말초혈액, 골수 및 제대혈에서 CD34 양성세포의 체외증폭과 표현형에 관한 연구

김신애,홍대식,김숙자,박성규,원종호,서원석,백승호,박희숙 대한조혈모세포이식학회 1997 대한조혈모세포이식학회지 Vol.2 No.1

One of the exciting areas for hematological research is the ex vivo expansion of hematopoietic stem cells. Bone marrow(BM) is most frequently used to transplant hematopoietic progenitor cells, but mobilized peripheral blood(MPB) and umbilical cord blood(UCB) provide alternative sources of progenitor cells for transplantation. The characteristics of the CD34^(+) cells from hematopoietic stem cell sources such as, peripheral blood, bone marrow, and umbilical cord blood are different each other. It is not well known what is the best condition and what source is superior ex vivo expansion. Also there are some arguments that the phenotypic changes of ex vivo expanded hematopoietic stem cells are still suitable for long-term engraftments. To study for this questions, CD34^(+) cells from MPB, BM and UCB were analyzed for in vitro colony formation, capacity of ex vivo expansion with hematopoietic growth factors, and characterized by immunophenotyping for several lineage-associated and maturation -related cell surface molecules at pre- and post-ex vivo expansion. The results were as follows: 1. The maximal increment of total nuclear cells in MPB, BM, and UCB were 42.63±10.39 folds, 64±23.52 folds, and 92.67±12.50 folds, respectively and the colony forming unit-colonies in MPB, BM, and UCB were increased maximally 17.60±1.90 folds, 18.00±1.41 folds, and 21.77±21.01 folds, respectively after 7 days liquid culture of CD34+ cells, with hematopoietic growth factors. 2. The absolute increment of CD34+ cells in MPB, BM, and UCB were 4.20±0.28 folds, 3.62±1.29 folds, and 14.35±3.32 folds, respectively in ex vivo expansion of CD34+ cells with combination of SCF, G-CSF IL-1, IL-3, and IL-6. 3. Myeloid progenitors in the CD34+ population studied by examining expression of CD33, CD45RA and CD13, CD34+ cells, either from MPB and BM, contained a large proportion of cells(≥25%) with myeloid cell- associated molecules(CD13) before liquid culture. The proportion of CD34+ cells in MPB expressing the B cell-associated molecules(CD19) was comparable to that found in BM and UCB(≤ 5%). Further, we observed a higher proportion of CD34+ cells expressing the CD71(transferrin receptor) is MPB(19.05±12.16%) compared with both BM(3.86±2.45%) and UCB(3.18±3.90%). 4. After 7 days liquid culture with hematopoietic growth factors(SCF+IL-1+IL-3+IL-6+G-CSF), we observed a higher proportion of CD34+ cells expressing the CD13, CD33, CD71 in MPB, BM and UCB. The data present here suggests that MPB, BM, and UCB can be the sources for ex vivo expansion of hematopoietic stem cells and circulating CD34^(+) cells, either from BM or UCB, display largely the same phenotypic profile, being different from resident CD34^(+) cells in MPB.

냉동 제대혈 세포의 체외 증폭

김삼용,김철희,배광봉,김현수,박상준,김종숙,윤환중,조덕연 大韓血液學會 1997 大韓血液學會誌 Vol.32 No.3

냉동 제대혈 세포의 체외 증폭

김삼용,김철희,배광봉,김현수,박상준,김종숙,윤환중,조덕연 충남대학교 암연구소 1998 癌共同硏究所 硏究誌 Vol.2 No.1

Background : Cord blood(CB), which has no HLA restriction, is an alternative to bone marrow for hematopoietic stem cell transplantation. The use of cord blood, however, is limited by the number of progenitor/stem cells necessary to reconstitute the older child or adult. Therefore, ex vivo expansion of CB could have tremendous impact on diverse clinical settings. We studied the ex vivo expansion of isolated population of CD34_(+) cells from cryopreserved CB cells. Methods : CD34 cells were isolated from cryopreserved CB mononuclear cells. Purified cells were cultured with various combinations of hematopoietic growth factors including erythropoietin(EPO), stem cell factor(SCF), granulocyte-colony-stimulating factor(G-CSF), gra-nulocyte, macrophage-colony-stimulating factor(GM-CSF), interleukin-1β(IL-1β), 1L-3, and IL-6. After 7, 10 or 14 days of culture, the fold increases of colony-forming unit- granu-locyte, macrophage(CFU-GM), burst-forming unit-erythroid(BFU-E), colony-forming unit-mix (CFU-Mix), and high proliferative potential colony-forming cell(HPP-CFC) were evaluated. Results : Ten-day culture with the combination of EPO, SCF, G-CSF, IL-1β, and IL-3 resulted in a median of 60-fold increase of CFU-GM, which was greater than those with the combinations of less than 5 growth factors. The addition of IL-6 or GM-CSF to this combination did not enhance CFU-GM expansion. Ten-day culture was significantly superior to 7-day culture for CFU-GM expansion. Prolongation of culture to 14 days, however, revealed decreased expansion of CFU-GM compared to 10 days. BFU-E and CFU-Mix were expanded to 2~5 folds in 7-day culture with the combination of EPO, SCF, and G-CSF. Further expansion was not achieved in 10-day culture and colonies disappeared in 14-day culture. HPP-CFC was expanded to a median of 7.5 folds in 7-day culture with the combination of EPO, SCF, G-CSF, IL-1β, IL-3, and IL-6. Neither 10-day or 14 day-culture enhanced expansion of HPP-CFU. Conclusion : Cryopreserved cord blood cells maintain ex vivo expansion potential. In our system, 10-day culture with the combination consisting of EPO, SCF, G-CSF, IL-1β, and IL-3 seems to be adequate for hematopoietic progenitor/stem cell expansion from cryopreserved cord blood cells.

증폭된 조혈전구세포에서 Fas 항원 및 Bax의 발현과아포토시스

김찬규,이남수,배상병,이규택,박성규,장금하,정희정,김숙자,원종호,박희숙,홍대식 대한혈액학회 2004 Blood Research Vol.39 No.2

Background:During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. Methods:CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony- forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. Results:CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. Conclusion:These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.

Proliferation of CD4+CD25high+Foxp3+ regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma

이신화,김용만,이하영,김대연,김종혁,남주현,김영탁 대한부인종양학회 2010 Journal of Gynecologic Oncology Vol.21 No.1

Objective: Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4+CD25high+Foxp3+ T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2). Methods: Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry. Results: Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4+ T lymphocytes increased, CD8+ T lymphocytes decreased, and the proportion of CD3−CD16+56+ NK cells was unchanged. The proportion of CD4+CD25high+Foxp3+ regulatory T lymphocytes in CD4+ T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group. Conclusion: This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.

골수세포의 단기배양을 통한 조혈전구세포의 증폭

오덕연,김현수,박상준,김종숙,최지영,윤한중,김삼용 大韓血液學會 1996 大韓血液學會誌 Vol.31 No.2

제대정맥내피세포와의 공조배양을 통한 제대혈조혈전구세포의 증폭

황진희,김성우,천재민,박남숙,박수진,박상은,윤환중,조덕연,김상용 대한혈액학회 2004 Blood Research Vol.39 No.3

Background:We examined an ex vivo expansion system for cord blood (CB) hematopoietic progenitor cells, which is based upon a co-culture of CD34+ cells with human umbilical endothelial cells (HUVECs) in the presence of stromal cell-derived factor-1 (SDF-1) and hematopoietic growth factors. Methods:Cord blood CD34+ cells were either incubated a liquid suspension culture or co-cultured on HUVEC monolayers with hematopoietic growth factors in the presence or absence of SDF-1. After 7~14 days of culture, cells were harvested and analyzed for fold- increase in nucleated cells, CD34+ cells, and colony-forming cells (CFCs) and apoptosis. Results:Seven-day suspension culture of CD34+ cells in the presence of a cytokine combination consisting of throbmopoietin, flk-2 ligand, and kit-ligand (TFK) led to a 43-fold increase of nucleated cells, a 19-fold increase of CD34+ cells, and 14-fold increase of CFCs, respectively. The addition of SDF-1 to TFK slightly further increased this expansion. A co-culture of CD34+ cells with HUVECs significantly enhanced the expansion of both CD34+ cells and CFCs compared with a liquid suspension culture. This was further increased by the addition of SDF-1. A co-culture of CD34+ cells on HUVECs transfected with null-adenoviral vector led to a better fold increase of haemtopoietic progenitor cells compared with a culture with non-transfected HUVECs. Adding SDF-1 to the co-culture diminished the annexn V- positive cells both in the supernatant and adherent cell layers. Conclusion:A co-culture of cord blood cells with HUVECs in the presence of hematopoietic growth factors and SDF-1 could be a new model for the efficient expansion of hematopoietic progenitors.

Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates

Kim, J.S.,Byun, N.,Chung, H.,Kim, H.J.,Kim, J.M.,Chun, T.,Lee, W.W.,Park, C.G. Academic Press 2016 Biochemical and biophysical research communication Vol.473 No.1

Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20<SUP>+</SUP> B cell purity rate of greater than 99%. Furthermore, small amounts of CD3<SUP>+</SUP>CD20<SUP>+</SUP>BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

Moharram Ahmadnejad,Naser Amirizadeh,Roya Mehrasa,Ahmad Karkhah,Mahin Nikougoftar,Arezoo Oodi 대한혈액학회 2017 Blood Research Vol.52 No.1

Background: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is consid-ered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. Methods: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cyto-kines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. Results: Maximum CB-CD34+ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34+ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 ex-pression was increased. A significant difference between the number of CD34+ and CD34−cells in the cytokine co-culture system was observed. Conclusion: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

인간 제대혈 림프구 체외 증폭시 조절 T 림프구의 변화 양상 및 특징

정의 ( Eui Jung ),김용만 ( Yong Man Kim ),송하영 ( Ha Young Song ),원혜성 ( Hye Sung Won ),김암 ( Ahm Kim ),강병문 ( Byung Moon Kang ) 대한산부인과학회 2006 Obstetrics & Gynecology Science Vol.49 No.10

목적: 조절 T 림프구는 CD4와 CD25를 표현하는 특정 T 림프구를 일컬으며, 자아에 대한 면역반응을 억제하는 역할을 하고 있다. 성인에서도 림프구는 그 자체가 실험실 배양 및 증폭이 까다롭다는 점 때문에 연구에 많은 제한을 받고 있지만 제대혈에서 분리해 낸 림프구, 특히 T 림프구는 적절한 배양 환경만 확립된다면 다양한 기능을 가진 여러 세포로 분화할 수 있고, 자기 복제능이 탁월하다. 본 연구는 만삭 산모와 정상 성인 여성의 말초 혈액, 그리고 제대혈내의 조절 T 림프구의 분포에 차이가 있는지 확인함으로써 산모와 태아간 면역 관용상태에 조절 T 림프구가 관여하고 있는지 알아보고, 또한 제대혈 림프구의 체외 증식이 조절 T 림프구의 분포에 어떤 영향을 미칠 것인가를 확인하여 조절 T 림프구를 다량으로 획득할 수 있는 방법을 확립해 장차 세포치료 등 임상적 이용을 앞당기고자 하였다. 연구 방법: 각각 10명의 비임신 성인 여성과 만삭 산모의 말초혈액 및 제대혈에 항 CD3 항체, 항 CD4 항체, 항 CD25 항체, 항 CD152 항체를 첨가한 후 FACSort flow cytometry를 이용하여 세포 표식자 분석을 시행하였다. 제대혈은 Ficoll-Hypaque 밀도 기울기 원심 분리법으로 단핵구 세포를 분리하여 항 CD3 항체와 IL-2를 이용한 체외증폭을 시행한 후, 배양 제 0, 4, 11, 21일에 세포 표식자 분석과 FoxP3의 발현도를 측정하였다. 또한 MACS(TM) kit를 이용하여 CD25-와 CD25+ T 림프구를 분리하여 CD25+ 림프구의 조절 T 림프구 여부를 FoxP3의 발현을 통해 확인하였다. 결과: 제대혈내의 CD4+CD25++ T 림프구는 정상 성인 여성과 산모의 말초혈액보다 높은 분포를 나타냈으며, 만삭 산모는 비임신 여성보다 CD4+CD25++ T 림프구가 유의하게 적었다 (p<0.05). 제대혈 림프구를 체외 배양했을 때 CD4+CD25++ 그리고 CD152+ T 림프구의 비율은 4일째 현저하게 높아지고 11일째 이후로 감소하는 소견을 보였고 (p<0.05), 이는 FoxP3의 발현 정도와도 일치한 결과를 보였다. 분리된 CD4+CD25+ T 림프구는 CD4+CD25- T 림프구에 비해 FoxP3 발현량이 의미있게 높았다 (p<0.05). 결론: 제대혈은 성인의 말초혈액보다 CD4+CD25++ 조절 T 림프구의 비율이 높으며, 체외 증폭하였을 때 조절 T 림프구의 비율이 4일째까지 증가하다가 이후로 감소되는 소견을 보인다. 이 결과는 향후 조절 T 림프구의 임상적 이용에 유용한 자료로 사용될 수 있다고 사료된다. Objective: Regulatory T cells, which expressing CD4 and CD25, have a crucial role in suppressing immune systems to self-antigens and preventing autoimmune diseases. This study aims to evaluate the role of regulatory T cells in maternal tolerance to the fetus by comparing the proportion of regulatory T cells in peripheral blood of pregnant and non-pregnant women with those in umbilical cord blood. Also we analyze the changes of proportions of regulatory T cells of umbilical cord blood according to ex vivo expansion. Methods: An immunophenotypic study on 10 peripheral blood of pregnant and non-pregnant women and 10 cord blood was performed by means of FACSort flow cytometry using anti-CD4, anti-CD25 and anti-CD152 antibodies. Fresh cord blood mononuclear cells (MNCs) were isolated by Ficoll-Hypaque density centrifugation. The MNCs were cultured in anti-CD3 Ab-coated flasks for 4 days. The cells were then transferred to non-coated flasks with IL-2 175 U/mL and were cultured for another 17 days. The expression of CD4, CD25, CD152 and FoxP3 PCR were analyzed in accordance with days in culture. We also performed FoxP3 PCR in isolated CD25+ and CD25- T cells using MACS(TM) kit. Results: Umbilical cord blood had a higher proportion of CD4+CD25++ regulatory T cells than adults, and term-pregnant women had a lower proportion than non-pregnant women (p<0.05). After ex vivo expansion in anti-CD3 Ab coated flask with IL-2, we observed a significantly increased expression of CD4, CD25, CD152 at 4(th) day after culture and decrease thereafter. The result was the same as the expression of FoxP3 PCR. Conclusion: Umbilical cord blood contained a high proportion of CD4+CD25++ regulatory T cells and regulatory T cells increased on culture day 4 and declined thereafter. Umbilical cord blood may serve as a readily available source of regulatory T cells for immunotherapy.