CB Cells Research Articles

Evaluation of biological features of cord blood units in different Taiwan ethnics.

Cord blood banks are established worldwide as a result of the increased use of umbilical cord blood (UCB) transplantation. The outcomes of this procedure relate to the cell dose of the UCB unit and the UCB collection. The aim of this study was to evaluate whether the differentiation of ethnicity influenced the biological features of the UCB units. A total of 1743 UCB units were measured for the biological features, including white blood cell count, red blood cell, Hb, CD34+ and volume of cord blood. Of these, 149 Mainlander, 1367 Hoklo, and 163 Hakka units were selected from the Buddhist Tzu Chi Stem Cell Center. The three groups were comparable for biological features of the UCB units. No differences were observed between the different ethnic groups in terms of white blood cell count, red blood cell, Hb, and volume of cord blood, except mean CD34+ in univariate analysis. However, the mean CD34+ cells appeared to be lower in Middle Taiwan in trend after adjustment for demographic factors. This preliminary study showed that ethnic distribution factor appeared to affect CB cell yields in Taiwan.

Co-culture of cord blood CD34+ cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.

Flow Cytometric Immunophenotyping of Umbilical Cord and Peripheral Blood Haematopoietic Progenitor Cells by Different CD34 Epitopes, CD133, P-Glycoprotein Expression and Rhodamine-123 Efflux

The flow cytometric enumeration of CD34+ cells, comprisingboth haematopoietic stem (HSC) and progenitor cells(HPC) is currently widely used to assess suitability of stemcell transplants for transplantation. However, to ensure thelong-term reconstruction in human recipients, the enumerationspecifically of those HSC possessing self-renewing anddifferentiation potential would be preferable. In the presentstudy we addressed a multiparametric flow cytometric profilingof haematopoietic stem cells of umbilical cord (CB)and peripheral blood (PB) using stem cells markers (epitope-specific CD34, CD133, rhodamine-123 (Rho123) efflux,p-glycoprotein) to assess their stem cell features. In contrastto CB, the CD34 class III compartment of PB did not includeall CD34 class I- and class II-expressing cells. In CB and PB,there was no clear correlation between CD34 epitope andCD133 expression. Furthermore, we detected CD133+ CD34-cells (0.07% of mononuclear cells) in CB. The functional abilityto exclude Rho123 was mainly restricted to CD34- andCD133-co-expressing cells. However, CB cells showed ahigher activity. Functionally active cells were characterisedby their expression of the verapamil-sensitive protein pumpp-glycoprotein 170 using the monoclonal antibody MRK16.It is therefore possible to use multiparametric flow cytometryto estimate the numbers of immature stem cells asCD34+ CD133+ (Rho^dull) MRK16+. This would allow a moreprecise assessment of transplant quality, especially with respectto their long-term engraftment potential, and supportshaemotherapy of malignant diseases.

Peripheral Blood, Cord Blood and Bone Marrow Cells Sorted into SP+, CD34+ and SP− Cells Are Distinct in Their ABCG2 and MDR1 Expression Levels, Which Are Most Increased in SP+ Cells and Substantially Decline after In Vitro Culture and Terminal Cell Differentiation.

Primitive hematopoietic stem cells (HSC) can be selected based on the rapid efflux of the DNA-binding dye Hoechst 33342 and are identified as side-population (SP+) cells. We and others have demonstrated that SP+ cells are negative for most lineage markers including CD34, are increased in long-term culture-initiating cell assays, reconstitute the bone marrow (BM) of lethally irradiated NOD/SCID mice and have been determined as early HSC (Engelhardt et al. Leukemia 2002; 16:1603, Guo et al. JHSCR 2003; 12:71). With further characterization of the SP phenotype, different members of the ABC-superfamily have been ascertained as an SP feature, of which ABCG2 and MDR1 are major efflux proteins. We analyzed the expression of ABCG2 and MDR1 (by RT- and Q-PCR) in an inducible system using different cell lines and in SP+ cells as compared to CD34+ and SP− cells from various cell sources (PB, CB and BM) before (d0) and after (d7) in vitro culture. The hypothesis was that 1. SP+ cells as compared to CD34+ and SP− cells show highest ABCG2 and MDR1 values before expansion and 2. expression levels substantially decrease with expansion and terminal cell differentiation. The tested cell lines CMK, AML 3.3 and AML 6.2 were strongly positive for ABCG2, less positive in HL60 and K562, and KG-1a was negative for both target genes. With use of PB, CB and BM SP+ cells before and after culture, ABCG2 expression was highest in SP+ cells, less in CD34+ and the least positive in SP− cells. This difference in ABCG2 was most prominent in CB cells, that showed a 7-fold higher expression in SP+ as compared to CD34+ cells, whereas this was similar for PB and BM SP+ and CD34+ cells. Of note, the differences in ABCG2 via matched pair analysis of SP+, CD34+ and SP− cells did not reach significance. After 7-days of expansion, ABCG2 expression was substantially reduced in all three hematopoietic cell subsets as compared to d0 values, reaching statistical significance with matched pair analysis for 34+ cells (p=0.001). Also for MDR1, the d0 expression was highest in SP+ as compared to CD34+ and SP− cells. Significant differences between SP+ and SP− cells and between CD34+ and SP− cells were observed (p<0.01), however not between SP+ and CD34+ values. MDR1 expression was also decreased on d7 as compared to d0 values, which reached significance for SP+ and CD34+ cells (p<0.05). Our results suggest that via ABCG2 and MDR1 expression, PB, CB and BM cells sorted into SP+, CD34+ and SP− cells show substantial differences in pump protein expression. Albeit ABCG2 and MDR1 expression was highest in SP+ as compared to CD34+ and SP− cells, which declined with cell differentiation in culture, the difference between SP+ and CD34+ cells was less distinct than expected. This suggests that ABCG2 and MDR1 serve as markers for primitive HSC, but also that 1. “contamination” of other cells within the SP+ cell population (e.g. early lymphocyte subsets) dilute the expression levels of ABCG2 and MDR1 in SP+ cells, and/or 2. transporter expression determination does not allowed to detect small differences in ontogeny and stem cells' primitiveness or 3. SP+ cells may not reflect - as much as initially postulated - very early HSC or are similar in its primitiveness as CD34+ cells.

Single Unit Cord Blood Transplant Supported by Third Party Highly Purified Mobilized Hematopoietic Stem Cells: Immune Reconstitution Studies.

Background and Objectives: Following cord blood transplants (CBT) there is a period of severe and often prolonged immune deficiency that results in long term susceptibility to infections. Immune reconstitution is an important factor for long term survival. We analyzed the immune reconstitution of adult recipients of a single unit CBTs supported by a low number of third party donor highly purified mobilized hematopoietic stem cells (dual CB/TPD transplants), as previously described (Magro et al. Haematologica 2006; 91:640–8). This strategy results in transient double chimerism of CB and TPD cells and early granulocyte recovery, initially of TPD predominance. Complete CB chimerism is regularly achieved within 100 days.The objective of this study is to evaluate immune reconstitution in this CBT.Patients and Methods: Data were obtained from 19 patients between July 2004 and July 2006. Data collection was initiated at different intervals (from day −7 to +720, quartiles Q1=35, Q2=90 and Q3=210). Samples were obtained on days +15, +35, +55, +90 and monthly thereafter up to two years. By four-color flow cytometric immunophenotyping we analyzed the subsets of peripheral blood lymphocytes: CD3+/CD4+ (T helper/inducer), CD3+/CD8+ (T suppressor/cytotoxic), NK cells (CD3−/CD56+/CD16+) and B cells, as well as cells with naïve, memory and effector T-cell immunophenotypes. TREC bearing cells were analyzed by quantitative PCR in sorted CD4+ and CD8+ T cells collected from 3 months post-transplant onwards.Results: CD56+ cells recovered early after transplantation, with median absolute number counts (ANC) of 69 (range 18–307), 170 (0–366) and 159 (32–531) cells/uL in days +15, +35 and +55 samples [normal controls 153 (71–438)], representing the largest subset within the first two months (decreasing proportions of 60%, 50% and 40%, respectively). ANC of CD4 and CD8 T cells remained low for several months, progressively increasing to reach normal ranges at different intervals. Naïve CD4 and CD8 cells (CD45RO−/CD27+) start to be detected by immunophenotyping after three months post-transplantation with median ANC of 17 (12–79) and 12 (5–103) cells/uL respectively and increasing thereafter [normal controls 860 (552–1072) and 331 (227–521)]. By the end of the first year values of T cell subsets were: CD4+, 823 (16–1123) cells/uL [normal controls 872 (470–1093)]; CD8 934 (56–1174) [normal controls 371 (208–808)], with persisting predominance of the naive phenotypes and proportions of memory phenotypes slowly increasing. B cells became detectable around day +90 with median ANC of 249 (0–1934) cells/uL, rapidly reaching values within the normal range [275 (133–684)]. Transient acute GVHD was developed by six of the 19 patients. All showed a transient drop in absolute numbers of NK, T and B cells. Chimerism analysis showed initial transient double chimerism of CB and TPD cells. Complete CB chimerism was achieved between days +15 and +94 (median, +35). Results of chimerism of lymphocyte subsets and TREC are not yet available.Conclusions: Following dual CB/TPD transplants we have observed early recovery of NK and B-cells and slow development of T cells subsets and of non-naive immunophenotypes.

A High Dose of Erythropoietin Induces the Expansion of CD34+CD133+ Cells in Human Umbilical Cord Blood.

Objectives: We compared the responsiveness to the high dose stimulation of human EPO between the transplant grafts in an in vitro culture system to clarify the characteristics of cord blood and to make clear the repopulation characteristics on human hematopoietic stem/progenitor cells.Methods: Cells (cord blood, bone marrow and peripheral blood stem/progenitor cells) were cultured for six days with G-CSF (100 ng/ml), EPO (10 U/ml), or without additives, and were sorted with antihuman CD133-1 monoclonal antibody. The obtained fractions were further labeled with antihuman CD34 monoclonal antibody and CD133-2 antibody as well as the lineage markers, and their expressions were analyzed. Cells were also assayed with ordinal colony-formation. For the analysis of long-term culture-initiating cells (LTC-ICs), Dexter's long-term culture method was carried out. The expression of the specific proteins was also analyzed with RT-PCR method.Results. The ratio of the CD34+ cell count to the total cultured mononuclear cell count was not changed between CB, BM and PBSC. However, the ratio of the CD133+ cell count to the total cultured mononuclear cell count was 1.4-fold that of when CB cells were cultured with a high dose of EPO. No significance was observed for BM and PBSC. The count of CD34+CD133+ cells was 1.9-fold that of when CB was cultured with EPO. Dexter's long-term culture system demonstrated that CB cultured with EPO contained more LTC-ICs and colony-forming cells with a high proliferation potential than that cultured without additives.Discussion: These results indicate that the CB has unique characteristics to respond to a high dose of EPO.

Delayed Immune Recovery after Umbilical Cord Blood Transplantation (UCBT) Is Characterized by Thymic Regeneration Failure.

UCBT is a potentially curative modality for many patients with relapsed or high-risk hematological malignancies who lack suitably matched hematopoietic progenitor cell donors. The advent of transplant approaches that combine multiple CB units to facilitate transplantation into adult recipients, as well as advances in conditioning regimens and supportive care, have significantly improved outcomes following UCBT. Despite these advances, infections and/or relapse associated with impaired T cell reconstitution remains an important source of morbidity and mortality. We prospectively assessed the quantitative and functional recovery of CD4+ and CD8+ T cells in 34 consecutively transplanted UCBT recipients. Functional studies of CD4+ and CD8+ T cells stimulated with a superantigen (staphylococcal enterotoxin B, SEB) and cytomegalovirus (CMV) antigens revealed that SEB-stimulated IFNγ responses by cytokine flow cytometry (CFC) were significantly lower in the UCBT recipients than in control bone marrow or peripheral blood stem cell transplant (SCT) recipients (median values of 0.99% (n=12) vs. 24.8% (n=24) in the CD4 compartment and 0.62% (n=14) vs. 6.37% (n=9) in the CD8 compartment, respectively). Despite this, many UCBT recipients had measurable donor-derived CMV-specific CD4+ and CD8+ T cells by 3 months (4 of 11, 44% in CD4+ T cells and 6 of 12, 50% amongst CD8+ T cells), indicating that primary immune responses were derived from the naïve CB graft precursors. Immunophenotypic studies demonstrated that a higher proportion of effector and late-effector memory cells (i.e., CCR7-CD45RA− and CCR7-CD45RA+ T cells) were present in UCBT recipients than SCT recipients. The median fraction of CCR7-negative T cells was 65% (n=12, UCBT recipients) vs. 30% (n=21, other SCT recipients) in the CD4 compartment, and 76% vs. 31%, respectively, in the CD8 compartment. Furthermore, the subgroup of UCBT recipients with a ratio of CCR7+ to CCR7-negative CD4+ T cells above the median at day 30 after SCT survived significantly longer (666 days vs. 224 days, p=0.03). Finally, we compared thymic regeneration (using a δ-chain T cell receptor excision, δTREC assay) in UCBT recipients with three other groups of SCT recipients, including longitudinally assessed autologous and allogeneic SCT recipients, as well another group of allogeneic SCT recipients assessed cross-sectionally. Thymic recovery was reduced at one and three months after UCBT, relative to all other SCT recipient groups. Median δTREC values (reported as δTRECs/million PBMC) at three months were 0.0 in UCBT recipients (n=12), 12 in autologous SCT recipients (n=11), 124 in allogeneic SCT recipients (n=27), and 2150 in a second group of allogeneic SCT recipients (n=31) (p<0.04 for all observations). These results demonstrate that T cell immune recovery in UCBT recipients is characterized by delayed functional recovery and a relative predominance of late memory (CCR7−) T cells, relative to other SCT recipients. However, naïve donor-derived CB cells are capable of primary pathogen-specific expansion in vivo. Finally, these data suggest that efforts to improve T cell immune reconstitution after UCBT should be directed at improving thymic regeneration, which is likely to be important in facilitating the recovery of naïve and early memory T cells.

CD80/CD28 co-stimulation in human brucellosis

Despite treatment, 10-30% of brucellosis patients develop chronic disease, characterized by atypical clinical picture and/or relapses. A defective T helper 1 (Th1) response and a low [corrected] percentage of CD4(+)/CD25(+) cells have been described in chronic brucellosis patients. CD80/CD28 co-stimulation is critical for an efficient Th1 response and has not been studied previously in human brucellosis. In order to investigate the role of CD80/CD28 co-stimulation, 13 acute brucellosis patients (AB), 22 chronic brucellosis patients (CB, 12/22 relapsing type-CB1 and 10/22 atypical type-CB2), 11 'cured' subjects and 15 healthy volunteers (controls) were studied. The percentage of CD4(+)/CD28(+) T lymphocytes and CD14(+)/CD80(+) monocytes were analysed by flow cytometry both ex vivo and after phytohaemagglutinin (PHA)-stimulation with or without heat-killed Brucella abortus (HkBA). Ex vivo analysis showed no differences between all groups studied. PHA stimulation up-regulated the percentage of CD80(+) monocytes in AB compared to 'cured' subjects and controls (P < 0.001), although the proportion of CD4(+)/CD28(+) cells did not alter. A higher percentage of CD80(+) monocytes was observed in the CB1 subgroup, compared to AB, 'cured' subjects and controls (P = 0.042, < 0.001 and < 0.001, respectively). CB2 was characterized by a lower percentage of CD80(+) monocytes in comparison to CB1 (P = 0.020). HkBA in PHA cultures down-regulated the percentage of CD80(+) monocytes compared to PHA alone in all groups, especially in AB and CB patients (P < 0.001 and P = 0.007, respectively). In conclusion, the diminished percentage of CD4(+)/CD25(+) T cells in CB is not associated with inadequate CD80/CD28 co-stimulation. We speculate that differential frequency of CD80(+) monocytes after PHA stimulation could serve as a qualitative parameter of disease status, related to the different clinical forms of chronic brucellosis.

Unrelated Cord Blood Banking and Transplantation: Implications for Obstetricians

Cord blood is recognised as a rich source of haematopoietic stem and progenitor cells and its use has increased greatly in recent years. Since first cord blood transplantation was performed in Paris in 1988, knowledge of the biologic characteristics of umbilical cord blood has improved, and the benefits of using cord blood stem cells have become apparent. In fact, cord blood has showed to have several advantages as compared to bone marrow: easy availability, lower risk of infectious diseases transmission such as CMV and EBV and lower risk of graft versus host disease. However, cord blood units progenitors content is usually only sufficient for smaller recipients, usually children weighing less than 40 Kg. Gluckman et al showed grafting occurring in 85 percent of patients receiving 37 million or more nucleated cells per kilogram of body weight. In spite of this, use of cord blood in greater recipients is increasing and an effort to improve cord blood haematopoietic content is mandatory. In fact, the main limitation factor for the wide use of CB as a source of hematopoietic progenitor for transplantation is cell dose. Some of the specific areas identified by some studies for improvement of CB cell content are donor selection and CB collection. Implications of obstetricians in these issues are very important and necessary. We have evaluated cord blood donors selection criteria and two different modes of collection in order to optimise the cord blood banking proceedings and increase the quality of the CB units stored. We will review the results of our previous studies in these areas and the results of literature. Keywords: Umbilical cord blood (UCB), infectious disease, cryopreservation, hematopoietic cell, pregnancy, Multiple gestation, IVF, prematurity, morbidity, single embryo transfer, PGD, blastocyst, cleavage stage embryo, ovulation induction

Targeted Expansion and Regulation of Genetically Modified Cord Blood Stem/Progenitor Cells in vitro

目的 探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和安全性.方法 构建逆转录病毒载体MGI-F2JAK2,内含有JAK2基因的功能催化区和两个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(2xF36v,F2)组成.AP20187可与F36v特异结合引起JAK2二聚化而激活细胞内信号传导.该载体同时含有绿色荧光蛋白(GFP)报告基因,用作检测细胞增殖的标记.应用MiniMACS免疫磁珠分选系统纯化分离脐血CD34+细胞,用含JAK2的逆转录病毒上清转染脐血CD34+细胞.转导后的CD34+细胞在SCF、FL、TPO、IL-6细胞因子的联合培养条件下,以不加或加入AP20187分别作为对照组和实验组.定期检测CD34+细胞基因转移后GFP动态变化、细胞免疫标记、造血祖细胞集落培养和裸鼠致瘤试验.结果 分选的CD34+细胞纯度为91%以上,基因转移率为49.3%±6.2%;实验组AP20187+SCF+FL+TPO+IL-6(ASFTI)与对照组SCF+FL+TPO+IL-6(SFTI)组均可获得CD34+细胞大量扩增.随着培养时间的延长,实验组扩增的CD34+细胞GFP阳性率由基线水平逐渐上升于第11周时达到95%以上,而对照组GFP阳性率逐渐下降到基线水平以下并逐渐消失.在培养6周左右,实验组CD34+CD38-、CD34+CD38+细胞亚群扩增倍数分别为(228.26±32.31)、(321.48±40.52),分别与对照组相比,差异有显著性.ASFTI组转基因CD34+细胞于12周后仍可产生造血祖细胞集落(BFU-E、CFU-GM、CFU-Mix).扩增后CD34+细胞检测染色体核型正常,裸鼠实验无致瘤特性.结论 转染JAK2基因的人脐血CD34+细胞协同其他细胞因子可以体外长期扩增脐血干祖细胞,对今后开展干细胞治疗某些遗传性血液病有潜在的应用价值。

Unrelated donor cord blood transplantation for childhood severe aplastic anemia after a modified conditioning

Treatment of severe aplastic anemia (SAA) patients who lack human leukocyte antigen (HLA)-matched donors and failed immunosuppressive therapy (IST) is challenging. Recently, umbilical cord blood transplantation (CBT) after non-myeloablative therapy has been reported in adult but not in childhood SAA. However, most cases resulted in mixed donor chimerism and incomplete hematological recovery. We reported an 11-yr-old girl with recurred SAA 5 yr after IST who underwent unrelated donor CBT after a modified regimen. This patient had renal and cardiac dysfunction, and lacked suitable bone marrow donors. The 3.9 x 10(7)/kg CB cells from an HLA one-locus mismatched unrelated donor were infused after conditioning with total body irradiation (5 Gy), melphalan (120 mg/m(2)), and fludarabin (120 mg/m(2)). Hematological recovery was favorable in complete chimerism. A major complication was only skin graft-versus-host disease (grade I). CB could be an alternate stem cell source for childhood SAA after modified preparative regimen.

Populations of hippocampal inhibitory neurons express different levels of cytochrome c

Cytochrome c (CC) immunoreactivity was quantified in functionally distinct rat hippocampal inhibitory neuron populations using double immunocytochemistry and laser scanning confocal microscopy to measure the CC expression level as well as the amount of mitochondria within the cells, which is a sign of neuronal activity. The CC signal showed a similar distribution to cytochrome c oxidase histochemical staining. Strongly stained somata, dendrites and axon terminal clouds were dispersed over the low intensity neuropil staining. The staining was granular and electron microscopic investigation confirmed that the signal was localized in mitochondria. Intensively labeled neurons, showing the morphological features of inhibitory cells, were most frequently found in the principal cell layers, stratum oriens of the CA1-3 areas, stratum lucidum and hilus. These neurons contained not only a higher number of mitochondria than the principal cells but the intensity of the mitochondrial staining was evidently stronger. Among the examined interneuronal populations, parvalbumin-immunoreactive neurons were intensively labeled for CC. Calbindin D28k- (CB), somatostatin- and cholecystokinin-labeled cells showed heterogeneous CC levels, whereas calretinin-immunoreactive cells never showed a strong CC signal. CB cells in stratum oriens and alveus layers, lucidum and the hilus were strongly labeled for CC. CB cells in such regions are known to project to the medial septum and contain somatostatin. We have demonstrated that the CA1 interneurons that project to the medial septum (hippocampo-septal neurons) express a high level of CC. Thus, similar to the parvalbumin-containing basket and axo-axonic cells, the hippocampo-septal neurons potentially have a high average activity level.

Direct evidence for ex vivo expansion of human hematopoietic stem cells

AbstractTo characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Post-engraftment infections in adult patients transplanted with single cord blood units supported by co-infusion of mobilized purified hematopoietic stem cells from a third party donor

Abstract We have co-infused mobilized purified hematopoietic stem cells (HSC) from a third party donor to shorten neutropenia in single unit cord blood transplantation (CBT). We describe post-engraftment infectious complications in 33 consecutive adults with high-risk hematologic malignancies. Median age was 30 (range 16–59), 22 were male. Patients were conditioned with TBI or busulfan, fludarabine, cyclophosphamide and ATG. GVHD prophylaxis included CyA and steroids. Non-bacterial prophylaxis included fluconazol, acyclovir, trimethoprim-sulfamethoxazole and azithromycin or Fansidar (toxopositive cases). Median total infused CB cell dose was 2.26 x 107/kg (1.31–3.7). Pre-CBT toxoplasma serology was positive in 12, negative in 13 (4 received HSC from a seropositive donor) and unknown in 8 cases. Pre-CBT CMV serology was positive in 30 cases. Pre-CBT, 7 patients were HBsAb(+), HBsAg(−) and 1 HBsAg(+) HBsAb(−). ANC>500/uL was achieved by 32/33, median time 10 days (9–36). Full CB chimerism was achieved in 32/33 cases. Most clinically significant infections occurred after ANC recovery (no major neutropenic infections).There were 34 episodes of CMV reactivation (3 patients developed CMV peumonitis and died Short and long term results in adult CBT are improving. However we and others are reporting high incidence of post-engraftment unusual infections which seem related to delayed recovery of cell-mediated immunity. CMV, for which early diagnostic techniques and antiviral agents are available, heads the list. Our toxoplasma cases have prompted us to use prophylaxis in seropositive patients. HBV reactivation among HBsAb(+) HBsAg(−) patients raises the issue of prophylaxis during CBT versus close serological monitoring and pre-emptive therapy. Leishmaniasis is endemic among our city's dogs, so we actively search for it in FUO (BM examination and culture). Polyomavirus can be found in the urine of up to 50% of BM recipients, viruria preceding symptoms. The value of prospective monitoring and the role of antivirals are unknown. Other unusual infections (as tripanosomiasis in our country) require high index of awareness and consideration in FUO protocols (BM, PB smears). Adult CB recipients are at very high risk of fastidious non-bacterial infections requiring wide pre-BMT screening, close analytical and clinical monitoring, prophylactic/preemptive strategies or early aggressive therapy and innovative immunotherapeutic approaches.

Flow cytometric assessment of hematopoietic cell subsets in cryopreserved preterm and term cord blood, influence of obstetrical parameters, and availability for transplantation

The aim of this study was to characterize the lymphocyte and the hematopoietic stem and progenitor cell (HPC) subsets of cryopreserved premature cord blood (PCB) compared to term cord blood (TCB) by flow cytometry, to study the influence of birth conditions, and to assess its availability for transplantation. Four-color flow cytometric analysis was performed on 43 PCB and 40 TCB cryopreserved samples using a panel of 24 different mAbs, directed against lymphoid and HPC surface markers. The CB volume was estimated by the weight of the newborn to determine the absolute MNC and CD34(+) cell content/CB sample. Clinical and obstetrical data were recovered. Statistical comparisons and a multiple regression analysis were performed. No consistent differences were found in the mononuclear cell (MNC) or CD34(+) cell concentration (x10(6)/L) between PCB and TCB. The percentage of primitive HPC (CD34(+)CD38(-), CD34(+)CD38(-)CD90(-)HLA-DR(-), CD34(+)CD38(-)CD90(-)HLA-DR(+)) and primitive lymphoid progenitors (CD34(+)CD7(+), CD34(+)CD7(+)CD19(-)CD117(-)) were higher in PCB than in TCB. Correspondingly, TCB had an increased percentage of committed HPC. No sample of PCB contained >2 x 10(7) MNC/kg (and only 48% had >1 x 10(5) CD34(+) cells) for a recipient of 20 kg body wt, as the minimum threshold recommended for CB transplantation. Obstetrical factors modulated mainly lymphocyte subsets and fewer HPC subpopulations. Acute fetal distress increased CD34(+) cells, especially the immature subsets. Maternal treatment with dexamethasone and intrauterine growth retardation decreased CD3(+) cells. No other obstetrical factors played a detrimental effect on CB cells if used for transplantation. PCB is richer in immature cells both in lymphocyte and HPC populations, and its use for transplantation, at least in special cases, should be reconsidered.

Direct Evidence for Ex Vivo Expansion of Human Hematopoietic Stem Cells.

Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro.To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol , 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Alternative Donor Stem Cell Transplantation after Reduced Intensity Conditioning Regimen for Patients with Bone Marrow Failure.

Allogeneic hematopoietic stem cell transplantation (HSCT) with HLA geno-identical sibling donor is the treatment of choice for children and young adults with constitutional or acquired (SAA) bone marrow failure (BMF). However, results of unrelated HSCT for BMF have been poorer due to high transplant related mortality mainly related to rejection and GVHD. Generally, a myeloablative regimen HSCT is used for acquired and some constitutional BMF but not for Fanconi anemia (FA) patients for whom a low dose conditioning regimen is employed. We have driven the hypothesis that immunosuppressive reduced intensity conditioning regimen should decrease TRM, decreasing GVHD and allowing engraftment. In a Phase I-II trial, 20 patients (pts) with BMF were enrolled and transplanted between 2002 and 2004. Thirteen pts had a constitutional aplasia: FA n=11, congenital megakaryocytopenia (CMK) n=1, Rothmund-Thomson syndrome n=1 and 7 pts had SAA among those two had paroxystic nocturnal hemoglobinuria (PNH). There were 12 male and 8 female. Median age was 8 years for constitutional BMF and 26 years for SAA. The HSC source was bone marrow for 11 pts, PBSC for 1 pt and cord blood for 8 pts. Ten of the twelve BM or PBSC donors were HLA matched for 10 loci (A, B, C, DRB1, and DQB1) and eight cord blood donors were HLA mismatched with 2 generic differences and were used for FA. All pts received the same conditioning regimen consisting of Busulfan (3mg/kg x 2), cyclophosphamide (10mg/kg x 4), fludarabine (30mg/m2 x3) and ATG (2.5mg/kg x4). The mean of nucleated stem cells infused and CD34 + cells was 2.8x108/kg and 5.9x106/kg respectively for the 12 pts who received BM stem cells and PBSC and 6.4x107/kg and 4.6x106/kg respectively for the eight pts who received CB cells. Acute GVH disease prophylaxis consisted of ciclosporine A (CsA) for pts with constitutional BMF and CsA and short course methotrexate for 6 of the 7 pts with acquired BMF, (one received tacrolimus instead of CsA due to thrombotic pre-existing co-morbidity). One pt (with CMK) died on day 0 from cerebral haemorrhage. Eighteen pts out of 19 had WBC recovery with a median time of 23 days (11–42); one FA pt did never reach sustained engraftment and died at D+291 from adenovirus infection. Three others had late graft rejection: in a context of acute GvHD and EBV infection and pulmonary aspergillosis for two pts with SAA who received BM graft and with acute GvHD and adenovirus infection for one FA pt who received CB graft. The conditioning was well tolerated without severe mucositis even in FA patients, sixteen patients experienced transient liver abnormalities. Nine patients developed reversible haemorrhagic cystitis at a median of 47 days post-transplant. There were 3 bacterial, 10 viral and 5 fungal infections with a cumulative incidence of TRM at one year of 45 ±24%. The cumulative incidence of acute GVH (II–IV) was 50 ±23%. Overall survival (OS) at one year was 55±11 %. It was 86%± 13 for SAA and 38% ± 13 for constitutional BMF. In spite of the short follow-up and few patients included, reduced intensity conditioning regimen provides encouraging results for patients with SAA. For constitutional BMF, low toxicity was observed, however the overall results seem similar to those reported in the literature using other RIC regimen and are probably related to other factors than the conditioning regimen.

Uptake of Protamine Sulphate Complexed Fluorescent Nano-Particles Is Defined by Cell Cycle Status in Primary Human CD34+ Cells: Use of a Multi-Color p27 kip1 Based Flow Cytometric Assay.

The use of iron based nano-particles for multi-modal imaging is gaining interest, since it allows high resolution non-invasive in vivo imaging of human hematopoietic homing and engraftment events in xenograft models. The uptake of ferridex nano-particles complexed to cationic protamine sulphate is believed to be non-specific through mechanisms like endocytosis, but this has not been well defined for hematopoietic stem cells (HSC). In defining ex vivo cultivation strategies for manipulation of human HSC, a key factor is the responsiveness of the most primitive cells to the in vitro conditions, with the aim of maintaining viability without inducing terminal differentiation. Here, we present a novel flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo protocol, aimed at facilitating labeling of human stem/progenitor cells using protamine sulphate complexed nano-particles for subsequent in vivo imaging. We used intracellular staining for the cell cycle inhibitor p27kip1, which is present in the highest levels in non-cycling cells, as the primary flow cytometric marker in combination with CD34, CD133 and Alexa 488, 647 and 750 conjugated ferridex nano-particles and the membrane dye PKH26. An assay was developed to simultaneously assess the molecular events occurring in individual human cord blood Lin− or CD34+ cells while they were cultured for up to 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates with or without nano-particles. Co-expression of p27kip1, CD34 or CD133 in the cultured cells slowly decreases from 86.1% CD34+p27kip1 (T=0) to 76.7%+/−12.2% (T=72) and from 89.6% CD133+p27kip1+ (T=0) to 54.1%+/−10.4% (T=72). We suggest that this slow decrease represents cells dividing and potentially differentiating over the time course of the ex vivo cultivation period. Assessing uptake of fluorescent conjugated nano-particles over a 72 hr period showed that the uptake of particles in CD34+ and CD133+ cells declined significantly after the first 24 hrs., from 32.5+/−3.7% nano-positive CD34+ cells to 19.2+/−2.9% at 48 hours ex vivo with a more significant decline to only 8.3+/−3.7% nano positive CD34+ cells in the culture after 72 hours ex vivo. The same decline in uptake over time was observed in cultured human CB cells that were positive for CD133. PKH26 co-staining demonstrated that the majority of cells that undergo cell division within the first 24 hours of ex vivo culture are the most likely to uptake the nano-particles. In summary, using a multi color p27kip1 based flow-cytometry assay, we found that human Lin−, CD133+, and CD34+ cells uptake Fe-Pro in a fashion which is not entirely cell cycle independent as previously suggested. These data indicate that cell cycle or metabolic status may influence the ability of human hematopoietic stem and progenitor subsets to uptake the protamine sulphate-complexed nano-particles. These findings emphasize the need to carefully develop ex vivo conditions for nano-particle labeling of primary human stem cells in order to perform accurate in vivo imaging of the most primitive human hematopoietic stem and progenitor cells.

Transplantation of Ex Vivo Expanded Cord Blood Progenitor Cells: First Experience in Two Children Affected by Hemoglobinopathies.

The main limitations of CB transplantation (CBT) are represented, in comparison to bone marrow transplantation, by a lower probability of engraftment and a slower hematopoietic recovery which can favor the occurrence of fatal complications. Several studies have clearly documented that the higher the number of cells infused, the faster the rate of engraftment and the lower the risk of transplant-related mortality. In order to increase the number of CB cells infused, approaches based on ex vivo expansion of CB progenitor cells are, in principle, particularly promising. In this regard, we developed a clinical protocol to expand the number of CB hematopoietic progenitor cells (HPCs), based on the use of a cytokine cocktail including interleukin-6 (10 ng/ml), stem cell factor (50 ng/ml), Flt-3 ligand (50 ng/ml) and thrombopoietin (10 ng/ml), in the presence of a serum-free medium and 10% AB allogeneic plasma. This combination induced significant and sustained ex vivo expansion of primitive CB HPCs. After clinical grade immunoselection (CliniMACS, Miltenyi Biotech), CD34+ cells were cultured at a concentration of 5x103 cells/ml for 2 weeks. Cytokines were added at the onset of culture and replaced twice a week. The purity of CD34+ cells (median and range) after immunoselection of thawed CB samples was 79.5% (47–96). After culture, the median fold expansion of total nucleated cells (NC) and CFU-GM was around 2 logs, while that of CD34+ cells was around 1 log. The expanded cells had levels of endotoxin below the required limit of 0.05IU/mL (E.Ph.) and were negative for mycoplasma, bacterial and fungal contamination.Two children, one with thalassemia and one with sickle cell disease (SCD), were given ex vivo expanded CD34+ cells from an HLA-identical sibling. In both cases the conditioning regimen included busulfan, thiotepa and fludarabine, while GVHD prophylaxis consisted of cyclosporine-A. The first patient, a 6-year-old boy with thalassemia, was given 2 CB units derived from 2 HLA-identical fraternal (i.e. non syngeneic) twins (1 unmanipulated CB unit and 1 CB unit infused after ex vivo expansion). The fold expansions of NC and CD34+ cells were 623 and 56.6 respectively. The patient had neutrophil recovery (i.e. above 500/cmm) on day +16, while the platelet recovery (i.e. above 30,000/cmm) occurred on day +31. The second patient, a 7-year-old girl with SCD, was given 2/3 of the CB unit unmanipulated and 1/3 after ex vivo expansion. The fold expansions of NC and CD34+ cells were 189.3 and 30.5 respectively. Neutrophil and platelet recovery occurred on day +18 and +33 respectively. Neither acute nor chronic GVHD occurred in both patients, who are alive and disease free at 12 and 5 months after CBT respectively. In the first patient, chimerism evaluation showed the presence of erythroid and myeloid colonies, as well as of T cells, derived from the ex vivo expanded cells up to 7 months after CBT. Mesenchymal stem cells remained of patient origin in both cases. These preliminary results indicate that our expansion procedure is safe and able to ex vivo expand CB HPCs; the expanded cells seem to accelerate hematopoietic recovery and, for the first time, we demonstrate that these cells contribute to sustain hematopoietic recovery after CBT. Altogether these results could widen the applicability of CBT in adults.

Optimal Ex-Vivo Expansion (EvE) of Cord Blood (CB) Natural Killer (NK) Cells, NK Cytotoxicity and NK Receptor (NKR) Expression: Potential for CB Adoptive Cellular Immunotherapy (ACI).

CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several triggering receptors such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta, et al, Curr Opinion in Immunol, 2004; Marcenaro et al, Eur J Immunol, 2003). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood, 2001; Locatelli, et al Blood,1999). We demonstrated the ability to EvE CB in short term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2A (Ayello/Cairo et al, BBMT, 25a, 2004). In this study, we compared short-term cultures (48 hrs) with prolonged cultures (4 to 10 days) on expansion, maturation and expression of NCR, NKG2, KIR and cytolytic mechanisms in previously cryopreserved CB that were TCTE. CB was cryopreserved and thawed by the NHLBI/COBLT method. (Kurtzberg/Cairo, Transfuison, 2005). Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free medium alone or with anti-CD3 (50 ng/ml, IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) (ABCY). NK receptor expression (CD94, NKG2C, NKG2D, NKp44, NKp46, KIR2DS4) and intracellular perforin and granzyme B activity were determined by flow cytometry. NK and LAK cytotoxicity was measured by europium release assay. Significant increases were seen in NK activating KIR2DS4 at day 10 vs 2 in ABCY both in CD3−/16+/56+ dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9±0.2%, p<0.001, respectively). C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4 ±0.43 vs 23.7±2.0 %, p<0.001). A significant increase was seen in NK (CD3−/16+/56+dim) KIR3DL1 subset at day 10 vs 2 (38.3±2.8 vs 18.9±6.3 7%, p<0.05). In contrast, NCR expression in CD3−/16+/56+ dim NKp44 subset was significantly decreasedat day 10 vs 2 of EvE with ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). A significant decrease was seen in CD3−/16+/56+ dim NKp46 expression following day 10 vs 2 (8.5±0.2 vs 23.5±1.2 %, p<0.001). Perforin expression demonstrated a significant decrease in ABCY at day 10 vs 2 (55.7±1.8 vs 84.3±1.3%, p<0.001) yet increasing levels of granzyme B from day 2 to 10 (25.8±1.8 vs 45.1±1.7%, p<0.0001). A significant increase in CB NK and LAK cytotoxicity was seen in ABCY on day 10 vs 2 (NK: 71.5 ±1.6 vs 53.8±10.3%, p<0.001; LAK: 63.2±0.24 vs 31.8±1.8%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed and at a later date EvE and activated for 7–10 days to yield viable NK subsets. EvE in ABCY at 10 days yielded increased expression of NK KAR (CD56+dim and bright) and granzyme B expression but decreased NK C-lectin CD94/NKG2D, NCR NKp46 and NKp48 and perforin expression.