Human Pluripotent Stem Cells as a Model for Neuroscience Research

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:

http://www.stemcell.com/en/Forms/Multimedia/Archived-Webinar-Derivation-of-Metabolically-Active-Hepatocytes-from-Human-Pluripotent-Stem-Cells.aspx

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.

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.

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.

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.

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.