Stem-cell therapy 3: Cell-based neovascularization therapy for peripheral arterial disease 

Author affiliations: Institute of Medical Research and Development, Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at Kobe, Japan

A neovascularization therapy developed by Translational Research Center for Medical Innovation (TRI) researchers in Japan and based on stem cells is showing great promise for treating chronic critical limb ischemia, offering hope to patients who are out of options.

1. INTRODUCTION

1.1 Pathology, epidemiology and treatment of chronic critical limb ischemia

A circulation disorder caused by the narrowing or occlusion of arteries in the limbs, peripheral arterial dis­ease (PAD) is estimated to affect more than 200 million people globally¹, ranging from asymptomatic to severe. The most common underlying disease is arteriosclerosis obliterans (ASO) caused mainly by athero­sclerosis; other underlying diseases include thromboangiitis obliterans (also known as Buerger’s disease), vasculitis and autoimmune diseases.

Chronic critical limb ischemia (CLI) is defined as advanced PAD for which sufferers experience pain at rest and have ulcers or gangrene on their lower limbs (Fontaine classification stage III or more severe, or Rutherford classification category 4 or more severe) that persist for two weeks or longer. The end stage of PAD, CLI (also called chronic limb-threatening ischemia recently) generally has a very poor prognosis, being comparable with those of some advanced malignancies. It has a mortality rate of 25%, a survival rate of 30% after major amputation, and a CLI persistence rate of 20% at 1 year. Furthermore, CLI patients have a 5-year survival rate of 40–50%². The annual incidence of CLI is 500–1,000 per million in Europe and the United States. An estimated 250,000 amputations of lower limbs are performed annually because of CLI¹. The global number of patients with PAD has increased by 23.5% between 2000 and 2010², and there is concern that the number of patients may increase rapidly in the future. Amputation of lower limbs not only seriously deteriorates the patient’s quality of life but also causes major social and economic loss.

The currently recommended therapeutic interventions for CLI include pain control, risk factor man­agement, treatment of ulcers or gangrene, and, for suitable patients, revascularization by bypass surgery or endovascular repair³. However, revascularization is unsuitable for approximately 25% to 40% of CLI pa­tients because they lack the vein grafts needed for bypass surgery or have multiple extensive artery lesions or comorbidities^1,4,5. The primary goal of CLI treatment is to save the patient’s life and limbs. Given that patients who are not suitable for revascularization or have refractory conditions have a very poor prognosis, it is socially and medically imperative to de­velop a treatment strategy for such ‘no-option’ CLI patients.

1.2 Neovascularization therapy using CD34⁺ cells for CLI patients

Basic and clinical research into endothelial progenitor cells (EPCs) took off after 1997, when Asahara and co-workers demonstrated that they are a subset (the CD34⁺ fraction) of human peripheral blood mononuclear cells (PBMCs)⁶. Neovascularization therapy involving the transplantation of EPCs has attracted attention as a novel therapy for ischemic diseases. Clinical studies of neovascularization therapy using autologous bone mar­row–derived CD34⁺ cells are being performed globally for diseases such as angina pectoris, acute myocardial infarction, dilated cardiomyopathy, ce­rebral infarction and CLI.

In 2003, we initiated a world-first phase I/IIa clinical study of lower-limb neovascularization therapy using granulocyte colony stimulating fac­tor (G-CSF)-mobilized autologous CD34⁺ cells in CLI patients and dem­onstrated safety and clinical efficacy⁷. Beginning in 2008, we conducted an investigator-initiated study of a CD34⁺ cell sorter in compliance with good clinical practice and again demonstrated efficacy and safety⁸. A mul­ticentre, randomized comparative study (sponsor initiated) was started in December 2017 with the aim of obtaining regulatory approval for CD34⁺ cells as a regenerative medicine product (Fig. 1). This chapter overviews the background to these studies as well as the prospects for CD34⁺ cells as a regenerative medicine product.

2. NONCLINICAL STUDIES

2.1 Characteristics and kinetics of EPCs

Angiogenesis, which involves the proliferation and migration of pre-exist­ing mature vascular endothelial cells, was originally proposed as the vas­cularization mechanism in adults. However, the identification of EPCs in adults as a subset (CD34⁺ cells) of PBMCs by Asahara and co-workers in 1997⁶ led to the new concept of vasculogenesis, which differs from angio­genesis in that it involves progenitor cells rather than mature endothelial cells. Neovascularization in adults is now thought to occur through inter­actions between angiogenesis and vasculogenesis.

EPCs exist within the peripheral blood as mononuclear cells expressing the hematopoietic stem cell surface antigen (CD34) and the early hema­topoietic stem cell surface antigen (CD133)⁹. When isolated and cultured in an endothelial cell growth medium at an appropriate density, these cells differentiate into spindle-shaped adherent cells that express CD34, CD31, vascular endothelial growth factor (VEGF) receptor 2 (also known as kinase insert domain receptor), Tie-2 and E-selectin. Also, their uptake of acetylated low-density lipoprotein has been confirmed^6,10.

Basic research using mouse or other animal models of bone-marrow transplantation has shown that EPCs are abundant in the bone marrow in adults. EPCs are forcefully mobilized from the bone marrow to the peripheral blood in conditions such as ischemia, inflammation, wounds, and tumorigenesis, or following the administration of cytokines such as G-CSF and granulocyte and macrophage colony stimulating factor, and hormones such as estrogen. They reach the neovascularization site by cir­culating blood, where they contribute to the formation of new blood ves­sels^11,12. These findings have been useful for establishing a method for col­lecting and isolating EPCs for neovascularization therapy (Fig. 2).

2.2 Nonclinical studies on EPC transplantation

Kalka and co-workers obtained EPCs by culturing PBMCs from healthy subjects, and they transplanted them into the nude mouse model of hind-limb ischemia. When EPCs were intravenously injected after two days of hind-limb ischemia, accumulation of transplanted cells at the isch­emia site was histologically confirmed, indicating that they contributed to neovascularization in collaboration with mouse-derived vascular endo­thelial cells. Transplantation significantly improved the capillary density (observed histologically) and blood perfusion in the ischemic limbs (as measured by laser Doppler flowmetry). As a result, the limb salvage rate from necrosis due to severe ischemia was 59% in the EPC transplanta­tion group, compared with only 7–8% in the control group¹³. Murohara and co-workers reported similar positive results after they collected EPCs from human umbilical cord blood and cultured and transplanted them into nude rats with hind-limb ischemia¹⁴. Losordo and co-workers also obtained favourable results after they collected and isolated CD34⁺ cells from the peripheral blood of healthy subjects who had received a subcu­taneous injection of G-CSF and transplanted them into nude rats with hind-limb ischemia (unpublished data).

Nonclinical studies have also shown that EPC transplantation ther­apy is effective for treating other diseases such as myocardial infarc­tion^15,16, cerebral infarction¹⁷ and refractory fracture¹⁸. These results the­oretically support not only EPC transplantation but also transplantation of bone-marrow mononuclear cells (BMMCs, a cell group that includes hematopoietic and mesenchymal cells as well as EPCs). EPCs trans­planted into the animal model of ischemia were directly integrated into new blood vessels in the ischemic tissue by vasculogenesis to form the vascular endothelium. The transplanted EPCs were found to produce various cytokines and vascular growth factors involved in angiogenesis, such as VEGF, hepatic growth factor, angiopoietin-1, stromal-cell-de­rived factor-1 alpha, insulin-like growth factor-1 and endothelial nitric oxide synthase^19–21, promoting the proliferation of the existing vascu­lar endothelium and cellular migration (the paracrine effect). Moreover, EPCs have been shown to release not only angiogenesis-related proteins but also RNAs and exosomes containing microRNA, which contribute to the paracrine effect via gene-control mechanisms (Fig. 2)^22,23.

While transplantation can be performed using EPCs (CD34⁺ cells) isolated from BMMCs or PBMCs and purified, many methods for trans­planting mononuclear cells (BMMC or PBMC transplantation) without isolating and purifying EPCs have been attempted in nonclinical studies. However, several studies have highlighted the risks associated with trans­planting mixed cell groups, including ossification by osteoblasts, chon­drification by chondroblasts and fibrosis by fibroblasts. The nonclinical study by Yoon and co-workers also showed that myocardial calcification occurred with high frequency when whole bone marrow cells were trans­planted intramyocardially into the rat model of acute myocardial isch­emia²⁴. We have also demonstrated that intramyocardial transplant of high-dose PBMCs into the rat model of acute myocardial ischemia led to intramyocardial hemorrhage with infiltration of many inflammatory cells and a reduced improvement in neovascularization and cardiac function. In contrast, transplantation of purified CD34⁺ cells alone was associated with an absence of adverse reactions, high levels of neovascularization and sustained recovery of cardiac function¹⁶. The in vitro EPC colony-forming assay developed by Masuda and co-workers showed that EPC colonies are formed from CD34⁺ cells at a high frequency, whereas colonies could not be obtained from CD34⁻ mononuclear cells even when using 100 times more cells, demonstrating a marked difference in vascularization potential between the EPC and non-EPC fractions²⁵. These fundamental research and nonclinical studies suggest that transplantation of purified EPCs is su­perior to BMMC or PBMC transplantation in terms of low invasiveness, therapeutic effect and safety.

3. CLINICAL STUDIES

3.1 Physiological significance of EPCs

Reports have shown that patients with diabetes mellitus and patients with many risk factors for arteriosclerosis have fewer EPCs circulating in their peripheral blood than healthy individuals and that EPCs in these patients have lower proliferative and migratory capacities^26,27. Arteriosclerosis pa­tients with fewer EPCs in circulation have many risk factors for poor car­diovascular prognosis and reduced vascular endothelial function²⁸. Thus, EPCs have been shown to be one of the important intrinsic factors that determine the prognosis of patients with arteriosclerosis.

3.2 Phase I/IIa clinical study of neovascularization therapy for CLI patients

Based on the results from the nonclinical studies, we conducted a mul­ticentre, single-blinded, dose-escalation phase I/IIa clinical trial of intra­muscular transplantation of G-CSF-mobilized CD34⁺ cells in CLI pa­tients, which started in 2003⁷. The study enrolled 17 CLI patients (five with ASO, including one on hemodialysis, and 12 with Buerger’s dis­ease). Mononuclear cells mobilized from the bone marrow to the periph­eral blood by five days of G-CSF treatment were collected by leukapher­esis with high efficiency, and CD34⁺ cells were further isolated using a magnetic-activated cell sorter (CliniMACS^®) (Figs. 3 and 4). The isolat­ed CD34⁺ cells, which had a mean purity of 92% and a mean cell viabil­ity of 87%, were transplanted to the ischemic lower limbs intramuscular­ly under lumbar spinal anesthesia (Fig. 4). No death or major amputation of lower limbs was reported for any patient in the year following treat­ment, and all patients maintained independent walking function. Heal­ing of ulcers or necrosis, relief of ischemic pain and improvement over time in physiological measures (for example, toe brachial pressure index (TBI), transcutaneous oxygen partial pressure (TcPO₂) were observed, and 88% of patients were free of CLI one year after treatment²⁹. Long-term follow up over the four years following treatment revealed no deaths in the first two years. Four patients died of cardiac diseases after the first two years, but a relationship with the cell therapy was ruled out. No ma­jor amputation of lower limbs was reported. The proportion of patients free of CLI remained at over 80% (Fig. 5). Significant improvements were noted in TBI at four years and in TcPO₂ at three years compared with pre-treatment data²⁹.

In the United States, Losordo and co-workers initiated a multicentre, randomized, double-blind, placebo-controlled study (study ACT34-CLI) in 2007, using transplantation of G-CSF-mobilized CD34⁺ cells in 28 pa­tients with CLI caused by ASO³⁰. The incidence of minor and major amputations of lower limbs tended to be lower in the CD34⁺ cell transplan­tation group than in the placebo group at 6 months (P = 0.125) and 12 months (P = 0.054) after treatment. No adverse events related to the cell therapy were reported during the one-year follow-up period.

3.3 Investigator-initiated study of CD34⁺ cell sorter

Following the early clinical studies described above, we sought regulato­ry approval of the CD34⁺ cell sorter Isolex^®. We began an investigator-initiated study in 2008 in 11 CLI patients (seven with Buerger’s disease and four with ASO who were not on dialysis), in compliance with good clinical practice for medical devices, and completed it in March 2012. This exploratory phase II study was the first investigator-initiated regener­ative medicine study in Japan and is still the only Japanese clinical study of lower-limb neovascularization therapy by cell transplantation in which data reliability was assured in accordance with good clinical practice. This investigator-initiated study showed favourable safety and efficacy, as had been previously demonstrated in the phase I/IIa study described above. Measures of signs and symptoms, as well as quality-of-life indices, clini­cal severity, pain at rest, blood flow improvement indices, and walk test results, were evaluated frequently. Pain at rest improved soon after trans­plantation of CD34⁺ cells. Physiological improvement was confirmed, and improvement in Rutherford classification, the clinical severity index, was demonstrated at approximately 6 months (Fig. 6)⁸.

3.4 Sponsor-initiated study aiming for regulatory approval of CD34⁺ cells as a regenerative-medicine product

Based on the positive results from the phase I/IIa clinical study and the exploratory investigator-initiated study of the medical device, we cur­rently aim to establish a neovascularization therapy of lower limbs us­ing G-CSF-mobilized autologous CD34⁺ cells as the novel standard of care. When we initiated the exploratory investigator-initiated study in 2008, the company that was intending to file an approval application was trying to develop a CD34⁺ cell sorter and to file an application for a medical device, but following the completion of our study, the compa­ny decided to develop CD34⁺ cells instead and file an application for a therapeutic cell product.

If a CD34⁺ cell sorter were developed, it would be installed at each medical institution, and each step — G-CSF administration, apheresis, isolation of CD34⁺ cells, and cell transplantation — would be complet­ed within the medical institution as a self-contained manufacturing pro­cess (Fig. 7). On the other hand, if a therapeutic cell product were de­veloped, G-CSF administration and apheresis would be implemented at each medical institution, and the apheresis product would then be trans­ported to an external cell-processing centre. The CD34⁺ would be isolated at the cell-processing centre and transported to each medical institution, where transplantation (treatment) would be performed (Fig. 7). Develop­ing a therapeutic cell product has the advantages of easy and wide dissem­ination (because it is not necessary for every medical institution to install the cell sorter or employ technicians experienced in cell sorting) and a sta­ble supply of high-quality products (because cell products are manufac­tured by experienced technicians under a more-controlled environment at a cell-processing centre than at general medical institutions). Disadvantages include a high manufacturing cost.

After the company developing the product had changed its plans, the cell-manufacturing process was commissioned to the Foundation for Biomedical Research and Innovation at Kobe. Preparation for a clinical study was initiated in compliance with the structure and facility regulations, cell-manufacturing control, and quality standards based on the Ordinance on Standards of Manufacturing Control and Quality Con­trol for Cellular and Tissue-based Products (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice). In 2013, the Pharmaceutical Affairs Law was amended, and the Law on Securing Quality, Efficacy and Safety of Products including Pharmaceuticals and Medical Devices (Pharmaceutical and Medical Devices Act) was enacted. In ad­dition to pharmaceuticals and medical devices, a new category of regenerative-medicine products was introduced for regulatory review. Follow­ing consultation with the Pharmaceuticals and Medical Devices Agency (PMDA) regarding the amendment, we changed our product from the pharmaceutical category to the regenerative-medicine product catego­ry. After repeated consultations with the PMDA, the next-phase spon­sor-initiated study was designed to be conducted as a multicentre, ran­domized, comparative study, aiming for regulatory approval of CD34⁺ cells under the category of regenerative-medicine products. The study was initiated in December 2017, and the first patient was enrolled in the same month. It is currently ongoing. In March 2018, this regenerative-medicine product was designated by the Ministry of Health, Labour and Welfare as an appropriate product for the Sakigake Strategy, which con­sists of the Sakigake Designation System and the Scheme for Rapid Au­thorization of Unapproved Drugs. This strategy identifies breakthrough medical devices, pharmaceuticals and regenerative-medicine products that are designed to treat serious diseases and are likely to be developed first in Japan. Such products are then given priority in consultations and reviews to reduce the review time for approval.

4. FUTURE PROSPECTS

The efficacy and safety of this cell therapy have been demonstrated not only for CLI and other ischemic diseases such as refractory angina pecto­ris^31–34, acute myocardial infarction^35,36, dilated cardiomyopathy^37–42, and cerebral infarction⁴³, but also in refractory fracture⁴⁴ and liver cirrhosis patients⁴⁵. The approval of CD34⁺ cells as a regenerative-medicine prod­uct is expected to pave the way for extending application to diseases oth­er than CLI.

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