Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self‐renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34⁺ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway—PD0325901 (PD)—significantly reduces the expansion of CD34⁺ and CD34⁺ CD38^? cells, while there is no change in the expression of stemness‐related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34⁺ cells. Notably, when ERK pathway is blocked in UCB‐MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst‐forming unit‐erythroid colony (BFU‐E) as well as enhancement of erythroid glycophorin‐A marker. These results are in total conformity with up‐regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down‐regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self‐renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB‐haematopoietic progenitor cells.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56⁺CD3⁻ NK cell products could be routinely generated from freshly selected CD34⁺ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34⁺ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56⁺ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34⁺ cells for cancer immunotherapy.     

Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34⁺ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 ⁺ cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 μM), 10 μM RES was demonstrated to be the most favorable for ex vivo CD34 ⁺ cells expansion. In the primary cultures, 10 μM RES favored higher expansion folds of CD34 ⁺ cells, CD34 ⁺CD38 ⁻ cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 ⁺CD38 ⁻CD45R ⁻CD49f ⁺CD90 ⁺ cells) in 10 μM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 ⁺ cells derived from primary cultures with 10 μM RES exhibited significantly higher total cells and CD34 ⁺ cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 μM RES group were both higher than those of without RES group, demonstrating that CD34 ⁺ cells expanded with 10 μM RES possessed better biological function. Furthermore, the addition of 10 μM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 ⁺ cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.     

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Dose‐intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34⁺ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum‐free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800‐fold expansion in cell number was achieved for UCB, and up to 4,000‐fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10‐fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave‐type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832–840 © 2009 Wiley Periodicals, Inc.     

Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34⁺ cells but also increases CD34⁻ cell numbers. These CD34⁻ cells influenced the ex vivo expansion of CD34⁺ cells. In this work, the effects of periodically removing CD34⁻ cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34⁻ cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34⁺ cells in the CD34⁻ cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34⁻ cells with SR1 addition improved the biological function of expanded CD34⁺ cells and significantly increased the percentage of self‐renewal symmetric division of CD34⁺ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34⁻ cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34⁻ cells plus cooperation with SR1 improved the expansion of CD34⁺ cells, maintained better biological function of expanded CD34⁺ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

SR1 increased self‐renewal symmetric division of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 increased ex vivo expansion of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 further downregulated AhR signalling     

Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34⁺ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin^-CD34⁺CD38^lowCD45RA^-CD90⁺ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.     

A stromal-free,serum-free system to expand ex vivo hematopoietic stem cells from mobilized peripheral blood of patients with hematologic malignancies and healthy donors

Background aimsThe number of hematopoietic stem cells (HSCs) is critical for transplantation. The ex vivo expansion of mobilized peripheral blood (MPB) HSCs is of clinical value for reconstitution to meet clinical need.MethodsThis study proposed a simple, defined, stromal-free and serum-free culture system (SF-HSC medium) for clinical use, which is composed of Iscove's modified Dulbecco's medium, cytokine cocktails and serum substitutes. This study also characterized the cellular properties of expanded MPB CD133⁺ HSCs from patients with hematologic malignancies and healthy donors by surface antigen, colony-forming cell, long-term culture-initiating cell, gene expression and in vivo engraftment assays.ResultsThe expanded fold values of CD45⁺ white blood cells and CD34⁺, CD133⁺, CD34⁺CD38^?, CD133⁺CD38^?, CD34⁺CD133⁺, colony-forming and long-term culture-initiating cells at the end of 7-day culture from CD133⁺ MPB of hematologic malignancies were 9.4-fold, 5.9-fold, 4.0-fold, 35.8-fold, 21.9-fold, 3.8-fold, 11.8-fold and 6.7-fold, and values from healthy donor CD133⁺ MPB were 20.7-fold, 14.5-fold, 8.5-fold, 83.8-fold, 37.3-fold, 6.2-fold, 19.1-fold and 14.6-fold. The high enrichment of CD38^? cells, which were either CD34⁺ or CD133⁺, sustained the proliferation of early uncommitted HSCs. The expanded cells showed high levels of messenger RNA expression of HOBX4, ABCG2 and HTERT and had the in vivo ability to re-populate NOD/SCID mice.ConclusionsOur results demonstrated that an initial, limited number of MPB CD133⁺ HSCs could be expanded functionally in SF-HSC medium. We believe that this serum-free expansion technique can be employed in both basic research and clinical transplantation.     

Residues 39-56 of Stem Cell Factor Protein Sequence Are Capable of Stimulating the Expansion of Cord Blood CD34+ Cells

Background

Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC.

Methods

Eleven polypeptides were synthesized, which were designed to cover the full-length of rhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptides on human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometer whereas CD34⁺ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture.

Results

Of all polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 39–56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34⁺ cells derived from human umbilical cord blood (UCB). Addition of P0 increased the numbers of total mononucleated cells and CD34⁺ cells, by ~2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansion assay.

Conclusion

Residues 39–56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34⁺ cells. The peptide P0 is a potential candidate for further development as a synthetic substitute for rhSCF in laboratory and clinical applications.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Background aimsNaturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1–5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed.MethodsSeveral methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model.ResultsWith the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo–expanded Treg were CD4⁺25⁺FOXP3⁺127^lo and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro.ConclusionsIn our NOD-SCID IL-2Rγ^null xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo–expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.     

Enhancement of wound closure in diabetic mice by ex vivo expanded cord blood CD34+ cells

Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34⁺ cells (CB-CD34⁺ cells), freshly isolated CB-CD34⁺ cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34⁺ cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34⁺ cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34⁺ cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34⁺ cells and the cytokine cocktail were shown to promote recruitment of CD31⁺-endothelial cells in the wounds. Both the CB-CD34⁺ cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34⁺ cells or freshly isolated CB-CD34⁺ cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34⁺ cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34⁺ cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation.     

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)‐derived HSC expanded in a serum‐free/stromal‐based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB‐cells reached the ninth generation, whereas BM‐cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7–3.8% of BM CD34⁺ cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 ± 6.3% to 30.6 ± 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34⁺CD38^? cells present at the end of culture arose from proliferating CD34⁺CD38⁺ cells that downregulated CD38 expression, while in CB, a CD34⁺CD38^? population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft. J. Cell. Physiol. 220: 102–111, 2009. © 2009 Wiley‐Liss, Inc.     

High‐dose wogonin exacerbates DSS‐induced colitis by up‐regulating effector T cell function and inhibiting Treg cell

Wogonin exerts anti‐tumour activities via multiple mechanisms. We have identified that high‐dose wogonin (50 or 100 mg/kg) could inhibit the growth of transplanted tumours by directly inducing tumour apoptosis and promoting DC, T and NK cell recruitment into tumour tissues to enhance immune surveillance. However, wogonin (20–50 μM) ex vivo prevents inflammation by inhibiting NF‐κB and Erk signalling of macrophages and epithelial cells. It is elusive whether high‐dose wogonin promotes or prevents inflammation. To investigate the effects of high‐dose wogonin on murine colitis induced by dextran sodium sulphate (DSS), mice were co‐treated with DSS and various doses of wogonin. Intraperitoneal administration of wogonin (100 mg/kg) exacerbated DSS‐induced murine colitis. More CD4⁺ CD44⁺ and CD8⁺ CD44⁺ cells were located in the inflamed colons in the wogonin (100 mg/kg) treatment group than in the other groups. Frequencies of CD4⁺ CD25⁺ CD127^? and CD4⁺ CD25⁺ Foxp3⁺ cells in the colons and spleen respectively, were reduced by wogonin treatment. Ex vivo stimulations with high‐dose wogonin (50–100 μg/ml equivalent to 176–352 μM) could synergize with IL‐2 to promote the functions of CD4⁺ and CD8⁺ cells. However, regulatory T cell induction was inhibited. Wogonin stimulated the activation of NF‐κB and Erk but down‐regulated STAT3 phosphorylation in the CD4⁺ T cells. Wogonin down‐regulated Erk and STAT3‐Y705 phosphorylation in the regulatory T cells but promoted NF‐κB and STAT3‐S727 activation. Our study demonstrated that high‐dose wogonin treatments would enhance immune activity by stimulating the effector T cells and by down‐regulating regulatory T cells.     

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in <Emphasis Type="Italic">Chlamydia</Emphasis>-triggered reactive arthritis

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8⁺ T cells in HLA-B27⁺-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8⁺ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8⁺ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27⁺ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8⁺ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8⁺ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8⁺ T cells could be increased by stimulating CD8⁺ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8⁺ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27⁺ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.     

Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34⁺ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34⁺ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.This work was supported by the Zhejiang Scientific Foundation (no. 2003C23015).