The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

<p>Abstract</p> <p>Background</p> <p>Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.</p> <p>Results</p> <p>The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior.</p> <p>Conclusions</p> <p>The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.</p

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

MEF2C Enhances Dopaminergic Neuron Differentiation of Human Embryonic Stem Cells in a Parkinsonian Rat Model

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field

Heat-shock proteins in infection-mediated inflammation-induced tumorigenesis

Inflammation is a necessary albeit insufficient component of tumorigenesis in some cancers. Infectious agents directly implicated in tumorigenesis have been shown to induce inflammation. This process involves both the innate and adaptive components of the immune system which contribute to tumor angiogenesis, tumor tolerance and metastatic properties of neoplasms. Recently, heat-shock proteins have been identified as mediators of this inflammatory process and thus may provide a link between infection-mediated inflammation and subsequent cancer development. In this review, the role of heat-shock proteins in infection-induced inflammation and carcinogenesis will be discussed

Time constraints do not limit group size in arboreal guenons but do explain community size and distribution patterns

To understand how species will respond to environmental changes, it is important to know how those changes will affect the ecological stress that animals experience. Time constraints can be used as indicators of ecological stress. Here we test whether time constraints can help us understand group sizes, distribution patterns and community sizes of forest guenons (Cercopithecus/Allochrocebus). Forest guenons typically live in small to medium sized one-male multi-female groups and often live in communities with multiple forest guenon species. We developed a time-budget model using published data on time budgets, diets, body sizes, climate, and group sizes to predict maximum ecologically tolerable group and community sizes of forest guenons across 202 sub-Saharan African locations. The model correctly predicted presence/absence at 83% of these locations. Feeding-foraging time (an indicator of competition) limited group sizes, while resting and moving time constraints shaped guenon biogeography. Predicted group sizes were greater than observed group sizes but comparable to community sizes, suggesting community sizes are set by competition among guenon individuals irrespective of species. We conclude that time constraints and intra-specific competition are unlikely to be the main determinants of relatively small group sizes in forest guenons. Body mass was negatively correlated with moving time, which may give larger bodied species an advantage over smaller bodied species under future conditions when greater fragmentation of forests is likely to lead to increased moving time. Resting time heavily depended on leaf consumption and is likely to increase under future climatic conditions when leaf quality is expected to decrease