A trap for iPSC
A new method of cell separation brings the era of cell therapy closer
LifeSciencesToday based on Georgia Tech materials:
Adhesive Differences Enable Separation of Stem Cells to Advance Potential TherapiesThe newly developed process of separating a mixture of different cell types, based on easily distinguishable differences in their adhesive properties, allows increasing the number of stem cells obtained by reprogramming.
Facilitating the conduct of new research, this process can help both in improving the reprogramming method itself and in modeling pathological processes characteristic of certain diseases.
The reprogramming method allows a small percentage of cells – most often skin or blood cells – to become human induced pluripotent stem cells (hiPSCs), from which a wide range of other cell types can then be obtained. A huge advantage of this method is the possibility of using it to reprogram the patient's own cells, which, sooner or later, will make clinically available methods of regenerative therapy based on transplantation, for example, cardiomyocytes for the treatment of cardiovascular diseases or neurons for the treatment of Alzheimer's disease or Parkinson's disease, without the risk of their immune rejection.
However, the practical use of reprogrammed cells is hindered by the low efficiency of the method: in the resulting mixture of different cell types, cells of clinical interest make up only a small percentage. Isolation of a pluripotent stem cell population from this mixture is currently a laborious process and requires a certain level of skill, which limits the use of the method itself and hinders the development of treatments based on pluripotent stem cells.
To solve this problem, scientists at the Georgia Institute of Technology (Georgia Tech) have developed a regulated process in which cell separation occurs based on the degree of their adhesion to the substrate inside a tiny microfluidic device.
(Photo: gatech.edu )The adhesive properties of human induced pluripotent stem cells differ significantly from those of other cell types, which makes it possible to isolate cells with therapeutic potential with 99 percent purity.
Adult human fibroblasts with intracellular proteins involved in the adhesion of these cells to the extracellular matrix. The stress fibers of actin are purple, the focal adhesion protein vinculin is green, which together determine how strongly the cells adhere to the matrix surface. The nuclei of cells are colored blue. These fibroblasts can be reprogrammed into human induced pluripotent stem cells, during which the adhesion proteins are restructured. (Photo: Ankur Singh)The new separation process, which takes less than 10 minutes, is based on a fundamentally different approach than labeling technologies, in particular, with antibodies.
And the possibility of separating intact cell colonies allows you to avoid cell damage, raising their survival rate above 80 percent. The resulting cells retain normal transcription profiles, differentiation potential, and karyotype.
"Our separation principle is based on the physical phenomenon of adhesion controlled by the corresponding biology," explains the head of the study, Andres Garcia, PhD, professor at the Woodruff School of Mechanical Engineering and the Petit Institute for Bioengineering and Bioscience of Georgia Tech. "This is a very powerful technological platform that is easily implemented and allows for the process of cell separation on an industrial scale."
The new technology is described in an article published in the online edition of the journal Nature Methods (Ankur Singh et al., Adhesion strength-based, label-free isolation of human pluripotent stem cells). The work was supported by the National Institutes of Health (NIH) USA, the National Science Foundation (National Science Foundation) and other foundations of the USA.
"These scientists have developed a fast and effective method for isolating a population of medically important cells, based on a new understanding of the adhesive properties of human induced pluripotent stem cells," Paula Flicker from the National Institute of General Medical Sciences (NIH), who partially funded this research, says about the new technology research. "Their work represents an innovative conversion of fundamental biological discoveries into a strategy with therapeutic potential."
According to the Director of the Stem Cell Engineering Center Georgia Institute of Technology Associate Professor of the Department of Biomedical Engineering at Georgia Tech and Emory University Todd McDevitt, PhD, the improved separation method is very important for the development of viable therapies based on human induced pluripotent stem cells obtained by reprogramming.
"In scientific research, dependence on labeling reagents for separation is not a serious problem," continues Dr. Mcdewitt, one of the co-authors of the article. "But if we are going to commercialize and create cell therapy for people, we need a technological approach that gives an unambiguous result and allows us to increase production."
The new separation technology, called micro stem cell high-efficiency adhesion-based recovery (muSHEAR), will standardize the work of laboratories, providing stable results that do not depend on the skill level of their personnel. "Thanks to engineering, technology and research work, we now have a method with the prospect of widespread adoption," says Dr. Mcdewitt.
The muSHEAR method grew out of understanding how cell morphology changes during the reprogramming process. Using a rotating disk device, the scientists tested the adhesive properties of human induced pluripotent stem cells, parent somatic cells, partially reprogrammed and fully reprogrammed cells that have already begun differentiation. For each cell type, they determined the adhesive signature – the force needed to separate cells from a substrate coated with specific proteins.
The researchers tested their technology on microfluidic devices developed in collaboration with Professor of the School of Chemical and Biomolecular Engineering (School of Chemical and Biomolecular Engineering) Georgia Tech Han Lu (Hang Lu).
In these tests, cultured cells were first allowed to attach to the substrate, and then exposed to a buffer fluid flow. Cells with less adhesion to the substrate were separated from it at lower flow rates. By changing the flow rate, the researchers were able to separate the unnecessary cell types in this case and obtain a stem cell culture with a purity equal to 99 percent – and this is from mixtures in which hiPSCs accounted for only a few percent of the total number of cells.
"At different stages of reprogramming, we see differences in the molecular composition and distribution of cellular structures, on which the strength of adhesion depends," explains Professor Garcia. "If we know the range of adhesive forces of each cell type, we can use these narrow ranges to select specific populations."
The microfluidic system, which uses inexpensive disposable cassettes for operation, can be easily switched to the mode of obtaining an increased cell volume and ensuring specific separation, Professor Garcia notes.
In addition to directly obtaining stem cells for further clinical use, the new separation method can help scientists in scientific research, including in improving the reprogramming method that brought its author Shinya (Xinya) Yamanaka (Shinya Yamanaka) The Nobel Prize in Medicine for 2012.
"Cell reprogramming has been and remains a black box," Dr. Mcdewitt sums up... "there are a number of really interesting scientific questions connected with this process, and by separating the cells undergoing the reprogramming process, we will be able to make new discoveries about how it proceeds."
Portal "Eternal youth" http://vechnayamolodost.ru16.04.2013