Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 μl) or phosphate‐buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI‐PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure–volume loops after 4 weeks. There is no significant difference in infarct size between MI‐matrigel (MI‐M; 21.48 ± 1.49%, n= 10) and MI‐PBS hearts (20.98 ± 1.25%, n= 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI‐M (0.72 ± 0.02 mm, n= 10) compared with MI‐PBS (0.62 ± 0.02 mm, n= 10). MI‐M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high‐power field [HPF; 400×], n= 6) than MI‐PBS hearts. c‐Kit⁺ stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c‐Kit⁺ cells per HPF [630×], n= 5, P < 0.05) and CD34⁺ cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34⁺ cells per HPF [630×], n= 5, P < 0.01) were significantly more numerous in MI‐M than in MI‐PBS in the infarcted hearts (n= 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34⁺ and c‐Kit⁺ stem cells.     

CX43 change in LPS preconditioning against apoptosis of mesenchymal stem cells induced by hypoxia and serum deprivation is associated with ERK signaling pathway

This study was designed to investigate the effect and mechanism of lipopolysaccharide (LPS) preconditioning on survival and connexin 43 (CX43) expression in rat bone marrow mesenchymal stem cells (bMSCs) under hypoxia and serum deprivation (Hypoxia/SD) conditions. Whole marrow cells were obtained from the femora and tibiae of SD rats, and bMSCs were isolated by density gradient centrifugation and attachment culture. Surface antigens were determined by FACS before the experiment using antibodies conjugated directly against anti-rat CD34, anti-CD45, anti-CD29, and anti-CD44. Passage 3 bMSCs were used for all experiments. The effect of LPS preconditioning on bMSCs apoptosis in response to Hypoxia/SD was investigated by an Annexin V-FITC/PI binding assay and a mitochondrial membrane potential (△Ψ_m) assay. Cyc-c released into the cytosol from mitochondria and CX43 in bMSCs was determined by Western blot before and after LPS preconditioning. Subsequently, extracellular signal-regulated kinase (ERK) was inhibited with PD98059 to analyze the role of ERK in modulating CX43 expression after LPS preconditioning. The bMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 and CD44 and negative for CD34 and CD45. The Hypoxia/SD conditions induced significant apoptosis of bMSCs. Compared with the Hypoxia/SD group, cells treated with LPS prevented △Ψ_m from falling significantly. LPS inhibited Hypoxia/SD-induced Cyc-c release. These results were consistent with the total analysis of apoptosis of MSCs. Compared with the control group, the level of CX43 expression in the Hypoxia/SD group and LPS + Hypoxia/SD group decreased significantly at each time point. The level of CX43 expression in the Hypoxia/SD group was lower than that in the LPS + Hypoxia/SD group, while the difference was not significant between the PD98059 + LPS + Hypoxia/SD group and the PD98059 + Hypoxia/SD group (P > 0.05). Compared with the LPS + Hypoxia/SD group, CX43 level in the PD98059 + LPS + Hypoxia/SD group and PD98059 + Hypoxia/SD group decreased significantly (P < 0.05). These results demonstrated that Hypoxia/SD conditions could induce apoptosis of bMSCs markedly. Low-dose LPS preconditioning may preserve the mitochondrial function by maintaining the mitochondrial transmembrane potential and inhibiting Cyc-c release in Hypoxia/SD-induced bMSCs apoptosis. LPS preconditioning also had a stabilizing effect on the cell membrane by inhibiting the decrease of CX43, and this modulating mechanism may be related to the ERK signaling pathway.     

CD14 monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

Here, the effect of CD14⁺ monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-γ (IFN-γ) secretion capacities of CD4⁺ and CD8⁺ T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E₂ (PGE₂) as an important soluble mediator. CD14⁺ monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1β, either exogenously added or produced by CD14⁺ monocytes in culture, could trigger expression of high levels of PGE₂ by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE₂ expression, but also reversed the promotional effect of CD14⁺ monocytes and partially restored CD4⁺ and CD8⁺ T cell proliferation and IFN-γ secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.     

CD45-positive cells are not an essential component in cardiosphere formation

The cardiosphere (CS) is composed of a heterogeneous population of cells, including CD45⁺ cells that are bone marrow (BM)-derived. However, whether the CD45⁺ cells are an essential cell component in CS formation is unknown. The current study was undertaken to address this question. Cardiospheres (CSs) were harvested from 1-week post-myocardial infarction (MI) or non-MI hearts of C57BL/6 J mice. The process of CS formation was observed by timelapse photography. To analyze the role of BM-derived CD45⁺ cells in CS formation, CD45⁺ cells were depleted from populations of CS-forming cells by immunomagnetic beads. We recorded the number of CSs formed in culture from the same amount (10⁵) of intact CS-forming cells, from CD45⁺-cell-depleted CS-forming cells and from CD45⁺ cells alone (n=6–9/cell type). CS-forming cells selectively aggregated together to form CSs by 35 h after plating. The depletion of CD45⁺ cells from CS-forming cells actually increased the formation of CSs (67±10 CSs/10⁵ cells) compared with non-depleted CS-forming cells (51±6 CSs/10⁵ cells, P<0.0001). Purified CD45⁺ cells from CS-forming cells did not form CSs in culture. Thus, BM-derived CD45⁺ cells including BM progenitors are neither necessary nor sufficient for CS formation.     

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.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

Efficacy of cascade-primed cell infusion as an adjuvant immunotherapy with concurrent chemotherapy for patients with non–small-cell lung cancer: A retrospective observational study with a 5-year follow-up

BackgroundClinical studies have shown the efficacy of combination therapy for various malignancies. In this study, the characteristics, safety and feasibility of use of cascade-primed (CAPRI) cells for the combination treatment of non–small-cell lung cancer (NSCLC) were evaluated both in vitro and in vivo.MethodsSixty-five patients with stage II–IV NSCLC were recruited. Of these patients, 31 patients received CAPRI cell therapy combined with chemotherapy (CAPRI group), and the other 34 patients constituted the control group and received chemotherapy alone. This study primarily aimed to evaluate the overall survival (OS), progression-free survival (PFS), short-term responses and treatment efficacy.ResultsCD83, CD1a, CD80 and CD86 marker levels were significantly upregulated in CAPRI cells. Interferon-γ expression levels were highest in CD3⁺CD8⁺ cells (33.77% ± 4.40%). Furthermore, interleukin-2 levels were highest in CD3⁺CD56⁺ cells (26.73% ± 6.63%), whereas perforin expression levels were similar in CD3⁺CD8⁺ and CD3⁺CD56⁺ cells. Furthermore, CAPRI cells had a better anti-tumor potential in CD3⁺CD56⁺ cells and displayed the highest expression levels of CD107a to H460 and A549 cell lines. The 5-year OS was significantly greater in the CAPRI group than in the control group (P = 0.008), and the PFS of two groups exhibited a significant difference (P = 0.007). Median OS (48 versus 31.6 months; P = 0.004) and PFS (48 versus 36.4 months; P = 0.016) differed between these two groups. Moreover, treatment-associated toxicities were mild and well-tolerated by patients with NSCLC.ConclusionCAPRI cell therapy potentially prolongs the survival of patients with NSCLC when combined with chemotherapy.     

Efficacy of postoperative adjuvant transfusion of cytokine-induced killer cells combined with chemotherapy in patients with colorectal cancer

Purpose

To assess the activity and safety of postoperative adjuvant immunotherapy with transfusion of cytokine-induced killer (CIK) cells combined with chemotherapy in patients with colorectal cancer.

Methods

We retrospectively studied 96 consecutive patients with colorectal cancer who were treated with resection between January 2010 and December 2012 as well as adjuvant chemotherapy. Twenty-one of these patients accepted at least 1 cycle of CIK cell transfusion for immunotherapy (CIK group). Disease free survival (DFS), immune cells and treatment related side effects were assessed. The patients were followed up until May 2013.

Results

By the end of follow-up, 10 patients (10.42 %) had died. Eighteen patients (18.75 %) had withdrawn. All the patients in the CIK group are still alive, and only 1 patient had withdrawn. Patients in the CIK group had significantly longer DFS than those in the control group [HR = 0.28, 95 % CI (0.09, 0.91), p = 0.034]. The 2-year DFS rates of patients in the CIK group and the control group were 59.65 ± 24.80 % and 29.35 ± 6.39 %, respectively. The CD4⁺/CD8⁺ ratios were significantly lower during the period of chemotherapy than those before chemotherapy (p = 0.0038), while the ratios were significantly higher during the period of CIK cell transfusion than those before CIK therapy (p = 0.0484). There were no immediate adverse reactions to the CIK cell transfusions.

Conclusion

Adjuvant transfusion of CIK cells prolongs DFS in patients with colorectal cancer.     

Expression of Treg Subsets on Intestinal T Cell Immunity and Endotoxin Translocation in Porcine Sepsis After Severe Burns

In the present study, the changes of the regulatory T cells (Treg) expression, endotoxin translocation, and the relationships in intestinal lymph nodes were investigated in porcine sepsis induced by severe burns. Flow cytometry, western blot, and Tachypleus amebocyte lysate were applied to study after the burn injury model was built. We found that the upregulated Treg expression was negatively related to the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio (r = ?0.832, P < 0.05) after burn injury-induced sepsis. While Treg expression and portal venous plasma endotoxin translocation levels were positively correlated (r = 0.876, P < 0.05) when compared with the control group. Moreover, we detected a transforming of T cell subsets from T helper 1 cells to T helper 2 cells. Therefore, intestinal Treg cells expression exerts immunosuppressive effects on other intestinal T lymphocytes and was closely related to endotoxin translocation in porcine sepsis after severe burns injuries. Above all, the intestinal Treg cells may play an important role in the intestinal immune barrier system after severe burns injuries.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Mobilization and engraftment of peripheral blood stem cells in healthy related donors >55 years old

Background aimsThe increasing scarcity of young related donors has led to the use of older donors for related allogeneic hematopoietic stem cell transplantation (HSCT). This study analyzed the influence of age on the results of mobilization of peripheral blood stem cells (PBSCs) in healthy donors as well as on the engraftment and outcome of HSCT.MethodsA retrospective analysis from a single center was performed comparing the results of PBSC mobilization from related healthy donors according to their age.ResultsThe study included 133 consecutive related donors. The median age was 50 years (range, 4–77 years); 70 (53%) donors were males, and 44 (33%) were >55 years old. All donors were mobilized with granulocyte colony-stimulating factor for 5 days. The peak CD34⁺ cell count in peripheral blood was higher in younger than in older donors (median, 90.5 CD34⁺ cells/μL [range, 18–240 CD34⁺ cells/μL] versus 72 CD34⁺ cells/μL [range, 20–172.5 CD34⁺ cells/μL], P = 0.008). The volume processed was lower in younger than in older donors (16,131 mL [range, 4424–36,906 mL] versus 18,653 mL [range, 10,003–26,261 mL], P = 0.002) with similar CD34⁺ cells collected (579.3 × 10⁶ cells [range, 135.14 × 10⁶–1557.24 × 10⁶ cells] versus 513.69 × 10⁶ cells [range, 149.81 × 10⁶–1290 × 10⁶ cells], P = 0.844). There were no differences in time to recovery of neutrophils and platelets or in the incidences of acute and chronic graft-versus-host disease, overall survival, non-relapse mortality and relapse incidence.ConclusionsDonors >55 years old mobilized fewer CD34⁺ cells and required a greater volume to collect a similar number of CD34⁺ cells. The outcome of HSCT was not influenced by donor age. Donor age should not be a limitation for related allogeneic HSCT.     

Intratumoral CD8+ T/FOXP3+ cell ratio is a predictive marker for survival in patients with colorectal cancer

The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8⁺ T cells and CD4⁺ regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)⁺ cells were considered to be Tregs in the present study. The number of intratumoral FOXP3⁺ cells (itFOXP3⁺ cells) was positively correlated with lymph node metastases (P = 0.030). itCD8⁺ T/itFOXP3⁺ cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8⁺ T cells or itFOXP3⁺ cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8⁺ T/itFOXP3⁺ cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8⁺ T/itFOXP3⁺ cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3⁺ cells positively correlated with transforming growth factor-beta TGF-β production at the tumor site (P = 0.020). In conclusion, itCD8⁺ T/itFOXP3⁺ cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8⁺ T/itFOXP3⁺ cell ratio may be an independent prognostic factor. And, tumor-producing TGF-β may contribute to the increased number of itFOXP3⁺ cells.     

Retention of basic electrophysiologic properties by human sweat duct cells in primary culture

Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V _a ) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na⁺−K⁺ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na⁺ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (V_a) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na⁺ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl⁻ substitution at the apical side by the impermeant anion gluconate depolarized the V _a of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (ΔV _a =−8.3±1.2mV,n=5) or depolarized (ΔV _a =8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na⁺ and Cl⁻ conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared to MPD cells. This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation.     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

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.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.     

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.     

Conditioned medium from umbilical cord mesenchymal stem cells improves nasal mucosa damage by radiation

Objectives

To explore therapeutic effects of conditioned medium from human umbilical cord mesenchymal stem cells (hUC-MSCs) on nasal mucosa radiation damage both in vivo and in vitro.

Results

The mucus cilia clearance time (7 and 30 days), degree of mucosal edema (7, 30, 90 and 180 days), cilia coverage (180 days) of concentrated conditioned medium group improved compared with radiotherapy control group. The proliferation and migration abilities of irradiated and non-irradiated nasal epithelial cells significantly increased after culture in bronchial epithelial cell growth medium (BEGM) containing 10% conditioned medium of hUC-MSCs compared to cells cultured in BEGM alone.

Conclusions

Soluble factors secreted by hUC-MSCs may promote nasal epithelial cell proliferation and migration. Intranasal administration of hUC-MSC conditioned medium effectively repairs nasal mucosa radiation damage.

     

Effect of Mg, Si, and Sr on growth and antioxidant activity of the marine microalga Tetraselmis suecica

Efforts to increase the productivity of microalgal cultures have been focused on the improvement of photobioreactors, but little attention has been paid to the nutritional requirements of microalgae in order to improve culture media formulation. In this study, the main goal was obtaining a high productivity for Tetraselmis suecica (Chlorophyta) in semicontinuous culture by adding magnesium (Mg), silicon (Si), and strontium (Sr) at concentrations from 0.01 to 10 mM; at the time, the effect on steady-state cell density, biochemical composition, and antioxidant activity of T. suecica was evaluated. Because productivity is higher in high-density cultures, the work was focused many times to cell density. Mg (3 mM) and Sr (0.1 mM) added separately reached the highest steady-state cell density (7.0?×?10⁶?±?0.4 cells mL^?1) in comparison to control (4.2?±?0.1 cells mL^?1), but simultaneous addition had a synergic effect, achieving 8.7?×?10⁶?±?0.6 cells mL^?1. Silicon (3 mM) significantly affected the steady-state cell density, reaching 6.0?±?0.3 cells mL^?1 and increased the cell ash-free dry weight, reaching 127?±?7.9 pg cell^?1 in comparison to control (102.7?±?5.0 pg cell^?1), resulting in an ash-free dry weight productivity of 0.75?±?0.07 g?L^?1 day^?1. The highest fatty acids content and antioxidant activity, measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method were obtained with Sr 10 mM. Sr treatments showed a high correlation (R ²?=?0.98) between DPPH inhibition and polyphenolic content, explaining its high antioxidant activity. Therefore, the addition of Mg, Si, and Sr to culture medium of T. suecica is recommended to achieve high steady-state cell density in semicontinuous cultures.