Human Pluripotent Stem Cells as a Model for Neuroscience Research
Company: STEMCELL Technologies Inc.
Job Title: Senior Scientist
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. STEMCELL Technologies recently hosted a webinar 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.
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.
Use of defined and optimized reagents for the entire workflow facilitates the establishment of a robust model and increase experimental reproducibility. STEMCELL Technologies offers tools to support each step of the process, from TeSR™-E7™ reprogramming medium through to mTeSR™1 maintenance medium and STEMdiff™ differentiation reagents.
This session is no longer taking submissions. To be notified of new Ask the Expert sessions, join our mailing list.
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:
Can you describe how you would use this system to model multifactorial disease? Are you referring to the ability to make many cell types within one system?
iPS cells derived from a patient's somatic cells will retain the mutation or mutations that he/she carries. Thus, researchers can use this approach to study multifactorial/multigenetic diseases, including those with an unknown genetic identity eg. autism spectrum disorders.
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.
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.
How do you determine the best somatic cell type to start with? Fibroblasts are probably the easiest to obtain in a sample, but are there advantages to other types?
In theory, all somatic cells can be reprogrammed. Usually one would choose a cell source that is easier to obtain and expandable so that you can obtain a large number of starting cells. Fibroblasts fit both criteria and have been widely used. As a result, there are many papers and protocols available for reprogramming fibroblasts. Blood cells are also increasing in popularity, because blood draw is usually a routine procedure, and there are many reagents available for enrichment and expansion of hematopoietic cell types.
We are looking for the most effective way to reprogram factor free iPS cells.
Currently, the most efficient non-integrating system is Sendai virus. For effective reprogramming, the reprogramming system and the reprogramming medium are both important variables. TeSR™-E7™(http://www.stemcell.com/en/Products/All-Products/TeSRE7.aspx) is an optimized xeno-free and defined reprogramming medium that enables the generation of high quality iPS cell colonies with reduced differentiation and fibroblast growth.