Repopulating activities of human cord blood cells separated by a stem cell collection filter in NOD/SCID mice: a comparative study of filter method and HES method

BACKGROUND: Volume reduction and removal of RBCs are essential for cost-efficient cord blood (CB) banking. It has previously been shown that a newly developed device, a stem cell-collection filter (SCCF), can reduce the CB volume and remove RBCs efficiently, giving high recovery rates for CD34+ cells, colony-forming cells, and long-term culture-initiating cells with short operation time. The aim of this study was to compare the quality of CB cells separated by SCCF and HES by analyzing repopulation in NOD/SCID mice. STUDY DESIGN AND METHODS: A total of 1 x 10(6) or 5 x 10(6) nucleated cells derived from SCCF- or HES-separated, cryopreserved, thawed, and washed CB were transplanted into NOD/SCID mice. Eight weeks after transplantation, bone marrow cells of the recipient mice were examined by flow cytometry and hematopoietic progenitor assay for the engraftment of human cells. RESULTS: Mice given human CB cells, separated by SCCF, showed degrees of engraftment similar to those in mice given HES-separated CB cells. There was no significant difference in the lymphohematopoietic reconstitution pattern in the two groups of mice. CONCLUSION: SCCF processing does not appear to reduce the number of repopulating cells in NOD/SCID mice or alter the number of HPCs. It is now shown that these cells can be captured by SCCF and removed, and that they will engraft.     

Nonobese diabetic-severe combined immunodeficient mice transplantation of volume-reduced and thawed umbilical cord blood transplants following closed-system immunomagnetic cell selection

BACKGROUND: Protocols for the expansion of human umbilical cord blood (UCB) progenitors begin with the selection of CD34+ cells from stored frozen and thawed units. Use of an immunomagnetic selection procedure within a closed blood bag system for volume-reduced UCB transplants was evaluated, and the influence of CD34 cell selection on in vivo engraftment potential was studied. STUDY DESIGN AND METHODS: Eleven thawed buffy coat-processed UCB units were processed within a standard blood bag with a washing solution. In six independent experiments, the same dosage of 2 x 104 CD34+ cells from paired selected and nonselected samples was transplanted into NOD-SCID mice. In two experiments, cells from the negative fraction were also transplanted. RESULTS: The purity of CD34+ cells after selection was correlated with the removal of supernatant after the first washing step and therefore with adequate removal of damaged or dead cells (r=0.86, p < 0.01). Mice transplanted with unselected UCB cells had more human cells within their marrow than animals transplanted with selected cells (8.6 +/- 5.9% selected group vs. 19.8 +/- 14.2% unselected group; p=0.04), whereas no engraftment could be observed transplanting cells from the two negative fractions. A higher percentage of human CD45+ cells in the unselected group were found to be positive for CD38, CD14, CD33, and CD19, indicating a higher potential for these unselected progenitors to differentiate into myeloid cells and B cells. CONCLUSIONS: Processing of volume-reduced and thawed UCB transplants within a closed-bag system before immunomagnetic CD34+ cell selection allows for the preparation of CD34+ cells of significant purity at technically useful cell recoveries. However, these experiments indicate a potential impairment of engraftment capacity for the CD34+ cell-enriched fraction.     

脐血干细胞的分离方法比较

目的:为合理选择脐血干细胞的分离方法提供参考.方法:采用改良HES(羟乙基淀粉)沉降分离法和Ficoll分离法分离脐血干细胞,比较两种方法分离前后的有核细胞数、分离后的CD34 细胞数、分离后的有核细胞回收率及台盼蓝拒染率.结果:改良HES沉降分离法和Ficoll分离法分离前的脐血量、有核细胞浓度和细胞数差异无统计学意义(P＞0.05);分离后改良HES法有核细胞数、CD34 细胞数、有核细胞回收率高于Ficoll 分离法(P＜0.05),而显示有核细胞活力的台盼蓝拒染率两种方法比较差异无统计学意义 (P＞0.05).结论:改良HES分离法的有核细胞回收率高,分离速度快、污染机会小、终体积小,是脐血干细胞分离较好的方法.     

不同来源人造血干/祖细胞在NOD/SCID小鼠体内归巢能力的差异

目的 比较不同来源的人造血干／祖细胞在NOD／SCID小鼠体内归巢能力的差异性，并探讨其体内归巢能力与膜表面归巢相关分子CXCR4表达水平的相关性。方法 采用流式细胞术（FACS）检测新鲜脐血、冻存脐血、动员后外周血（mPB）及骨髓来源的CD34^＋细胞表面CXCR4表达水平；将荧光染料CFSE标记的人CD34^＋细胞移植人接受照射的NOD／SCID小鼠，移植后20小时检测已归巢于NOD／SCID小鼠骨髓及脾脏中不同来源的人CD34^＋细胞，计算其相应的骨髓及脾脏归巢效率；并将小鼠股骨制成组织切片，荧光显微镜下观察人CD34^＋细胞在小鼠骨髓腔内的分布。结果 新鲜脐血、冻存脐血、mPB和骨髓CD34^＋细胞膜表面CXCR4表达阳性率分别为（49.52±1．12）％。（46．12±2．29）％，（48．50±2．48）％和（65．39±1．27）％，CD34^＋细胞在NOD／SCID小鼠骨髓的归巢效率分别为（3．00±0．44）％，（2．84±0．46）％，（4．06±0．70）％及（5．76±0．52）％；在脾脏的归巢率分别为（1．88±0．12）％，（1．80±0．15）％，（1．90±0．22）％，（2．16±0．34）％。归巢的CD34^＋细胞主要分布于小鼠股骨的骨内膜区域。结论 脐血CD34^＋细胞膜表面CXCR4水平低于mPB和骨髓。经冻存复苏后脐血CD34^＋细胞膜表面CXCR4水平略有下调。脐血CD34^＋细胞在照射NOD／SCID小鼠的骨髓归巢效率低于mPB和骨髓。     

Establishing a cord blood banking and transplantation program in Mexico: a single institution experience

BACKGROUND: Over the past decade, umbilical cord blood (UCB) banking and transplantation have increased significantly worldwide. The experience in developing countries, however, is still limited. In January 2005 the Mexican Institute of Social Security (IMSS) initiated its UCB banking and transplantation program. This study reports on the experience generated at this institution during the first 2 years of activities. STUDY DESIGN AND METHODS: A public UCB bank was established at La Raza Medical Center, IMSS, in Mexico City. Good manufacturing practices and standard operating procedures were used to address donor selection, as well as UCB collection, processing, and cryopreservation. Based mainly on human leukocyte antigen (HLA) and total nucleated cell (TNC) content, specific UCB units were thawed, processed, and released for transplantation. RESULTS: Based on stringent selection criteria, 360 UCB units were collected from January 2005 to December 2006. A total of 201 (56%) units (minimum volume, 50 mL without anticoagulant) were processed and stored. Median values for specific parameters were as follows: volume, 89.9 mL; viability, 94.8%; TNCs, 0.91 x 10(9); CD34+ cells, 3.13 x 10(6); and colony-forming cells, 1.20 x 10(6). During this period, 10 units had been released for transplantation to seven patients (six children and one adult). Engraftment was observed in five patients; four of them were still in remission (114-293 days after transplant). In spite of showing sustained engraftment, one patient died on Day +88. Two patients showed no engraftment and died 29 to 30 days after transplant. CONCLUSION: The results obtained during this initial period are encouraging and indicate that the UCB banking and transplantation program at IMSS will help to improve already existing hematopoietic cell transplant programs in Mexico. The experience generated at IMSS may be helpful to other institutions, particularly those in developing countries.     

A large number of mature and functional dendritic cells can be efficiently generated from umbilical cord blood–derived mononuclear cells by a simple two‐step culture method

BACKGROUND: Advances in the past two decades in dendritic cell (DC) biology paved the way to exploit them as a promising tool in cancer immunotherapy. The prerequisite for DC vaccine preparations is large‐scale in vitro generations of homogeneous, mature, and functional DCs. Frequent improvements are being made in the existing in vitro DC production protocols to achieve this goal. In our previous study we reported a large‐scale generation of mature, functional DCs from umbilical cord blood (UCB) CD34+ cells. Here we report that this method can be used for the efficient generation of DCs from UCB mononuclear cells (MNCs) and thus the hematopoietic stem cell isolation step is not essential. STUDY DESIGN AND METHODS: MNCs or CD34+ cells isolated from the same cord blood (CB) samples were used for the generation of DCs. DCs were characterized for morphology, phenotype, and functional assays including antigen uptake, chemotaxis, and mixed leukocyte reaction. Similarly DCs generated from the MNCs of same fresh and frozen CB units were compared. RESULTS: The morphologic, phenotypic, and functional characterization of the DCs generated from various sets show that they were comparable in nature irrespective of the starting population used. CONCLUSION: We conclude that the CD34+ isolation step is not essential for the generation of mature, functional DCs and thus can be eliminated. More importantly, we show that DCs can be generated with equal efficiency from the MNCs of frozen CB units. Our culture method will be useful for exploiting the potential of UCB as an additional source for allogeneic DCs in the clinical settings.     

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots

BACKGROUND: The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability.
STUDY DESIGN AND METHODS: The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration.
RESULTS: Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 ± 0.22 percent, the results after thawing were 0.98 ± 0.65 and 0.57 ± 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition.
CONCLUSIONS: The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.     

Competitive repopulation assay of two gene-marked cord blood units in NOD/SCID/gammac(null) mice.

In multiunit cord blood transplantation, hematopoietic stem cells from each unrelated cord blood (UCB) unit competitively reconstitute the hematopoietic system in a recipient. To evaluate the fate of the progeny of each UCB unit and to determine the effects of graft-versus-graft reaction, we established a novel competitive repopulation assay using NOD/SCID/gammac(null) mice in which human T lymphocytes develop from CD34+ cells. CD34+ cells from each UCB unit were labeled with recombinant lentivirus vectors carrying genes encoding either enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP). Hematopoietic chimerism composed of both EGFP+ and EYFP+ cells was stably maintained up to 6 months after transplantation with purified CD34+ cells; the ratio of EGFP+ to EYFP+ cells in peripheral blood and bone marrow posttransplantation was equivalent to the ratio of these cells at transplantation. However, when mononuclear cells from two UCB units were cotransplanted with CD34+ cells, engraftment was highly competitive, with cells from only one or the other of the two UCB units surviving. Further subfractionations of mononuclear cells indicate that the skewed chimerism that is often observed in clinical multiunit cord blood transplantation may be mediated by the cooperation of both CD4+ and CD8+ T cells. The assay established here will be a useful tool for analyzing hematopoietic reconstitution in clinical multiunit cord blood transplantation.     

Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood

BACKGROUND: The results of current processing procedures for reducing volume and recovering HPCs from umbilical cord blood (UCB) before cryopreservation vary. STUDY DESIGN AND METHODS: Dextran was added to bags containing UCB, followed by sedimentation for 30 minutes. The processed UCB was then frozen. RBCs, nucleated cells, MNCs, CD34+ cells, CFUs and long-term culture-initiating cells (LTC-ICs), viability, and sterility were evaluated. Fractionations in ficoll-hypaque and hydroxyethyl starch (HES) were also run in parallel for comparison. RESULTS: The nucleated cell (NC) recovery and RBC depletion were 86.1 percent and 94.3 percent, respectively (n = 50). Sedimentation with dextran also enabled the recovery of 80.7 percent MNCs and 82.6 percent CD34+ cells (n = 30). Postsedimentation samples displayed no impairment of CFU growth (n = 42, 108.7% CFU-C, 104.6% CFU-GEMM, 107% CFU-GM, and 95.7% BFU-E). Long-term cultures on five paired samples before and after sedimentation generated similar numbers of CFU-C each week (p = 0.88). Limiting dilution analysis of 12 paired pre/postsedimentation samples showed comparable median proportions of LTC-ICs (1/6494 vs. 1/5236; p = 0.18). The cell viability of 24 samples of thawed UCB after sedimentation was 90.3 percent (77.5-96%) and the recovery of CFU-C, CFU-GEMM, CFU-GM, and BFU-E of 11 postsedimentation samples was 93.4 percent, 84.9 percent, 92.3 percent, and 83.4 percent, respectively. NC recovery was significantly higher after treatment with dextran than with ficoll-hypaque (n = 30; 88.5% vs. 29.1%; p<0.005) and HES treatment (n = 21; 88.5% vs. 76.4%; p = 0.004). However, MNCs, CD34+ cells, CFUs, LTC-ICs, and RBCs were comparable. Two cycles of dextran sedimentation recovered 93.9 percent of NCs with cell viability of 98.6 percent (96.5-100%), whereas 11.7 percent of RBCs were retained (n = 20). The final yield volume was 33.5 (28-41) mL. CONCLUSION: In a semi-closed system, dextran sedimentation enabled volume reduction of UCB without significant quantitative and qualitative losses of HPCs.     

双份脐血间CD34^＋细胞与CD3^＋细胞相互作用对分化增殖能力的影响

目的双份脐血移植的植入动力学机制目前尚无定论,推测双份脐血中的淋巴细胞与优势份脐血的产生相关。本实验将双份脐血的CD34^＋细胞与CD3^＋细胞混合培养,观察CD3^＋细胞对CD34^＋细胞的增殖分化有无影响。方法建立液体和半固体培养体系,将免疫磁珠分选纯化的双份脐血间的CD34^＋细胞和CD3^＋细胞混合培养6d和14d。以流式细胞计数观测CD34^＋细胞培养后的分化指标（CD33,CD41,CD71）;计数集落形成单位（GM—CFU、BFU-E、GEMM—CFU）分析CD34^＋细胞的增殖情况。结果液体共培养后各份CD34^＋细胞表面分化指标的变化。脐血CD34^＋细胞分选富集的纯度为（98．70±0．72）％。3d实验组和对照组的各项分化指标无差异（P〉0．05）;6d的CD33、CD71实验组明显低于对照组,而CD41明显高于对照组（P〈0．05）。半固体共培养后CD34^＋细胞增殖能力的变化。实验组的红系集落形成单位（BFU—E）及粒单细胞集落形成单位（GM—CFU）数低于对照组（P〈0．05）,而混合细胞集落形成数（GEMM—CFU）高于对照组（P〈0．05）。结论将两份脐血的CD34^＋细胞和CD3^＋细胞体外混合培养对CD34^＋细胞的增殖分化能力有影响,推测双份脐血间的相互作用可部分地通过CD3^＋细胞介导。     

人脐血及骨髓淋巴细胞免疫表型的比较及其意义

为比较脐血和骨髓淋巴细胞及祖细胞分化抗原，通过流式细胞术（FCM）双标法对38份脐血及10份骨髓免疫细胞表型进行了分析研究。研究发现：（1）脐血及骨髓淋巴细胞中均测到稚淋巴细胞（CD3^-CD4^ )，且前中含量较多，但脐血细胞毒T细胞含量（CTL，CD3^ CD16^ 56^ )低于骨髓；（2）脐血中NK细胞（CD3^-CD16^ 56^ )比例高于骨髓；（3）脐血有核细胞中CD34^ 细胞的比值接近于骨髓，但脐血CD34^ 细胞中髓系祖细胞（CD34^ CD13^ ,CD34^ HLA-DR^ )及淋巴系祖细胞（CD34^ CD19^ )含量均低于骨髓，结论：（1）脐血免疫细胞具有不成熟性，这估计是脐血移植后GVHD程度轻的主要原因；（2）脐血淋巴细胞中NK细胞含量较高，推测脐血移植后移植物抗白血病效应（GVL）并不会降低；（3）脐血CD34^ 细胞中髓系祖细胞及淋巴系祖细胞比例均低于骨髓，可能是脐血移植后造血及免疫重建速度较慢的原因之一。     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment

BACKGROUND: Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at -80 degrees C, was tested on antigen-presenting dendritic cells (DCs) and cells used in their derivation. STUDY DESIGN AND METHODS: DCs cultured in serum-free media from adherent or CD14+ apheresis MNCs (n = 36) in the presence of GM-CSF + IL4 +/- TNFalpha were frozen and stored at -80 degrees C in 6-percent HES, 5-percent DMSO, and 4-percent HSA. Apheresis MNCs, CD14+ monocytes, and lymphocytes were similarly frozen and later thawed for culture. Cells were assayed for viability, DC phenotype, mixed lymphocyte reaction, and antigen presentation before and 3, 6, 9, 12 or more months after freezing. RESULTS: DCs retained viability (82 +/- 2.3%) for at least 24 months. Mature and immature phenotype and function were preserved. Thawed MNCs and CD14+ cells differentiated to DCs and lymphocytes maintained high functional viability (92 +/- 3%) comparable to prefreeze levels. CONCLUSION: A simple -80 degrees C freezing and storage method that combines extracellular (HES) and intracellular (DMSO) agents is practical and preserves functional viability of DCs, MNCs, CD14+ monocytes, and lymphocytes.     

人脐血间充质干细胞促进脐血CD34+细胞在NOD/SCID小鼠体内植入的实验研究

目的探讨人脐血间充质干细胞(MSC)联合人脐血CD34+细胞移植,能否促进CD34+细胞在NOD/SCID小鼠体内植入及加速其造血恢复.方法NOD/SCID小鼠于60Co 2.5 Gy照射后24h内由尾静脉输注胎儿脐血CD34+细胞1×105/只(低细胞量移植组)或1×106/只(高细胞量移植组),联合移植组同时输注脐血MSC 1×106/只.动态观察移植后小鼠外周血白细胞、血红蛋白和血小板恢复情况,于移植后第8周用流式细胞术检测存活小鼠骨髓中人CD45+、CD45+CD3+、CD45+CD19+和CD45+CD33+细胞的含量.结果①低细胞量移植时,联合移植组的植入率明显高于单纯移植组,分别为26.02%和16.52%(P＜0.05);高细胞量移植时,联合移植组和单纯移植组的植入率相近,分别为43.71%和39.23%(P＞0.05).②高细胞量联合移植组和单纯移植组的存活率分别为80%和70%;低细胞量联合移植组和单纯移植组的存活率分别为70%和50%.③无论高细胞量还是低细胞量组,联合移植小鼠白细胞、血红蛋白和血小板的恢复明显早于单纯移植组.④移植8周后,小鼠骨髓中人CD45+CD19+、CD45+CD33+细胞含量在低细胞量移植时,联合移植组高于单纯移植组;但在高细胞量移植时,两组之间差异无统计学意义.CD45+CD41a+细胞的含量无论在低细胞量和高细胞量移植时,联合移植组均高于单纯移植组.各组小鼠骨髓中CD45+CD3+细胞的含量均较少,且各组之间差异无统计学意义.结论①低细胞量移植时,人脐血MSC联合移植可提高人脐血CD34+细胞在小鼠体内的植入率.②人脐血MSC与人脐血CD34+细胞联合移植,加速NOD/SCID小鼠各系造血恢复,提高移植小鼠存活率.③MSC联合移植可促进人脐血CD34+细胞在NOD/SCID小鼠体内向粒系、B淋巴系和巨核系定向分化.     

不同时相移植人骨髓间充质干细胞对人脐血CD34^＋细胞植入NOD／SCID小鼠的影响

为了观察不同时相移植人骨髓间充质干细胞（MSC）对脐血（UCB）CD34^＋细胞移植的NOD／SCID小鼠造血重建的影响，明确最佳的移植时机，将体外培养扩增的人骨髓MSC分别于UCBCD34^＋细胞移植同时、移植前48小时及移植后48小时输入经^60Coγ射线照射的NOD／SCID小鼠，观察共移植后42天内小鼠外周血白细胞和血小板变化，并于移植后42天处死小鼠，用FACS检测外周血、骨髓和脾脏人源细胞含量。结果表明：（1）MSC和UCBCD34^＋细胞同时输注可明显降低外周血白细胞和血小板下降幅度，缩短白细胞和血小板恢复时间；二者不同时输注均不降低白细胞和血小板下降幅度，且输注UCBCD34^＋细胞后48小时输注MSC时外周血血小板恢复时间明显晚于同时输注者。（2）与单纯UCBCD34^＋细胞移植相比较，不同时相输注MSC均可促进UCBCD34^＋细胞的植入，三个共输注组间促进骨髓各系造血植入效应无明显差异。结论：人骨髓MSC与UCBCD34^＋细胞共移植时，以同时移植效果最佳，此结果为MSC的临床应用提供了实验依据。     

Cord blood banking and transplantation at the Mexican Institute of Social Security: the first 5 years

BACKGROUND: In January 2005, the Cord Blood Bank (CBB) at the Mexican Institute of Social Security initiated activities. Herein, we describe the experience generated during this period (January 1, 2005‐December 31, 2009). STUDY DESIGN AND METHODS: Good manufacturing practices and standard operating procedures were used to address donor selection, as well as umbilical cord blood (UCB) collection, processing, and cryopreservation. Based mainly on HLA and nucleated cell content, specific UCB units were thawed, processed, and released for transplantation. RESULTS: A total of 589 UCB units were stored, representing 54% of the total number of units collected. Forty‐eight units (8.14% of the stored units) were released for transplantation of 36 patients. Twenty‐six patients (72% of cases) corresponded to patients with acute leukemia, five (14%) to patients with marrow failure, and the rest (five; 14%) to patients with hemoglobinopathies and other syndromes. The median number of nucleated cells infused per patient was 6.71 × 10⁷/kg and the median number of CD34+ cells was 4.8 × 10⁵/kg. Current engraftment data indicate that engraftment occurred in 56%, and no engraftment in 44%, of cases. Engraftment was more frequent (59%) in patients that received more than 3 × 10⁷ total nucleated cells (TNCs)/kg body weight, than in those receiving fewer than 3 × 10⁷ TNCs/kg (40%). Myeloid engraftment was observed 7 to 54 days posttransplant (median, 23 days), whereas platelet engraftment was detected on Days 12 to 87 posttransplant (median, 38 days). To date, the disease‐free survival rate was 41% and the overall survival was 47%, with survival periods of 126 to 1654 days. CONCLUSION: Although the experience presented herein is still limited and the period of analysis is still short, the results obtained during these 5 years are encouraging.     

脐血造血干/祖细胞免疫表型的流式细胞术双标法分析及其意义

目的比较脐血和骨髓中造血干/祖细胞(HSPC)的免疫表型差异.方法使用流式细胞术(FCM)双标法对38份脐血及10份骨髓HSPC进行免疫表型分析.结果①脐血有核细胞中CD34+细胞所占比例与骨髓中相近,约为0.5%;②脐血CD34+细胞中CD34+CD38-[(17.C4±5.37)%]、CD34+HLA-DR-[(32.65±10.71)%]及CD34+H-CAM+(CD44+)[(77.84±7.69)%]亚群含量均高于骨髓[含量分别为(8.26±3.19)%、(14.05±1.67)%和(70.02±6.40)%],CD34+CD13+、CD34+CD19+亚群比例低于骨髓.结论脐血与骨髓CD34+细胞比例相近,但前者较原始的干细胞含量更高,故脐血是极具潜力的HSPC来源;而脐血CD34+细胞中髓系及淋系祖细胞含量低于骨髓,可能是脐血移植后造血及免疫重建缓慢的原因之一.     

人骨髓基质细胞促进脐血CD34+细胞的体外扩增和对NOD/SCID小鼠的植入

目的 研究人骨髓基质细胞(MSC)促进脐血CD34+细胞体外扩增及植入能力的作用.方法 分离、培养正常人的MSC作为滋养层细胞.在TPO、SCF、FL和G-CSF刺激下,比较有、无MSC滋养层细胞对扩增脐血CD34+细胞后,CD34+细胞和CFU数的增加倍数,以及植入非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠的能力.结果 以骨髓MSC为滋养层细胞的培养体系可以更加有效地扩增脐血CD34+细胞.体外扩增1周总细胞数(TNC)、CD34+细胞和CFU的均数分别增加111.6、19.3和58.0倍;体外扩增2周后TNC、CD34+细胞和CFU的均数分别增加532.8、41.3和563.5倍.脐血细胞输入NOD/SCID小鼠6周后,移植未扩增脐血细胞的对照组小鼠骨髓细胞中人CD45+细胞比例仅为1.2%～3.7%;移植单纯细胞因子刺激扩增组小鼠骨髓中,人CD45+细胞比例为7.6%～12.1%;以MSC为滋养层扩增的脐血细胞移植组,人CD45+细胞比例达到45.3%～59.1%.结论 以人骨髓MSC为作为饲养层细胞,不但可以更加有效扩增脐血CD34+细胞,而且可以促进脐血细胞植入NOD/SCID小鼠的能力,有潜在的临床应用价值.     

成人移植双份脐血植入状态的定量监测

背景:脐血因所含的有核细胞数量有限,主要应用于儿童患者,近年来,人们尝试将两份脐血混合输入,用于成人血液系统疾病的治疗。目的:定量监测双份异基因脐血用于成人白血病患者移植后两份脐血的植入状态、嵌合体类型、相对数量的动态变化及演变规律。设计:以脐血干细胞移植的供受者为观察对象,供受者移植前及受者移植后不同时间段的系列血样提取DNA作为检测标本,短串联重复序列遗传位点为观察指标。单位:深圳市血液中心输血医学研究所免疫遗传重点实验室。对象:纳入2005-06在北京大学深圳医院住院治疗的急性髓细胞白血病患者,男性,43岁,体质量75kg。首次化疗完全缓解后6个月移植两份人类白细胞抗原(HLA)各一个位点不合的非血缘脐血,脐血1有核细胞数为2.5×107kg-1;脐血2有核细胞数为1.53×107kg-1。脐血来源于广州脐血库。患者对治疗方案知情同意。方法:采用荧光标记复合扩增短串联重复位点嵌合体定量检测技术,对急性髓细胞白血病成人患者移植两份(脐血1有核细胞数为2.5×107kg-1,脐血2有核细胞数为1.53×107kg-1)HLA各一个位点不相合的异基因脐血前后的序列血样进行了9个短串联重复序列位点的检测,利用供、受者之间的差异位点定性判断脐血是否植入以及嵌合体类型;而后根据377XLDNA测序仪上荧光扫描后两供者差异基因检出峰的峰面积计算脐血植入后患者体内两份脐血的相对数量,定量分析供体细胞植入程度及演变规律。并与采用HLA差异基因对植入状态的分析结果进行对比。主要观察指标:在成人移植双份脐血后,观察患者及两供者9个位点的短串联重复序列基因在患者体内的转变过程,对植入状态进行定量及定性的描述。结果:移植后15d两份脐血植入状态为完全双份供者嵌合体,患者体内脐血1的相对细胞数量占51.3%,脐血2占48.7%;30d时脐血1上升为70.0%,脐血2下降为30.0%。52d时只检测到脐血1的基因,植入状态转为完全单份供者嵌合体,有核细胞数少的一份脐血被排斥,有核细胞数多的一份长期植入。结论:荧光标记复合扩增短串联重复序列嵌合体定量检测可精确地描述两份脐血的植入程度及变化过程,为临床脐血的应用及供者的选择提供了一个准确、可靠的实验依据,证明双份HLA各一个位点不合的脐血同时用于成人的移植是可行的。     

A theoretically optimized method for cord blood stem cell cryopreservation

The objective of this study was to develop an optimal cryopreservation method for human umbilical cord blood hematopoietic progenitor cells as evidenced by improved retention of in vivo engraftment ability and multilineage differentiation. An extended understanding of the osmometric/permeability characteristics of cord blood stem cells was accomplished by measuring permeability of the cryoprotectant dimethyl sulfoxide (DMSO) at below-ambient temperatures (10 degrees and 3 degrees C). These data were combined with previously published osmotic and permeability data and the water-NaCl-DMSO phase diagram in conjunction with a mathematical model to determine an optimal initial DMSO concentration, cooling rate, and liquid nitrogen plunging temperature. Cells cryopreserved with the theoretically optimized procedure were then compared with cells frozen using standard methods for the ability to engraft in irradiated NOD/SCID mice. The optimal procedure was determined to include a 0.7 molal (approximately 5%) DMSO concentration at a cooling rate of 4 degrees C/min, and a plunging temperature of -44 degrees C. The optimized protocol resulted in significantly higher engraftment of human CD45(+) cells (17.2 +/- 1.6% vs. 8.4 +/- 1.6%), CD19(+) B lymphocytes (11.3 +/- 1.2% vs. 5.8 +/- 1.2%), and CD34(+) cells (1.9 +/- 0.09% vs. 0.6 +/- 0.09%) compared to cells frozen using a standard method. Engraftment of CD33(+) cells was not significantly different (4.0 +/- 0.3 vs. 3.2 +/- 0.6, respectively). This study demonstrated that the use of a theoretically determined optimal cryopreservation method is superior to standard methods for maintaining UCB PCBs with multilineage repopulation potential in NOD/SCID mice.