Daynene M. Vykoukal

Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction

AIMS We assessed whether freshly isolated human adipose tissue-derived cells (fhADCs) or cultured human adipose tissue-derived stem cells (hASCs) have beneficial effects on cardiac function after myocardial infarction (MI), whether the injected cells can survive long term, and whether their effects result from direct differentiation or paracrine mechanisms. METHODS AND RESULTS Myocardial infarction was experimentally induced in severe combined immunodeficient mice, and either fhADCs, cultured hASCs, or phosphate-buffered saline was injected into the peri-infarct region. Myocardial function improved significantly in mice treated with hASCs or fhADCs 4 weeks after MI. Immunofluorescence revealed that grafted hASCs and fhADCs underwent cardiomyogenic differentiation pathway, as indicated by expression of connexin 43 and troponin I in a fusion-independent manner. Some of the injected cells integrated with host cardiomyocytes through connexin 43, and others were incorporated into newly formed vessels. Human adipose tissue-derived stem cells survived in injured hearts up to 4 months, as detected by luciferase-based bioluminescence imaging. Vascular density was significantly increased, and fewer apoptotic cells were present in the peri-infarct region of cell-injected mice. CONCLUSION This is the first study to systematically compare the effects of fhADCs and hASCs on myocardial regeneration. Both cell types engraft into infarcted myocardium, survive, and improve myocardial function, suggesting that fhADCs, like hASCs, are a promising alternative cell source for myocardial repair after MI.

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)].

A three-dimensional dielectrophoretic particle focusing channel for microcytometry applications

In this paper, we have designed and fabricated a microfluidic channel to focus biological cells using dielectrophoresis for cytometry applications. The device consists of an elliptic-like channel fabricated by isotropic etching of soda lime glass wafers and a subsequent wafer-bonding process. Microelectrodes are patterned on the circumference of the channel to generate ac fringing fields that result in negative dielectrophoretic forces directing cells from all directions to the center of the channel. An analysis using a thin shell model and experiments with microbeads and human leukemia HL60 cells indicate that biological cells can be focused using an ac voltage of an amplitude up to 15 V/sub p-p/ and a frequency below 100 kHz, respectively. This design eliminates the sheath flow and the fluid control system that makes conventional cytometers bulky, complicated, and difficult to operate, and offers the advantages of a portable module that could potentially be integrated with on-chip impedance or optical sensors into a micro total analysis system.

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.

Dielectric characterization of complete mononuclear and polymorphonuclear blood cell subpopulations for label-free discrimination

Dielectric spectroscopy is a powerful technique for the elucidation of a number of important cell biophysical properties, and it can provide information about cell morphology, physiological state, viability and identity. A high-impact application for dielectric cell analysis would be microfluidic flow-through impedance sensing to perform what is perhaps the most routinely ordered medical diagnostic assay, a complete blood count with white blood cell differential enumeration. To assess the biophysical feasibility of such an analysis, we obtained reference dielectric measurements of the complete complement of purified leukocyte subpopulations using the dielectrophoretic crossover frequency method. The sensitivity of this method can detect subtle changes in cell morphology and physiology, so we developed a leukocyte isolation protocol based on a suite of negative selection techniques to yield cell subpopulations that were minimally processed and in an as near native state as possible. This is the first reported study of the dielectric properties of all the major leukocyte subpopulations that includes separate analysis of the polymorphonuclear neutrophil, basophil and eosinophil cell types. We show that T-lymphocytes, B-lymphocytes, monocytes and granulocytes possess distinct membrane dielectric properties and that the morphologically similar granulocyte subpopulations can be identified via their dielectric and size properties. Finally, we discuss the application of our findings to label-free systems for the analysis of leukocytes.

Quantitative Detection of Bioassays with a Low-Cost Image-Sensor Array for Integrated Microsystems†

A considerable global need exists for simple, portable, inexpensive, and integrated assay and diagnostic approaches that are appropriate for use in minimal-infrastructure, resource-poor settings, such as those found in the developing world, as well as in resource-limited environments, such as those encountered by emergency first responders, primarycare physicians, patients at home, forensic investigators, and military field personnel. Continuing advances in microfluidics have enabled the demonstration of prototype lab-ona-chip devices that offer to help answer this challenge and improve access to chemical and biological sample analysis by paving the way for the introduction of low-cost, portable point-of-need assay systems. Although such systems would have immediate applications in many fields, there are at present relatively few commercially available examples of the technology. 5] In clinical and industrial laboratory analyses, the most widely used and generally accepted methods to quantify particulate chemical or biochemical analytes employ optical detection approaches based on absorbance, fluorescence, or luminescence. Although lab-on-a-chip implementations of optical methods have been demonstrated, detection is typically carried out off-chip by using conventional microscope optics and digital-camera systems or custom and relatively expensive chip-scale optoelectronics. 7] The translation of these established methods into truly portable total-analysis microsystems has been hindered in part by a lack of reasonably priced, sensitive, and compact optical detectors that can be interfaced readily with microfluidic sample handling. In response to this limitation, we demonstrate the feasibility of applying an inexpensive, readily available complementary metal–oxide–semiconductor (CMOS) image sensor, originally intended for use in mass-market digitalcamera applications, for integrated optical detection in a variety of microfluidic systems. Specifically, we report the direct integration of this chip-scale sensor with our digital fluid-handling system to track nanoliter-volume reagent droplets by contact imaging. We also show the more general application of the sensor as a quantitative photometer for the integrated optical detection of colorimetric and bioluminescence assays implemented in various lab-on-a-chip architectures. Point-of-need assay platforms will complement, rather than supplant, existing laboratory-based analysis methods and hardware that form the foundation of clinical diagnostics and academic research. Indeed, within the realm of analytical (and in particular, diagnostic) technology and instrumentation, the development of inexpensive point-of-need assays is a rather specific, but not inconsequential endeavor. Over 95% of deaths due to major infectious diseases occur in developing countries. Although these diseases are largely treatable with drug therapy, the lack of available, infrastructure-appropriate diagnostic assays means that healthcare workers in these settings are unable to identify who is, or—just as critically—is not, in need of treatment. Simple, portable, inexpensive assay systems are also of value in natural-disaster and other emergency situations that arise indiscriminately in all countries. Technology and instrumentation for performing chemical and biochemical analyses have, thus far, typically been advanced to meet the demands of comparatively wellfunded laboratories. State-of-the-art assay systems employing flow-injection analysis (FIA), automated microplate processors and readers, or other mechanized sample-handling technologies, 12] provide impressive capabilities for continuous or parallel processing and the assaying of hundreds or thousands of samples per hour (sample preparation and readout times combined can average well under one second per sample). Thus, they are especially useful in centralized laboratories where high-throughput is essential. Whereas such systems are currently the mainstay of clinical diagnostics, drug discovery, and many research laboratories (and will probably remain so for the foreseeable future), the primary target of lab-on-a-chip assay approaches is to provide “sample in, answer out” assay capabilities by integrating sample handling and processing steps into a closed microfluidic, often single-use, architecture that is more ideally suited for localized assay applications. It is probable that many analyses will remain the province of dedicated centralized laboratories, but it is also anticipated that therapies and biomarkers discovered with high-through[*] Dr. D. M. Vykoukal, Dr. G. P. Stone, Dr. P. R. C. Gascoyne, Dr. E. U. Alt, Dr. J. Vykoukal Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center 7435 Fannin Street, Houston, TX 77054 (USA) Fax: (+ 1)713-834-6103 E-mail: jody@mdanderson.org

Liposome-mediated transfection with extract from neonatal rat cardiomyocytes induces transdifferentiation of human adipose-derived stem cells into cardiomyocytes

Objective. Recent studies indicate that direct cell‐to‐cell interaction is involved in transdifferentiation of adult stem cells into cardiomyocytes. We investigated whether transdifferentiation of human adipose‐tissue‐derived stem cells could be achieved by transfecting the cells with a nuclear neonatal cardiomyocyte extract using a liposome‐based transfection system. Material and methods. In this study, we isolated stem cells derived from human subcutaneous adipose tissue. These cells were transfected with nuclear protein extracts from either isolated cardiomyocytes or whole hearts of neonatal rats. Results. We found that transfection induced expression of the cardiac markers α‐sarcomeric actin, Nk×2.5, troponin I and troponin T after 1–3 weeks. Whole‐heart protein extracts showed the additional capacity to induce differentiation into endothelial‐like and smooth muscle‐like cells. Conclusion. We demonstrate that transfection with nuclear protein extracts from neonatal rat cardiomyocytes can induce a cardiomyogenic differentiation pathway in human stem cells.

Engineered Dielectric Microspheres for Use in Microsystems

We present a new class of microspheres that have been designed with specific dielectric properties to enable their manipulation and identification in microsystems using dielectrophoretic methods. Self-assembled monolayers of alkanethiol and phospholipid were applied to gold-coated polystyrene microspheres. Microsphere populations having layers of different thicknesses were distinguishable and manipulatable by dielectrophoresis in a characteristic, frequency-dependent manner as predicted by theory. These proof of principle studies are the first step toward the development of an engineered bead-based dielectrophoretic microsystem for multiplexed molecular separation and analysis.