Ex vivo Expansion and Clonal Maintenance of CD34+Selected Cells from Cord Blood and Peripheral Blood

김순기,길혜윤,송순욱,최종원,박상규 대한소아청소년과학회 2005 Clinical and Experimental Pediatrics (CEP) Vol.48 No.8

제대혈 및 말포혈로부터 분리한 CD34 양성 세포의 체외 증폭 및 클론 유지인하대학교 의과대학 소아과학교실*,인하대학교병원 중앙연구소†, 진단검사의학교실 ,울산대학교 의과대학 소아과학교실§김순기*·길혜윤†·송순욱†·최종원 ·박상규§목 적 : 조직적합 항원의 불일치로 인하여 골수이식을 할 수 없는 경우에 점점 더 제대혈이 사용되고 있다. 그러나 제대혈의 조혈모세포의 수가 적기 때문에 이를 증가시킬 대책이 필요한 바, 여러 성장인자를 조합하여 체외증폭하여 말초혈의 체외증폭과 비교하였다. 방 법 : 저자들은 제대혈 및 말초혈로부터 분리한 CD34+ 세포를 혈청이 아닌 배양체에서 체외 증폭하여 비교하였다. Miltenyi 방법으로 분리한 CD34+는 조혈성장인자들과 함께 체외 증폭 시켰다. 증폭 당일, 4일 후, 7일 후 및 14일에 증폭된 세포를 가지고 burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) 및 colony-forming units of megakaryocytes (CFU-Mk)의 생성 능력을 알아보았다. 결 과 : 말초혈에 비하여 제대혈로부터 분리한 CD34+ 세포의 증폭 능력이 2배로 컸다. 체외에서7일 및 14일 동안 증폭된 제대혈이 더 많은 BFU-E를 생성하였고, 4일 및 7일 동안 증폭된 제대혈이 더 많은 CFU-Mk를 생성하였다.결 론:MGDF, FL 및 IL-3를 포함한 성장인자의 자극 하에서 제대혈의 체외 증폭이 더 많은 BFU-E 및 CFU-Mk를 생성하였으므로, 이를 이용한 체외 증폭을 시도하는 것의 가능성을 시사하고 있다. Purpose : Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. Methods : The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony- forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. Results : The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. Conclusion : The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

Immunomodulatory Properties of Mesenchymal Stem Cells and Their Therapeutic Applications

이택기,송순욱 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.2

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from most adult tissues, including bone marrow, adipose, liver, amniotic fluid, lung, skeletal muscle and kidney. The term MSC is currently being used to represent both mesenchymal stem cells and multipotent mesenchymal stromal cells. Numerous reports on systemic administration of MSCs leading to functional improvements based on the paradigm of engraftment and differentiation have been published. However, it is not only difficult to demonstrate extensive engraftment of cells, but also no convincing clinical results have been generated from phase 3 trials as of yet and prolonged responses to therapy have been noted after identification of MSCs had discontinued. It is now clear that there is another mechanism by which MSCs exert their reparative benefits. Recently, MSCs have been shown to possess immunomodulatory properties. These include suppression of T cell proliferation, influencing dendritic cell maturation and function, suppression of B cell proliferation and terminal differentiation, and immune modulation of other immune cells such as NK cells and macrophages. In terms of the clinical applications of MSCs, they are being tested in four main areas: tissue regeneration for cartilage, bone, muscle, tendon and neuronal cells; as cell vehicles for gene therapy; enhancement of hematopoietic stem cell engraftment; and treatment of immune diseases such as graft-versus-host disease, rheumatoid arthritis, experimental autoimmune encephalomyelitis, sepsis, acute pancreatitis and multiple sclerosis. In this review, the mechanisms of immunomodulatory effects of MSCs and examples of animal and clinical uses of their immunomodulatory effects are described.

Combined treatment of dedritic cells, chemotherapy and radiation therapy for murine fibrosarcoma

이현숙,이승규,이시원,신정현,송순욱,김우철,김순기,이문희,최광성 대한피부과학회 2008 초록집 Vol.46 No.20

Therapeutic effect of human clonal bone marrow-derived mesenchymal stem cells in severe acute pancreatitis

정경희,이택기,손미권,송순욱,홍순선 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.5

Severe acute pancreatitis (SAP), a commonnecroinflammatory disease initiated by the premature activationof digestive enzymes within the pancreatic acinarcells, is associated with significant morbidity and mortality. In this study, we investigated whether human bone marrowderivedclonal mesenchymal stem cells (hcMSCs), isolatedfrom human bone marrow aspirate according to our newlyestablished isolation protocol, have potential therapeuticeffects in SAP. SAP was induced by three intraperitoneal(i.p.) injections of cerulein (100 lg/kg) and sequentialLPS (10 mg/kg) in Sprague-Dawley (SD) rats. hcMSCs(1 9 106/head) were infused on 24 h after LPS injection viathe tail vein. The rats were sacrificed 3 days after infusion ofhcMSCs. We observed that infused hcMSCs reduced thelevels of serum amylase and lipase. Infused hcMSCs amelioratedacinar cell necrosis, pancreatic edema, and inflammatoryinfiltration. Also, infused hcMSCs decreased thelevel of malondialdehyde, and increased the levels of glutathioneperoxidase and superoxide dismutase. The numberof TUNEL positive acinar cells was reduced after hcMSCsinfusion. In addition, hcMSCs reduced the expression levelsof pro-inflammation mediators and cytokines, and increasedthe expression of SOX9 in SAP. Taken together, hcMSCscould effectively relieve injury of pancreatitis as a promisingtherapeutics for SAP.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

김시나,이현주,전명신,이택기,송순욱 대한면역학회 2015 Immune Network Vol.15 No.5

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the β-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Helicobacter pylori 감염과 관련된 철 결핍성 빈혈에서 Lactoferrin Sequestration의 역할

문광빈,강창규,최연호,한혜승,송순욱,Moon, Kwang-Bin,Kang, Chang-Kyu,Choe, Yon-Ho,Han, Hye-Seung,Song, Sun-Uk 대한소아소화기영양학회 2002 Pediatric gastroenterology, hepatology & nutrition Vol.5 No.1

배경: Lactoferrin은 위장 점막에서 H. pylori의 철분의 공급원으로 알려져 있다. 본 연구는 H. pylori에 감염된 위장 점막의 조직검체에서 lactoferrin 값을 측정하고 철 결핍성 빈혈의 유무에 따라 lactoferrin 발현의 주요 위치를 정하는 방법을 이용하여 lactoferrin과 철 결핍성 빈혈을 동반한 H. pylori감염 사이의 연관성을 밝히기 위해 시행하였다. 방법: 상부 위장관 내시경을 시행한 55명의 환아를 세 군으로 분류하였다: 정상군(NL, n=19), H. pylori 감염군(HP, n=15), H. pylori 감염이 동반된 철 결핍성 빈혈군(IDA, n=21). 조직 병리학 소견은 Updated Sydney System을 이용하여 정도를 나누었다. 위장 점막의 lactoferrin 값은 면역 측정법으로 측정하였다. lactoferrin의 발현 위치와 양을 측정하는 것은 면역조직화학적 염색방법을 이용하였다. 결과: 위 전정부에서의 lactoferrin의 값은 조직 검체의 H. pylori 밀도나 다형핵세포의 침습정도, 만성 염증에 비례하여 의미있게 증가되었다. HP와 IDA군에서는 정상군에 비하여 lactoferrin의 수치가 통계학적으로 유의하게 증가되었다(p=0.0001). IDA군은 HP군에 비하여 lactoferrin의 수치가 높은 경향이 있었다(p=0.2614). 면역조직화학적 방법에 의한 lactoferrin 발현의 주요부위는 상피세포내의 분비샘과 호중구에 있었다. Lactoferrin은 정상군에서는 약하게 염색되었고 HP와 IDA군에서는 강하게 염색되었다. 결론: IDA군에서 위장 점막내 lactoferrin 격리가 현저하였으며 이 소견은 H. pylori 감염이 철 결핍성 빈혈에 영향을 미치는 기전을 밝히는데 중요한 단서로 작용할 것이다. Purpose: It is known that lactoferrin serves as a source of iron for H. pylori in gastric mucosa. This study was undertaken to investigate the relationship between lactoferrin and H. pylori infection coexistent with iron-deficiency anemia by determining the lactoferrin levels in gastric biopsy specimens, and by locating the major sites of lactoferrin expression, according to the presence or absence of iron-deficiency anemia. Methods: Fifty-five adolescents that underwent gastroduodenoscopy were divided into three groups: NL (n=19) for normal controls, HP (n=15) for patients with H. pylori, and IDA (n=21) for patients with H. pylori gastritis and coexisting iron-deficiency anemia. Histopathologic features were graded from to marked on the basis of the Updated Sydney System. The gastric mucosal levels of lactoferrin were measured by immunoassay. Immunohistochemical technique was used to allow identification of the location and quantification of the lactoferrin expression. Results: Lactoferrin levels in the antrum increased significantly, in proportion to, H. pylori density, polymorphonuclear cell infiltration, and chronic inflammation in the histologic specimens. Patients in the HP and IDA groups showed significantly increased mucosal levels of lactoferrin compared with that observed in the normal group (p=0.0001). The lactoferrin level in IDA group tended to be higher than that in the HP group (p=0.2614). The major sites of lactoferrin expression by immunohistochemistry were in glands and neutrophils within epithelium. Lactoferrin was stained weakly in NL, and strongly in HP and IDA. Conclusion: The lactoferrin sequestration in the gastric mucosa of IDA was remarkable, and this finding seems to give a clue that leads to the clarification of the mechanism by which H. pylori infection contributes to iron-deficiency anemia.

갑상선에 생긴 원발성 편평세포암에 관한 고찰

최정석,임재열,주영채,송순욱,김영모 대한이비인후과학회 2012 대한이비인후과학회지 두경부외과학 Vol.55 No.1

Background and ObjectivesZZSquamous cell carcinoma of thyroid is uncommon and accounts for less than 1% of all primary thyroid malignancies. Clinical features mimic the natural course of anaplastic carcinoma. This study reviewed the clinical course of six cases of primary squamous cell carcinoma of thyroid. Subjects and MethodZZWe diagnosed six cases of primary squamous cell carcinoma of thyroid diagnosed from 1999 to 2006 at the College of Medicine Department of Pathology. Clinical data, treatment modality, and pathologic test results from medical records were retrospectively analyzed. ResultsZZWe found five women and one man (with the mean age of 52.1 years) with squamous cell carcinoma of thyroid. The main presenting features were abruptly enlarging neck swelling and obstructive symptom. Pre-operative needle aspiration biopsy revealed papillary carcinoma in five cases. Only one patient was diagnosed as squamous cell carcinoma through pre-operative needle aspiration biopsy. Three patients had massive adjacent organ invasion, and four patients had lymph node metastasis according to the pathology review. There were no cases of distant metastasis at the time of treatment. All patients received surgery and adjuvant therapy (radiation therapy, chemotherapy, radioiodine therapy). Three patients are still alive with a mean follow up period of 47.3 months (range, 44-49 months). The other three patients died within one year post-operatively. ConclusionZZPrimary squamous cell carcinoma of thyroid should be considered in patients diagnosed with papillary carcinoma and who exhibit aggressive clinical behavior. Complete tumor resection and radiotherapy should be performed if thyroid squamous cell carcinoma is confirmed.

Molecular Characterization of Neurally Differentiated Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

이택기,이현주,조윤경,전명신,송순욱 대한면역학회 2014 Immune Network Vol.14 No.1

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

Synergistic Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma on Bone Regeneration of Calvarial Defects in Rabbits

오남식,윤정호,유재흥,최성호,이명현,이상진,송순욱 한국조직공학과 재생의학회 2012 조직공학과 재생의학 Vol.9 No.1

Bone tissue regeneration techniques include tissue engineering approaches which employ mesenchymal stem cells as an osteogenic agent for bone repair. Recent studies have demonstrated that tissue engineering scaffolds and growth factors can support cell proliferation, bone formation, and bone tissue repair in lost bone tissue. Furthermore,many studies have suggested that platelet-rich plasma (PRP) can improve bone regeneration due to the numerous growth factors that it contains. This study was performed to investigate the influence of bone marrow-derived mesenchymal stem cells (BMMSCs) and PRP on bone regeneration of calvarial defects in rabbits. Hydroxyapatite (HA) was used as a scaffold for bone regeneration. There were three groups in this experiment: 1) HA loaded with BMMSCs (HS group), 2) HA loaded with PRP (HP group), and 3) HA loaded with BMMSCs and PRP (HSP group). Bone tissue regeneration techniques include tissue engineering approaches which employ mesenchymal stem cells as an osteogenic agent for bone repair. Recent studies have demonstrated that tissue engineering scaffolds and growth factors can support cell proliferation, bone formation, and bone tissue repair in lost bone tissue. Furthermore,many studies have suggested that platelet-rich plasma (PRP) can improve bone regeneration due to the numerous growth factors that it contains. This study was performed to investigate the influence of bone marrow-derived mesenchymal stem cells (BMMSCs) and PRP on bone regeneration of calvarial defects in rabbits. Hydroxyapatite (HA) was used as a scaffold for bone regeneration. There were three groups in this experiment: 1) HA loaded with BMMSCs (HS group), 2) HA loaded with PRP (HP group), and 3) HA loaded with BMMSCs and PRP (HSP group). Two circular bony defects (6 mm in diameter) were made in rabbit calvaria using a trephine bur. BMMSCs and PRP with a HA scaffold (diameter 5.5 mm, height 3 mm) were applied to each defect. The animals were sacrificed after 2 weeks, 4 weeks and 8 weeks. The level of their ability of osteogenesis was evaluated through histological and histomorphometric analyses. High-quality bone regeneration was observed in the HSP group. The percentage of new bone area around the scaffolds was higher in the HSP group than it was in the other groups (HS and HP group), especially at 8 weeks (HS, 72.5±15 %; HP, 85.8±14 %; HSP, 98.8±2.5%). In addition, the level of bone maturation was higher in the HSP group than in the other groups. The results of this study show that PRP has a positive effect on bone generation. HA with a combination of BMMSCs and PRP can enhance bone regeneration. In addition, the growth factor capacity of PRP may affect the differentiation of BMMSCs and promote bone formation.

Allogeneic clonal mesenchymal stem cell therapy for refractory graft-versus-host disease to standard treatment: a phase I study

이현규,양승아,김인호,이제환,민창기,김준형,김철수,송순욱 대한약리학회 2016 The Korean Journal of Physiology & Pharmacology Vol.20 No.1

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinicalgrade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1×106 cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.