제대혈 내피기원세포 및 간엽줄기세포의 분화에 대한 연구

김은석(Eun-Seok Kim),김현옥(Hyun-Ok Kim) 대한구강악안면외과학회 2005 대한구강악안면외과학회지 Vol.31 No.1

Stem cell therapy using mesenchymal stem cells(MSCs) transplantation have been paid attention because of their powerful proliferation and pluripotent differentiating ability. Although umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, the presence of mesenchymal stem cells (MSCs) in UCB has been controversial and it remains to be validated. In this study, we examine the presence of MSCs in UCB harvests and the prevalence of them is compared to that of endothelial progenitor cells. For this, CD34+ and CD34- cells were isolated and cultured under the endothelial cell growth medium and mesenchymal stem cell growth medium respectively. The present study showed that ESC-like cells could be isolated and expanded from preterm UCBs but were not acquired efficiently from full-terms. They expressed CD14-, CD34-, CD45-, CD29+, CD44+, CD105+ cell surface marker and could differentiate into adipogenic and osteogenic lineages. Our results suggest that MSCs are fewer in full-term UCB compared to endothelial progenitor cells.

Blood Vessel Regeneration using Human Umbilical Cord-derived Endothelial Progenitor Cells in Cyclophosphamide-treated Immune-deficient Mice

조재진 KOREAN ACADAMY OF ORAL BIOLOGY 2011 International Journal of Oral Biology Vol.36 No.3

Endothelial cells are a vital constituent of most mammalian organs and are required to maintain the integrity of these tissues. These cells also play a major role in angiogenesis, inflammatory reactions, and in the regulation of thrombosis. Angiogenesis facilitates pulp formation and produces the vessels which are essential for the maintenance of tooth homeostasis. These vessels can also be used in bone and tissue regeneration, and in surgical procedures to place implants or to remove cancerous tissue. Furthermore, endothelial cell regeneration is the most critical component of the tooth generation process. The aim of the present study was to stimulate endothelial regeneration at a site of acute cyclophosphamide (CP)-induced endothelial injury by treatment with human umbilical cord-derived endothelial/mesenchymal stem cells (hEPCs). We randomly assigned 16 to 20-week-old female NOD/SCID mice into three separate groups, a hEPC (1 × 10 5 cells) transplanted, 300mg/kg CP treated and saline (control) group. The mice were sacrificed on days 5 and 10 and blood was collected via the abdominal aorta for analysis. The alanine transaminase (ALT), aspartate aminotransferase (AST), serum alkaline phosphatase (s-ALP), and albumin (ALB) levels were then evaluated. Tissue sections from the livers and kidneys were stained with hematoxylin and eosin (HE) for microscopic analysis and were subjected to immunohistochemistry to evaluate any changes in the endothelial layer. CP treatment caused a weight reduction after one day. The kidney/body weight ratio increased in the hEPC treated animals compared with the CP only group at 10 days. Moreover, hEPC treatment resulted in reduced s-ALP, AST, ALT levels compared with the CP only group at 10 days. The CP only animals further showed endothelial injuries at five days which were recovered by hEPC treatment at 10 days. The number of CD31-positive cells was increased by hEPC treatment at both 5 and 10 days. In conclusion, the CP-induced disruption of endothelial cells is recovered by hEPC treatment, indicating that hEPC transplantation has potential benefits in the treatment of endothelial damage.

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy

Monsanto, Megan M.,White, Kevin S.,Kim, Taeyong,Wang, Bingyan J.,Fisher, Kristina,Ilves, Kelli,Khalafalla, Farid G.,Casillas, Alexandria,Broughton, Kathleen,Mohsin, Sadia,Dembitsky, Walter P.,Sussman, Grune & Stratton 2017 Circulation research Vol.121 No.2

<P><B><U>Rationale:</U></B></P><P>The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration.</P><P><B><U>Objective:</U></B></P><P>Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy.</P><P><B><U>Methods and Results:</U></B></P><P>Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm<SUP>3</SUP> pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit<SUP>+</SUP> cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit<SUP>−</SUP> mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit<SUP>+</SUP> population is further enriched by selection for a CD133<SUP>+</SUP> endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry.</P><P><B><U>Conclusions:</U></B></P><P>Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.</P>

골막기원세포의 조골세포 분화과정에서 나타나는 혈관내피전구세포의 증식

김종렬(Jong-Ryoul Kim),송정호(Jung-Ho Song),김욱규(Uk-Kyu Kim),박봉욱(Bong-Wook Park),하영술(Young-Sool Hah),김진현(Jin-Hyun Kim),김덕룡(Deok Ryong Kim),조영철(Yeong-Cheol Cho),성일용(Iel-Yong Sung),변준호(June-Ho Byun) 대한구강악안면외과학회 2009 대한구강악안면외과학회지 Vol.35 No.4

Purpose : The purpose of this study was to examine the expression of various angiogenic factors during osteoblastic differentiation of periostealderived cells and the effects of osteogenic inductive medium of periosteal-derived cells on the proliferation of endothelial progenitor cells. Materials and methods : Periosteal-derived cells were obtained from mandibular periosteums and introduced into the cell culture. After passage 3, the cells were divided into two groups and cultured for 21 days. In one group, the cells were cultured in the DMEM supplemented with osteogenic inductive agent, including 50g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate. In the other group, they were cultured in DMEM supplemented without osteogenic inductive agent. VEGF isoforms, VEGFR-1, VEGFR-2, and neuropilin-1 mRNA expression was observed. Human umbilical cord blood-derived endothelial progenitor cell proliferation was also observed. Results : The expression of VEGF isoforms was higher in osteogenic inductive medium than in non-osteogenic inductive medium. The expression of VEGFR-2 was also higher in osteogenic inductive medium than in non-osteogenic inductive medium. However, the expression of VEGFR-1 and neuropilin-1 was similar in both osteogenic inductive medium and non-osteogenic inductive medium. In addition, conditioned medium from differentiated periosteal-derived cells stimulated human umbilical cord blood-derived endothelial progenitor cell numbers compared to conditioned medium from non-differentiated periosteal-derived cells. Conclusion : These results suggest that in vitro osteoblastic differentiation of periosteal-derived cells has angiogenic capacity to support endothelial progenitor cell numbers.

지방조직내 혈관내피세포와 전구세포의 효율적인 분리와 분화

김정남,이중호,오득영,유결,전영준,문석호,서제원,안상태,김상헌,이종원 한국조직공학과 재생의학회 2011 조직공학과 재생의학 Vol.8 No.1s

Recently, it has been documented that human adipose tissue (AT) has mesenchymal stem cells which can be differentiated into multiple cell lineage including endothelia cells, and there are many attempts to differentiate these cells into endothelial cells for the experimental and clinical study about angiogenesis. However, it seems to be that endothelial cells and their precursor cells in AT could be discarded in the process of extracting adipose derived stromal cells (ADSCs). In this study, we evaluated that endothelial differentiation of discardable cells. After the isolation of ADSCs, 6 hour and 24 hour non adherent floating cells were collected and characterized by the flow cytometry analysis using CD29, CD31, CD34 and CD90 cell surface markers. Also, we cultured these cells in the endothelial medium and RT-PCR for endothelial-specific mRNA was performed. Lastly, CD31 staining and Dillabeled ac-LDL uptake study were performed to confirm endothelial differentiation. In vitro experiment indicated that ADSCs showed a spindle like shape, but 6 hour and 24 hour non adherent floating cells showed a cobblestone like shape. When 6 hour and 24 hour non adherent floating cells were incubated for up to 1 weeks in endothelial medium, they detected to express a variety of endothelial-specific mRNA. Differentiated cells were also found to be able to uptake ac-LDL and stain CD 31. These results suggest that a subset of 6 hour and 24 hour non adherent floating cells derived from AT can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages,which could be applied to in vivo vasculogenesis.

Tissue Regeneration with Human Umbilical Cord Blood Functional Cells

Eun-Seok Kim,Hyun-Ok Kim,Hong-Ryun Kim 대한구강악안면병리학회 2007 대한구강악안면병리학회지 Vol.31 No.5

Recovery 01' original form ancl function from c1amaged organs 01' tissues is olltmost goal of regenera tive meclicine. Va riolls methods such as moleclll ar biology. drug c1elivery system, biomaterials. tissue engineering have been s tllcliecl and appl iecl in that field . 1'he core factor of all 01' these kinds of efforts might be the cells including stem cells 01' progenitor cell s . AclllJt progenitor 01' stem cells have many advantages for therapeutic meclicine, inclucling free form ethi cal probl em. easiness in collection and clllture. Bone marrow‘ fat tissue, peripheral blood. placenta‘ ancl umbilicaJ cord bloocl a re preferable source 01' acllllt stem cells 01' progenitors. ]-]uman umbilical cord bloocl. taken form vein of corcl after baby c1elivery‘ are known to contain many progenitor cells. Since Boyse et al reportecl bone marrow transplantation with hllman cord blood CD34+ cells f'or leukemia. functional cells in human cord blood have been the cells of great interest. 1n this study‘ the a u thors i s이 ated peripheral bloocl mononuciear celJs. endothelial progenitors‘ late outgrowth vascular endothelial-like cells‘ ancl mesenchymal stem cell- like cells from human umbilical cord blood and a pplied in bony defects, myocardiac infarction and limb ischemic lesl0n Al I of these fllnctional cells showed favorable healing capacity and their effects primarily based on enhanced anglOgenesls Conclllsively. a lthollgb the precise cha racteristics are not well-described‘ the current stucly reveals that various types of functional cell s of human umbilical cord blood have some stem cell or progenitors features and play an important r ole in ti ssue regeneration

Role of Stromal Cell-Derived Factor-1 in Endothelial Progenitor Cell-Mediated Vascular Repair and Regeneration

Li Ji-Hua,Li Yang,Huang Dan,Yao Min 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.5

Endothelial progenitor cells (EPCs) are immature endothelial cells that participate in vascular repair and postnatal neovascularization and provide a novel and promising therapy for the treatment of vascular disease. Studies in different animal models have shown that EPC mobilization through pharmacological agents and autologous EPC transplantation contribute to restoring blood supply and tissue regeneration after ischemic injury. However, these effects of the progenitor cells in clinical studies exhibit mixed results. The therapeutic efficacy of EPCs is closely associated with the number of the progenitor cells recruited into ischemic regions and their functional abilities and survival in injury tissues. In this review, we discussed the regulating role of stromal cell-derived factor-1 (also known CXCL12, SDF-1) in EPC mobilization, recruitment, homing, vascular repair and neovascularization, and analyzed the underlying machemisms of these functions. Application of SDF-1 to improve the regenerative function of EPCs following vascular injury was also discussed. SDF-1 plays a crucial role in mobilizing EPC from bone marrow into peripheral circulation, recruiting the progenitor cells to target tissue and protecting against cell death under pathological conditions; thus improve EPC regenerative capacity. SDF-1 are crucial for regulating EPC regenerative function, and provide a potential target for improve therapeutic efficacy of the progenitor cells in treatment of vascular disease.

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function

Jung, Seok Yun,Choi, Jin Hwa,Kwon, Sang‐,Mo,Masuda, Haruchika,Asahara, Takayuki,Lee, You‐,Mie Wiley Subscription Services, Inc., A Wiley Company 2012 Journal of cellular biochemistry Vol.113 No.5

<P><B>Abstract</B></P><P>Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb <I>Angelica gigas</I> is known to possess potent anti‐inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood‐derived AC133+ cells that produce functional EPC progenies. Decursin dose‐dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle‐shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin‐2, angiopoietin receptor Tie‐2, Flk‐1 (vascular endothelial growth factor receptor‐2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose‐dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor‐induced mobilization of circulating EPCs (CD34 + /VEGFR‐2+ cells) from bone marrow and early incorporation of Dil‐Ac‐LDL‐labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild‐type‐ or bone‐marrow‐transplanted mice. Accordingly, decursin attenuated EPC‐derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. J. Cell. Biochem. 113: 1478–1487, 2012. © 2012 Wiley Periodicals, Inc.</P>

CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

Lee, Jun Hee,Lee, Sang Hun,Yoo, So Young,Asahara, Takayuki,Kwon, Sang Mo American Heart Association, Inc. 2013 Arteriosclerosis, thrombosis, and vascular biology Vol.33 No.7

<P><B>Objective—</B></P><P>Although endothelial progenitor cells (EPCs) have been reported to promote neovessel formation during vascular injury, the function of supporting cells of EPCs and their interaction with EPCs during EPC isolation remain unclear.</P><P><B>Approach and Results—</B></P><P>We investigated the functional properties of 2 types of EPCs, also known as endothelial colony-forming cells (ECFCs), CD34<SUP>−</SUP>/CD34<SUP>+</SUP> cell–derived ECFCs (hybrid-dECFCs) and CD34<SUP>+</SUP> cell–derived ECFCs (stem-dECFCs), isolated using different methods, to elucidate the role of CD34<SUP>−</SUP> cell populations as cell-supporting niches. Using EPC colony-forming and insert coculture assays, we found that CD34<SUP>−</SUP> accessory cells dynamically modulate hematopoietic stem cell–derived endothelial cell progenitor commitment via angiogenic cytokines secreted by CD34<SUP>−</SUP>/CD11b<SUP>+</SUP> macrophages. On the basis of these findings, we isolated 2 types of ECFCs and investigated their bioactivities. We found that stem-dECFCs showed remarkably retarded cell growth, enhanced senescence, and decreased characteristics of ECFCs, whereas hybrid-dECFCs showed greater proliferative properties but delayed senescence. In a murine hind-limb ischemia model, hybrid-dECFCs showed significantly enhanced blood perfusion, capillary density, transplanted cell survival and proliferation, and angiogenic cytokine secretion compared with stem-dECFCs. In particular, the migratory capacity of hybrid-dECFCs was significantly enhanced, in part mediated via an augmented phosphorylation cascade of focal adhesion kinase and Src, resulting in a highly increased incorporation capacity of hybrid-dECFCs compared with stem-dECFCs. CD34<SUP>−</SUP> accessory cells of hybrid-dECFCs might be niche-supporting cells that facilitate cell survival, increase the secretion of angiogenic cytokines, and increase incorporation.</P><P><B>Conclusions—</B></P><P>This study provided important insight into blood vessel formation and repair in ischemic diseases for ECFC-based cell therapy.</P>

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Vahid Siavashia,Reyhaneh Sariri,Seyed Mahdi Nassiri,Masoumeh Esmaeilivand,Simin Asadian,Hadi Cheraghi,Mazyar Barekati-Mowahed,Reza Rahbarghazi 한국통합생물학회 2016 Animal cells and systems Vol.20 No.3

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angiomodulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50 ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.