Iscove's Modified Dulbecco's Medium Research Articles

Ultrastructural Insights in the Interface between Generated Renal Tubules and a Polyester Interstitium

In regenerative medicine, stem/progenitor cells are emerging as potential candidates for the treatment of renal failure. However, the mechanism of regeneration of renal tubules from stem/progenitor cells is not well-elucidated. In this study, a new method was developed for the generation of tubules replacing coating by extracellular matrix proteins. Renal stem/progenitor cells are mounted between layers of polyester fleece. This artificial interstitium supports spatial development of tubules within 13 days of perfusion culture in chemically defined Iscove's modified Dulbecco's medium (IMDM) containing aldosterone as the tubulogenic factor. Whole mount label by soybean agglutinin (SBA) showed that generated tubules exhibited a lumen and a continuously developed basal lamina. Immuno-labeling for cytokeratin Endo-A demonstrated the presence of isoprismatic epithelial cells, and laminin gamma1, occludin, and Na/K-ATPase alpha5 labeling revealed typical features of a polarized epithelium. To get first insight in the interface between tubules and polyester interstitium, transmission electron microscopy (TEM) was performed. The results showed that the generated tubules exhibited polar differentiation with a continuously developed basal lamina consisting of a lamina rara interna, lamina densa, and lamina rara externa. Collagen type III was found to be the linking molecule between the basal lamina and the surrounding polyester fibers by immuno labeling studies. Thus, the findings demonstrate that the spatial development involves the interface between the tubular basal lamina and the polyester interstitium of tubules and is not restricted to the epithelial portion.

High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels

A simple and non-mechanical enzymatic method for the isolation and large-scale in vitro culture of bovine retinal endothelial cells (BRECs) is described. A sufficiently high yield of retinal microvessels was obtained from bovine eyes by brisk stirring of the retina with Minimum Essential Medium for 3 min, washing the pellet five times by repeated centrifugation at 300 × g and sieving in a single step. Pericyte contamination was eliminated by increasing the incubation period in an enzyme mixture and passaging the cells at a low concentration of trypsin for 1–2 min. The cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum. The cells were cobble-stone shaped and stained positive for von Willebrand Factor (vWF) and cluster differentiation factors, CD31 and CD146. These cells stained negative for α-smooth muscle actin (ASMA) and glial fibilary acidic protein (GFAP), forming characteristic capillary tube-like structures on matrigel. Here we optimized the method for the isolation of pure retinal endothelial cells without using complex procedures and sophisticated instruments. This method is simpler, more rapid and cost-effective, and also results in a higher yield of endothelial cells than other methods. Retinal endothelial cells cultured in this way will be used to study the pathogenesis of vascular eye diseases such as diabetic retinopathy and other neovascularization diseases. They can also serve as an excellent in vitro model system for drug analysis.

The tubulogenic effect of aldosterone is attributed to intact binding and intracellular response of the mineralocorticoid receptor

Abstract Little is known about the extra- and intracellular stimuli inducing renal stem/progenitor cells to develop into three-dimensionally structured tubules. To study this specific development in a controlled environment, we used an advanced culture technique. Embryonic tissue derived from neonatal rabbit kidney was placed in a perfusion culture container at the interface of an artificial interstitium made of a polyester fleece. Culture was carried out in chemically defined Iscove’s Modified Dulbecco’s Medium (IMDM) for 13 days. Development of tubules was histochemically detected on cryosections labeled with Soybean Agglutinin (SBA). The experiments showed that aldosterone exerts a specific tubulogenic effect. Application of aldosterone (1 × 10−7 M) raised numerous SBA-labeled tubules, while in the absence of the steroid hormone the development of tubules was lacking. Specificity of hormone action was analyzed by the use of aldosterone antagonists. Administration of spironolactone (1 × 10−4 M) and canrenoate (1 × 10−5 M) completely inhibited the development of tubules. Finally, disrupting the intracellular molecular complex of the mineralocorticoid receptor (MR) and heat shock proteins by geldanamycin (2 μg/ml) prevented the development of tubules. Our results suggest that the tubulogenic effect induced by aldosterone is attributed to both hormone binding and an undisturbed intracellular response of the MR.

Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction

Adipose-derived stem cells (ADSCs) are capable of differentiating into cardiomyogenic and endothelial cells in vitro. We tested the hypothesis that transplantation of ADSCs into myocardial scar may regenerate infracted myocardium and restore cardiac function. ADSCs were isolated from the fatty tissue of New Zealand white rabbits and cultured in Iscoves modified dulbeccos medium. Three weeks after ligation of left anterior descending coronary artery of rabbits, either a graft of untreated ADSCs (UASCs, n = 14), 5-azacytidine-pretreated ADSCs (AASCs, n = 13), or phosphate buffer saline (n = 13) were injected into the infarct region. Transmural scar size, cardiac function, and immunohistochemistry were performed 5 weeks after cell transplantation. ADSCs in culture demonstrated a fibroblast-like appearance and expressed CD29, CD44 and CD105. Five weeks after cell transplantation, transmural scar size in AASC-implanted hearts was smaller than that of the other hearts. Many ADSCs were differentiated into cardiomyocytes. The AASCs in the prescar appeared more myotube-like. AASCs in the middle of the scar and UASCs, in contrast, were poorly differentiated. Some ADSCs were differentiated into endothelial cells and participate in vessel-like structures formation. All the ADSC-implanted hearts had a greater capillary density in the infarct region than did the control hearts. Statistical analyses revealed significant improvement in left ventricular ejection fraction, myocardial performance index, end-diastolic pressure, and peak +dP/dt, in two groups of ADSC-implanted hearts relative to the control hearts. AASC-implanted hearts had higher peak -dP/dt values than did control, higher ejection fraction and peak +dP/dt values than did UASC-implanted hearts. ADSCs transplanted into the myocardial scar tissue formed cardiac islands and vessel-like structures, induced angiogenesis and improved cardiac function. 5-Azacytidine pretreatment before implantation is desirable for augmenting myogenesis. Transplantation of 5-azacytidine-treated ADSCs into the myocardial scar was more efficient than that of untreated ADSCs in preservation of cardiac function.

MOLECULAR EPIDEMIOLOGY OF MALARIA IN CAMEROON. XXIII. EXPERIMENTAL STUDIES ON SERUM SUBSTITUTES AND ALTERNATIVE CULTURE MEDIA FOR IN VITRO DRUG SENSITIVITY ASSAYS USING CLINICAL ISOLATES OF PLASMODIUM FALCIPARUM

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.

Development of serum-free media for a recombinant CHO cell line producing recombinant antibody

A serum-free medium (SFM-MDM) without any animal-driven protein was developed for the production of recombinant antibody in suspension cultures of recombinant Chinese hamster ovary cell line, CS*13-1.00. The formulation of SFM-MDM was based upon the Iscove's modified Dulbecco's medium (IMDM), and Long™R 3IGF-1 and ferric citrate were supplemented instead of insulin and transferrin. Among various protein hydrolysates, yeast extract was selected as a serum-substituting additive for the improvement cell growth and productivity of recombinant antibody. Through about 3600 h of adaptation period from conventional SFM to SFM-MDM, integral of viable cell density and specific antibody productivity increased from 7.60 × 10 6 cells d and 19.14 μg (10 6 cells d) −1 to 7.79 × 10 6 cells d and 54.04 μg (10 6 cells d) −1, respectively, resulted in final antibody concentration of 420.98 mg l −1, which was about three times higher than that obtained from commercial medium, 486F. Also, supplementation of various vitamins including biotin, pyridoxal·HCl, and ascorbic acid was observed to enhance specific productivity, which led to high final antibody concentration. These results suggested the fundamental and effective strategy for development of a serum-free medium and to enhance cell growth and production of target proteins.

Differentiation of Human ES Cell into the Hemangioblast.

We hypothesize that during early human embryo development a common endothelial/hematopoietic precursor is formed. This precursor, named the hemangioblast, is an early endothelial/hematopoietic precursor responsible for angiogenesis and both primitive and definite hematopoiesis. The aims of this work are 1) to establish the ES/EB model system for genotypic studies of early vasculo/hematopoiesis. 2) to phenotype human hemangioblast and establish standard culture/storage condition that will stabilize the phenotype of these cells for a defined period and permit consistent, predictable outcomes of experiments. Human ES (H-1 line) cells (NIH code WA01) has been maintained on g -irradiated MEF cells supplemented with DMEM/F12 media containing 4ng/ml b-FGF and 15% knockout serum by changing media everyday and passaging cells every 3–4 days. EBs were generated either in liquid or 1% methylcellulose cultures (1 x 104 ES cells per 35-mm Petri dish) in medium containing Iscove modified Dulbecco medium (IMDM), 15% FCS, 2 mM glutamine, 450 μM MTG, 50 μg/mL ascorbic acid, and 20% BIT (1% bovine serum albumin (BSA), 10 μg/mL insulin, and 200 μg/mL transferrin. In order to induce EB formation two days old hES cells were plated in 1% methyl cellulose in IMDM media supplemented with 50mg/ml ascorbic acid, 10mg/ml insulin, 200 mg/ml transferrin, 20% Bits, 15% FCS, 450mM MTG, 2mM-L-glutamine for 3days. To generate hemangioblasts, 1 x 104/mL EB cells were replated in 1% methylcellulose in the presence of IMDM, 10% PDS (bovine platelet-poor plasma-derived serum; Biomedical Technologies, Stoughton, MA), 2 mM glutamine, 450 μM MTG, 25 μg/mL ascorbic acid, 20% BIT, 5 ng/mL human vascular endothelial growth factor (hVEGF), 50 ng/mL SCF, 10 ng/mL human fibroblast growth factor 2 (hFGF-2), and 2 U/mL hEPO. RNA from day 0, 2, 3 and 4 days old EBs was collected for gene regulation studies. We have chosen to study the key regulatory genes (FLK-1, Runx-1, SCL, HEX, GATA-1) during this process. RNA collected from undifferentiated hES cells has been used as control. We observed expression of stemness genes: Sox-2 and Oct-4, in the undifferentiated ES cells while there was an upregulation in expression of Brachury on day 2 and FLK-1 and Hex genes in day 3 of differentiation. In hemangioblast forming cells (BL-CFC), we observed upregulation of FLK-1, SCL, Runx and downregulation of Brachury and Hex. This sptio-temporal expression profile of genes suggest that during hemangioblast formation and at the initial stages of differentiation hemangioblast fate is decided by Hex, while after day 2 the control is taken by FLk-1, SCl and Runx genes. Ongoing studies will define signaling pathways involved in this process. Understanding of molecular processes involved in hemangioblast formation will allow directed generation of this progenitor from human ES cells in the future.

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.

Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.METHODS: vSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P＜0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P＜0.01). Albumin increased significantly on d 16 (t = 6.68, P＜0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P＜0.01). Urea(4.72±1.03 μmol/L) was detected on d 20 (t = 4.272,P＜0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276, P＜0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCBderived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases. MSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining. By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20+/-1.16 microg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28+/-3.11 microg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02+/-0.15 microg/mL, and to 3.63+/-0.30 microg/mL on d 28 (t = 11.748, P<0.01). Urea (4.72+/-1.03 micromol/L) was detected on d 20 (t = 4.272, P<0.01), and continued to increase to 10.28+/-1.06 micromol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCB-derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

Macrophage-like synoviocytes display phenotypic polymorphisms in a serum-free tissue-culture medium

Synovial macrophages play an outstanding role in many rheumatic diseases. However, traditional serum-containing tissue-culture techniques hamper in vitro studies due to fibroblast activation not found in vivo. The objective of this study was to examine dissociated synovial cells in a macrophage-selective, serum-free tissue-culture medium. Osteoarthritis synovial tissue (n=11) was cultured in Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum (FBS) and compared to a serum-free, insulin-supplemented medium. After 9-11 and 19-21 days in vitro, immunohistochemistry was performed for macrophage/lymphocyte markers and cell division. Cytokine profiles were determined by RT-PCR. In serum, cells with a bipolar morphology rapidly proliferated. Respectively, 14.34+/-12.94% and 13.25+/-12.66% expressed CD68 and HLA-DR. These markers further decreased after one passage. In serum-free medium, proliferation was infrequent, and cells with diverse morphologies expressed 83.10+/-6.80% and 55.03+/-6.88% CD68 and HLA-DR respectively. CD14 was rare, and lymphocytes were missing. Both cultures expressed interleukin-6 and interleukin-8. This novel serum-free method permits the culture of distinct CD68/HLA-DR associated phenotypes.

AML1/RUNX1 Increases During G1 to S Cell Cycle Progression Independent of Cytokine-dependent Phosphorylation and Induces Cyclin D3 Gene Expression

AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G(1) to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.

Cell cycle analysis of the CD133 + and CD133 − cells isolated from umbilical cord blood

Background: Umbilical cord blood cells (stem/progenitor cells) exhibit high proliferative capacities leading to a large expansion of cells in appropriate cell culture conditions. The aim of this study was to evaluate by flow cytometry the cycling status of CD133 + and CD133 − cells depending on various culture conditions, such as sera, stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 6 (IL-6). Methods: An immunomagnetic system was used for cell separation. CD133 + and CD133 − cells were seeded in Iscove's Modified Dulbecco's Medium (IMDM) with different serum concentrations and were stimulated with SCF (100 ng/ml), IL-3 (50 ng/ml) and IL-6 (50 ng/ml). Results: Our experiments demonstrated that immediately after separation, 96.75±0.58% of CD133 + cells and 97.04±1.76% of CD133 − cells were in G 0/G 1-phase, while 2.02±0.38% and 0.88±0.52% were in the S-phase, respectively. Our data documented that CD133 + cells are more active than CD133 − cells after the first week of cultivation ( p<0.01). Statistically significant difference was found for CD133 + cells vs. CD133 − cells after second week of cultivation in G 0/G 1- and S-phases under all tested conditions. A combination of 12.5% FCS+12.5% HS yielded the highest cell expansion for CD133 + cells; this was concomitant with highest percentage of S-phase and G 2M-phase. Our data show that the medium with 25% HS was the best for cell expansion and cycling of the CD133 − cells for the first week, followed by the 12.5% FCS+12.5% HS. After 2 weeks of cultivation, obviously 12.5% HS and 12.5% FCS+12.5% HS exhibited similar S-phase amounts in CD133 − cells. A decrease of HS concentrations seemed to stimulate CD133 − cells' S-phase after the second week. Conclusions: Our data indicate that the source and the concentration of the serum used for cultivation have an impact on both cell populations: CD133 + cells are most comfortable with a combination of FCS and HS; CD133 − cells prefer media-containing HS. Cell cycle status may be an important factor for defining cultivation strategies for stem cell expansion.

Expression of hematopoietic inhibitory factors in mouse fibroblasts, macrophages and endothelial cells.

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT-PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated. The results showed: (1) six cytokines, MIP-1alpha, MIP-2, TGF-beta, TNF-alpha, IFN-gamma and Tbeta(4), inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-alpha, INF-gamma and Tbeta(4), was seen in all three types of stromal cells, while TGF-beta mRNA was only seen in endothelial cells and macrophages, and MIP-1alpha mRNA in endothelial cells and fibroblasts. The inhibitors TGF-beta, MIP-1alpha, and Tbeta(4)have an inhibitory effect on the growth of CFU-GM, but TNF-alpha, INF-gamma and MIP-2 do not.

Hematopoietic stem/progenitor cell harvesting from umbilical cord blood

Umbilical cord blood contains hematopoietic stem cells from which mature, functional cells are derived w1x. These pluripotent stem cells form colonies in vitro in semi-solid culture medium, and colony-forming units–granulocyte-macrophage (CFU-GM) and burst-forming units– erythroid (BFU-E) are indirect indicators of the stem cell population. Another marker of hematopoietic stemyprogenitor cells is the CD34 antigen, a membrane glycoprotein that regulates the adhesion of stem cells to stromal cells w2x. To quantify stem cells (i.e. CFU-GM and CD34q cells) in cord blood, 27 gravidas with uncomplicated pregnancies who were in labor at term were enrolled after giving informed consent. After delivery, the umbilical vein was punctured and residual placental blood recovered in a modified blood collection bag containing 20 ml of citrate phosphate dextrose adenine as an anticoagulant (Terumo Penpol Ltd, Puliyarakonam, Trivandrum, India). Approximately 3 ml of collected cord blood was mixed with a triple volume of Iscove’s modified Dulbecco’s medium (IMDM)

Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into multiple mesodermal tissues. We previously reported that monoclonal antibodies to the low-affinity nerve growth factor receptor (alpha-LNGFR) stain bone marrow (BM) mesenchymal cells. We now show that LNGFR antibodies label primitive MSCs with high specificity and purity in adult BM, and compare these cells to those isolated by plastic adherence (PA) and CD45(-)anti-glycophorin A(-) selection. Low-density mononuclear cells (LD-MNCs) from normal BM were separated by PA or immunomagnetic selection for NGFR(+) or CD45(-)alpha-glycophorin A(-) cells. The three fractions were grown in Iscove's modified Dulbecco medium + 20% fetal bovine serum +/- basic fibroblast growth factor (bFGF) in order to assess their proliferative capacity and evaluate their phenotype during culture. The clonogenic potential of the MSCs was assessed using a colony-forming unit fibroblast (CFU-F) assay, whereas multipotential differentiation was determined after culture in adipocytic and osteoblastic conditioned media. The NGFR(+) mesenchymal cells grown without growth factors showed persistent NGFR expression (rapidly down-regulated after the addition of bFGF) and persistent CFU-F activity. The NGFR(+) fractions were rich in clonogenic precursors: CFU-F median frequency was 1584/1 x 10(6) cells (range 325-13,793) in the NGFR(+) cells and 35/1 x 10(6) cells (range 27-112) in the LD-MNCs. The NGFR(-) fraction never showed any residual CFU-F activity. Compared with the other two fractions, the NGFR(+) cells (+/- bFGF) showed a 1 to 3 log greater expansion in the number of fibroblastic cells and a greater capacity to give rise to adipocyte colonies and induce osteoblastic differentiation, and they had similar effects in supporting the growth of hematopoietic precursors. The data suggest that positive selection using low-affinity NGFR antibodies makes it possible to obtain homogeneous multipotent MSCs.

Bone marrow stromal cells of bcl-2 transgenic mice express widespread Bcl-2 protein and reduce apoptosis in a serum-free medium

P225 Stem cell transplantation is a vibrant area of research with the goal of treatment of uncurable diseases. Bone marrow stromal cells (MSCs, including marrow stem and progenitor cells) have been employed to improve neurological functional recovery after stoke and injury. Grafted cell death is a common feature of intracerebral transplantation. Bcl-2 has been shown to protect a variety of cell types from apoptoic cell death. We tested the hypothesis that MSCs harvested from bcl-2 transgenic mice promote survival and decrease apoptosis of cells in vitro. MSCs were harvested from adult male C57BL/6 transgenic mice (n=4) overexpressing bcl-2, and from normal control C57BL/6 mice (n=4). Cells were cultured in regular medium, Iscove s Modified Dulbecco s medium (IMDM) supplemented with 10% fetal bovine serum (FBS); and then isolated by their adherence to the plastic dishes from non-adherent hematopoietic cells at 72 h of incubation. To examine the effect of bcl-2 on cell death induced by serum deprivation. MSCs were washed and incubated with the serum-free medium. Immunostaining and Western blot were used to identify Bcl-2 protein expression, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Immunocytochemical staining indicated that 100% of MSCs harvested from bcl-2 transgenic mice express Bcl-2 protein. Western blot showed that Bcl-2 protein was 8 times more intense in the MSCs harvested from transgenic mice than that from normal control mice at 12 days cultured in regular medium. After 48 h cultured in the serum-free medium, 5.8% of MSCs from bcl-2 transgenic mice were presented apoptoic morphology, compared with 39% of MSCs from normal controls. Our finding demonstrates that the overexpression of bcl-2 gene in the MSCs reduces apoptotic cell death.

Antimetastatic efficacy of niosomal pentoxifylline and its combination with activated macrophages in murine B16F10 melanoma model.

The aims of the present study were a) to enhance the effectiveness of antimetastatic agent, Pentoxifylline (PTX) by encapsulation in niosomes and b) to investigate the anticancer activity by combination therapy involving activated macrophages and PTX solution/PTX niosomes. Niosomes were prepared by lipid film hydration method. Particle size distribution revealed bimodal distribution with median vesicle size of 462 nm. The entrapment efficacy of PTX niosomes was found to be 9.64%. A cumulative release of 82.43% from niosomal suspension was observed at the end of 21 hours. Intravenous administration of niosomal PTX (6 mg/kg and 10 mg/kg) resulted in significant reduction in lung nodules in an experimental metastatic B16F10 model suggesting accumulation of PTX in a distant target organ-lung. Light microscopic observations of histologic sections showed a decrease in number of tumor islands in the lung. Macrophages activated by intraperitoneal injection of Iscove's Modified Dulbecco's Medium (IMDM) containing 20% fetal calf serum (FCS) followed by in vitro incubation with muramyl dipeptide (MDP) were more effective in controlling tumor spread than those activated by FCS alone. Combination therapy of activated macrophages and PTX solution/niosomal PTX showed no additive or synergistic effect in controlling tumor spread. Carbon clearance studies revealed that PTX inhibits the phagocytic ability of activated macrophages, thereby resulting in the failure of combination therapy.

Establishment of an adherent cell layer from human umbilical cord blood

In addition to bone marrow and peripheral blood, stem cells also occur in human umbilical cord blood (HUCB), and there is an increasing interest in the use of this material as an alternative source for bone marrow transplantation and gene therapy. In vitro hematopoiesis has been maintained for up to 16 weeks in HUCB cultures, but the establishment of an adherent, stromal layer has consistently failed. Adherent cell precursors among mononuclear cells from HUCB were sought for in long-term cultures. Mononuclear cells obtained from cord blood after full term, normal deliveries were cultivated at different concentrations in Iscove's modified Dulbecco's medium (IMDM) with weekly feeding. An adherent layer was detected in 16 of 30 cultures, 12 of which were plated at cell concentrations higher than 2 x 10(6) cells/ml. In contrast to bone marrow cultures, in which the stroma is detected early, in most (10/16) positive cultures from HUCB the adherent layer was identified only after the fourth week of culture. The cells never reached confluence and detached from the plate approximately four weeks after detection. May-Grünwald-Giemsa staining of positive cultures revealed fibroblast- or endothelial-like adherent cells in an arrangement different from that of bone marrow stroma in 13 samples. In two of these, the adherent cells were organized into characteristic, delimited cords of cells. Unlike bone marrow cultures, fat cells were never observed in the adherent layers. A rapid development of large myeloid cells in the first week of culture was characteristic of negative cultures and these cells were maintained for up to 12 weeks. HUCB contains adherent cell precursors which occur in lower numbers than in bone marrow and may be at a different (possibly less mature) stage of differentiation.

Bone marrow stromal cells of bcl-2 transgenic mice express widespread Bcl-2 protein and reduce apoptosis in a serum-free medium

P225 Stem cell transplantation is a vibrant area of research with the goal of treatment of uncurable diseases. Bone marrow stromal cells (MSCs, including marrow stem and progenitor cells) have been employed to improve neurological functional recovery after stoke and injury. Grafted cell death is a common feature of intracerebral transplantation. Bcl-2 has been shown to protect a variety of cell types from apoptoic cell death. We tested the hypothesis that MSCs harvested from bcl-2 transgenic mice promote survival and decrease apoptosis of cells in vitro. MSCs were harvested from adult male C57BL/6 transgenic mice (n=4) overexpressing bcl-2, and from normal control C57BL/6 mice (n=4). Cells were cultured in regular medium, Iscove s Modified Dulbecco s medium (IMDM) supplemented with 10% fetal bovine serum (FBS); and then isolated by their adherence to the plastic dishes from non-adherent hematopoietic cells at 72 h of incubation. To examine the effect of bcl-2 on cell death induced by serum deprivation. MSCs were washed and incubated with the serum-free medium. Immunostaining and Western blot were used to identify Bcl-2 protein expression, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Immunocytochemical staining indicated that 100% of MSCs harvested from bcl-2 transgenic mice express Bcl-2 protein. Western blot showed that Bcl-2 protein was 8 times more intense in the MSCs harvested from transgenic mice than that from normal control mice at 12 days cultured in regular medium. After 48 h cultured in the serum-free medium, 5.8% of MSCs from bcl-2 transgenic mice were presented apoptoic morphology, compared with 39% of MSCs from normal controls. Our finding demonstrates that the overexpression of bcl-2 gene in the MSCs reduces apoptotic cell death.