Creating a Kidney: How Stem Cells Might Be Used to Bioengineer a Vital Organ

Driving Kidney Cell Differentiation Using Soluble Factors

What soluble factors would be best to use when trying to differentiate human induced pluripotent stem cells (hiPSCs) into kidney cells, specifically the kidney cells of the nephron, the functional unit of the kidney? To differentiate hiPSCs into nephron cells efficiently, the soluble factors that are added to the cell media need to prompt the hiPSCs to turn into nephron cells. A few studies have investigated differentiating iPSCs into nephron cell types, but the techniques are inefficient, resulting in a mixture of cell types with only 60% to 90% being the desired cell type. In this part of the science project, you will decide which soluble factors, and combinations of soluble factors, would be best to test when prompting hiPSCs to turn into different types of nephron cells.

1. Look at Table 1, which summarizes the soluble factors used in key publications of research on turning ESCs or iPSCs into different nephron cell types. Note: Table 1 summarizes this information so you do not need the actual papers, which are not publicly available, to do this bioengineering science project.
   1. What soluble factors did the different researchers add to make either podocytes (cells in the glomerulus) or renal tubule cells? You can look at Figure 1 (in the Introduction in the Background tab) to review the structure of, and different cell types in, the nephron.
      1. For example, two groups of researchers (Kramer et al., 2005, and Ren et al., 2010) were able to make podocyte cells. One of these groups (Kramer et al., 2005) did not report adding any soluble factors, while the other one (Ren et al., 2010) reported adding two different soluble factors, activin A and retinoic acid (RA). Three groups (Kramer et al., 2005; Kobayashi et al., 2005; and Morizane et al., 2009) made renal tubule cells using one, two, or no soluble factors.
      2. Although one research group (Kramer et al., 2005) added no soluble factors, whether their efficiency could be improved by adding soluble factors should be investigated.
      3. In the bottom row of Table 1, where the Dankers et al. research from 2011 is listed, several soluble factors are named that are necessary for embryonic kidney development in the body, also called in vivo. These factors are not known to have been tested on cells grown in a laboratory, or in vitro, but they are likely candidates for prompting hiPSCs to differentiate into some nephron-like cells.
      4. Note: Extensive studies have not yet been published on turning pluripotent stem cells into cells of the Bowman's capsule, and so it is omitted from this bioengineering science project. See the "Variations" for how you could explore bioengineering this part of the nephron.
   2. Although you do not need the research papers to do this science project, if you want to learn more about a particular paper, click on the link under the "Research Paper" column to see the paper, as an abstract or in its entirety. What can you understand about the paper and its reported findings by reading the abstract?
      1. If you are interested in ways to obtain these papers, you may be able to use the guide Resources for Finding and Accessing Scientific Papers to do so.
      2. If you choose to read the papers, you may also find you need some help from someone experienced with stem cells or bioengineering to help you find out How to Read a Scientific Paper.

*Wnt4 was not actually a soluble factor, but was transfected into the cells so that the cells made their own Wnt4. In this bioengineering science project, Wnt4 can still be explored and treated as a soluble factor.

** Conditioned media is made by exposing media to cells so that factors secreted by the cells are collected in the media. In the Ren et al. paper from 2010, conditioned media was made and tested from ureteric buds, which are the structures that develop into the entire kidney, except for the nephrons. For the purposes of this engineering science project, conditioned media can be ignored, but it can be explored in the Variations section.

2. Learn more about the soluble factors in Table 1 by using at least two of the following databases (listed in steps 2.a.–2.c.). Before you start using the databases, make a data table in your lab notebook like Table 2, below, for you to organize the information that you will be collecting on the different soluble factors. What are the functions of these different soluble factors? How might they be used to differentiate stem cells into different kidney cell types? Note that you may be unable to answer all of the questions in Table 2 for all of the soluble factors, and that not every database may have information on every soluble factor, but put as much relevant information into your data table as you can as you search through the databases.
   1. The NCBI Gene Database:
      1. Use the Science Buddies NCBI Gene & SNP Tutorial to help you navigate the NCBI Gene Database.
      2. For your searches, use the abbreviated gene names listed in Table 2, below.
      3. Make sure to choose the top search result that lists your soluble factor name, such as "INHBA," and that it is for humans (Homo sapiens).
      4. What does the database tell you about each soluble factor? Fill in your data table with any relevant information from the NCBI Gene database.
         • For example, depending on the information provided for a given soluble factor, you might fill in the data table as follows: Under "Does it regulate proliferation or apoptosis?" you may write "Yes, inhibits cell growth and proliferation." Under "Does it regulate differentiation?" you may write "Yes, regulates cell differentiation." Under "What other key functions does it have?" you may write "Regulates stem cell maintenance and progesterone production."
         • If you run across terms you are unfamiliar with (such as possibly "progesterone"), you should always look them up and try to figure out how they fit in with what else you know about the soluble factor.
   2. Pathway Commons:
      1. In the search bar on the homepage, type in the abbreviated gene name of the soluble factor you want to explore (such as "INHBA").
      2. On the results page, choose the "interactions" option. This will take you to a network visualizer which visually shows the connections between the soluble factor and other genes/factors. The soluble factor you searched for will be in the middle of the interaction graph.
      3. Click on the circle with the soluble factor's name. This should cause text to pop with summary information about the soluble factor.
      4. You can repeat this process to learn more about the other factors shown in the network visualizer to interact with the soluble factor you searched for.
         • What other factors does the factor you searched for directly regulate, and which ones does it more distantly regulate?
         • Can you tell how they regulate each other?
      5. Fill in your data table with any relevant information from the Pathway Commons on the different soluble factors.
   3. Embryonic and Induced Pluripotent Stem Cells & Lineage-specific Markers:
      1. This gives a visual overview of lineage-specific markers during the differentiation of pluripotent stem cells. Click on any factor's name (and then click on its name in the window that pops up) to be taken to a webpage with more information on it.
      2. Fill in your data table with any relevant information from this database on the different soluble factors.

*Apoptosis is programmed cell death.

** During development, the tubules in the nephron (and many other tissues throughout the body) undergo a process called epithelial-to-mesenchymal transition, or EMT, where one type of tissue (epithelial tissues) differentiates into another type (mesenchymal tissues).

3. After you have filled out your data table of soluble factors using at least two of the databases in step 2, look at how different and how similar the soluble factors are.
   1. Do many of the soluble factors regulate the same function, such as differentiation? Do any factors regulate three of the same functions? If any two factors regulate three of the same functions, they may act similarly.
4. Review Table 1 to see which soluble factors have been used previously in the named studies in certain combinations. Can you substitute any of the factors from a previously used combination with a similar factor, based on your data table?
   1. For example, Wnt4, HGF, and Activin A have been previously used in a combination (Kobayashi et al., 2005). Is there another factor similar to Wnt4 that you could use instead of Wnt4 in this combination? Or is there another factor similar to HGF or Activin A that could be substituted instead?
   2. In your lab notebook, create a data table of the different combinations that could be created based on how similar the soluble factors are.
      1. Make columns listing the "Nephron Cell Type" and "Existing Combinations of Soluble Factors," as listed in Table 1. Then include columns listing the "Substitutions" that could be made for each factor and the "Potential Combinations" that could be made based on the substitutions (as discussed in step 5a for Wnt4, HGF, and Activin A).
5. Using different combinations of soluble factors may improve upon the current nephron cell differentiation protocols discussed in Table 1. Based on the data table you made in step 2, what would be feasible, new soluble factor combinations to test to turn hiPSCs into podocytes or renal tubule cells? Do you expect some combinations to work better than others? Why? Might some combinations work well to make nephron cells, or nephron precursors (cells that can easily turn into nephron cells), in general?

Using the Extracellular Matrix to Drive Kidney Cell Differentiation

What Extracellular Matrix (ECM) proteins would be best to use when trying to differentiate hiPSCs into nephron cells? The ECM is a mesh-like network of proteins that surrounds cells in the body, giving structural support to the cells and promoting communication among them. To efficiently differentiate hiPSCs into nephron cells, not only do the hiPSCs need the appropriate soluble factors in their media, but the substrate they are grown on can also affect how they differentiate. (The studies summarized in Table 1 did not investigate different substrates, but generated nephron cell types by initially creating cell formations called embryoid bodies, which were cultured by floating them in the media, without touching a substrate, for two to five days. After being in the floating culture, they were cultured on gelatin, which is a standard culture substrate made up of fragments of collagen.) Differentiating hiPSCs on certain ECM proteins may produce nephron cells more efficiently, and this research could be used as the basis for designing synthetic scaffolds on which to grow kidney cells for transplants. To interact with ECM proteins (including laminins, collagens, vitronectin, fibronectin, and more), the hiPSCs need specific cell receptors on the surface of their cells. In this part of the science project, you will examine which cell receptors, called integrins, are made by kidney cells during development.

1. Table 3 lists integrins and the ECM proteins they bind.
   1. Integrins are heterodimers, meaning they are made up of two different subunits, specifically an alpha and a beta subunit. Only certain alpha and beta subunits can combine. The combination determines what ECM proteins they can bind.
   2. Note: Not all integrin subunits are listed in Table 3. Table 3 includes integrin subunits that have data in the NIH Gene database and that form integrin heterodimers with well-established information on the ECM proteins they bind. Also, the integrin heterodimers in Table 3 may bind additional ECM proteins that are not listed.

2. In your lab notebook, create a data table with a column called "Integrin Subunits" and list each of the integrin subunits in it (all alpha and beta subunits listed in Table 3), with one row for each subunit. Make four additional columns labeled "Expression in Adult Kidney", "Expression in 10wk Embryonic Kidney", "Expression in 16wk Embryonic Kidney", and "Expression in 20wk Embryonic Kidney". You will be filling in this data table in the following steps.
3. Learn about the expression of certain integrin subunits by using NIH's Gene Database.
   1. Type the name of the gene you want to learn more about in the search box.
      1. For alpha subunits, enter "ITGA" followed by the number. For example, for α5 enter ITGA5. (For integrin αV, use ITGAV.)
      2. For beta subunits, enter "ITGB" followed by the number.
   2. This will take you to a search results webpage. Choose the "Gene" result. It is usually the top result on the page.
   3. On the gene's page, scroll down to the "Expression" section. This section shows you how much the gene 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.
   4. For this science project, you are only interested in each subunit's expression in the kidney.
      1. To see the expression in adult kidney tissue, choose "HPA RNA-seq normal tissues" from the pull-down menu. The results will show in both a graph and in a table. In your data table, record the kidney RPKM values for the subunit. [Note: RPKM is a normalized way of looking at gene expression.]
      2. To see the expression in embryonic kidney tissue, choose "Tissue-specific circular RNA induction during human fetal development" from the pull-down menu. Record the kidney RPKM values for the subunit at 10, 16, and 20 weeks of embryonic development in your data table.
   5. Repeat steps 3a–3d for all for all integrin subunits in Table 3.

Image Credit: Public domain
Figure 2. The expression data for a specific integrin subunit in NIH's Gene database will look similar to this one for the integrin α5 subunit.

4. Looking at your data table, which integrin subunits are most highly expressed, or have the highest values, in the kidney cells?
   1. Set an expression level threshold that results in a list of several "highly expressed" integrin subunits.
5. Go back to Table 3 and either print out or copy it into your lab notebook. Highlight, or mark in some other way, the alpha and beta integrin subunits that you determined to be highly expressed, and the integrin heterodimers that are highly expressed in both their alpha and beta integrin subunits.
6. Which ECM proteins are bound by the highly expressed integrins? Which ECM proteins are bound by multiple highly expressed integrins? On Table 4 mark which ECM proteins are bound by at least two highly expressed integrins.
7. Are the marked ECM proteins also highly expressed in the kidney during development? If so, this may make them even better candidates to use in bioengineering a kidney.
   1. Repeat steps 2-3 but this time use the ECM proteins you marked in step 6 instead of integrin subunits.
      1. Use Table 4 to help you search for the correct ECM protein names on the NIH's Gene site. Like integrins, some ECM proteins are also made up of multiple subunits that you will want to investigate.
      
      

      ECM Protein(s)	Name(s) to Search For
      Laminins	Laminins are made up of three subunits, an alpha, beta, and gamma, which are listed in order in their names in Table 4. For example, Laminin-521 is made up of an alpha 5, beta 2, and gamma 1 subunit. For alpha subunits, search for "LAMA" followed by the number. For example, for alpha 5 you would search for "LAMA5." For beta subunits, search for "LAMB" followed by the number. For gamma subunits, search for "LAMC" followed by the number.
      Collagens	Collagens are made up of alpha subunits. For these subunits, search for "COL" followed by the number of the collagen and then the specific subunit. Collagen I is made up of alpha 1 and 2 subunits. For these subunits, search for "COL1A1" and "COL1A2," respectively. Collagen IV is made up of alpha 1, 2, 3, 4, 5, and 6 subunits. For these subunits, search for "COL4A1," COL4A2," etc.
      Vitronectin	Search for "VTN"
      Fibronectin	Search for "FN1"

      Table 4. When searching the NIH's Gene database, use these abbreviated ECM protein names.
      
      
   2. In the data table you made in step 6, underline or highlight which ECM proteins are also highly expressed.

Creating the Right Microenvironment to Make a Kidney

Now that you have determined the ideal media conditions to test and the ideal substrates to grow the cells on, in this part of the science project you will be pulling your results together to create the overall best conditions for bioengineering a kidney, starting with a nephron.

1. Based on your findings, what would you hypothesize to be the best soluble factors and ECM proteins to use for differentiating hiPSCs into certain nephron cell types?
   1. Review the data tables you made in "Driving Kidney Cell Differentiation Using Soluble Factors" and in "Using the Extracellular Matrix to Drive Kidney Cell Differentiation".
   2. Make a new data table that combines the results from these data tables. Specifically, have one column listing "Nephron Cell Type" (as listed in Table 1), one listing "Potential Combinations of Soluble Factors," and one listing "Potential ECM Proteins."
      1. Because the ECM proteins you selected were for the kidney as a whole, and not specific kidney cell types, you may want to test the same ECM proteins for all of the different nephron cell types. You can test ECM proteins individually or in combinations.
   3. What results would you expect from your experimental plan? What problems do you anticipate, and how might you be able to solve them? Why might some conditions be more efficient than others?
2. Based on your findings, what do you hypothesize would be the best overall conditions in which to bioengineer a nephron or an entire kidney?
   1. If you could make a substrate or a kind of "skeleton" to grow the organ on, what ECM proteins should be used to coat it? Why do you think some would be better than others?