Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Cotransplantation of human umbilical cord-derived mesenchymal stem cells and umbilical cord blood-derived CD34+ cells in a rabbit model of myocardial infarction

The objective of the study is to investigate the effect of hypoxic preconditioning on the immunomodulatory properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and the effect of cotransplantation of hUC-MSCs and human umbilical cord blood (hUCB)-derived CD34⁺ cells in a rabbit model of myocardial infarction. hUC-MSCs with or without hypoxic preconditioning by cobalt chloride were plated in a 24-well plate, and then cocultured with hUCB-CD34⁺ cells and PBMCs for 96 h at 37 °C in a 5 % CO₂ incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34⁺ cells + PBMC, A2 = hUC-MSCs + hUCB-CD34⁺ cells + PBMC, Negative Control = hUCB-CD34⁺ cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured by ELISA. A rabbit model of MI was established using a modified Fujita method. The animals were then randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or hUC-MSCs + CD34⁺ cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone. Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively. Stem cell differentiation and neovascularization in the infracted area were characterized for the presence of cardiac Troponin I (cTnI) and CD31 by immunohistochemical staining, and the extent of myocardial fibrosis was evaluated by hematoxylin and eosin (H&E) and Masson’s trichrome. IFN-γ was 27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups, respectively, which indicated that hUCB-CD34⁺ cells induced immune reaction of peripheral blood mononuclear cells, whereas both hUC-MSCs and HP-MSCs showed an immunosuppressive effect, which, however, was attenuated by hypoxic preconditioning. The Cotrans group had less collagen deposition in the infarcted myocardium and better heart function than the hUC-MSCs or PBS group. The presence of cTnI-positive cells and CD31-positive tubular structures indicated the differentiation of stem cells into cardiomyocytes and neovascularization. The microvessel density was 12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans group, respectively (P < 0.01). As a conclusion, both hUC-MSCs and HP-MSCs have an immunosuppressive effect on lymphocytes, which, however, can be attenuated by hypoxic preconditioning. Cotransplantation of hUC-MSCs and hUCB-CD34⁺ cells can improve heart function and decrease collagen deposition in post-MI rabbits. Thus, a combined regimen of hUC-MSCs and hUCB-CD34⁺ cells would be more desirable than either cells administered alone. This is most likely due to the increase of cardiomyocytes and enhanced angiogenesis in the infarcted myocardium.     

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.     

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of db/db mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

Histone deacetylase inhibition by sodium valproate regulates polarization of macrophage subsets

Recent studies suggest that change of macrophage phenotype (M1/M2) is associated with autoimmune diseases. Sodium valproate (VPA) is a class I histone deacetylase (HDAC) inhibitor, which has immunomodulatory function in graft-versus-host disease. However, its impact on macrophage polarization has not been defined. We evaluated the effects of VPA on both mouse macrophage cell line RAW264.7 and primary mouse bone marrow macrophages (BMMs). Exposure to VPA significantly repressed the production of interleukin 12 (IL-12), and tumor necrosis factor α by lipopolysaccharide (LPS)-induced macrophage activation, in contrast, promoted IL-10 expression. VPA also affected the costimulatory molecule expression on LPS-stimulated RAW264.7 and BMMs (downregulation of CD40 and CD80, and upregulation of CD86). Specifically, VPA inhibited macrophage-mediated T helper 1 (Th1) effector but enhanced Th2 effector cell activation. Together, our preclinical study demonstrates that VPA significantly affects the phenotype and function of macrophage, indicating an important role of HDAC activity in immune regulation and inflammation. It also provides a rationale to evaluate VPA activity for the treatment of macrophage dysfunction-associated diseases.     

Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells

Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have multi-lineage differentiation potential, thus highlighting the feasibility of using UCB-MSCs as a valuable source of stem-cells for cell-based therapy. However, there are no well-defined markers for assessment of the multi-potency of UCB-MSCs. Thus, we focused on the identification of suitable markers by examining cell surface protein expressions of UCB-MSCs as their multi-lineage differentiations progressed. The expression of CD105, one of the cell surface proteins, was significantly decreased in differentiated osteoblasts, chondrocytes, adipocytes, and respiratory epithelium, and the portion of CD105-positive cells from 99.4 ± 0.1% to 3.5 ± 1.4%, 3.5 ± 2.3%, 16.7 ± 3.6%, and 2.1 ± 1.5%, respectively. As to such indicators as alkaline phosphatase (ALP), glycosaminoglycan (GAG), oil Red O, and surfactant protein C (SPC), they showed increases, confirming differentiation of UCB-MSCs into osteoblasts, chondrocytes, adipocytes, and respiratory epithelium. This is the first study to demonstrate a negative correlation between expression of CD105 over the time course of multi-lineage differentiation and the degree of differentiation of UCB-MSCs. We propose that CD105 is a useful novel marker to characterize differentiation status of isolated human UCB-MSCs, which will be useful to facilitate the application of such cells in stem-cell therapy.     

Regulation of human umbilical cord blood-derived multi-potent stem cells by autogenic osteoclast-based niche-like structure

Stem cell niches provide the micro-environment for the development of stem cells. Under our culturing regimen, a kind of osteoclast-centralized structure supports the proliferation of MSCs, derived from human cord blood, once they reside on osteoclasts. MSCs in this structure expressed Oct4 which is a marker of embryonic stem cells. Floating daughter cells of MSCs colony showed abilities to differentiate into osteocyte, adipocyte, and neuronal progenitor cells. Compared with the easy senescence of MSCs without this niche-like structure in vitro, these results suggested that osteoclasts might play an important role the development and maintenance of Umbilical cord blood (UCB)-derived MSCs and might provide a means to expand UCB-MSCs in vitro, more easily, through a stem cell niche-like structure.     

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.     

Histone deacetylase and DNA methyltransferase in human prostate cancer

CpG island hypermethylation and chromatin remodeling play important roles in repression of various genes during malignant transformation. We hypothesized that histone deacetylases (HDACs) and DNA methyltransferases (DNMTase) are associated with prostate cancer and we examined the enzyme activity, gene, and protein expression of HDAC1 and DNMT1 in cell lines and tissues. We found that DNMTase and HDACs activities were two- to threefold higher in cell lines compared to benign prostatic hyperplasia (BPH-1) cell line. Treatment of cells with 5-aza-2'-deoxycytidine decreased the activity of HDAC and DNMTase. The mRNA expression of these genes in BPH-1 cells and BPH tissues was lower than that in prostate cancer cells and tissues. HDAC1 and DNMT1 protein expression was higher in prostate cancer compared to BPH. This is the first report to demonstrate that DNMT1 and HDAC1 levels are up-regulated in prostate cancer compared to BPH, suggesting their roles in inactivation of various genes, by DNA-methylation-induced chromatin-remodeling, in prostate cancer.     

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.