We recently finished our Ask the Expert discussion on Human Pluripotent Stem Cells as a Model for Neuroscience Research. This week we had several interesting questions and informative responses. Specific information was provided on using iPSCs for creating disease models. There were also questions regarding reprogramming, embryonic body culture, monolayer culture and neural patterning.
Induced pluripotent stem (iPS) cell-based models hold tremendous potential for the study of human neurological disease. Advances in technologies, including improvements in the ease and efficiency of generating neural progenitor cells from iPS cells, have resulted in increased adoption of these models by the neuroscience community.
There are many options available to researchers interested in developing iPS-based models to complement traditional methods of neuroscience research. Points of consideration include the type of somatic cell used to generate the iPS colonies, the reprogramming system, the conditions under which iPS cells are maintained, the protocols used for neural induction and terminal differentiation, and the end-point assays used to investigate disease phenotype.
This Ask the Expert Session was Sponsored by STEMCELL Technologies and hosted by Dr. Vivian Lee. Vivian Lee, PhD, is a Senior Scientist in STEMCELL Technologies’ Research and Development department, responsible for the development of products for neural tissues and pluripotent stem cells. Before joining STEMCELL, Vivian was a Principal Investigator at the Medical College of Wisconsin, studying the genetic regulation of neural stem cell development.
STEMCELL Technologies recently hosted a webinar on this topic in which the applications, features and workflow for this research model were presented, together with an example of how Dr. Marina Bershteyn (UCSF) uses iPS cell-derived neural cells to model lissencephaly.
Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – Human Pluripotent Stem Cells as a Model for Neuroscience Research.
Can you compare/contrast the use of EB vs. Monolayer culture methods for someone who is trying to decide which method to use- what would you base your decision on?
The EB protocol is slightly more labour intensive because there are more plating steps and neural rosette selection is required. However, the formation of morphologically distinct neural rosettes provides a quick and reliable readout of the success of neural induction, and rosette selection provides researchers with the ability to enrich for CNS-type neural progenitor cells. The monolayer is quick and easy to set up but neural rosettes are not usually obvious due to the high cell density. Thus, assays such as immunocytochemistry, flow cytometry, or RT-PCR are required to measure the success of neural induction. STEMdiff™ Neural Induction Medium supports efficient neural induction using both protocols.
I am working to create a model system for liver diseases and toxicity testing. Do you have a StemDiff protocol for other disease model systems or just neurological disease?
For differentiation of ES and iPS cells to hepatocytes we recommend using the STEMdiff™ Definitive Endoderm Kit to generate definitive endoderm, in conjunction with Dr. David Hay’s protocol for downstream differentiation (Hay et al. Stem Cells, 2008). Cells differentiated using the STEMdiff™ Definitive Endoderm Kit express high levels of endoderm markers, including CD184 (CXCR4), SOX17, FOXA2 and c-Kit, and lack expression of ectoderm, mesoderm and pluripotency markers. The definitive endoderm produced using this kit is multipotent and capable of further differentiation towards the hepatic lineage (and pancreatic and pulmonary, for that matter). If you’re interested to hear more about how this kit has been used by Dr. David Hay to generate metabolically active hepatocytes, please view this complimentary view-on-demand webinar:
What methods are you using for neural patterning of progenitor cells and do you think it could be applied to other types of progenitors?
If you are referring to neural differentiation of human ES/iPS cells to generate neural progenitor cells (NPCs), the most common methods involve either embryoid body or monolayer culture systems. The same approaches have been used to generate progenitors from all three germ layers (ectoderm, mesoderm, endoderm), but specific growth factors or small molecules are normally required to direct differentiation to a particular lineage.
If you are referring to downstream differentiation of hPSC-derived NPCs to different types of neurons and glia, NPCs can be patterned to produce neurons from different regions of the nervous system, such as cortical neurons, midbrain dopaminergic neurons, or spinal motoneurons. In this case, specific patterning factors (e.g. FGF-8, SHH, RA, etc.) are usually required in addition to the initial neural induction step. Region-specific patterning using specific sets of growth factors or small molecules is also used to generate anterior or posterior endoderm derivatives from hPSC-derived definitive endoderm.