Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Umbilical Cord Vein

Background

Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child.

Objective

To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype.

Methods

Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence.

Results

Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed.

Conclusions

The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation.     

Esophageal pathology in patients after treatment for head and neck cancer

Objective

To determine the prevalence of esophageal pathology following treatment for primary head and neck cancer (HNCA).

Study Design

Case series with planned data collection.

Setting

Academic medical practice.

Subjects and Methods

Subjects comprised HNCA survivors. Esophagoscopy was prospectively performed on 100 patients at least three months after treatment for HNCA. Patient demographics including cancer stage, cancer treatment, use of reflux medications, symptoms surveys, and esophageal findings were prospectively determined.

Results

The mean age of the cohort was 64 (± 10) years; 75 percent were male. The mean time between the end of treatment and endoscopy was 40 (± 51) months. Eighty-one percent of HNCA was advanced stage (3 or 4). The distribution of site of the primary HNCA was as follows: oropharynx (38%), larynx (33%), oral cavity (17%), unknown primary (10%), hypopharynx (1%), and nasopharynx (1%). Treatment modalities included surgery alone (15%), surgery with radiation (34%), radiation alone (6%), chemoradiation alone (24%), and chemoradiation with surgery (20%). The findings on esophagoscopy included peptic esophagitis (63%), stricture (23%), candidiasis (9%), Barrett metaplasia (8%), gastritis (4%), and carcinoma (4%). Only 13 percent had a normal esophagoscopy.

Conclusion

Esophageal pathology is extremely common in patients treated for HNCA. These findings support routine esophageal screening after HNCA treatment.     

Ketone bodies inhibit the viability of human neuroblastoma cells

Purpose

Recent studies have shown that brain tumor cells, unlike normal brain cells, are largely dependent upon glucose for energy and are not able to use ketone bodies as a primary energy source. These findings are thought to be because of decreased expression of succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT), a key enzyme involved in ketone body metabolism. Because of their neural crest origin, we hypothesized that neuroblastoma cells would also be unable to use ketone bodies as a primary energy source.

Methods

Human foreskin fibroblasts (control) and human neuroblastoma cells (SK-N-AS) were grown in standard media with glucose (glc+), standard media without glucose (glc−), glucose-free media with acetoacetate, or glucose-free media with β-hydroxybutyrate. Cell viability was determined with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] assay and apoptosis with fluorescence-activated cell sorting analysis. Immunoblotting was performed to SCOT protein.

Results

Neuroblastoma cell viability was significantly decreased in the acetoacetate and hydroxybutyrate media by 52% and 61%, respectively, compared with control media. In addition, neuroblastoma cells showed significantly more apoptosis in the ketone media. Viability and apoptosis in the normal fibroblasts were not affected by the culture media. The expression of SCOT protein was significantly less in human neuroblastoma cells compared with the control fibroblasts.

Conclusions

Unlike human fibroblasts, neuroblastoma cells were unable to use ketone bodies as an energy source, likely because of their decreased expression of SCOT protein. Dietary manipulation using ketone bodies in accordance with SCOT expression may be a novel therapeutic strategy for neuroblastoma.     

Expression of apoptosis-related genes after fetal tracheal occlusion in rabbits

Background/purpose

Late-gestation lung remodeling is associated with alveolar type II cell apoptosis early in the saccular stage (day 28 in fetal rabbits). Intrauterine tracheal occlusion (TO), a potent stimulus of fetal lung growth and maturation, significantly increases type II cell apoptosis. The aim of this study was to determine the effect of fetal TO on the spatiotemporal expression of key apoptosis-related signaling molecules.

Methods

Tracheal occlusion of fetal rabbits was performed at gestational day 25 (term, 31 days), and apoptotic gene expression was studied between days 26 and 28.

Results

At days 26 and 27, the protein levels of Fas and Fas-ligand (FasL) in lung lysates were similar in TO fetuses and sham-operated controls. At day 28, however, synchronous with the onset of TO-induced pulmonary distension and type II cell apoptosis, the FasL protein content was 8-fold higher in TO lungs compared with controls (P < .01), whereas Fas levels were comparable. In contrast, Bax and Bcl-2 protein levels were similar in TO and control fetuses at all time-points. TO significantly increased the cellular concentration of immunoreactive FasL in type II cells and bronchial epithelial Clara cells. Furthermore, bronchoalveolar lavage fluid (BAL) from TO fetuses at day 28 induced significantly more type II cell apoptosis in vitro compared with control BAL, an effect that was inhibited by neutralizing anti-FasL antibody.

Conclusions

Our findings show that TO results in time-specific increase of both cellular and soluble FasL in fetal lungs and implicate the Fas/FasL pathway as a pivotal autocrine and/or paracrine regulator of TO- induced type II cell apoptosis.     

Expression of Pancreatic Endocrine Markers by Prolactin-Treated Rat Bone Marrow Mesenchymal Stem Cells

Background

Mesenchymal stem cells (MSCs) are an attractive source for generation of cells with β-cell properties. Previous studies have demonstrated the ability of prolactin to induce an increase in β-cell mass and maturation, which suggests beneficial effects of its use in MSC differentiation protocols.

Objective

To evaluate the expression of endocrine differentiation markers in rat MSCs treated in vitro with prolactin.

Methods

Mesenchymal stem cells from bone marrow of Wistar rats were isolated, expanded, and characterized. Differentiation of MSCs was induced in medium containing 23 mmol/L of glucose, and nicotinamide, 2-mercaptoethanol, and exendin-4, in the presence or absence of 500 ng/mL of rat recombinant prolactin. Expression of endocrine markers and prolactin receptor genes was evaluated using real-time polymerase chain reaction, and compared between culture stages and presence vs absence of prolactin in the culture medium. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was also evaluated at immunofluorescence microscopy.

Results

Isolated cells were mostly MSCs, as confirmed at fluorescent-activated cell sorting and cytochemistry. Pax6, Ngn-3, Isl1, NeuroD1, Nkx2.2, and Nkx6.1 exhibited varied expression during culture stages. The long form of the prolactin receptor messenger RNA was induced in prolactin-treated cultures (P < .05). The somatostatin gene was induced in early stages of differentiation (P < .05), and its expression was induced by prolactin, as confirmed using immunofluorescence.

Conclusion

Culture of rat bone marrow MSCs in differentiation medium induces expression of pancreatic endocrine-specific genes, and somatostatin and prolactin receptor expression was also induced by prolactin.     

Prolongation of Islet Graft Survival Using Concomitant Transplantation of Islets and Vascular Endothelial Cells in Diabetic Rats

Background

The transplantation of isolated islets of Langerhans is nearing acceptance as treatment of type 1 diabetes mellitus. Because the arterial and venous connections of the pancreas are disrupted during islet isolation, islets must be revascularized after transplantation.

Objective

To observe whether increased numbers of vascular endothelial cells in islets can affect the angiogenesis and function of the grafts.

Materials and Methods

Rats with streptozocin-induced diabetes were divided into 3 groups. The rats in group 1 received islet grafts under the capsule of the left kidney; rats in group 2 received combined vascular endothelial cell and islet transplants; and rats in group 3 served as controls. After the transplantation procedure, blood glucose and insulin concentrations were evaluated daily. Hematoxylin-eosin and immunohistochemical staining was used to detect expression of vascular endothelial growth factor antibodies in the diabetic rat kidneys. The mean microvascular density was also calculated.

Results

At 3 days posttransplantation, blood glucose and insulin concentrations returned to normal in group 2, however, they declined only slightly in group 1, and moderate hyperglycemia was present. There was a significant difference in blood glucose and insulin concentrations between the 2 groups after 3 days (P < .05). The mean (SD) microvascular density in group 2 was markedly higher than that in group 1 (12.58 [1.81] vs 10.38 [0.97] P = .04).

Conclusion

This study suggests that concomitant transplantation of isolated islets with endothelial cells can prolong islet graft survival in diabetic rats.     

Chitosan/Gelatin Hydrogel Prolonged the Function of Insulinoma/Agarose Microspheres In Vivo During Xenogenic Transplantation

Purpose

A chitosan/gelatin solution with glycerol 2-phosphate disodium salt hydrate in liquid phase at room temperature becomes a hydrogel at 37°C. The material can be used as an injectable cell carrier into the human body for gelation in situ. We hoped that the chitosan/gelatin hydrogel provided extra protection for insulinoma/agarose microspheres during xenogenic transplantation.

Materials and Methods

Mouse insulinoma was microencapsulated in agarose as microspheres, which were macroencapsulated in chitosan/gelatin hydrogel. Insulin secreting profiles were first demonstrated in vitro. Diabetic rats were injected subcutaneously with insulinoma/agarose microspheres or insulinoma/agarose microspheres suspended in chitosan/gelatin solution. The nonfasting blood glucose concentrations (NFBG) of diabetic rats were measured perioperatively. Rats were humanely killed 1 month postoperatively and the hydrogel was retrieved for histological examination.

Results

The insulinoma/agarose microspheres continually secreted insulin for 1 month when macroencapsulated in chitosan/gelatin hydrogel in vitro. The NFBG of diabetic rats injected with insulinoma/agarose microspheres decreased to euglycemic status albeit hyperglycemia was restored within 10 days. The NFBG of diabetic rats injected with chitosan/gelatin hydrogel, which contained insulinoma/agarose microspheres, was maintained at less than 200 mg/dL for 25 days. The histological section revealed immune cell infiltration and accumulation within the hydrogel and around the iusulinoma/agarose microspheres that may have contributed to the slowly increasing NFBG after day 25.

Conclusion

This study showed that chitosan/gelatin hydrogel can be used as a cell carrier for an injectable bioartificial pancreas; the hydrogel prolonged the function of cells encapsulated in agarose microspheres during xenogenic transplantation.     

The foreshortened malleus: Anatomic variant, not pathologic sign

Objective

To quantitatively study the appearance of the malleus as viewed through the external ear canal clinically to determine whether its shortened appearance in some ears is associated with otitis media.

Study Design

Postmortem material analysis.

Setting

University temporal bone laboratory.

Subjects and Methods

A total of 41 adult crania without clinical otitis. The mastoid size indicator of previous childhood otitis media was quantified radiographically. On digitized photographs of the tympanic membranes (64 ears useable), two sets of measurements were performed: 1) the distance from the malleus' lateral process to the umbo and to the annulus; and 2) angles formed anteriorly and posteriorly at the umbo.

Results

The two metrics of malleus foreshortening did not correlate with one another, that is, the ratio manubrium-length/tympanic diameter did not correlate with the ratio posterior/anterior umbo angles (for right ears, r = −0.34; for left, r = 0.30). Mastoid size did not correlate with either metric of malleus foreshortening. As to right-left symmetry, the size of mastoid pneumatization had good correlation (r = 0.68, 95% confidence interval 0.47-0.82); but, r = 0.21 for lengths, r = 0.34 for angles, each confidence interval included zero.

Conclusion

Because the data showed no correlation of the physical appearance of the malleus with the mastoid size indicator of otitis media, and right-left symmetry was only hinted, we contend that a foreshortened malleus lacks clinical relevance. Foreshortened malleus is an anatomic variant, not a sign of pathology.     

FTY720-loaded poly(DL-lactide-co-glycolide) electrospun scaffold significantly increases microvessel density over 7 days in streptozotocin-induced diabetic C57b16/J mice: preliminary results

Background

Nanofiber scaffolds could improve islet transplant success by physically mimicking the shape of extracellular matrix and by acting as a drug-delivery vehicle. Scaffolds implanted in alternate transplant sites must be prevascularized or very quickly vascularized following transplantation to prevent hypoxia-induced islet necrosis. The local release of the S1P prodrug FTY720 induces diameter enlargement and increases in length density. The objective of this preliminary study was to evaluate length and diameter differences between diabetic and nondiabetic animals implanted with FTY720-containing electrospun scaffolds using intravital imaging of dorsal skinfold window chambers.

Methods

Electrospun mats of randomly oriented fibers we created from polymer solutions of PLAGA (50:50 LA:GA) with and without FTY720 loaded at a ratio of 1:200 (FTY720:PLAGA by wt). The implanted fiber mats were 4 mm in diameter and ∼0.2 mm thick. Increases in length density and vessel diameter were assessed by automated analysis of images over 7 days in RAVE, a Matlab program.

Results

Image analysis of repeated measures of microvessel metrics demonstrated a significant increase in the length density from day 0 to day 7 in the moderately diabetic animals of this preliminary study (P < .05). Furthermore, significant differences in length density at day 0 and day 3 were found between recently STZ-induced moderately diabetic and nondiabetic animals in response to FTY720 local release (P < .05, Student t test).

Conclusions

Driving the islet revascularization process using local release of factors, such as FTY720, from biodegradable polymers makes an attractive system for the improvement of islet transplant success. Preliminary study results suggest that a recently induced moderately diabetic state may potentiate the mechanism by which local release of FTY720 from polymer fibers increases length density of microvessels. Therefore, local release of S1P receptor-targeted drugs is under further investigation for improvement of transplanted islet function.     

Digital photography using the intraoperative microscope in neurosurgery

Background

The ability to obtain high-resolution intraoperative microscopic images has previously been reserved for those with expensive microscope-mounted cameras, standard optical or digital. The authors review the technique and utility of intraoperative digital photography through the microscope.

Methods

We present a simple technique for obtaining high-quality digital micrographs without the need for special equipment that may be expensive, complex, and/or dedicated to a specific operative microscope.

Results

Images and video may be obtained using a standard personal digital camera.

Conclusions

Use of the technique presented provides the surgeon with the ability to acquire high-quality intraoperative microphotographs and video.     

Streptozotocin-Induced Diabetic Rat-Derived Bone Marrow Mesenchymal Stem Cells Have Impaired Abilities in Proliferation, Paracrine, Antiapoptosis, and Myogenic Differentiation

Background

Diabetes has been widely recognized as a major risk factor for cardiovascular disease. With the development of the regenerative medicine, autologous bone marrow-derived mesenchymal stem cells (BMSCs), transplantation can effectively improve cardiac function after myocardial infarction. However, the BMSCs used in most previous studies are derived from young or normal donors. Little is know about the biological characters change of BMSCs in diabetes mellitus.

Methods

BMSCs were taken from the streptozotocin (STZ)-induced diabetic rats and normal control rats. Cell proliferation was evaluated by CCK-8 assay. Production of vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-1 were measured by enzyme-linked immunosorbent assay. Apoptosis under hypoxia and serum deprivation culture conditions were detected by Hoechst 33342 stain and flow cytometry. Myogenic differentiation, induced by 5-azacytidine was assessed by using immunocytochemical staining for the expression of sarcomeric α-actin and desmin.

Results

Diabetic rat models were successfully induced by intraperitoneal injection of STZ. The proliferative abilities of BMSCs derived from diabetic rats decreased significantly compared with that from normal rats (P < .05). Similar results were also presented in the cytokines (VEGF and IGF-1) release (P = .02 and P < .01, respectively) that the ability of antiapoptosis and myogenic differentiation decreased obviously between diabetes group and the normal control group (P < .01).

Conclusion

BMSCs from STZ-induced diabetic rats could be successfully harvested and expanded in vitro culture condition; their morphology was very similar to normal control group, with minor changes. However, the proliferative and differentiation properties of diabetic BMSCs, as well as cytokine release and antiapoptosis ability, were significantly impaired.     

Comparison of laparoscopy-assisted and open pylorus-preserving pancreaticoduodenectomy for periampullary disease

Background

Although many reports have described laparoscopic pancreatic surgery, pancreaticoduodenectomy (PD) has not been widely accepted. The present study aimed to compare laparoscopy-assisted and open pylorus-preserving pancreaticoduodenectomy (PPPD) to investigate the feasibility, safety, and tumor clearance.

Methods

Fifteen patients with periampullary disease underwent laparoscopy-assisted PPPD, in which resection was performed laparoscopically and the reconstruction was performed through a small midline incision. These patients were compared with 15 patients who, during the same period, underwent conventional open PPPD.

Results

Mean operative time and mean blood loss were similar between groups. No significant differences in the incidence of complications or hospital stay were noted between groups. Surgical margin and number of lymph nodes found in the resected specimen did not differ between groups.

Conclusions

Laparoscopy-assisted PPPD is on the same level with conventional open surgery in terms of perioperative outcomes or treatment efficacy.     

Does Pretransplantation Etiology Have Any Effect on Exercise Results in Heart Transplant Patients?

Objective

The objective of this study was to analyze the effect of pre-transplantation etiology and post-transplantation exercise on pulmonary function tests, functional capacities, psychological symptoms and quality of life among heart transplant patients.

Methods

An eight-week exercise program was applied to 35 heart transplant patients with histories of ischemic heart failure (HF; n = 20) or dilated HF (n = 15). All patients were evaluated before and after exercise in terms of breathing function tests, functional capacity (FVC; maximal oxygen consumption, pVO2), psychological symptoms (Beck Depression Scale (BDS), Spielberger's State-Trait Anxiety Inventory (STAI)) and quality of life (Short Form 36, SF-36).

Results

At the end of the exercise compared to the pre-exercise period significant improvements were observed in all FVC%, FeV1%, FeV1/FVC%, pVO2, SF 36 scores reflecting physical function, physical role, pain, general health, vitality, social function, and emotional role (P < 0.05) among heart transplant patients who were operated due to ischemic or dilated heart failure. In contrast, no significant improvement was observed in the BDS and STAI scales (P > 0.05). There was no significant etiology-related difference between the groups in terms of the evaluated parameters (P > 0.05).

Conclusion

We demonstrated improvements in function tests, functional capacity and quality of life for both ischemic and dilated heart transplant patients following a supervised exercise program. We concluded that the positive effect achieved by exercise was not related to pre-transplantation etiology. Whatever the preoperative etiology, a regular exercise program is recommended for heart transplant patients in the rehabilitation unit.     

Rat enterocyte cultures on human amniotic membranes

Background

Rat enterocytes were cultured on human amniotic membranes.

Methods

Intestine of neonatal DA rats was digested using collagenase and dispase according to the technique developed by Evans. The harvested enterocytes were cultured on human amniotic membranes using standard cell culture techniques.

Results

After the second day of culture, some intestinal epithelial units started to gradually detach from the membrane, dispersing as single cells and disappearing within a few days. On the contrary, other units showed signs of cell proliferation. The cultured cells underwent morphologic changes, survived, and remained attached to the amniotic membrane for 3 weeks. Paraffin sections of the membrane showed cultured cytokeratin-positive cells attached to the membrane as a monolayer.

Conclusions

Human amniotic cell membranes help to maintain rat enterocytes in culture for a long time period (3 weeks), possibly via secretion of trophic factors. This technique may provide a valuable tool to study the development and the properties of these epithelial cells in a culture environment.     

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

Introduction

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions.

Materials and method

Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein.

Results

The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes.

Conclusion

Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.     

Production of islet cell sheets using cryopreserved islet cells

Background

To establish novel islet-based therapies, our group has recently developed technologies to create a contiguous, monolayered sheet made from freshly dispersed islet cells. Islet cell sheets generated from freshly isolated cells are easily transplantable for engraftment into subcutaneous sites in rodents. The use of a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dishes with laminin-5 coating is an important feature of this process. To expand the utility of this protocol, the present study was performed to assess whether sheets generated using cryopreserved islet cells maintained viability and normal cellular phenotypes.

Methods

Dispersed islet cells obtained from Lewis rats were, cryopreserved using University of Wisconsin solution and 10% dimethyl sulfoxide. Specially coated plastic dishes were prepared by covalently immobilizing PIPAAm onto the culture plastic, followed by a coating of rat laminin-5. After 1 month of cryopreservation, the thawed cells were plated onto the PIPAAm-coated dishes.

Results

Viability of the thawed islet cells as assessed by trypan blue exclusion test was 86% ± 5%. Thawed dispersed islet cells favorably attached to PIPAAm dishes could be harvested as a contiguous cell sheet using a simple change in culture temperature conditions. Electron microscopy showed the harvested islet cell sheet to retain cell-cell connections and numerous secretion granules.

Conclusions

The present data indicated that dispersed islet cells, which were appropriately frozen and thawed, represent another viable cells source to create functional islet sheets for tissue engineering and potential clinical applications.     

Perioperative use of continuous renal replacement therapy for orthotopic liver transplantation

Purpose

We present a retrospective study describing the perioperative use of continuous renal replacement therapy (CRRT) for orthotopic liver transplantation (OLT).

Materials and Methods

We retrospectively reviewed the clinical course of patients who underwent OLT with the perioperative use of CRRT. The following variables were recorded: Gender, age, indication for transplantation, time when CRRT was initiated, postoperative need for CRRT, and the patient and organ (liver, kidneys) outcome up to 1 year after transplantation.

Results

Among 105 patients who underwent OLT from 2006 to 2010; we used CRRT in 12 cases (11.4%) perioperatively, including 9 (8.3%) patients intraoperatively. Perioperative CRRT was employed for volume, electrolyte, and/or pH management. All patients who underwent CRRT perioperatively were alive at 1 month, 10 (83.3%), at 3 and 6 months and 9 (75%) at 1 year after OLT. Only 1 surviving patient (8.3%) required renal replacement therapy at 1 month after surgery. Renal replacement therapy was not required in any surviving patient up to 12 months posttransplantation.

Conclusion

Perioperative and especially intraoperative use of CRRT therapy can potentially improve the outcomes of patients undergoing OLT.     

The Effect of Malononitrilamides (FK778) on Phenotypic Properties of Human Peripheral Dendritic Cells

Background

FK778, a malononitrilamide analogue of lefunomide, is currently a promising immunosuppressive drug. Because the cellular and molecular mechanisms underlying the effects of FK778 are not entirely clarified. We studied its effects on human peripheral dendritic cells.

Methods

Peripheral blood mononuclear cells (PBMC) from 12 healthy volunteers were isolated by density separation over Ficoll solution. After resuspension in adaptive immunotherapy medium (AIM)-V medium, they were cultured without exogenous growth factors. The study group was treated with FK 778 (50 μg/mL) or Rapamycin (10 ng/mL). The phenotype of dendritic cell was ascertained by indirect immunoflurescence for analysis by flow cytometry.

Results

Compared with the Rapamycin-treated controls, the expressions of CD80, CD83, CD86, HLA-DA, CD54, CD62, CCR5, and CCR7 in the FK778-treated myeloid dendritic cells and the expression of CD80, CD83, CD86, HLA-DA, and CD54 in the FK778-treated plasmacytoid dendritic cells were significantly down-regulated.

Conclusion

FK778 inhibited the differentiation and maturation of dendritic cells.     

Human cytokine expression profile in various conditioned media for in vitro expansion bone marrow and umbilical cord blood immunophenotyped mesenchymal stem cells

Introduction

Bone marrow (BM) represents the major source of mesenchymal stem cells (MSCs); however, umbilical cord blood (UCB) MSCs have some advantages over BM, such as a higher differentiation capability and noninvasive collection methods.

Objectives

We compared antigen expression and cytokine-secretion by MSC from BM and UCB expanded with media supplemented with fetal bovine serum (FBS) or human platelet lysate (HPL).

Materials and Methods

We compared protocols for the expansion of hMSC starting from samples of BM or UCB by morphological analysis, calculation of population doubling numbers, and cytometry techniques using monoclonal antibodies (BD Biosciences). Using the last technique, cytokines were detected in brain homogenate supernatant fluids of MSC cultured in various media, using the Bio-Plex cytokine assay system (BD Biosciences).

Results

Calculating the number population doubling (PD) and colony-forming unit-¹fibroblast (CFU-F) assays showed significantly better expansion with HPL compared with a selected batch of FBS and within fewer days: PD about 5 for 10%HPL versus 25 for fibroblast growth factor2 (FGF2) medium. By flow cytometry, we observed a greater number of BM MSCs compared with UCB MSCs, as well as differences in the expression of some MSC antigens, particularly CD105, CD90, and CD31. Analysis of cytokines: FGFb, RANTES, VEGF, IL-6, IL-8, G-CSF, and GM-CSF showed only some of them to be expressed: namely, IL-6, IL-8, and VEGF. MSCs derived from UCB showed low concentrations of these cytokines compared with MSCs derived from BM.