Comparison of phenotypic and functional dendritic cells derived from human umbilical cord blood and peripheral blood mononuclear cells

Dendritic cells (DC) are important accessory cells that are capable of initiating an immune response. Generation of functional DC has potential clinical use in treating diseases such as cancer. In this report, we have demonstrated the generation of functional DC from mononuclear cells isolated from human umbilical cord blood cells (UCBC) and peripheral blood cells (PBC) using a defined medium Prime Complete Growth Medium (PCGM) (GenePrime LLC, Gaithersburg, MD). DC generated using PCGM showed the typical phenotype of DC as determined by flow cytometry and electron microscopy. Further analysis of the DC using confocal microscopy showed localization of the antigen and major histocompatibility complex (MHC) molecules in the cytoplasm 3-5 days following tumor antigen loading into DC. Subsequently, the tumor antigen-MHC complex was localized on the surface of DC. DC generated from UCBC or PBC also increased (p < 0.001) the allogeneic mixed lymphocyte reaction, confirming their immune accessory functions compared to a control mixed lymphocyte reaction (MLR) without DC added. Interestingly, DC generated using PCGM medium also significantly enhanced the hematopoietic colony (CFU-C)-forming ability. Furthermore, addition of 5% DC derived from cord blood loaded with tumor antigen also significantly (p < 0.001) increased peripheral and cord blood-derived antigen-specific cytotoxic T lymphocyte (CTL)-mediated killing of human leukemic cells (K562) and breast cancer cells (MDA-231). Thus, these results show that functional DC generated from cord blood using a defined medium are a useful source of accessory cells for augmenting CTL-mediated cytotoxicity and have potential use in cellular therapy for human leukemia and breast cancer.     

A simple two-step culture system for the large-scale generation of mature and functional dendritic cells from umbilical cord blood CD34+ cells

BACKGROUND: In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood–derived CD34+ cells is described.
STUDY DESIGN AND METHODS: CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables.
RESULTS: This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting.
CONCLUSION: A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy.     

Efficient generation of functional hepatocyte‐like cells from menstrual blood‐derived stem cells

In recent years, the advantages of menstrual blood‐derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum‐supplemented or serum‐free culture media, were also investigated. The sequential differentiation was monitored by real‐time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT‐4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin‐18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte‐specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte‐like cells. We recommend a complementary serum‐free differentiation protocol for enrichment of in vitro production of functional MenSC‐derived hepatocyte‐like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

人脐带和骨髓源间充质干细胞对脐血单个核细胞增殖能力的影响

目的：探讨人脐带间充质干细胞（UCMSCs）与骨髓间充质干细胞（BMMSCs）在体外对造血干细胞的支持作用。方法分别从人脐带和骨髓中分离、培养间充质干细胞,通过免疫细胞化学染色等方法对其进行表型鉴定;采用流式细胞仪测定脐血单个核细胞的周期分布,采用甲基纤维素法测定脐血单个核细胞混合集落形成单位（CFU-Mix）,比较 UCMSCs和BMMSCs对脐血单个核细胞细胞周期、CFU-Mix形成能力的影响。结果成功培养获得 UCMSCs和BMMSCs,鉴定结果符合预期;与非共培养组细胞相比,UCMSCs和 BMMSCs共培养均能促进脐血单个核细胞进入增殖周期,并增加其形成CFU-Mix的能力（P＜0．05）,但UCMSCs和BMMSCs共培养组之间比较差异无统计学意义（P＞0．05）。结论成功从人脐带和骨髓组织中培养获得间充质干细胞,两种来源的间充质干细胞均能提高脐血单个核细胞的体外增殖能力及 CFU-Mix形成能力,均具有造血支持作用。     

In vitro protection of umbilical cord blood–derived primitive hematopoietic stem progenitor cell pool by mannose‐specific lectins via antioxidant mechanisms

BACKGROUND: Earlier we reported that an oral administration of two mannose‐specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice. STUDY DESIGN AND METHODS: Cord blood–derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum‐ and growth factor–free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin‐coupled paramagnetic beads. RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long‐term culture‐initiating cell (LTC‐IC) and extended LTC‐IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin‐coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays. CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost‐effective HSPC enrichment tools.     

A simple,xeno‐free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation

Oligodendrocyte progenitors (OPCs) are ranked among the most likely candidates for cell‐based strategies aimed at treating neurodegenerative diseases accompanied by dys/demyelination of the central nervous system (CNS). In this regard, different sources of stem cells are being tested to elaborate xeno‐free protocols for efficient generation of OPCs for clinical applications. In the present study, neural stem cells of human umbilical cord blood (HUCB‐NSCs) have been used to derive OPCs and subsequently to differentiate them into mature, GalC‐expressing oligodendrocytes. Applied components of the extracellular matrix (ECM) and the analogues of physiological substances known to increase glial commitment of neural stem cells have been shown to significantly increase the yield of the resulting OPC fraction. The efficiency of ECM components in promoting oligodendrocyte commitment and differentiation prompted us to investigate the potential role of gelatinases in those processes. Subsequently, endogenous and ECM metalloproteinases (MMPs) activity has been compared with that detected in primary cultures of rat oligodendrocytes in vitro, as well as in rat brains in vivo. The data indicate that gelatinases are engaged in gliogenesis both in vitro and in vivo, although differently, which presumably results from distinct extracellular conditions. In conclusion, the study presents an efficient xeno‐free method of deriving oligodendrocyte from HUCB‐NSCs and analyses the engagement of MMP‐2/MMP‐9 in the processes of cell commitment and maturation. Copyright © 2015 John Wiley & Sons, Ltd.     

Therapeutic effect of long‐interval repeated intravenous administration of human umbilical cord blood‐derived mesenchymal stem cells in DBA/1 mice with collagen‐induced arthritis

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen‐induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long‐interval repeated intravenous administration of human umbilical cord blood‐derived (hUCB)‐MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long‐interval repeated intravenous administration of hUCB‐MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB‐MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin‐O staining, and toluidine blue staining. We used real‐time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB‐MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB‐MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB‐MSCs also significantly decreased IL‐1 beta protein levels in multimedium and multihigh groups and IL‐6 protein levels in all hUCB‐MSCs‐treated groups. Furthermore, mRNA levels of IL‐1 beta and IL‐6 were decreased significantly in all hUCB‐MSCs‐treated groups, whereas the expression of anti‐inflammatory cytokine IL‐10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long‐interval repeated intravenous administration of hUCB‐MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose‐dependent manner.     

Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product.
STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-γ (IFN-γ). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated.
RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-γ and soluble CD40L/IFN-γ were used.
CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.     

Comparison of in vitro‐cultivation of human mesenchymal stroma/stem cells derived from bone marrow and umbilical cord

Cell‐mediated therapy is currently considered as a novel approach for many human diseases. Potential uses range from topic applications with the regeneration of confined tissue areas to systemic applications. Stem cells including mesenchymal stroma/stem cells (MSCs) represent a highly attractive option. Their potential to cure or alleviate human diseases is investigated in a number of clinical trials. A wide variety of methods has been established in the past years for isolation, cultivation and characterization of human MSCs as expansion is presently deemed a prerequisite for clinical application with high numbers of cells carrying reproducible properties. MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro. Accordingly, different isolation, culture and upscaling techniques contribute to the heterogeneity of MSC characteristics and the, sometimes, controversial results. Therefore, this review discusses and summarizes certain experimental conditions for MSC in vitro culture focusing on adult bone marrow‐derived and neonatal umbilical cord‐derived MSCs in order to enhance our understanding for MSC tissue sources and to stratify different procedures. Copyright © 2016 John Wiley & Sons, Ltd.     

Collagen scaffold combined with human umbilical cord‐derived mesenchymal stem cells promote functional recovery after scar resection in rats with chronic spinal cord injury

Effective therapeutic strategies for treating chronic spinal cord injury (SCI) are currently unavailable. Scar tissue in the lesion area is a main inhibitory factor for axonal regeneration and repair of chronic SCI. In this study, scar tissue was surgically resected from adult rats with 12 week chronic SCI and then collagen scaffold (NeuroRegen Scaffold; NRS) and human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) were implanted into the resected cavity to repair chronic SCI. The results demonstrated that the locomotor function of rats was not affected by surgical scar resection, indicating its safety in treating chronic SCI. Implanting NRS and hUC‐MSCs promoted locomotion in rats and improved cortical motor‐ and somatosensory‐evoked potentials. Furthermore, implanting NRS and hUC‐MSCs promoted neurofilament‐ and β‐tubulin‐III‐positive neural regeneration and remyelination, elicited β‐tubulin‐III‐positive neuron production in the lesion area and blocked astrocyte growth outside the lesion area. In conclusion, implanting NRS in combination with hUC‐MSCs provided a beneficial microenvironment for neural regeneration, showing significant therapeutic effects for chronic SCI.     

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix‐derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)‐derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non‐invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large‐scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate‐based culture supplement (UltraGRO^TM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC‐like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO^TM‐supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO^TM‐supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13‐fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic‐free microcarrier‐based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.     

Reduced sirtuin 1/adenosine monophosphate‐activated protein kinase in amyotrophic lateral sclerosis patient‐derived mesenchymal stem cells can be restored by resveratrol

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neuron system. Our previous study has shown that bone marrow‐mesenchymal stem cells (BM‐MSCs) from ALS patients have functional limitations in releasing neurotrophic factors and exhibit the senescence phenotype. In this study, we examined sirtuin 1/adenosine monophosphate‐activated protein kinase (SIRT1/AMPK) activities and identified significant decreases in the ALS‐MSCs compared with normal healthy control originated BM‐MSCs. This decline was restored by pretreatment with resveratrol (RSV), measured using quantitative polymerase chain reaction, NAD/NADH assay, and immunoblot analysis. Neuroprogenitor markers were increased in RSV‐treated ALS‐MSCs (RSV/ALS‐MSCs). The differentiated ALS‐MSCs (ALS‐dMSCs) exhibited a cell body and dendritic shape similar to neurons. RSV/ALS‐MSCs showed significantly increased differentiation rate as compared with the untreated ALS‐dMSCs. The neurite numbers and lengths were also significantly increased. This was confirmed with immunoblot analysis using neuron specific markers such as nestin, NF‐M, Tuj‐1, and Map‐2 in RSV/ALS‐dMSCs. Thus, this study shows that ALS‐MSCs showed down‐regulation of AMPK/SIRT1 signalling, which was recovered by treatment with RSV. This data suggest that RSV can be one of the candidate agents for improving therapeutic efficacy of ALS patients' originated MSCs.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Functionalization of porous BCP scaffold by generating cell‐derived extracellular matrix from rat bone marrow stem cells culture for bone tissue engineering

The potential of decellularized cell‐derived extracellular matrix (ECM) deposited on biphasic calcium phosphate (BCP) scaffold for bone tissue engineering was investigated. Rat derived bone marrow mesenchymal stem cells were cultured on porous BCP scaffolds for 3 weeks and decellularized with two different methods (freeze–thaw [F/T] or sodium dodecyl sulfate [SDS]). The decellularized ECM deposited scaffolds (dECM‐BCP) were characterized through scanning electron microscopy, energy dispersive X‐ray spectrometer, and confocal microscopy. The efficiency of decellularization was evaluated by quantifying remaining DNA, sulfated glycosaminoglycans, and collagens. Results revealed that F/T method was more effective procedure for removing cellular components of cultured cells (95.21% DNA reduction) than SDS treatment (92.49%). Although significant loss of collagen was observed after decellularization with both F/T (56.68%) and SDS (70.87%) methods, F/T treated sample showed higher retaining amount of sulfated glycosaminoglycans content (75.64%) than SDS (33.28%). In addition, we investigated the cell biocompatibility and osteogenic effect of dECM‐BCP scaffolds using preosteoblasts (MC3T3‐E1). Compared to bare BCP scaffolds, dECM‐BCP_F/T scaffolds showed improved cell attachment and proliferation based on immunofluorescence staining and water soluble tetrazolium salts assay (p < .001). Moreover, dECM‐BCP scaffolds showed increased osteoblastic differentiation of newly seeded preosteoblasts by up‐regulating three types of osteoblastic genes (osteopontin, alkaline phosphatase, and bone morphogenic protein‐2). This study demonstrated that functionalization of BCP scaffold using cell‐derived ECM could be useful for improving the bioactivity of materials and providing suitable microenvironment, especially for osteogenesis. Further study is needed to determine the potential of dECM‐BCP scaffold for bone formation and regeneration in vivo.