作者：Sung-Whan Kima, Hoon Hanb, Gue-Tae Chaea, Sung-Hoon Leec, Sun Boc, Jung-Hee Yoond, Yong-Soon Leec, Kwang-Soo Leee, Hwon-Kyum Parke, Kyung-Sun Kangc 作者单位：a Department of Pathology, College of Medicine, The Catholic University, Seoul, Korea;b The Seoul Cord Bank, Histostem Co., Ltd, Seoul, Korea;c Laboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health,d Department of Veterinary Radiology, College of Veterinary Medicine, Seou
【摘要】
Buerger¡¯s disease, also known as thromboangiitis obliterans, is a nonatherosclerotic, inflammatory, vasoocclusive disease. It is characterized pathologically as a panangiitis of medium and small blood vessels, including both arteries and adjacent veins, especially the distal extremities (the feet and the hands). There is no curative medication or surgery for this disease. In the present study, we transplanted human leukocyte antigen-matched human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) into four men with Buerger¡¯s disease who had already received medical treatment and surgical therapies. After the stem cell transplantation, ischemic rest pain suddenly disappeared from their affected extremities. The necrotic skin lesions were healed within 4 weeks. In the follow-up angiography, digital capillaries were increased in number and size. In addition, vascular resistance in the affected extremities, compared with the preoperative examination, was markedly decreased due to improvement of the peripheral circulation. Because an animal model of Buerger¡¯s disease is absent and also to understand human results, we transplanted human UCB-derived MSCs to athymic nude mice with hind limb ischemia by femoral artery ligation. Up to 60% of the hind limbs were salvaged in the femoral artery-ligated animals. By in situ hybridization, the human UCB-derived MSCs were detected in the arterial walls of the ischemic hind limb in the treated group. Therefore, it is suggested that human UCB-derived MSC transplantation may be a new and useful therapeutic armament for Buerger¡¯s disease and similar ischemic diseases.
【关键词】 Cord blood Mesenchymal stem cells Buergers disease Cell transplantation
INTRODUCTION
Buerger¡¯s disease, also known as thromboangiitis obliterans, is a rare disease characterized by a combination of acute inflammation and thrombosis (clotting) of the arteries and veins in the hands and feet . The obstruction of blood vessels in the hands and feet reduces the availability of blood to the tissues, causes pain, and eventually damages or destroys the tissues. It often leads to skin ulcerations and gangrene of fingers and toes. Rarely, in advanced stages of the disease, it may affect vessels in other parts of the body.
The etiology of Buerger¡¯s disease is unknown, but a strong association with cigarette smoking is a striking characteristic . However, so far, these clinical trials have produced no satisfactory methods to save patients.
Therapeutic angiogenesis in patients with ischemic heart and lower limb ischemic disease was also studied by many researchers . The EPCs were harvested from peripheral blood, autologous bone marrow, and human umbilical cord blood.
Many clinical trials were done on patients with critical limb ischemia by using vascular endothelial growth factor, gene transfer, and autologous implantation of bone marrow mononuclear cells, including EPCs .
The treatment of ischemic vascular disease of the limbs remains a significant challenge. Unfortunately, if medical and surgical salvage procedures fail, amputation is an unavoidable result for these patients.
This study is the first report of a clinical trial on patients with Buerger¡¯s disease using MSCs derived from human UCB. Recently we also reported that UCB-derived MSCs could show functional and morphological improvement in a female patient with chronic spinal cord injury .
In the present study, we tried clinical applications for the patients with Buerger¡¯s disease to demonstrate the efficacy of UCB-derived MSCs for improvement of peripheral circulation and rest pain. Furthermore, in our animal model, we proved that transplantation of UCB-derived MSCs augmented arteriogenesis in the ischemic limb of immunodeficient nude mice.
MATERIALS AND METHODS
Patients
Four patients were tried for human UCB-derived MSCs transplantation because they had chronic limb ischemia, including severe rest pain and nonhealing ischemic ulcers, and they had already received currently available medical treatment and surgical interventions such as sympathectomy, bypass surgery, and even amputations. The profile of the patients is as follows: Patient 1, a 53-year-old man who had already received a variety of medical treatments and surgical operations, including a lumbar sympathectomy, bypass surgery, and amputations of digits of the feet and hand; Patient 2, a 44-year-old man with intractable pain in his fingers and an ulcer on his right thumb and for whom a lumbar sympathectomy and other medical treatments had failed; Patient 3, a 39-year-old man who already had a below-knee amputation on his lower left extremity; Patient 4, a 36-year-old man with severe rest pain and an ulcer on his foot who had received a variety of medical treatments. Of particular note, Patient 4 had previously received a bone marrow-derived MSC transplantation at another hospital. Taking his history into account, we waited for 6 months for any sign of possible delayed effects, but there was no change in his rest pain and skin lesions. All patients had to take strong pain killers (for example, three or four injections of pethidine) to sleep at night. The human leukocyte antigen (HLA) typings were done to get a proper match between the patients and preserved umbilical cord blood. These clinical trials were approved by the Korean Food and Drug Administration as emergent cases following informed consent from the patients.
Selection of HLA-Matched UCB Units
HLA allele types of cryopreserved human UCB units comparable to those of each patient were identified by polymerase chain reaction (PCR)-sequence-specific oligonucleotide probe (SSOP) and PCR-sequence-specific oligonucleotide (SSO) methods and selected from the inventory. The detailed HLA matching information is described in Table 1.
Table 1. HLA matching of umbilical cord blood-derived multipotent stem cells for Buerger¡¯s disease patients
MSC Isolation and Expansion
As described in our previously published papers on UCB-derived MSCs ) and then seeded in T-25 flasks at a concentration of 4 x 106 to 5 x 106 cells per cm2. Cultures were maintained at 37¡ãC in a humidified atmosphere containing 5% CO2. Three days later, the suspended cells were transferred into new flasks. The medium was changed every 7 days thereafter. Cells were passaged by trypsinization (0.005% trypsin/ ethylenediaminetetraacetic acid; Gibco) upon reaching 80%¨C90% confluence and replated at 5 x 104 cells per cm2. Cells were cultured and expanded for 4 weeks from primary culture and prepared under two passages before being used for clinical trials.
Transplantation Procedure
We delivered our UCB-derived MSCs (1 x 106) into just the proximal and adjacent area to the lesions of the patients. The depth of injection was approximately to the level of the subcutaneous tissues and muscles. We used a 23-gauge needle syringe for the effective delivery of the cells. An immunosuppressant was not given to the patients.
Follow-Up Angiography
We preformed a pretransplantation angiography of the patients, as well as 1-month and 4-month follow-up angiographies, to evaluate the vascular status in the affected areas.
Animal Experiments
Animal Model. Nude mice (BALB/cAnNCrjBgi-nu; Charles River Diagnostics) aged 7 weeks were anesthetized with 150 mg/ml Ketamine i.p. for operative resection of left femoral artery and subsequently for angiography imaging. Immediately before sacrifice, the mice were injected with an overdose of Ketamine.
MSC Transplantation. Immediately after the resection of one femoral artery, 1.3 x 106 culture-expanded MSCs were injected (i.m.) into the ischemic position of the hind limb (n = 8). Control groups were identically injected with medium (n = 3) or saline (n = 7; as control for medium effect).
Physiological Assessment of Transplanted Animals. Mice were anesthetized with Ketamine as described previously. The left ventricle was fixed proximally and canulated distally with a 26G polyethylene catheter. Warmed heparinized saline (10 U/ml, 0.4 ml total volume) was injected into the aortic catheter. Iodine was then injected into the aortic catheter. The skin was removed from the mouse hind limbs to avoid imaging the dermal vasculature. Images were acquired by using single-enveloped Kodak X-OMAT TL film at 500 mA, 50 kV, and 0.5-second exposure.
Probe. Genomic DNA of human liver cancer cells was extracted with the DNeasy tissue kit (Qiagen, Hilden, Germany, ml). Afterward, a DNA band of 224 bp was eluted with the Qiaquick gel extraction kit (Qiagen). The PCR product was DIG-labeled with the PCR DIG probe synthesis kit (Roche Diagnostics, Basel Switzerland, p>
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