Effect of Cell Derived Matrices on growth and differentiation of human Wharton's jelly derived Mesenchymal Stem Cells.

Cell derived matrices (CDMs) are scaffolds constructed by decellularization of cellular matrices from different tissues and organs. Since cell derived matrices mimic the ECM of native tissues, CDM plays an essential role in the preparation of bioscaffolds. CDM scaffolds from Mesenchymal Stem Cells (MSCs) have been reported to support cell adhesion and proliferation of its own cells. Therefore, in this study we aimed to test if growth of human Wharton's jelly derived MSCs (hWJ-MSCs) may be enhanced when cultured on their own cell derived matrices. To do this, MSCs were induced to generate ECM using ascorbic acid. Thus, obtained matrices were decellularized and characterized quantitatively for changes in their biochemical components (total protein, collagen, glycosaminoglycans) and qualitatively for fibronectin, laminin and collagen (I & IV) by immunostaining. Our results show the retention of essential ECM components in the decellularized WJ-CDM. The influence of WJ-MSC-derived CDM on proliferation and differentiation of WJ-MSCs were evaluated by comparing their growth on collagen and fibronectin only coated plates. A non-coated tissue culture polystyrene plate (TCPS/WC) served as control. Our cell proliferation results show that no significant changes were observed in the proliferation of MSCs when cultured on WJ-MSC derived CDM as compared to the bio-coated and non-coated cultures. However, gene expression analysis of the differentiation process showed that osteogenic and adipogenic differentiation potential of the WJ-MSCs was significantly increased upon culturing them on WJ-MSC-CDM. In conclusion, the present study reveals that the WJ-MSCs cultured on WJ-MSC-CDM may augment osteogenic and adipogenic differentiation.

Differential immunomodulation of human mesenchymal stromal cells from various sources in an inflammation mimetic milieu.

Mesenchymal stromal cells (MSCs) are very advantageous in the field of regenerative medicine because of their immunomodulatory properties. However, reports show that these properties vary from source to source. Hence, understanding the source-dependent specificity of MSCs and their immunomodulatory abilities will enable optimal use of MSCs in cell-based therapies. Here, we studied human MSCs from three different sources, adipose tissue (AT), bone marrow (BM) and Wharton's jelly (WJ), with respect to phenotypic responses of human peripheral blood mononuclear immune cells (hPBMCs/MNCs) and the concurrent changes in cytokine expression in MSCs, under mitogen-stimulated co-culture conditions. We used cytometric analysis to study the immunoregulatory properties of MSCs on MNCs and cytokine profiling of MSCs using a customized PCR array and solid-phase sandwich enzyme-linked immunosorbent assay. Our results reveal differential modulation of immune cells as well as MSCs upon activation by the mitogen phytohemagglutinin, independently and in co-culture. Notably, we observed source-specific MSC-cytokine signatures under stimulated conditions. Our results show that AT-MSCs up-regulate VEGF, BM-MSCs up-regulate PTGS-2 and WJ-MSCs increase expression of IDO considerably compared with controls. This remarkable modulation in source-specific cytokine expression was also validated at a functional level by quantitative protein expression studies. In our hands, even though MSCs from AT, BM and WJ sources exhibit characteristic immunomodulatory properties, our results highlight that MSCs sourced from different tissues may exhibit unique cytokine signatures and thus may be suitable for specific regenerative applications.

Effect of Human Platelet Lysate in Differentiation of Wharton's Jelly Derived Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are highly preferred in clinical therapy for repair and regeneration of diseased tissues for their multipotent properties. Conventionally, MSCs have been cultured in media supplemented with animal derived serum, however, it is ideal to expand MSCs in media containing supplements of human origin for clinical therapy. Currently, a number of human derived products are being studied as an alternative to animal sources. Amongst these, platelet lysate (PL) has gained interest in the culture of MSCs without affecting their phenotypic property. OBJECTIVE: In this study, we used various concentration of PL (2.5, 5, 7.5 & 10%) in the growth medium of MSCs to identify the least concentration of PL that could be an effective alternative to animal products. METHODS: MSCs were isolated from Wharton's Jelly by using explant method and expanded in various concentration of PL supplemented medium against the standard FBS containing medium. WJ-MSCs were characterised as per the minimal criteria proposed by International Society for Cell therapy (ISCT), Proliferation study by BrdU assay, gene expression study by qRT-PCR, sterility test for bacteria, Mycoplasma by PCR and endotoxin detection by LAL assay. RESULTS: Whartons jelly derived MSCs (WJ-MSCs) cultured using standard medium supplemented with various concentration of PL exhibited enhanced proliferation and differentiation potential, unaltered immunophenotypic property and genetic stability when compared with the commercial medium containing 10% FBS. CONCLUSION: The least concentration of PL for an ideal expansion of MSCs was found to be 2.5% and was comparable to FBS.

Role of Human Wharton's Jelly Derived Mesenchymal Stem Cells (WJ-MSCs) for Rescue of d-Galactosamine Induced Acute Liver Injury in Mice.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) are multipotent precursor cells having self-renewal ability making them a candidate for use in regenerative medicine. Acute liver injury results in sudden loss of hepatic function leading to organ failure. Liver transplantation is often required to salvage patients with acute liver failure. Due to shortage of organs, identification of alternate method is the need of the hour. In view of this, an attempt has been made to check the regenerative ability of WJ-MSCs (wharton's jelly derived MSC) in mice models for acute liver injury. METHODS: Swiss albino mice weighing 25 ± 5 g were used in this study. The control mice (Group I), was given saline. Group II mice received d-Galactosamine (d-GalN-800 mg/kg; i.p). Group III mice similar with Group II, received WJ-MSCs (5 × 105 cells/0.5 ml DMEM) through tail vein, 24 h after d-GalN administration and Group IV mice received MSC alone. RESULTS: Parameters, indicative of hepatotoxicity and oxidative stress were analyzed. A two-fold elevation in the marker enzymes of liver toxicity such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (SAP), and total serum bilirubin (TBIL) confirms hepatocellular injury, while a greater than four-fold increase in malondialdehyde (MDA) formation, along with around 40% fall in superoxide-dis-mutase (SOD) activity was indicative of oxidative stress and loss of hepatocellular membrane integrity induced by d-GalN. The above biochemical and pathological changes were significantly restored in mice that received WJ-MSCs indicating hepatoprotective and probable regenerative property. CONCLUSION: The present study showed that WJ-MSC treatment is able to rescue/ameliorate the hepatotoxicity induced by d-GalN in mice.

Isolated caudate lobe resection: technical challenges.

BACKGROUND: Isolated caudate lobe resection remains a technical challenge even in the best hands. This is due to the difficult approach and its location between major vessels. This retrospective study aims to analyze our experience with isolated caudate lobe resections. METHODS: Of the 402 patients who underwent liver resections between January 2002 and December 2011, we identified 13 caudate lobectomies. We analyzed the operative parameters, hospital stay, morbidity and follow up of these patients. RESULTS: There were nine males and four females, age ranging between 30 and 72 years. The indications were hepatocellular carcinoma in nine patients, hilar cholangiocarcinoma in two, solitary fibrous tumor in one, and a regenerative nodule in one patient. Left-sided approach was employed in seven cases, right-sided approach in three cases and a combined approach in three cases. Operating time ranged between 125 and 225 min and blood loss ranged between 210 and 630 mL. There was no mortality in the post-operative period. No local recurrence was noted in the follow-up period ranging from 6 months to 7 years. CONCLUSION: Caudate lobe resections, although technically challenging, can be successfully performed with minimal blood loss. Surgery offers potential cure in isolated caudate lobe tumors. The location and size of the tumor decides the approach.

Role of endoscopic band ligation in management of non-variceal upper gastrointestinal bleeding.

BACKGROUND: Acute non-variceal upper GI bleeding (NVUGIB) is a challenging emergency condition. Early endoscopic therapy has been recommended as the first-line of treatment for upper GI bleeding (UGIB) as it has been shown to reduce recurrent bleeding. We aimed to determine the various causes of NVUGIB and discuss the role of band ligation. PATIENTS AND METHODS: A total of 74 patients with NVUGIB who had been treated with endoscopic band ligation (EBL), between November 2006 and December 2011, were included in the study. Bleeding lesions included Dieulafoy lesion (DL), Mallory-Weiss tears (MWTs), duodenal ulcer, post-surgical anastomosis bleed and gastric ulcer after polypectomy. After the basic life support was provided, all patients underwent emergent and elective endoscopy. RESULTS: The study comprised 49 (66.2%) men and 25 (33.8%) women. The mean age was 48.2 +/- 6.4 years for men and 40.6 +/- 2.2 years for women. MWTs and DL constituted the majority of bleeding lesions, of these 26 and 17, respectively required EBL. Other causes were: prepyloric ulcer 11; duodenal ulcer 9; ulcers in antrum 5; post-polypectomy bleed 3; anastomosis bleed 1; and malignant lesions 2. Bleeding stopped after endoscopic therapy in 96.5% of patients. The single failure was in bleeding from a pre-pyloric lesion which was treated by using injection sclerotherapy with 1:10,000 adrenaline solution where EBL was not successful. CONCLUSION: EBL provides safe and effective modality for haemostasis in patients with NVUGIB. EBL could be considered as an alternative method of choice for treatment of endoscopic haemostasis in patients with NVGIB.

Biliary cystadenomas: a case for complete resection.

Introduction and Objective. Biliary cystadenoma is a rare benign neoplasm of the liver with less than 200 cases being reported allover the world. We report a series of 13 cases highlighting the radiological findings and problems related to its management. Materials and Methods. Records of thirteen patients who underwent surgery for biliary cystadenomas, between March 2006 and October 2011, were reviewed retrospectively. Results. Majority of the patients were females (11 out of 13), with a median age of 46 (23-65) years. The most frequent symptom was abdominal pain (92%). Seven patients had presented with history of previous surgery for liver lesions. Five patients had presented with recurrence after partial resection for a suspected hydatid cyst and two after surgery for presumed simple liver cyst. Ten of the 13 patients had complete resection of the cyst with enucleation in 3 patients, 2 of whom in addition required T-tube drainage of the bile duct. There has been no recurrence during the follow-up period ranging from 3 months to 5 years. Conclusion. Biliary cystadenoma must be differentiated from other benign cysts. Hepatic resection or cyst enucleation is the recommended treatment option.

Characteristics of gallbladder cancer in South India.

Gallbladder cancer is common in north India. It is also a well established fact that gall bladder cancer is frequently associated with gallstone disease in north India, similar to reports from the West. The magnitude of the problem of gallbladder cancer in south India and its link to gallstone disease is not clearly established. The aim of the study was to determine retrospectively, the characteristics of individuals with GBC in south India and to determine its association with gallstone disease. Retrospective data was obtained from records of proven cases of gallbladder cancer and patients undergoing cholecystectomy for gallstone disease between Jan 2001 and Dec 2010. Data retrieved included age, gender, and clinical presentation, findings on imaging, histology and details of management. The number of proven cases of gallbladder cancer each year ranged from 8 to 17. There were 38 men and 23 women. Male female ratio was 1.6:1. There were more men in the successive decades. Right upper quadrant pain (42 %) followed by jaundice (27 %) and a presence of a palpable mass (12 %) were the common clinical presentation. Pre-operative diagnosis of gall bladder cancer was possible in 80 %. Twelve patients had co-existing gallstones (19.6 %). Forty patients (50 %) had stage IV disease; only 6 patients had Stage I operable disease (9.8 %). During the same time 758 patients had cholecystectomy for gallstone disease. Only one patient had an incidental gall bladder cancer, who had an extended cholecystectomy. Gallbladder cancer is uncommon in south India and its association with gallstone is also low.

Influence of copper, iron, zinc and fe (3) (+) haemoglobin levels on the etiopathogenesis of chronic calcific pancreatitis--a study in patients with pancreatitis.

Chronic pancreatitis is a serious condition associated with severe abdominal pain, and a significant percentage of patients progresses to irreversible calcification in pancreas. The present study evaluates the degree to which the levels of trace elements, copper, iron, selenium, zinc and haemoglobin-Fe(3+), in blood, serum and pancreas have any role to play in the calcification process associated with fibrosis in pancreas. Twenty-seven calcific (CCP) and 23 non-calcific chronic pancreatitis (CP) patients and equal number of age- and sex-matched normal volunteers (50) were enrolled in the study. Surgically removed pancreatic tissue and blood samples were analysed for copper, iron, selenium, zinc, protein, collagen and lipid peroxidation products in terms of malondialdehyde, protein carbonyls, glutathione, methemoglobin, methemoglobin reductase and ceruloplasmin activity levels. We could find that the pancreatic tissue levels of copper, iron, protein and collagen contents were significantly elevated in CCP patients when compared to CP patients. Serum levels of copper, free ionic copper and iron were also elevated in CCP patients. The serum and the pancreatic tissue level of zinc and selenium showed a significant decrease in CCP patients. The level of methemoglobin was elevated more significantly with the concomitant decline in the activity of methemoglobin reductase. There was a positive correlation between the pancreatic level of copper and iron with the collagen and protein levels. The results of the present study revealed that the levels of copper and iron, the pro-oxidants and zinc and selenium may influence calcification process in CCP patients. Hypoxia-related tissue injury due to the formation of oxidised haemoglobin may also contribute to the pathogenesis of calcification in pancreas.

Role of cytosolic calcium and actin polymerization on agonist-induced secretion by the platelets of liver cirrhosis patients.

OBJECTIVE: Variceal bleeding in cirrhosis is mainly due to platelet activation defect and secondary to coagulation defects. Secretion is an important process which release procoagulants for hemostasis. In the present investigation we have evaluated the secretory function of platelets in liver cirrhosis and also the simultaneous changes in cytosolic calcium (Ca2+) and the polymerization of actin in agonist- stimulated platelets in vitro. METHODS: Liver cirrhotic patients with (n=27) or without (n=23) bleeding complication were included in the study. Control subjects (n=50) were also utilized for the study to compare the analytical data. Platelets were activated by collagen in vitro and the secretory response was assessed by the levels of nucleotides, serotonin, pyrophosphate (PPi) and inorganic phosphate (Pi) secreted into the extracellular fluid of the platelet suspension at various time intervals. During the course of secretion the alteration in the polymerization of actin was monitored simultaneously with the changes in the cytosolic Ca2+ level. RESULTS: The secretory response of platelets to collagen was significantly low in both bleeders and non-bleeders when compared to that of normal subjects. During secretion, low level of actin polymerization and cytosolic Ca2+ level were observed in the platelets of bleeders than in non-bleeders and normal subjects. The low secretory capacity of cirrhosis platelets could be correlated with low levels of actin polymerization and cytosolic Ca2+. The alterations were highly significant in the platelets of bleeders when compared to those of non-bleeders. CONCLUSION: The defective secretory activity of platelets in cirrhosis bleeders might be partly due to low polymerization of G-actin to F-actin which is required for platelet shape change and for the release of procoagulants. Cytosolic Ca2+ level seems to influence actin polymerization and thereby impairs platelet secretory response to agonists in cirrhosis patients with bleeding complication.

Evaluation of neutrophil functions in obstructive jaundice patients.

Jaundiced surgical patients have a high incidence of post-operative complications and sepsis is a major factor in the high mortality and morbidity of obstructive jaundice patients after surgery. Cachexia and immune suppression have been shown to be involved in this post-operative complications. Since neutrophils dysfunction is recognized in recurrent infections in general, the aim of the study was to evaluate the functional status of neutrophils in jaundiced patients. Phagocytosis, bactericidal activity, mitogenic stimulation of T-lymphocytes and myeloperoxidase activity were measured in the neutrophils isolated from obstructive jaundice patients. Phagocytic and bactericidal activities of neutrophils were significantly low in obstructive jaundice patients. The number of cells stained for myeloperoxidase was significantly low in obstructive jaundice patients. There was a high level of stimulation and proliferation exhibited by lymphocytes when challenged by conconavalin-A and lipopolysaccharide in vitro. The functional impairment of neutrophils may be responsible for the immunity alteration in obstructive jaundice patients and this may be responsible for the post-operative complications in obstructive jaundiced patients.