The Hard Cell

William R Prather RPh, MD at Pluristem Therapeutics, Inc investigates the role of placental-derived mesenchymal stromal cells in the treatment of critical limb ischaemia and other diseases

MESENCHYMAL STROMAL CELLS (MSCs)

Isolated from anatomical locations, including the placenta and bone marrow, MSCs are multipotent stem cells with ectodermal, endodermal and mesodermal characteristics. Historically, MSCs in adult tissues were believed to be reservoirs of reparative cells, ready to mobilise and differentiate in response to wound signals or disease conditions. Recent evidence suggests that their efficacy may be related to their secretion of cytokines or other potent immune modulators. MSCs derived from the human placenta – placental-derived mesenchymal stromal cells (PMSCs) – can be used allogeneically and are unique in the way that they are expanded using a three-dimensional bioreactor.

PERIPHERAL ARTERY DISEASE & CRITICAL LIMB ISCHAEMIA

Peripheral artery disease (PAD), also known as peripheral arterial occlusive disease and peripheral vascular disease, is caused by the obstruction of large peripheral arteries resulting from inflammatory processes such as atherosclerosis. The advancement of PAD, aggravated by various conditions including hypercholesterolaemia, smoking and diabetes, may lead to acute or chronic ischaemia (1). Critical limb ischaemia (CLI) is the end stage of PAD. Approximately, seven million patients in the US over 40 years of age, or 4.3 per cent of the population, have been diagnosed with PAD with the disorder significantly progressing with age to 20 per cent of the US population over the age of 70 (2). In total, CLI affects approximately 1.1 million patients in the US and is anticipated to grow to approximately 1.4 million patients by 2015 (3).

The Rutherford Category is a commonly used measure for assessing PAD. CLI is classified as category 4 (pain at rest) or category 5/6 (tissue necrosis/gangrene) (4). Figure 1 illustrates a patient with category 5 CLI.

The ankle-brachial index (ABI) is a commonly used objective parameter to assess the severity of CLI. This is a ratio of the systolic pressure in the dorsalis pedis or posterior tibial artery in the leg divided by the systolic pressure in the brachial artery in the arm. Doppler probes are also used in vascular laboratories to measure the pulse volume waveform at segmental locations in the leg artery. Sites of arterial blockage can be identified by changes in the Doppler waveform from triphasic to biphasic to monophasic and then stenotic waveforms. However, angiography remains the gold standard for assessing the severity and anatomical pathology for CLI.

CURRENT THERAPIES FOR CLI

Minimal invasive endovascular therapies have been the most popular treatments for CLI, including angioplasty, stent placement and laser. The location and severity of the injury and the physician’s expertise are usually the determining factors in choosing the endovascular therapy. However, endovascular therapy is most successful with short, proximal lesions. One or two days are typically required for the patient to recover from these procedures, which are usually performed on an outpatient basis.

Limb preservation is the goal with most patients with CLI. Surgical revascularisation of patients may be cost-effective and can lead to a better quality of life for selected patients and is associated with lower perioperative morbidity and mortality than amputation. Angiographic findings, as well as the availability of a bypass conduit, determine the feasibility of surgical revascularisation. Commonly, three-year bypass patency rates of calf arteries range from 40 per cent for prosthetic bypasses, to 85 per cent for saphenous vein bypasses.

Conservative treatments for CLI patients include anti-platelet therapies and have shown a 25 to 49 per cent success rate with non-healing wounds and a 50 to 80 per cent rate of improvement in ischaemic rest pain (5).

MESENCHYMAL STROMAL CELL THERAPY

Endovascular therapies and revascularisation surgery are effective treatment options for a number of CLI patients. However, CLI progression in many patients is gradual and, although not at risk of imminent limb loss, tends to have a downhill course. Additionally, many patients with CLI have anatomic involvement in areas where endovascular therapy or surgery would be of no help. Data indicate that patients with chronic CLI have a three year limb loss rate of approximately 40 per cent (6). These facts dictate a need for a therapy with a novel mechanism of action that is directed to the basic pathology of limb ischaemia. MSCs have been found to efficacious via the stimulation of angiogenesis in the CLI-involved extremity.

Esato et al published the results of an eight patient pilot study in patients with PAD in whom surgical bypass procedures or longterm medication had failed (7). Four out of the eight patients had arteriosclerosis obliternas, while the other four cases suffered from severe Buerger’s disease affecting five to eight fingers or toes. Patients were treated with autologous bone marrow-derived mononuclear cells and evaluated after four weeks and one year. Seven out of eight reported improvement in ischaemic rest pain after four weeks. Increased collateralisation was observed by angiography in two of eight cases and six of nine limbs showed an improvement in the Fontaine stage, an assessment of the severity of PAD, after four weeks. These improvements were sustained for at least one year post-treatment.

Taguchi et al published a report on a patient suffering from non-healing ulcers on both hands that were treated with autologous bone marrow cells, resulting in the dilation of existing arteries and new collateral vessel formation – systolic pressures in the fingers were increased, skin colour changed from ischaemic red to pink and the healing ulcers were covered with skin (8).

Umbilical cord blood-derived MSCs were administered to four men with Buerger’s disease in a 2006 study by Kim et al (9). As a result, these patients, who previously received medical and surgical therapy, experienced significant relief in ischaemic rest pain with no adverse events associated with the implantation of MSCs. MSCs isolated from the human placenta, termed PMSCs, provide an allogeneic cell therapy that is standardised and potentially cost effective. PMSCs can be cultured in a bioreactor that provides a microenvironment, enabling the large-scale growth of these cells. PMSCs can be expanded in vitro without the loss of phenotype and without showing signs of karyotypic changes.

In mixed lymphocyte reaction (MLR) experiments, PMSCs cultured in vitro have been demonstrated to suppress the proliferation of T cells triggered by allogeneic peripheral and umbilical cord blood-derived T lymphocytes with these results sharing the same characteristics as bone marrow-derived MSCs. PMSCs can also escape allo-recognition and reduce the T cell response (10). As a result, PMSCs are considered to be immune privileged, immunomodulatory, and to not require human leukocyte antigen (HLA) matching.

PRECLINICAL & CLINICAL STUDIES USING PMSCsWITH CLI PATIENTS

In vivo pilot studies were performed to determine whether the implantation of PMSCs might reduce the ischaemic damage in an established mouse hind limb ischaemia model (11). Twenty male Balb/c mice were anesthetised, and a 1 to 1.5cm incision was made in the skin in the inguinal area. The femoral artery was ligated twice and transected distal to the ligature. The wounds were closed and the mice were allowed to recover. Five hours following the excision of the femoral artery, the animals received intramuscular (IM) injections of 1x106 PMSCs in the hip and paw distal to the ligature, with a control group similarly injected with PBS (saline).

In this study, the injection of PMSCs markedly improved the blood flow to the damaged limb (P=0.0008). Additionally, a statistically significant increase (P=0.021) was observed in capillary density, decreases were noted in oxidative stress and endothelial damage, and there was an increase in the functionality of the limb.

Nine days following injection, and observed throughout the entire study, the group treated with PMSCs showed increased blood flow using a laser Doppler from 24 ± 2.3 per cent to 80 ± 4.7 per cent. In the control, saline-treated group, blood flow ranged from 35 ± 2 per cent to 54 ± 4.5 per cent in the hip/implantation area (day zero versus day 21 respectively). Similar to the observed effect in the hip area, an increase in blood flow could also be demonstrated in the paw area. In the group treated with PMSCs, blood flow increased from 10 ± 0.7 per cent to 52 ± 5.5 per cent, while in the vehicle-treated group, blood flow increased from 12 ± 0.6 per cent to 46 ± 4.9 per cent (day zero versus day 21 respectively). These results are depicted in Figure 3.

Post mortem immunohistochemical analyses of the limbs treated with PMSCs indicated a statistically significant increase (P=0.021) in the number of new capillaries supplying the limb, suggesting that PMSCs have the ability to promote angiogenesis (see Figure 4).

Additionally, there was an observed decrease in oxidative stress and reduction in endothelial inflammation, considered a surrogate parameter for improved endothelial function, in the animals treated with PMSCs (see Figure 5).

There was also an improvement of limb function demonstrated in the group treated with PMSCs (2.1 ± 0.2 per cent) versus the control vehicle group (2.5 ± 0.2 per cent). When compared to controls, none of the mice injected with PMSCs exhibited any adverse clinical signs or symptoms in response to the IM administration of these cells. It was concluded that, under the conditions of the study, the administration of PMSCs induces increases in blood flow, likely resulting from angiogenesis as supported by histological evaluation of the damaged limb.

PMSCs are currently in clinical trials in the US and are completed in Europe in patients with CLI. Interim top-line results from these Phase I studies demonstrated that PMSCs are safe, well tolerated and effective. In the trials thus far, a total of 21 patients, representing 77 per cent of the cohorts required to complete the Phase I studies, experienced no serious adverse effects and reported an improvement in their quality of life and Rutherford Category. Additionally, 83 per cent of these patients recorded quantitative improvements in their blood flow measurements.

THE USE OF PMSCs IN OTHER INDICATIONS

Several animal models of disease using PMSCs have demonstrated that they are potentially effective in the treatment of a variety of maladies, including inflammatory bowel disease and multiple sclerosis, as an alternative to bone marrow transplant and, recently, stroke. A preclinical study in rats using PMSCs showed that they are a promising source for the treatment of ischaemic stroke (12). Animals treated with PMSCs demonstrated significant differences over a control group in the improvement of sensory and motor deficits, reduction in the development of the stoke lesion, and increase in the production of glial nerve tissue.

These effects occurred even though PMSCs were administered eight and 24 hours after the inducement of stroke. This suggests that the use of PMSCs in ischaemic stroke may allow patients a longer window of time for successful treatment after the onset of the stroke. Optimal current theory dictates that patients must be treated within four and a half hours after the onset of ischaemic stroke. PMSCs may increase the window from four and a half hours up to eight.

Thus, placenta derived MSCs (PMSCs) demonstrate the potential in both preclinical and clinical studies to effectively treat CLI and other diseases.

Note

To read William Prather’s full report on Mesenchymal Stem Cells, his paper titled ‘The role of placental-derived adherent stromal cell (PLX-PAD) in the treatment of critical limb ischemia’ is available to read in Cytotherapy 11(4): pp427-434, 2009.

References

1. Van den Bosch M et al, Peripheral arterial disease, Lancet 359, 9311: pp1,070-1,070, 2002
2. Selvin E et al, Prevalence of and Risk Factors for Peripheral Artery Disease in the United States, Results From the National Health and Nutritional Examination Survey, 1999-2000, Circulation 110: pp738-743, 2004
3. The Sage Group Report, 12th September, 2005
4. Rutherford R et al, Recommended standards for reports dealing with lower extremity ischemia: Revised version, J Vasc Surg 26: pp517-538, 1987
5. Cheshine NJ, Wolfe JH, Noone MA, Davies L and Drummond M, The economics of femorocrural reconstruction for critical leg ischemia with and without autologous vein, JVasc Surg 15: pp167-174, 1992
6. Albers M, Fratezi AC and De Luccia N, Assessment of quality of life of patient with severe ischemia as a result of infrainguinal arterial occlusive disease, J Vasc Surg 16: pp54-59, 1992
7. Esato K, Hamano K, Li TS, Furutani A, Seyama A, Takenaka H and Zempo N, Neovascularisation induced by autologous bone marrow cell implantation in peripheral arterial disease, Cell Transplant 11: pp747-752, 2002
8. Taguchi A et al, Therapeutics angiogenesis by autologous bone-marrow transplantation in a general hospital setting, Eur J Vasc Endovasc Surg, Mar 25(3): pp276-278, 2003
9. Kim SW et al, Successful stem cell therapy using umbilical cord blood-derived multipotent stem cells for Buerger’s disease and ischemic limb disease animal model, Stem Cells 24(6): pp1,620-1,626, 2006
10. Li CD et al, Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocytes proliferation, Cell Res 15(7): pp539-547, 2005
11. Tokai J et al, Search for appropriate experimental methods to create stable hind-limb ischemia in mouse, Exp Clin Med 31(3): pp128-132, 2006
12. Kranz A et al, Transplantation of placenta-derived mesenchymal stromal cells upon experimental stroke in rats, Brain Research 1,315: pp128-136, 2010