Comparison of mammosphere formation from stem-like cells of normal breast, malignant primary breast tumors, and MCF-7 cell line.

BACKGROUND: Mammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors. METHODS: This study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44+/CD24-/low and CD49f+/EpCAM-/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining. RESULTS: Breast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44+/CD24-/low and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues. CONCLUSIONS: Thus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.

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.

Functional variations between Mesenchymal Stem Cells of different tissue origins: A comparative gene expression profiling.

BACKGROUND: Mesenchymal Stem Cells (MSCs), regardless of the tissue sources, are considered as excellent candidates for cellular therapy as they are immune-privileged cells containing a multitude of therapeutic functions that aid in tissue regeneration and repair. For the effective application of these cells in cell therapy, it is important to understand and characterize their biological functions. OBJECTIVES: The present study attempts to characterize the variations in multipotent function such as cell surface antigen levels, proliferation, differentiation and stemness (pluripotency) potential of MSCs isolated from foetal [wharton's jelly (WJ), foetal and maternal side of placenta (PF and PM)] and adult tissue sources [bone marrow (BM) and adipose tissue (AT)] using gene expression by real time PCR (qRT-PCR). RESULTS: Amongst the different tissue sources, PM, PF and AT-MSCs exhibited significant increase (p < 0.001, p < 0.001 and p < 0.01 respectively) in CD 73 expression and therefore could have a role in immunomodulation. WJ-MSCs exhibited superior proliferation potential based on growth curve, PCNA and Wnt gene expression. BM-MSCs were superior in exhibiting trilineage differentiation. Enhanced stemness potential (Oct 4 and Nanog) was observed for both BM and WJ-MSCs. In addition, BM and WJ-MSCs expressed high levels of CD 90 making them suitable in bone repair and regeneration. CONCLUSION: Thus to conclude, out of the five different sources tested, BM an adult source and WJ-MSCs a foetal source were superior in exhibiting most of the biological functions indicating that these sources may be suitable candidates for cell repair and regeneration studies.

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.

Punigratane, a novel pyrrolidine alkaloid from Punica granatum rind with putative efflux inhibition activity.

A new pyrrolidine alkaloid named Punigratane was isolated from the rind of Punica granatum. This is the first report of a pyrrolidine-like structure from the rind. The activity of this compound was tested in a representative MDR Klebsiella pneumoniae strain which exhibited high efflux pump activity. At a concentration of 6 mg, this compound Punigratane was found to have efflux inhibition activity.

Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study.

The multipotent and immunosuppressive capacities of mesenchymal stem cells (MSCs) attract several scientists worldwide towards translational research focusing on treatment of diseases including liver failure. Though MSC's have been isolated from different sources, researchers do not concur on the best source for expansion and clinical translation. In this study, we have compared the isolation, proliferation and expansion of MSCs from umbilical cord blood (UCB), Wharton's Jelly (WJ), bone marrow (BM) and adipose tissue (AT). MSCs were isolated by density gradient separation from UCB, BM and AT and by both enzymatic and explant method for WJ. The MSCs are characterized by their ability to adhere to plastic, expression of positive (CD105, CD73, CD90, CD29, CD44) and negative (CD45, CD14, CD34) markers by flow cytometry and also by their in vitro adipogenic, osteogenic and chondrogenic differentiation. This comprehensive study clearly shows that WJ is better than UCB both in terms of rapidity, yield and ease of procedure. AT and BM are autologous sources for MSC's but the specimen collection involves cumbersome and painful procedures and an invasive approach. However being autologous, they are safe and probable candidates for therapeutic future applications.

Hematological changes following early ligation of splenic artery during splenectomy in shunt surgery for portal hypertension.

OBJECTIVE AND BACKGROUND DATA: Reduction in cellular elements of blood, secondary to hypersplenism is an established component of non-cirrhotic portal hypertension. Prior transfusion of blood or blood components is frequently required for safe surgical intervention. Due to thrombocytopenia, epidural catheter insertion for effective and durable analgesia is not possible. The aim of the present study was to objectively demonstrate the gain in blood components following early ligation of splenic artery for splenectomy in shunt surgery. METHODS: From Jan 2008 to July 2010, 30 patients underwent elective proximal spleno renal shunt for portal hypertension, for various indications and were analyzed prospectively. We followed the standard protocol of ligating the splenic artery in situ, first in the lesser sac. Proximal spleno shunt was done. After the surgical procedure and before extubation, an epidural catheter was placed for effective and durable analgesia. 5ml of venous blood was drawn in the following order of sequence: prior to induction of anesthesia, immediately after the ligation of splenic artery, 30 minutes after ligation of splenic artery and 30 minutes after splenectomy. Samples were sent for complete hemogram and values were analyzed in respective order. Patients requiring transfusion of blood or blood components during surgery were excluded from the study. RESULTS: 30 patients (M - 9, F- 21) with mean age of 29.4 years (11-60 years) were analyzed (NCPF- 20, EHPVO- 9, cirrhosis- 1). We objectively demonstrated a significant gain in RBCs (p = 0.016) and platelets (p = 0.000) using this standard protocol. As there were no intrinsic abnormalities in RBCs, red blood cell indices (MCV, MCH, MCHC) showed no changes as expected (p-0.9). CONCLUSION: By following this standard protocol, in addition to reduction in blood loss there was a significant gain in RBCs and platelets. This gain allows the surgeon to perform the surgical procedure safely and the anesthetist to secure an epidural catheter immediately after surgery for effective and durable analgesia without prior transfusion.