Development of a unique epigenetic signature during in vivo Th17 differentiation

Activated naive CD4⁺ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4⁺ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4⁺ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

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.     

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34⁺ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34⁺ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34⁺ cells. However, in contrast to what occurs in primary CD34⁺ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34⁺ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed.     

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.     

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.     

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture

Background aimsExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.MethodsWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34⁺ cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.ResultsThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34⁺ cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34⁺ cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.ConclusionsCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.     

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies

Background aimsBone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34⁺-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.MethodsThe need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34⁺ cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34⁺ cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells.ResultsGene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14⁺ subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively.ConclusionsOur data suggest that combining basal and expanded BM-derived CD34⁺ cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.     

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.     

Chromosomal stability during ex vivo expansion of UCB CD34(+) cells

Objectives: Ex vivo expansion is a feasible strategy, which may overcome limitation of the very low frequency of haematopoietic stem/progenitor cells, in umbilical cord blood (UCB). However, both quality of cells and safety of expanded population are critical issues to be addressed for their clinical application. Hence, in this study, we evaluated genetic stability of UCB‐derived CD34⁺ cells during ex vivo culture, based on karyotype analysis, as well as its effect on cell proliferation characteristics. Materials and methods: CD34⁺ cells were isolated from human UCB samples by immunomagnetic separation and were expanded ex vivo over a 28‐day period. Expansion of total nucleate cells, CD34⁺ cells and CD34⁺ CD38^? cells was investigated. Karyotype analysis of the expanded cells from six randomly selected UCB samples was performed to evaluate their genetic stability. Results: Chromosomal abnormality of expanded cells mainly appeared by day 14, but was seldom sustained until day 28. None of the chromosomal abnormal samples displayed neoplastic proliferation, and expanded cells with altered chromosomes did not show obvious transformation phenomena according to soft agar assay. Conclusions: Ex vivo expansion could lead to occurrence of chromosomal abnormality, although here it did not produce excessive proliferative advantage of the expended cells. Importantly, chromosomal alteration seemed not to be inheritable and unlikely to result in malignant transformation. However, further in‐depth evaluation of potential clinical risks of chromosomal abnormality is warranted.     

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background

Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34⁺ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/Principal Findings

Using commercially available G-CSF mobilized peripheral blood (PB) CD34⁺ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/Significance

This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34⁺ cells.     

Ex vivo expansion of human umbilical cord blood hematopoietic progenitor cells in a novel three-dimensional culture system

A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34⁺ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.     

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment

Background aimsAdvantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo.MethodsCD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.ResultsBoth FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes.ConclusionsAlthough FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.     

Substance P enhances mesenchymal stem cells-mediated immune modulation

Since clinical application of MSCs requires long-term ex vivo culture inducing senescence in MSCs and reducing the therapeutic activity of transplanted MSCs, numerous efforts have been attempted to sustain the active state of MSCs. Substance P (SP) is a neuropeptide that functions to activate the cellular physiological responses of MSCs, including proliferation, migration, and secretion of specific cytokines. In this study, we explored the potential of SP to restore the weakened immune modulating activity of MSCs resulting from long-term culture by measuring T cell activity and interleukin-2 (IL-2) secretion of CD4⁺ Jurkat leukemic T cells and primary CD4⁺ T cells. As the number of cell passages increased, the immunosuppressive function of MSCs based on T cell activity decreased. This weakened activity of MSCs could be restored by SP treatment and nullified by co-treatment of an NK1 receptor blocker. Higher levels of transforming growth factor beta 1 (TGF-β1) secretion were noted in the medium of SP-treated late passage MSC cultures, but IL-10 levels did not change. SP-treated MSC-conditioned medium decreased T cell activity and IL-2/Interferon gamma (IFN-g) secretion in T cells even in the activation by lipopolysaccharide (LPS) or CD3/CD28 antibodies, both of which were successfully blocked by inhibiting the TGF beta signaling pathway. This stimulatory effect of SP on late passage MSCs was also confirmed in direct cell–cell contact co-culture of MSCs and CD4⁺ Jurkat T cells. Collectively, our study suggests that SP pretreatment to MSCs may recover the immunosuppressive function of late passage MSCs by potentiating their ability to secrete TGF-β1, which can enhance the therapeutic activity of ex vivo expanded MSCs in long-term culture.     

Human CD141+ dendritic cells generated from adult peripheral blood monocytes

Human CD141⁺ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141⁺ DCs from cord blood CD34⁺ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141⁺ DCs. After culture of CD14⁺ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141⁺ DCs can be sufficiently generated from peripheral blood CD14⁺ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.     

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4⁺ T, CD19⁺ B, and splenic CD4^-CD19^- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19⁺ and CD4^-CD19^- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.