细胞功能测定

Human pluripotent stem cells (hPSCs) have been used to derive self-renewing neural progenitor (NP) cell lines. Here we describe methods to genetically modify these cells. Detailed methods for transfection and nucleofection in PSC-derived NP cells are presented. We have shown that nucl ...

Relatively safe, HIV-1-based lentiviral vectors have served as an efficient means of transducing human embryonic stem cells (hESCs). Here we describe the variety of lentiviral vector systems available with the basic strategy for designing viral vectors and methods for generating vi ...

Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regu ...

High-content screening (HCS) permits simultaneous observation and analysis of multiple cellular variables including cell morphology, survival, and differentiation in live cells at the single-cell level, at the level of the culture well, and across the entire culture. By combining h ...

This chapter covers gene expression analysis by microarray to study and characterize stem cells. In a case-study scenario, we describe basic bioinformatic methodologies used to answer common questions in microarray experiments involving one or more stem cell populations. Service ...

Chromatin immunoprecipitation (ChIP) is used to evaluate the interaction of proteins and genomic DNA. In eukaryotic cells, the DNA is highly compacted with the evolutionarily conserved histone proteins (which together with DNA form the nucleosome) and other chromosomal-associ ...

Genetic diversity among human genomes comes in many forms, including single nucleotide polymorphisms (SNPs) and small insertions and deletions on the order of one to several basepairs. More recently, large, 1 kb copy number changes have been identified as an important source of normal geno ...

A teratoma is a nonmalignant tumor comprised of a disorganized mixture of cells and small foci of tissue comprised of cells from all three of the embryonic germ-layers. By definition, a cell is pluripotent if it can differentiate into cells derived from all three of the embryonic germ-layers: ecto ...

Human pluripotent stem cells, human embryonic stem cells and induced pluripotent stem cells, represent an exciting new era in regenerative medicine and drug discovery. However, prior to their clinical translation, there is a need to gain an in-depth understanding of human pluripotent st ...

This chapter will describe the most common immunocytochemical method utilized in the stem cell field – using fluorescently tagged secondary antibodies to detect a primary antibody that is bound to an epitope on a molecule of interest. Secondary antibodies recognize the heavy chain of the p ...

Human pluripotent stem cells (PSCs) hold promise for treating a multitude of diseases. These fascinating cells are unique in their ability to both self-renew and differentiate into cells from all three germ layers. However, PSCs, as well as other cultured cells, are prone to genetic instabili ...

Classical cytogenetics by karyotyping has been utilized in clinical research laboratories for more than 50 years and remains the key method used in the stem cell laboratory to assess the genetic stability of stem cell cultures. It is currently the most readily accessible method for detecti ...

Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined w ...

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous cu ...

For certain applications, particularly experiments involving high-resolution imaging, it is necessary to culture cells on glass slides or cover glasses. This chapter describes techniques for successfully growing human embryonic stem cells (hESCs) on glass surfaces under thr ...

Human pluripotent stem cells (PSCs), which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs), represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such, it is becoming increasi ...

This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is striki ...

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) allows the derivation of �personalized stem cells. Transposon transgenesis is a novel and viable alternative to viral transduction methods for the delivery of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) to ...

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodefic ...

This chapter describes a protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with a cocktail of retroviral vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC. The cul ...