조혈모세포 기증 향상을 위한 활성화 방안에 관한 연구

박충민(Chung Min Park),안소희(So Hee An) 국가생명윤리정책원 2017 생명, 윤리와 정책 Vol.1 No.1

국민들의 생활수준이 향상되고 다양한 환경의 변화로 인해 질병양태가 과거의 전염성 질환 위주에서 비감염성 질환으로 전환되는 추세다. 특히 백혈병, 재생불량성 빈혈 등 혈액질환과 특정 대사질환 등의 조혈모세포이식을 요구하는 환자는 매년 약 2,800명씩 발생하고 있으나, 환자들은 조혈모세포 공여자를 어렵게 찾았음에도 불구하고 기증희망자의 거부로 받지 못하는 경우와 제때에 찾지 못해 생명을 잃는 등 진료에 어려움을 겪고 있다. 이에 본 연구자는 조혈모세포 관련 단체(모집기관, 의료기관, 이식조정기관, 정부기관) 전문가들에게 설문을 실시하여 조혈모세포 기증의 활성화에 대한 장애요인이 무엇인지를 살펴보고 이를 토대로 조혈모세포 기증사업의 방향을 제시하고자 한다. 또한 조혈모세포 기증희망자들의 기증희망신청부터 현 상태를 파악하여 기증동의까지에 미치는 영향이 무엇이고 기증희망신청 시 강조해야 할 부분이 무엇인지를 살펴보고자 한다. 마지막으로 조혈모세포 기증희망자와 실 기증자들이 조혈모세포 기증 시 영향을 주는 사항들을 파악하고 이를 분석하는 등 동의율 향상의 문제점과 해결방법을 제시하여 정부에서 이를 반영함으로써 조혈모세포 기증동의율을 향상시킬 뿐만 아니라 국고보조금 낭비를 막고 조혈모세포 이식을 기다리는 혈액암 등의 환자에게 새로운 생명의 기쁨을 주는 효과를 기대할 수 있다. 연구방법으로는 조혈모세포 기증희망자와 실 기증자들의 등록 시부터 현재 상태를 파악 및 추후관리 받은 사항과 기증 시 해소되어야 할 사항 등을 설문하고, 조혈모세포 활성화를 위한 전문가(모집기관, 의료기관, 이식조정기관, 정부기관)의 의견을 AHP분석방법으로 분석하였다. 이에 나타난 결론은 다음과 같다. 첫째, 조혈모세포 기증관련 기존 전문가들의 공통된 의견들처럼 조혈모세포는 뇌사자나 생체로부터 신체의 일부를 적출하는 고형장기와는 달리, 헌혈과 같은 혈액기증의 개념으로 관리 하는 분야로, 현재의 「장기 등 이식에 관한 법률」에서 독립되는 것이 바람직 할 것이다. 둘째, 조혈모세포기증의 구체적인 방법에 대한 대중매체(TV)를 활용한 지속적인 대국민 홍보가 필요하다. 셋째, 현재 기증희망자 등록가능나이는 만18세 이상 40세 미만으로 되어있어 40세 이상자 중 기증을 원하는 사람들이 있으나 등록을 못하고 있는 실정이다. 따라서 기증동의율을 위해 40세 이상 50세 미만 기증희망자 중 신체건강하고 본인이 희망할 경우 등록기관 방문자에 한해 신청을 할 수 있도록 하는 방안을 제시한다. 넷째, 기증 시 부모 및 배우자의 동의를 구하고 기증이 진행됨에 따라 모집기관에서는 한해 배정된 목표만을 위해 무작위 모집이 아닌 조혈모세포기증에 대한 정확한 교육 및 최종 등록 전 까지 부모 및 배우자 등의 의사 확인을 해야 할 것이다. 다섯째, 인도주의정신으로 사랑과 봉사를 몸소 실천하여 이웃의 생명을 위해 자기의 조혈모세포를 기꺼이 기증한 공여자들에 대한 배려는 마련되어져야 하며 또한 이들이 자긍심을 가질 수 있도록 하고 이들을 통하여 더 많은 공여자들이 나올 수 있도록 국가가 제도적으로나 예산을 확보하는 것도 중요하다. 여섯째, 조혈모세포 기증 동의율 향상을 위한 활성화는 개인이나 한 단체에서만 노력한다고 되는 것이 아니라 정부기관을 포함한 조혈모세포관련 모든 단체들이 힘을 모아 각자의 역할을 충실히 해줄 때 이룰 수 있다. 이를 위해 전문가들과 실무자들이 한자리에 모여 의견을 나눌 수 있는 심포지엄 또는 토론회를 가져야 할 것이며, 여기서 나오는 결과에 따라 추진되어야만 조혈모세포기증사업이 발전할 수 있을 것이다. Background : This study investigated obstacles to hematopoietic stem cells donation through surveys to hematopoietic stem cells-related professional organizations(recruitment agencies, hospitals, transplant coordinating agency, government agency), donation applicants, and donors. Through the analysis the direction of donation business is presented. Subject and method : Questionnaires were e-mailed to donation applicants and donors who registered through Korea Marrow Donor Program(KMDP) from 2012 to 2014. 380 donation applicants(rate of reply 63.3 %) and 285 donors(rate of reply 47.5%) answered the questionnaires. In addition, 34 hands-on workers(7 from recruitment agencies, 8 from hospitals, 10 from transplant coordinating agencies, 9 from government agencies) attended the questionnaires Conclusion : The following conclusion was obtained through the analysis. First, as a result of survey to the hematopoietic stem cells-related professionals, role of the professional organizations(recruitment agencies, hospitals, transplant coordinating agency, government agency) is the most crucial. Volunteer spirit is the most important and people who have acquaintance with relevant patients and experience with blood donation tend to apply for the donation. Recruitment agency should inform donation applicants clear information at each step of the donation process. Transplant coordinating agency and hospitals should improve service quality. The priority order of role of government agency is to create a unified legal basis, then public relations about bone marrow. The priority order among detailed elements is to create a unified legal basis, public relations about hematopoietic stem cells, to inform donation applicants clear information, to improve service quality, and volunteer spirit. Second, as a result of the most concerned issue of donation applicants is the stability of the operation and if it is resolved they are willing to donate. Their motivation is altruism and religious beliefs. Public relations about hematopoietic stem cells is the most important to increase the actual rate of donation. Continuous and precise information should be provided to keep donation applicants" attention. Third, as a result of survey to the donors, majority of them have experience with blood donation. Also, they have already identified the importance of hematopoietic stem cells donation through the media and register voluntarily. To contact information on the donation after registering helps to make decisions and the most influential one among them was the story of patients who need hematopoietic stem cells transplant. Suggestion : The following suggestions are made through the conclusion. First, a unified legal basis should be created in bone marrow donation. Second, continuous public relations about hematopoietic stem cells through media(TV) us needed to improve the perception of the public. Third, Thus, the way should be sought to increase the age for donation applicants. Fourth, clear information about the hematopoietic stem cells donation should be provided and the opinion of parents and the spouse should be checked before the final registration process. Fifth, benefits for hematopoietic stem cells donors should be provided because relying just on the altruism of donors has limitations in activating the donation. Sixth, all hematopoietic stem cells-related professional organizations should do their own roles to increase the consent rate of the hematopoietic stem cells donation.

Profiling of Differentially Expressed Genes in Human Stem Cells by cDNA Microarray

김철근,이종주,정대영,전진선,허현석,강호철,신준호,조윤신,차경준,김찬길,도병록,김경숙,김현수 한국분자세포생물학회 2006 Molecules and cells Vol.21 No.3

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation, although a wide variety of adult stem cells as well as embryonic stem cells have been identified and stem cell plasticity has recently been reported. To identify genes implicated in the control of the stem cell state as well as the characteristics of each stem cell line, we analyzed the expression profiles of genes in human embryonic, hematopoietic (CD34+ and CD133+), and mesenchymal stem cells using cDNA microarrays, and identified genes that were differentially expressed in specific stem cell populations. In particular we were able to identify potential hESC signature-like genes that encode transcription factors (TFAP2C and MYCN), an RNA binding protein (IMP-3), and a functionally uncharacterized protein (MAGEA4). The overlapping sets of 22 up-regulated and 141 downregulated genes identified in this study of three human stem cell types may also provide insight into the developmental mechanisms common to all human stem cells. Furthermore, our comprehensive analyses of gene expression profiles in various adult stem cells may help to identify the genetic pathways involved in self-renewal as well as in multi-lineage specific differentiation.

Bong-Han Corpuscles as Possible Stem Cell Niches on the Organ-Surfaces

김민수,홍주영,홍수,이병천,남창훈,우희종,강대인,소광섭,Kim, Min-Su,Hong, Ju-Young,Hong, Su,Lee, Byung-Cheon,Nam, Chang-Hoon,Woo, Hee-Jong,Kang, Dae-In,Soh, Kwang-Sup KOREAN PHARMACOPUNCTURE INSTITUTE 2008 Journal of pharmacopuncture Vol.11 No.1

목적 : 봉한소체가 성체줄기세포의 원천이며, 봉한관이 줄기세포 수송로일 가능성을 확인함. 방법 : 쥐의 내부 장기표면에서 봉한소체와 봉한관을 채취했다. 다양한 줄기세포 표지항체를 써서 면역조직학적 분석을 했다. 결과 : mesenchymal 줄기세포에 관한 Integrin ${\beta}1$, Collagen type 1, Fibronectin의 강한 발현을 확인했다. CD54는 발현되지 않았다. 조혈줄기세포에 관련하여 Thy 1의 발현이 있었다. 결론 : 골수조직과 유사하게 mesenchymal과 조혈줄기세포의 표지가 BHC에서 확인되었고, 봉한관에서는 vWF가 발현되어 줄기세포 수송로 가능성을 확인했다. Objectives : Showing that Bong-Han corpuscles(BHC) are suppliers of the stem cells in adulthood, and the Bong-Han ducts(BHD) are transportation routes of stem cells. Methods : BHC and BHD were obtained from the internal organ-surfaces of rats. The sliced BHC and BHD were immunostained with various stem cell markers. Extracellular matrices were also analyzed by immunohistochemistry. Result : The presence of mesenchymal stem cells was confirmed by the expression of Integrin beta 1, Collagen type 1 and Fibronectin. But CD54 was not expressed. The hematopoietic stem cell marker, Thy 1 was strongly expressed. BHDs showed Collagen type 1, Fibronectin, and vWF expression. Conclusion : Both hematopoietic and mesenchymal stem cell markers were expressed strongly in BHC similarly as in bone marrow. An endothelial cell marker(vWF) demonstrated the possibility of the stem cell transportation routes of BHD.

Current approaches in biomaterial-based hematopoietic stem cell niches

Bello, Alvin Bacero,Park, Hansoo,Lee, Soo-Hong Elsevier Science B.V. Amsterdam 2018 ACTA BIOMATERIALIA Vol.72 No.-

<P><B>Abstract</B></P> <P>Hematopoietic stem cells (HSCs) are multipotent progenitor cells that can differentiate and replenish blood and immune cells. While there is a growing demand for autologous and allogeneic HSC transplantation owing to the increasing incidence of hereditary and hematologic diseases, the low population of HSCs in cord-blood and bone marrow (the main source of HSCs) hinders their medical applicability. Several cytokine and growth factor-based methods have been developed to expand the HSCs <I>in vitro</I>; however, the expansion rate is low, or the expanded cells fail to survive upon engraftment. This is at least in part because the overly simplistic polystyrene culture substrates fail to fully replicate the microenvironments or niches where these stem cells live. Bone marrow niches are multi-dimensional, complex systems that involve both biochemical (cells, growth factors, and cytokines) and physiochemical (stiffness, O<SUB>2</SUB> concentration, and extracellular matrix presentation) factors that regulate the quiescence, proliferation, activation, and differentiation of the HSCs. Although several studies have been conducted on <I>in vitro</I> HSC expansion via 2D and 3D biomaterial-based platforms, additional work is required to engineer an effective biomaterial platform that mimics bone marrow niches. In this study, the factors that regulate the HSC <I>in vivo</I> were explained and their applications in the engineering of a bone marrow biomaterial-based platform were discussed. In addition, current approaches, challenges, and the future direction of a biomaterial-based culture and expansion of the HSC were examined.</P> <P><B>Statement of Significance</B></P> <P>Hematopoietic stem cells (HSC) are multipotent cells that can differentiate and replace the blood and immune cells of the body. However, <I>in vivo</I>, there is a low population of these cells, and thus their use in biotherapeutic and medical applications is limited (i.e., bone marrow transplantation). In this review, the biochemical factors (growth factors, cytokines, co-existing cells, ECM, gas concentrations, and differential gene expression) that may regulate the over-all fate of HSC, <I>in vivo,</I> were summarized and discussed. Moreover, different conventional and recent biomaterial platforms were reviewed, and their potential in generating a biomaterial-based, BM niche-mimicking platform for the efficient growth and expansion of clinically relevant HSCs <I>in-vitro</I>, was discussed.</P> <P><B>Graphical abstract</B></P> <P>On making an <I>in vitro</I> bone marrow (BM): The essential components. To make a bone marrow mimicking microenvironment for the growth and expansion of hematopoietic stem cells, several factors need to be taken into consideration. These factors include the BM’s in-vivo biochemical properties (growth factors hormones, co-existing cells, ECM, gas concentrations, regulated gene expression) and the biomaterial properties (type, dimension, topography, and mechanical strength).</P> <P>[DISPLAY OMISSION]</P>

Cytokine-free Expansion of Hematopoietic Stem Cells with Mixed Mesenchymal Stem Cells Isolated from Bone Marrow and Periosteum

( Yong Soo Choi ),( Sang Min Lim ),( Chang Woo Lee ),( Chul Soo Kim ),( Moon Whan Im ),( Moon Hee Lee ),( Dong Il Kim ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4

Most hematopoietic stem cells are located in bone marrow(BM) architecture in close proximity to the endosteal surface of bone. We investigated the synergistic effects of mesenchymal stem cells(MSCs) from BM and periosteum-derived progenitor cells(PDPCs) as mixed feeder cells on the ex vivo expansion of CD34+ cells from umbilical cord blood. Enriched CD34+ cells were cultured in a cytokine- and serum-free medium for 2 weeks with the respective mitomycin C-treated each or mixed feeder layers. Total cell expansion, immunophenotypes, and clonogenic ability were analyzed. In mixed feeder cultures, total nucleated cells were expanded by 26.4-fold at day 10. Furthermore, fold increase of CD34+/CD38-cells was found to be 67-fold. These results indicate that the PDPCs can be used as efficient feeder cells for the ex vivo expansion of CD34+ cells.

Expression and Characterization of Purinergic Receptor, P2Y10 in Hematopoietic Stem Cells

Lee Eun-Jong,Kim Dong-Ku 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & Developmental Biology(Supplement) Vol.29 No.2s

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughoutthe lifetime. In this study, we analyzed the expression of receptors of P2Y10, purinergic receptor families in murine hematopoietic stem cells, hematopoietic progenitor cells. In addition, the biological activity of P2Y10 was investigated with B lymphocyte cell line, Ba/F3 in effect to cell growth and cell cycle. From the analysis of expression in hematopoieticstem cell. and progenitor with RT-PCR, P2Y10 was strongly expressed in murine hematopoieticstem cells (c-kit+ Sca-l+ Lin-) and progenitor cell population, such as c-kit- Sca-l+ Lin-, c-kit+ Sca-l- Lin- and c-kit- Sca-l- Lin-. To investigate the biological effects by P2Y10, retroviral vector from subcloned murine P2Y10 cDNA was used fur gene introduction into Ba/F3 cells, and stable transfectant cells were obtained by flow cytometry sorting. In cell proliferation assay, the proliferation ability of P2Y10 receptor gene­transfected cells was strongly inhibited, and the cell cycle was arrested at G1 phase. These result suggest that the P2Y10 may be involved the biological activity in hematopoietic stem cells and immature B lymphocytes.

동종조혈모세포 및 배양확장된 간엽모세포 동시이식의 가능성

강석윤,김재홍,박준성,김현수,최진혁,임호영,박상진,양말숙,김영진,김효철 대한조혈모세포이식학회 2001 대한조혈모세포이식학회지 Vol.6 No.2

배경: 사람의 골수내에 두가지 종류의 모세포가 밝혀져 있고 혈액질환에서 간엽모세포 (mesenchymal stem cell)의 역할에 대한 연구가 활발해지고 있다. 이런 간엽모세포들은 조혈기능에 관여하는 싸이토카인을 분비하고, 이식시 조혈모세포들의 생착 및 기능적 분화에 많은 영향을 미치는 것으로 알려져 있으며, 세포의 형태적 변화나 기능의 소실없이 실험실에서 분리하고 배양확장 시킬 수 있는 기술들의 발달로 최근에는 이를 치료에 이용하는 연구들이 확대되고 있다. 현재까지 많은 연구에 있어 조혈모세포의 경우는 이식 후 공여자의 형태로 전환되지만 간엽모세포의 경우에 있어서는 지속적으로 환자의 형태로 남아있음을 보고하고 있다. 본 저자들은 다양한 혈액질환의 치료에 있어 현재까지 사용되고 있는 일반적인 동종조혈모세포 이식술 뿐만 아니라 조혈모세포와 간엽모세포를 동시에 이식하여 이식 후 생착에 도움을 주고 키메리즘에 변화가 있는지를 알아보고자 12명의 환자를 대상으로 연구를 진행하였다. 방법: 12명의 환자에서 9명은 혈연공여자로 부터 동종 조혈모세포이식술을 시행받았고 3명에 있어서는 조혈모세포와 간엽모세포를 동시에 이식하였다. 이식 전 환자 및 공여자의 말초혈액 또는 골수세포로 부터 얻어진 백혈구를 이용하여 키메리즘을 확인하였고 이식 후 3주부터 10개월이 지난 후 공여자의 형태로 전환되었는지를 확인하기 위해 환자의 골수를 채취하여 간엽모세포를 배양하였고 성일치 이식시에는 DNA microsatellite 다형성검사를 성불일치 이식시에는 Y-염샘체를 이용하여 키메리즘의 변화를 확인하였다. 결과: 동종 조혈모세포이식을 받은 환자 9명에 있어서는 이식 후 9일부터 19일까지에 걸쳐 생착 (평균 13.11일)이 되었고 이식 후 3주째부터 10개월째에 걸쳐 시행한 키메리즘 검사상 모든 환자에서 골수는 공여자의 형태로 전환됨이 확인되었으나 배양된 간엽모세포는 지속적으로 환자의 형태로 남아있었다. 동종 조혈모세포와 배양확장된 간엽모세포의 동시이식이 시행된 3명의 환자에서는 평균 10일째 생착이 되었고 이식 후 3주와 4주째 시행한 키메리즘검사에서 골수내 조혈모세포는 모두 공여자의 형태로 바뀌었지만 간엽모세포는 지속적으로 환자의 형태로 확인되었다. 결론: 현재의 이식방법으로는 동종 간엽모세포의 이식이 어렵고 동시이식의 효과도 불명확하므로 향후 기질세포의 생착을 위한 주입양과 주입방법, 환자자신의 골수내 환경을 마련하기 위한 전처치의 방법 등에 대한 연구가 지속되어야 할 것이며 또한 이를 조기에 발견할 수 있는 실험적도구의 개발에 노력해야 할 것으로 사료된다. Background: Human bone marrow contains two kinds of stem cells, hematopoietic stem cells (HSCs) which can differentiate into mature blood cells and multipotential mesenchymal stem cells (MSCs) which are capable of differentiating into a number of mesenchymal cell lineages. We developed an efficient method of isolation and ex vivo expansion of a homogenous population of MSCs from human bone marrow. These MSCs are shown to secrete hematopoietic cytokines and support hematopoietic progenitors ex vivo. Therefore, enrichment of donor MSC in the graft may be useful in the treatment of many hematologic disorders. Methods: To determine if donor derived MSCs can be engrafted in the recipients with hematologic disorders by allogeneic HSC transplantation (CD34+:2.0-16.0×10^(6)/kg) or cotransplantation of both HSC (CD34+:1.98-12.53×10^(6)/kg) and enriched MSC (1.44-13.79×10^(5)/kg) of donor origin, we investigated MSCs after conventional allogeneic HSC transplantation in 9 patients and in 3 patients with cotransplantation of both enriched MSC and HSC at 1 to 10 months after transplantation. Marrow derived MSCs before and after transplantation were genotyped either by polymerase chain reaction using Y chromosome in gender-mismatched or by microsatellite DNA polymorphism in gender-matched transplantation. Results: We found that MSCs isolated from recipients after allogeneic HSC transplantation or cotransplantation remained as the recipient types in all patients, not of donor genotype, despite successful engraftment by donor type hematopoietic cell. Conclusions: These findings indicate that with current allogeneic HSC transplantation or cotransplantation procedures, the stromal compartment of hematopoiesis is not transplantable in humans. Therefore, the reasons for difficulty of engraftment with donor MSCs needs to be further elucidated.

Endogenous Stem Cells in Homeostasis and Aging

임지은,손영숙 한국조직공학과 재생의학회 2017 조직공학과 재생의학 Vol.14 No.6

In almost all human tissues and organs, adult stem cells or tissue stem cells are present in a unique location, the so-called stem cell niche or its equivalent, continuously replenishing functional differentiated cells. Those endogenous stem cells can be expanded for cell therapeutics using ex vivo cell culture or recalled for tissue repair in situ through cell trafficking and homing. In the aging process, inefficiency in the endogenous stem cell–mediated healing mechanism can emerge from a variety of impairments that accumulate in the processes of stem cell self-renewal, function, differentiation capacity, and trafficking through cell autonomous intrinsic pathways (such as epigenetic alterations) or systemic extrinsic pathways. This review examines the homeostasis of endogenous stem cells, particularly bone marrow stem cells, and their dysregulation in disease and aging and discusses possible intervention strategies. Several systemic pro-aging and rejuvenating factors, recognized in heterochronic parabiosis or premature aging progeroid animal models, are reviewed as possible anti-aging pharmaceutical targets from the perspective of a healthy environment for endogenous stem cells. A variety of epigenetic modifications and chromosome architectures are reviewed as an intrinsic cellular pathway for aging and senescence. A gradual increase in inflammatory burden during aging is also reviewed. Finally, the tissue repair and anti-aging effects of Substance-P, a peptide stimulating stem cell trafficking from the bone marrow and modifying the inflammatory response, are discussed as a future anti-aging target.

Extracellular ATP Is Involved in the Induction of Apoptosis in Murine Hematopoietic Cells

Yoon, Mi-Jung,Lee, Hyun-Joo,Lee, Yong-Soo,Kim, Jae-Hwan,Park, Jin-Ki,Chang, Won-Kyong,Shin, Ho-Chul,Kim, Dong-Ku Pharmaceutical Society of Japan 2007 Biological & pharmaceutical bulletin Vol.30 No.4

<P>Extracellular nucleotides have multiple biological actions in processes such as proliferation, differentiation, chemotaxis, and cytokine secretion through P2X receptors on the cell surface. To determine the biological activity of adenosine triphosphate (ATP) and the expression of P2 nucleotide receptors in murine bone marrow-derived hematopoietic cells and stem cells/progenitor cells, we investigated the effects of ATP in assays of cell proliferation and cell death <I>in vitro</I>. Our results demonstrated that several subtypes of P2X receptors were expressed on hematopoietic cells and that P2X<SUB>7</SUB>, in particular, was partially expressed in hematopoietic stem cells/progenitor cells. In addition, stimulation of hematopoietic cells with high concentrations of ATP caused severe inhibition of cell proliferation despite the presence of cytokine stimulation. We analyzed the apoptotic effects of stimulation with several different dosages of ATP and confirmed the enhanced apoptotic activity in hematopoietic cells and progenitor cells. Antagonists, against P2X receptors and ATP, suramin and oxidized ATP, inhibited the induction of cell death for murine hematopoietic cells. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for regulating the cell fate of hematopoietic stem cells.</P>

사람의 동종 조혈모세포이식에서 CD4+CD25+ T세포의 분포와 이식편대숙주병

이대형,정낙균,정대철,김학기,조빈 대한소아청소년과학회 2008 Clinical and Experimental Pediatrics (CEP) Vol.51 No.12

Purpose:This study aimed to determine the frequencies of CD4+CD25+ T cells in donor graft and peripheral blood CD4+CD25+ T cells in recipients after hematopoietic stem cell transplantation (HSCT) and their association with graft-versus-host disease (GVHD). Methods:Seventeen children who underwent HSCT were investigated. CD4+CD25+ T cells in samples from donor grafts and recipient peripheral blood were assessed by flow cytometry at 1 and 3 months after transplantation. Results:CD4+CD25+ T cell frequencies in the grafts showed no significant difference between patients with and without acute GVHD (0.90% vs. 1.06%, P=0.62). Absolute CD4+CD25+ T cell number in grafts were lower in patients with acute GVHD than in those without acute GVHD (6.18×105/kg vs. 25.85×105/kg, P=0.09). Patients without acute GVHD showed a significant decrease in peripheral blood CD4+CD25+ T cell percentage at 3 months compared to those at 1 month after HSCT (2.11% vs. 1.43%, P=0.028). However, in patients with acute GVHD, CD4+CD25+ T cell percentage at 3 months was not different from the corresponding percentage at 1 month after HSCT (2.47% vs. 2.30%, P=0.5). Conclusion:The effect of frequencies of CD4+CD25+ T cells in donor grafts on acute GVHD after HSCT could not be identified, and the majority of peripheral blood CD4+CD25+ T cells in patients who underwent HSCT may be activated T cells related to acute GVHD rather than regulatory T cells. Further studies with additional markers for regulatory T cells are needed to validate our results. (Korean J Pediatr 2008;51:1336-1341) Purpose:This study aimed to determine the frequencies of CD4+CD25+ T cells in donor graft and peripheral blood CD4+CD25+ T cells in recipients after hematopoietic stem cell transplantation (HSCT) and their association with graft-versus-host disease (GVHD). Methods:Seventeen children who underwent HSCT were investigated. CD4+CD25+ T cells in samples from donor grafts and recipient peripheral blood were assessed by flow cytometry at 1 and 3 months after transplantation. Results:CD4+CD25+ T cell frequencies in the grafts showed no significant difference between patients with and without acute GVHD (0.90% vs. 1.06%, P=0.62). Absolute CD4+CD25+ T cell number in grafts were lower in patients with acute GVHD than in those without acute GVHD (6.18×105/kg vs. 25.85×105/kg, P=0.09). Patients without acute GVHD showed a significant decrease in peripheral blood CD4+CD25+ T cell percentage at 3 months compared to those at 1 month after HSCT (2.11% vs. 1.43%, P=0.028). However, in patients with acute GVHD, CD4+CD25+ T cell percentage at 3 months was not different from the corresponding percentage at 1 month after HSCT (2.47% vs. 2.30%, P=0.5). Conclusion:The effect of frequencies of CD4+CD25+ T cells in donor grafts on acute GVHD after HSCT could not be identified, and the majority of peripheral blood CD4+CD25+ T cells in patients who underwent HSCT may be activated T cells related to acute GVHD rather than regulatory T cells. Further studies with additional markers for regulatory T cells are needed to validate our results. (Korean J Pediatr 2008;51:1336-1341)