Taking Short Cuts: How Direct Reprogramming Can Transform One Type of Cell Straight into Another

Finding Transcription Factors in Your Target Cell Type

What types of cells are in the highest demand for tissue transplants? In other words, what types of cells are good targets for reprogramming efforts? For this science project, you can use any cell type shown in Table 1 as your target cell type. To simplify the directions, we will use kidney cells as the target cell type example throughout this Experimental Procedure. Kidneys are one of the most commonly transplanted organs, although many other types of tissues and organs are in high demand for transplants. In this part of the science project, you will investigate which transcription factors are made by your target cell type and are the most important for its identity.

1. Select a tissue type listed in Table 1. This table was generated from published scientific studies that investigated which transcription factors are made and are active in different tissue types. Transcription factors usually have long names that are abbreviated for simplicity's sake, and those abbreviations are listed for each tissue type in Table 1.
   1. For data on organs donated for transplants to give you an idea of which tissues are in high demand, search this national data report from the Organ Procurement and Transplantation Network and the U.S. Department of Health and Human Services.

2. In your lab notebook, copy and fill out Table 2 with the names of the important transcription factors listed in Table 1 with the tissue type that you selected. Confusingly, sometimes the same transcription factor can have multiple different names or abbreviations, or "aliases."
   1. If there are more than ten transcription factors listed for the tissue type you chose, just pick ten.
   2. Table 2 has been filled out using the kidney as an example, but you can fill it out with information on a different target cell type.
   3. You will fill out the other columns of Table 2 as you go through the Experimental Procedure.

How Specific Are Your Selected Transcription Factors?

You now have a list of transcription factors known to be active in your target cell type, but how specific are they? Are they active in a lot of other cell types too? In order to directly reprogram cells efficiently to your target cell type, the transcription factors used should be fairly specific to your target cell type. Alternatively, you could try to find a combination of transcription factors that, taken together, are uniquely active in your target cell type. Researchers often do this by using three or four transcription factors together. In this part of the science project, you will look at how your selected transcription factors are expressed (turned into mRNA and thus likely into protein) in a wide range of tissue types.

1. You can learn about the expression of your selected transcription factors of interest by using NIH's Gene database.
   1. In the search box enter the name of one of the transcription factors you wrote down in Table 2. Choose the "Gene" result for your transcription factor.
      1. If there are no results, try one of the transcription factor's aliases. If there are still no results, note this in your lab notebook and move on to another transcription factor in your list.
      2. If there are multiple results, make sure to choose the one that is for humans (Homo sapiens). If there are multiple Homo sapiens results, choose the one that mentions being a transcription factor.
   2. On the transcription factor's gene page, scroll down to the "Expression" section. This section shows you how much the transcription factor is expressed in different tissues. Choose the "See details" option in the top right corner to take you to a page similar to Figure 2.
   3. The results will be shown in both a graph and in a table, similar to Figure 1. The transcription factor expression is reported in RPKM, which is a normalized way of looking at gene expression. Compare the transcription factor's expression in your target cell type (kidney in our Table 2 example) with the other cell types displayed.
      1. Looking at the graph, does your target cell type express the highest amounts of your selected transcription factor of interest? Are there other target cell types that express the same amount or more of your transcription factor?
      2. The data often has error bars indicating the variation between samples. When deciding whether other target cell types have similar or higher expression, make sure to look for overlap within the region outlined by the error bars. Fill in your data table by listing cell types, besides the one you are targeting, that express the transcription factor. If there are many, just list the top ones.
      3. Also note in your data table if the transcription factor is highly expressed in any of the other cell types.
2. Repeat steps 1a–1c for the remaining transcription factors on your list.

Figure 1. The NIH Gene's data on HNF1A expression is shown as both a graph and a table. The greatest expression is seen in the kidneys, but the error bars show that there is a great deal of variability among samples. Given the variation, expression in the duodenum and small intestine are similar to the kidney expression.

Direct Reprogramming Concerns

Although some transcription factors look like they might work great at directly reprogramming cells to become a particular target cell type, it is important to make sure that these transcription factors will not cause problems for the cells. For example, some researchers used transcription factors that are associated with cancer to reprogram cells to become induced pluripotent stem cells (iPSCs). Although iPSCs are stem cells with great medical potential, because some are made using transcription factors associated with cancer, this has made people concerned about using them in medical applications. In this part of the science project, you will look at the normal function of your selected transcription factors.

1. Learn as much as you can about each of your selected transcription factors. You can do this using the NCBI Gene Database.
   1. Use the Science Buddies NCBI Gene & SNP Tutorial to help you navigate the database. As an example, you can use the transcription factor HNF1A for your searches.
      1. Usually the transcription factor protein name will be the same as its gene name, so just use the name listed in Table 1 for all searches.
      2. Make sure to choose the top result that lists your transcription factor name, such as "HNF1A," and is in humans (Homo sapiens).
   2. What does the database tell you about your selected transcription factor? Are there any diseases associated with it?
      1. Keep in mind that even if defects in the transcription factor's gene may cause diseases, this does not necessarily mean that making a cell produce the transcription factor will cause the same diseases.
   3. Add this information to the column titled "Known Function(s)" in Table 2 for each of your selected transcription factors. This has been done for HNF1A as an example.
2. Learn about the signaling pathways (biochemical pathways) that each of your selected transcription factors are involved in by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database.
   1. In the search box under "Enter keywords," type in the name of a transcription factor and click "Go." For example, search for HNF1A.
   2. Click on the "Thumbnail Image" for each pathway result.
   3. Look at the pathways for the transcription factor. It should be in red on the pathway diagrams, but if you cannot locate it, search for its name in the search box at the top of the page.
      1. What cellular processes is it involved in? What downstream events (events that the transcription factor leads to) might be triggered if a cell produces your selected transcription factor? Are any of these effects undesirable?
      2. To learn more about any protein in the pathway, click on its name (inside a box).
      3. Add information learned about the transcription factor to the column titled "Known Function(s)" in Table 2. As an example this has been done for HNF1A.

Selecting the Best Transcription Factors for Direct Reprogramming

You have now learned a lot about your selected transcription factors. Table 2 should now be completely filled out in your lab notebook for your target cell type. Overall, how good to you think your selected transcription factors could be at directly reprogramming some cells into your target cell type? In this part of the science project, you will evaluate your different selected transcription factors and decide which would be the best - and the worst - to use in direct reprogramming experiments.

1. How specific are the transcription factors for your target cell type? To answer this question, look at Table 2 in your lab notebook at the columns titled "Other Cell Types That Express It" and "Highly Expressed in Other Cell Types?" and think about the following questions: Are many of the selected transcription factors highly expressed in other cell types? If they are, is it usually the same cell type, or is it a wide range of cell types that they are expressed in?
   1. How might this affect your ability to directly reprogram cells to your target cell type using these transcription factors? How could these transcription factors turn cells into cells other than your target cell type?
   2. How might this affect the cell type you choose to start with, the starting material for your direct reprogramming experiments? If it is usually the same cell type that is also expressing your selected transcription factors, you may be able to use this to your advantage.
   3. Are there many graphs for your selected transcription factors that show low expression of your transcription factor in your selected cell type? How may this affect which transcription factors you choose for your direct reprogramming efforts?
   4. Look back at Table 1. Are any of your transcription factors listed as being active in other cell types? If so, does this agree or disagree with what you wrote down in Table 2 in the column titled "Other Cell Types That Express It"? How might this further affect your selection of transcription factors?
      1. Note: You can use Ctrl+F to find a word or specified text on this page.
2. Do any of the transcription factors cause diseases? Look at Table 2 in your lab notebook at the column titled "Known Function" to answer this question. How does this affect which transcription factors you will select for direct reprogramming experiments?
3. Overall, based on steps 2 and 3, evaluate which transcription factors may be the best ones to use in direct reprogramming efforts to make cells of your target cell type, and which ones may be the worst. You could narrow it down to about four transcription factors that may work well together. Also, think about how this may affect which cell type(s) you would like to use as your starting material.