Laser-assisted generation of human induced pluripotent stem cells.

This unit describes a procedure for generating human induced pluripotent stem cells (hiPSCs) using the Laser-Enabled Analysis and Processing (LEAP®) system, which combines high-throughput cell imaging with laser-mediated cell manipulation. Use of this system should not only improve the quality and uniformity of hiPSCs produced, but ultimately enable a more rapid, efficient, high-throughput, and automated production process.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

High-Resolution Genomic Profiling of Chromosomal Abnormalities in Human Stem Cells Using the 135 K StemArray.

Culturing stem cells for an extended period of time can lead to acquired chromosomal aberrations. Determining the copy number variant (CNV) profile of stem cell lines is critical since CNVs can have dramatic effects on gene expression and tumorigenic potential. Here, we describe an improved version of our StemArray, a stem-cell-focused comparative genomic hybridization (aCGH) microarray, which contains 135,000 probes and covers over 270 stem cell and cancer related genes at the exon level. We have dramatically increased the median probe spacing throughout the genome in order to obtain a higher resolution genetic profile of the cell lines. To illustrate the importance of using the StemArray, we describe a karyotypically normal iPSC line in which we detected acquired chromosomal variations that could affect the cellular phenotype of the cells. Identifying adaptive chromosomal aberrations in stem cell lines is essential if they are to be used in regenerative medicine.