) administration of PLX-PAD in our model significantly improved blood flow (BF) (P=0.0008), increased capillary density (P=0.021), reduced oxidative stress (P=0.034) and reduced endothelial damage (P=0.004), while increasing limb function versus the administration of a phosphate-buffered saline (PBS) control vehicle in the affected limb. CONCLUSIONS: Allogeneic placental-derived ASC may provide an off-the-shelf supply of therapeutic cells that would need no histocompatible tissue matching and be potentially less expensive and considerably more convenient than BM or adipose-derived MSC.

Safety and biodistribution profile of placental-derived mesenchymal stromal cells (PLX-PAD) following intramuscular delivery.

The administration of mesenchymal stromal cells (MSCs) provides an exciting emerging therapeutic modality for the treatment of peripheral arterial disease, a condition that is associated with critical limb ischemia as its end stage. Placental-derived MSCs, termed PLX-PAD cells, are stable adhesive stromal cells isolated from full-term human placentae, cultured on carriers, and expanded in a bioreactor called the PluriX. These cells can be expanded in vitro without phenotypic or karyotypic changes. We studied the safety and biodistribution properties of PLX-PAD cells following intramuscular administration in NOD/SCID mice. No significant clinical signs, hematological and biochemical parameters, or major pathological changes were found in PLX-PAD-treated animals in comparison to vehicle controls. Several animals in the control and PLX-PAD-treated groups developed thymic malignant lymphoma, first seen after one month, as expected in this mouse strain. In addition, both groups developed spontaneous mesenteric vessel inflammation. Real-time quantitative polymerase chain reaction (RT-qPCR) demonstrated that distribution of PLX-PAD cells was confined to the injection site. Placental-derived MSCs remained in this site with gradual decrease in concentration during a three-month period. In view of these data, we conclude that the administration of PLX-PAD cells is not associated with any adverse effects in NOD/SCID mice.

Spontaneous aortitis in the Balb/c mouse.

We examined whether high incidence rates (18%-56%) of inflammation in the root of the aorta detected in a Balb/c mouse model for hind limb ischemia were related to the surgical procedure. Twenty mice underwent ligation of the femoral artery; incidences of aortic root inflammation were compared to those observed in controls. We used a multiple-section sampling method to increase the sensitivity of the diagnostic rates. Although a cumulative incidence of 12.5% was found, no difference was seen in the overall incidence rates between the control and the surgically treated groups. Evaluation of blood levels of inflammatory cytokines showed that ligation of the femoral artery produced higher levels of interleukin-6 in the surgically transected group of mice. The development of spontaneous arteritis in this strain must be considered in future studies.

Placental-derived and expanded mesenchymal stromal cells (PLX-I) to enhance the engraftment of hematopoietic stem cells derived from umbilical cord blood.

For the past 40 years, bone marrow transplantation (BMT) has become standard therapy to re-establish marrow function in patients with damaged or defective bone marrow. A human leukocyte antigen-matched sibling is the donor of choice for patients needing transplantation of allogeneic hematopoietic stem cells (HSCs). As most patients do not have an acceptable matched, related donor, the National Marrow Donor Program has been established to match volunteer bone marrow donors with potential recipients who require BMT. Although transplantation of HSCs from an unrelated donor can be an effective therapy for a variety of malignant and non-malignant diseases, it remains complicated because of treatment-related morbidity and mortality, which has led to the investigation of alternative sources of HSCs such as umbilical cord blood (UCB). This review highlights the advantages and disadvantages of UCB and recent developments that address its disadvantages. This includes the use of a placenta-expanded mesenchymal stromal cell product (PLX-I) being developed by Pluristem Therapeutics, Inc. and our opinion about the potential of this product.

CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell-derived factor 1alpha), redirects the polarization of effector Th1 cells into CD4(+)CD25(-)Foxp3(-)interleukin (IL) 10(high) antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10-deficient mice. Anti-IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

Targeted overexpression of IL-18 binding protein at the central nervous system overrides flexibility in functional polarization of antigen-specific Th2 cells.

The current study shows that functional polarization of Ag-specific CD4(+) Th2 cells entering the CNS during the accelerating phase of experimental autoimmune encephalomyelitis is flexible and dependent on the cytokine milieu there. Thus, targeted cell/gene therapy by Ag-specific T cells overexpressing IL-18 binding protein overrides this flexibility and induces infectious spread of T cell tolerance. Using a congenic system, we demonstrated that at this time, Ag-specific Th2 cells accumulate at the CNS but then arrest of IL-4 production. A manipulation of targeted cell/gene delivery was then used to detect whether this function is dependent on the cytokine milieu there. Targeted overexpression of IL-18 binding protein, a natural inhibitor of IL-18, restored the ability of these Ag-specific Th2 cells to produce IL-4 and subsequently induce protective spread of Th2 polarization. These findings not only suggest a novel way of therapy, but also explain why shifting the balance of Ag-specific T cells toward Th2 suppresses ongoing experimental autoimmune encephalomyelitis, whereas a direct transfer of these cells is ineffective.