Stem Cell Studies

Analysis of pro-arrhythmic effects induced by different routes of administration of bone marrow stem cells

Anna Vittoria Mattioli — 2012-03-26

There has been repeated concern that intramyocardial (i.m.) delivery of cells could cause ventricular arrhythmias. The aim of the study was to evaluate the pro-arrhythmic effects of bone marrow stem cell (BMSC) injection and compare different routes of administration. An ischemia reperfusion injury was induced in New Zealand rabbits by temporal ligation of anterior descending coronary artery. Homologous BMSCs were isolated, cultured and re-suspended for injection. We compared different routes of BMSC injections, intramyocardial (i.m.) versus intravenous (i.v.) administration of cells. A control group was treated with i.m. injections of saline. The hourly number of supra- and ventricular premature contractions (VPCs), QT interval time and QTc time were recorded and calculated. At Day 7 after cell injections, VPCs were more frequent in the groups treated with i.m. BMSCs and i.m. saline compared with i.v. BMSCs (132 ±19; 54±14 and 34±9, respectively; P<0.01 within groups), whereas at Day 21 the number of VPCs was higher in the 2 groups treated with either i.m. or i.v. BMSCs compared with saline (96±23; 52±19 and 25±20, respectively; P<0.001 within groups). QTc time interval was prolonged during ischemia, and recovered in control and in the group treated with i.v. cells, whereas it remained longer in rabbits treated with i.m. BMSCs. These findings show that i.m. BMSC injections induced a high number of pre-arrhythmic events suggesting changes in cardiac electrophysiological properties. The i.v. administration of cells resulted in lower VPC beats and in a temporary QT prolongation. These results suggest that the combination of BMS cells and i.m. injections induced an electrical remodeling that contributed to the development of arrhythmias.

Stage specific differentiation of human embryonic stem cells into hepatocyte-like cells using conditioned medium from a human hepatoma cell line

Arundhati Mandal — 2012-04-02

Hepatocytes derived from human embryonic stem cells (hESC) promise to be an inexhaustible source of functional cells for use in biomedical research, drug discovery and treatment of liver diseases. We have developed a unique strategy to efficiently differentiate hESC into functional hepatocyte-like cells (HLC) in vitro. The robustness of our protocol was assessed by duplicating the process of differentiation in two of our in house derived hESC lines, Relicell^®hES1 and Relicell^®hES2, and in the well studied BG01 cell line. To induce early hepatic commitment, undifferentiated hESC were initially primed with conditioned medium from HepG2, a human hepatoma cell line, which resulted in an enriched population of definitive endoderm. We have also attempted to recapitulate the hepatogenetic events occurring in vivo by sequential application of growth factors involved in liver development, such as aFGF, HGF, oncostatin, dexamethasone and EGF. Our differentiation process yielded a homogenous population of HLC exhibiting the typical polygonal morphology of hepatocytes. This population expressed hepatic lineage markers including HNF4α, AFP and ALBUMIN, and drug metabolizing enzymes such as CYP3A4 (Phase I) and GSTA1 (Phase II). Flow cytometric analysis showed that over 70% of the differentiated cells expressed albumin and CK8/18. The differentiated HLC exhibited hepatic characteristics such as glycogen storage and production of albumin and urea. Our results indicate that functional HLC generated by this method can be utilized in regenerative medicine and as a screening platform in the discovery and development of new drugs.

The effect of physiological oxygen levels on GABAergic neuronal differentiation from mouse embryonic stem cells

Eunju Shin — 2012-05-14

Embryonic stem cells (ESCs) have the ability to generate any kind of cell in the body. They, therefore, have great potential for use in cell therapies for neurodegenerative disorders such as Huntington’s disease. Establishing a culture environment to mimic components of physiological conditions may help to maintain and differentiate ESCs more successfully. One of the important conditions is the level of oxygen. Traditionally, 20% oxygen (O₂) has been used to culture cells, but this is much higher than physiological levels (2% O₂). In this study, we used the mouse ESC line 46C (Sox1- GFP knock-in) to investigate the effect of physiological oxygen on proliferation of mESCs, and their differentiation to neural progenitors (where Sox1 is expressed) and mature GABAergic neurons. mESCs were cultured in either high (20%, H) or low (2%, L) oxygen levels for four days before induction of differentiation, and subsequently differentiated under either high or low oxygen, in a 2x2 factorial design (H-H, H-L, L-H, L-L). mESCs placed in low oxygen levels during the differentiation phase showed less proliferation (a decreased proportion of Ki67⁺ cells), complete loss of the self-renewing population (Oct4⁺ cells), and a decrease in Sox-1⁺ neural precursors. Consistent with this, neurons generated under low levels of oxygen showed a more mature morphology with an increased number of primary neurites and increased levels of GABA neurotransmitter. There was no significant difference in the percentage of neurons generated from either condition. We conclude that mESC culture in low oxygen conditions promotes maturation during neuronal differentiation and helps eliminate the residual Oct4⁺ population. The adoption of low oxygen environments during neuronal differentiation may, therefore, decrease teratoma formation and increase the potential for ESC use in cell therapies for neurodegenerative disease.

Defining heterogeneity of human non-metastatic breast cancer tumor by identifying individual cell types using cellular and molecular markers

Pravin D. Potdar — 2012-07-11

Breast cancer is a complex and heterogeneous disease with a high degree of intratumor heterogeneity and diversity. In the present study, we isolated morphologically distinct cell types with tumorigenic potential from breast cancer tumor. The cells show epithelial, endothelial, mesenchymal and mixed cell types which were characterized by using specific molecular markers. Our data showed that these CD44+ cells distinctly expressed mesenchymal phenotype with self-renewal potential and proliferative capacity. These cells also expressed oncogenic, cytokine and chemokine markers indicating their cancer initiating stem cell nature. Interestingly, these cancerinitiating cells exhibit an A3624G BRCA2 mutation. This study reports for the first time the heterogeneous population of malignant cells in the breast cancer tumor which are responsible for cancer development. It represents a suitable in vitro model to allow us to study the mechanism of breast cancer development, and to identify and design a specific molecular target for breast cancer therapies even at advanced stage of disease.