In our previous blog we likened somatic cell reprogramming to a career change . Once a somatic cell has been converted to an induced pluripotent stem cell (iPSC), the next stage in the process can be thought of as an appraisal or performance review. This is essential to confirming that the reprogrammed cell is behaving as expected.
Before a new iPSC line can be registered and banked, extensive characterisation is required. This involves rigorous testing to demonstrate that the iPSCs are pluripotent - able to give rise to cells from all three germ layers. Following this the differentiation capacity of the cells is validated. The aim of characterisation is to ensure consistent and reproducible results across laboratories, and to address possible safety issues of stem cell-based therapies early in development.
In this blog we will discuss the different methods available to determine the pluripotency of your new iPSC cell line and fully ensure it is characterized for registration and banking.
Determining the pluripotency of iPS cells
There are several methods for assessing pluripotency potential. These include detection of an alkaline phosphatase which is specifically related to pluripotency, evaluation of various cellular markers via immunostaining techniques, and use of the widely-cited teratoma assay. In addition, several bioinformatic methods have evolved, providing a more cost-effective methodology to the latter.
Alkaline phosphatase (AP) expression
AP expression is easy to determine since the enzyme can convert a soluble colorimetric reagent to a precipitated state, yielding a rapid visual readout. Placental alkaline phosphatase (hPLAP) is a form of AP that is related to pluripotency and is easily distinguished from other APs through the incorporation of a high temperature step into immunostaining protocols. hPLAP can withstand high temperatures (68oC) which inactivate endogenous APs. This allows its expression to be easily characterized and used as an assay to assess pluripotency.
iPSC maker expression
Marker expression is another approach which is used to confirm pluripotency and is typically performed once hPLAP expression has been identified. Immunostaining techniques are used to detect a panel of markers specific to iPSC physiology and to maintaining these cells in an undifferentiated state. In humans these markers include octamer-binding protein 4 (Oct4), the transcription factors Nanog and Sox2, tumor-rejection antigens Tra-1-60 and Tra-1-81 and stage-specific embryonic antigen-3 and -4 (SSEA3 and SSEA4), while SSEA1 is commonly used as a negative control. In mice the main markers of pluripotency are Oct4, Nanog, Sox2 and SSEA1. It is important to point out that such immunostaining data requires careful interpretation. For example, Oct4 exists as two splice variants, Oct4A and Oct4B, yet only Oct4A is related to pluripotency. It is essential that any Oct4 antibodies that are employed recognise only Oct4A to avoid false positive results.
One of the most frequently used methods for evaluating pluripotency is the teratoma assay. This is based on the ability of iPSC to form teratomas (tumours composed of cells derived from all three of the germ layers) in immunodeficient mice. Although currently considered to be a gold standard approach, the teratoma assay is time-consuming, technically challenging and difficult to standardise. A further drawback is that it is not scalable to the increasing number of iPSC lines that are being created. For these reasons there is a significant need for the development of a cost-effective and animal-free alternative. PluriTest and ScoreCard , developed in 2011, are two potential alternatives.
PluriTest and ScoreCard are bioinformatic assays which provide a molecular signature for pluripotency based on gene expression profiles. The PluriTest database contains transcriptional profiles derived from over four hundred diverse stem cell preparations by microarrays. The ScoreCard database was produced by applying three genomic assays (gene expression profiling, DNA methylation mapping and transcript counting of lineage marker genes in embryoid bodies) to approximately thirty previously derived human embryonic stem cell lines and iPS cell lines. Both assays provide a reference against which unknown cell lines can be compared and have seen considerable success. ScoreCard has recently evolved to incorporate qPCR, enabling faster, more quantitative assessment of functional pluripotency.
Assessing the stability and differentiation of iPSC cell lines
Once it has been established that an iPSC line is pluripotent, it is necessary to demonstrate that the cells can form tissues derived from the three germ layers of the embryo . If the differentiation test is performed in vitro , the cells are cultured in suspension until they form aggregates known as embryoid bodies. Pluripotent cells will spontaneously differentiate into cell types derived from the mesoderm, ectoderm or endoderm. During an in vivo differentiation test, the cells are injected into severe combined immunodeficient (SCID) mice. Pluripotent cells will proliferate and differentiate, ultimately forming a teratoma.
Prolonged culture of iPSC is associated with genetic abnormalities and so karyotype analysis is used to show that new cell lines have maintained genetic stability. By arresting the cells in their metaphase and then staining, chromosomal abnormalities can be observed. DNA fingerprinting can also be used to investigate the genetic stability of a new cell line through the analysis of short tandem repeats (STRs), which will be unique to each cell line and allow for cell identification. This form of genetic assessment can add an additional level of confidence to conclusions drawn from karyotyping.
A diverse range of assays and techniques are available to assess the pluripotency of a newly isolated iPSC line. Employing these methods will help you to assess this and ensure that the cell line can differentiate into tissue derived from the one of the three germ layers.
Axol Bioscience is the place to go for all things iPSC-related. We offer a comprehensive range of iPSC-derived cells along with expertly optimised growth media for their successful culture and propagation, and we also supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. Our expertise includes reprogramming cells to iPSCs and differentiating them to various cell types. Through our custom service offerings , we can also take cells provided by you and carry out the reprogramming and differentiation, saving you both time and money.view our custom cell reprogramming service