Efficient cell specific differentiation systems for iPSC
Company: Thermo Fisher Scientific, Inc.
Job Title: Director of Research and Development
Introduction
The promise of human pluripotent stem cells will be realized only when these cells are successfully coaxed into different cell types found in the human body, through the process of directed differentiation. This is critical to getting the desired cell types and numbers needed for drug screening, translational Cell Therapy and regenerative medicine applications. Most of the existing methods of differentiation are suboptimal, involving laborious mechanical and manual steps leading to issues of reproducibility and reduced efficiency in downstream processing of functionally mature lineages. The complex developmental process of differentiation and the challenges associated need to be efficiently deciphered in order to successfully direct the hPSC differentiation to target cell types.
During this Ask the Experts session, we will be discussing the challenges associated with hPSC differentiation to neural and cardiac lineages, how Thermo Fisher Scientific can address how these processes can be efficiently simplified with tools and cGMP cell culture media systems for robust, efficient and scalable differentiation of these two critical cell lineages. Use of these reagent systems will enable researchers to precisely control and direct the differentiation to terminal lineages in a relatively easy manner, and speedily with high efficiency.
This Ask the Expert session will be hosted by Dr. Mohan C Vemuri, the Director of Research and Development for Cell Biology at Thermo Fisher Scientific. In this capacity, Dr. Vemuri leads R&D activities in stem cell product development in the areas of human iPSC, adult stem cells, immune cells and cell lineage specific differentiation in GMP environment for research use and subsequently for use in Cell Therapy with regulatory compliance.
Question 1
I am looking for a system that can detect differentiation using imaging techniques that are not invasive to the culture. Do you have any recommendations?
There are two possible approaches you could use for a system to detect differentiation using non-invasive imaging techniques. The first is simple phase contrast microscopy. Using this approach you would look for the spike appearance of differentiating cells. Using imaging tools you can mask the undifferentiated cells and mark the differentiated cells. You may want to look in to an imaging tool hosted by the NIH which would also allow you to quantify this method. The second method I would recommend is utilizing a tool offered by Life Technologies – the Alkaline Phosphatase Live Stain. This is a non-invasive technology by which undifferentiated cells can be stained in 20 minutes or less by fluorescence. Cells that are not picked by this stain are differentiated cells. The Alkaline Phosphatase Live Stain is a quick and easy tool to use for detecting differentiated cells.
Click for more information about the Alkaline Phosphatase Live Stain - http://www.lifetechnologies.com/order/catalog/product/A14353?CID=search-product
Click to see a selection of tools for live and fixed cell staining of pluripotent stem cells - http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/pluripotent-stem-cell-detection/pluripotent-stem-cell-antibodies.html?icid=cvc-stemcelldetection-c2t1
Question 2
Our lab is differentiating iPSCs into cardiomyocytes and we have initial success for a few times then our efficiency drops. The only thing that is changing is cell density. Do you think the cell density is affecting our efficiency and if so, what kind of passaging do you recommend for the best efficiency?
iPSC quality of the starter population and seeding densities are critical for cardiac differentiation depending on the protocol you are adopting for your differentiation to cardiomyocytes. If you have expanding populations in the beginning during differentiation, the stochastic ratios alter and could lead to less robust methods of generating desired cell types. Similarly if the starting quality of iPSCs is poor, it will eventually result in poor differentiation of desired lineages.
One product you could try to improve your efficiency is the new PSC Cardiomyocyte Differentiation kit offered by Life Technologies. This kit offers a simple and reproducible three-step protocol to generate cardiomyocytes. Early cardiomyocytes can be generated as early as eight days and can be matured for the length of time required. Cells differentiated using this kit are applicable to studies including cardiotox screening, cardiac development, disease modeling, etc.
Learn more about the PSC Cardiomyocyte Differentiation Kit - http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-differentiation/culture-systems-reagents-cardiomyocyte-differentiation.html
Question 3
I am trying to culture human iPSCs without a feeder layer and keep them undifferentiated. I am using a commercial iPSCs media. I am trying to do this without matrigel, but the cells keep differentiating. Do I need to add Matrigel or is there a way to do this without?
(A) iPSC feeder free cultures would primarily require a matrix ( substrate) and robust culture media systems to enable and sustain stem cell state and at the same time allow expansion of the iPSC within the culture system. Without a substrate and culture media system, the cells will crash. You could consider using alternative substrates including Geltrex®, CellStart™ or Vitronectin as part of your culture system. These products are manufactured in GMP conditions and the reproducibility of performance is well validated. The only way PSCs could be grown without a substrate/matrix would be to grow them as small aggregates in suspension cultures.
Learn more about 3D matrices for stem cell research - http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/extracellular-matrices-psc-culture.html
Question 4
What would be an efficient protocol for hESCs differentiation into epidermal cells, with an optimal subculture afterwards?
Epidermal cells consist of a variety of cells including any of the cells making up the skin such as keratinocytes, melanocytes, melanoblast (precursor of melanocyte) Langerhans cells, and Merkel cells. It is important to identify the desired cell type for differentiation. Most methods for keratinocytes and melanocytes are still developing in the literature. That said, there are some preferred methods which have shown success, but would likely require further optimization.
The two references below may be helpful to you:
• Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells (PMID: 21555586) - http://www.pnas.org/content/108/21/8797.full
• Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis (PMID: 21856949) - http://www.pnas.org/content/108/36/14861.long
Question 5
Hi! What is the best differention medium for hepatogenic differentiation of iPS cells derived from human skin fibroblasts? Thanks.
Several researchers are working on iPSC to hepatic differentiation. Commonly, the first step required is definitive endoderm differentiation. Following the definitive endoderm fate, the cells need to be taken through posterior foregut fate and then towards hepatic. There is no one culture media to accomplish these different steps available today. Currently this process involves the use of specific media systems at each of the step during the differentiation process.
The following reference may be useful to you, however we have not performed this protocol in our labs and cannot comment on reproducibility.
• Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells (PMID: 23681950) - http://stemcellstm.alphamedpress.org/content/2/6/409.full.pdf+html
Question 6
What are your thoughts on single-cell sequencing vs. population averaging methods in confirming/evaluating differentiation?
Each method has its own advantages and some disadvantages.
Biological phenotype is a result of a complex cellular architecture and multiple interactions of several cell types (sometimes single cells) based on their environmental niche with in the human body. Recently, advances in single cell genomic profiling approaches has created an opportunity to see and understand these developmental opportunities which were not previously available.
Population averaging methods provide the benefit of assessing the collective activity/performance of cells, but does not account for where activity may be originating in the cell. In this case, specificity is lost, but output of the population is still valuable.
In summary, the best approach for you should be determined based on what you need to measure. For example, if you are interested in clonal expansion or differentiation abilities of one cell, the single cell approach can provide the specificity needed. Should you require an understanding of multiple cell type interactions, as would be the case with studying hepatic differentiation for example, then population averaging methods would be most valuable.
Question 7
Rapid protocol for differentiation of iPSCs into dopaminergic neurons?
Rapid in this case may not be as important as efficient. The developmental biology of TH+ neurons is not understood well enough to ‘speed up’ the development of different types of neurons including dopaminergic or cortical motor neurons. A recent study found that Dibutyryl cyclic-AMP (dbcAMP) was shown to induce up to 85% in vitro differentiation of neural stem cells in to neurons, but still required the same differentiation timeline as that of in vivo human brain development of these neurons. When looking for an efficient method to differentiate, you may consider starting with enriched dopaminergic precursor cells. It has been shown that the better the quantity of enriched precursors (for example FoxA2+/Lmx1a+) the better the efficiency of the final phenotype for dopaminergic neurons ( for example, TH+/Nurr1+?FoxA2+) which in turn creates a starting population which is enriched for desired lineages.
Question 8
Can you share your thoughts on the use of small molecules in differentation of iPSCs?
Small molecules are being increasingly used in exploring the differentiation ability of iPSCs. While several advances have been made in recent years, this approach has more to offer in the future and certainly holds a great promise for stem cell research. Advantages of using small molecules for differentiation of iPSCs include: their ability to swap growth factor pathways, increased stability in liquid formats decreasing GMP and regulatory hurdles, and finally they tend to be less expensive when compared to growth factors.
The following articles may be of interest to assess the increasing role of small molecules in understanding stem cell biology:
a. Chemical approaches to stem cell biology and therapeutics. - http://www.ncbi.nlm.nih.gov/pubmed/24012368
Li W, Li K, Wei W, Ding S.
Cell Stem Cell. 2013 Sep 5;13(3):270-83. doi: 10.1016/j.stem.2013.08.002. Review.
b. Small molecules, big roles -- the chemical manipulation of stem cell fate and somatic cell reprogramming. - http://www.ncbi.nlm.nih.gov/pubmed/23420199
Zhang Y, Li W, Laurent T, Ding S.
J Cell Sci. 2012 Dec 1;125(Pt 23):5609-20. doi: 10.1242/jcs.096032. Review
This post is sponsored by Thermo Fisher Scientific, Inc..