Training Stem Cells to Differentiate Properly

Pluripotent stem cells have the ability to differentiate into a whole host of adult cell types. Unfortunately this ability to differentiate into any adult cell type also comes with it the tendency to form tumors. Controlling stem cell differentiation requires that you give a little “push” in the right direction. What is the nature of that push? It varies from stem cell to stem cell and it also depends on what type of cell you want you stem cells to make. Therefore, pluripotent stem cell differentiation is sometimes a matter of art as much as a matter of science.

A research group at Stanford University School of Medicine have designed an experimental protocol that uses the signals in the body to direct the differentiation of stem cells to a desired end.

Stanford University professor Michael Longaker, who is also the director of the Institute for Stem Cell Biology and Regenerative Medicine at Stanford University, explained it this way: “Before we can use these cells, we have to differentiate or ‘coach,’ them down a specific developmental pathway.” Longaker continued: “But there’s always a question as to exactly how to do that, and how many developmental doors we have to close before we can use the cells. In this study, we found that, with appropriate environmental cues, we could let the body do the work.”

Allowing the patient’s body to direct differentiation of pluripotent stem cells could potentially speed approval of stem cell-based treatments by the US Food and Drug Administration (FDA). If Longeker’s protocol pans out, it could eliminate long period of extended laboratory manipulation in order to force stem cells to differentiate into the desired cell type.

“Once we identify the key proteins and signals coaching the tissue within the body, we can try to mimic then when we use the stem cells,” said Longaker. “Just as the shape of water is determined by its container, cells respond to external cues. For example, in the future, if you want to replace a failing liver, you could put cells in a scaffold or microenvironment that strongly promotes liver cell differentiation and place the cell-seeded scaffold into the liver to let them differentiate in the optimal macroenvironment.”

Longaker does not work on liver, but bone. As a pedatric plastic and reconstructive surgeon who specializes in craniofacial malformations, finding ways to coax pluripotent stem cells to make bone is his research Holy Grail. “Imagin being able to treat children and adults who require craniofacial skeletal reconstruction, not with surgery, but with stem cells,” opined Longaker.

In this experiment, Longaker and his colleagues removed a four-millimeter circle of bone taken from the skulls of anesthetized mice and implanted a tiny, artificial scaffold coated with a bone-promoting protein called BMP-2 (bone morphogen protein-2) that was seeded with one million human pluripotent stem cells.

According to Longaker, these implants formed bone and repaired the defect in the skulls of the mice even the original stem cells were not differentiated when added to the wound. These human stem cells made human bone that was then replaced by mouse bone as time progressed. This shows that the repair was physiologically normal.

This bone growth was stimulated by the presence of BMP-2 and the microenvironment that induced the stem cells to differentiate into bone-making cells that made normal bone.

In this experiment, Longaker and his group used human embryonic stem cells and induced pluripotent stem cells and both stem types seemed to work equally well at repairing the skull defect.

Teratomas (tumors made by pluripotent stem cells) were observed but only rarely (two of the 42 animals that received the stem cell implants developed tumors). Interestingly, the few teratomas that formed developed in two laboratory animals that received embryonic stem cell implants and not induced pluripotent stem cell implants. This is surprising, since most stem cells researchers consider induced pluripotent stem cells to be more tumorigenic than embryonic stem cells. Standard tests of these stem cells (implantation under the kidneys of immunodeficient mice) showed that they did produce teratomas under these conditions.

Longaker commented: “We still have work to do to completely eliminate teratoma formation, but we are highly encouraged.” Longaker also thinks that by combining this technique with other strategies, he and his group might be able to completely prevent teratoma formation. For example, including other cell types that can act as shepherds for the stem cells as they differentiate into the desired cell type can also increase differentiation into a desired cell type.

Longaker said, “I want to see how broadly applicable this technique may be.” He was referring to tissues that do not heal well. For example, cartilage heals very poorly if at all. Longaker wonders if you could “add some cells that can form replacement tissue in this macroenvironment while you’re already looking at the joint.”

Induced Pluripotent Stem Cells do not Form Neural Stem Cells as well as Embryonic Stem Cells

Induced pluripotent stem cells show tremendous promise for regenerative medicine. However in a February 15th article in the Proceedings of the National Academy of Sciences, showed that induced pluripotent stem cells (iPSCs) were inefficient at forming the cells of the brain in comparison to their embryonic stem cell counterparts.

The senior author of the article , Su-Chun Zhang, (professor, University of Wisconsin-Madison School of Medicine and Public Health), said:  “Embryonic stem cells can pretty much be predicted,” and “Induced cells cannot. That means that at this point there is still some work to be done to generate ideal induced pluripotent stem cells for application.”

This study compared the ability of five different embryonic stem cell lines to 12 different iPSC lines to form nerve cell precursors.  Embryonic stem cells are considered the “gold standard” for all pluripotent stem cells, which are cells that can differentiate into all of the 220 cell types in the human body.  Zhang’s group found that the induced cells differentiated into progenitor neural cells and further into the different kinds of functional neurons that make up the brain, but they did not faithfully reproduce all the differentiation capabilities of embryonic stem cells.  This suggests that there are unknown factors at play that may limit the use of iPSCs when it comes to modeling diseases in the laboratory.  Such unknowns would also limit their use in clinical settings for such things as cell transplants.

Despite their unpredictability, Zhang notes that iPSCs can still be used to make pure populations of specific types of cells, which makes them useful for some applications like testing potential new drugs for efficacy and toxicity.  Zhang also noted that the limitations identified by his group are technical issues likely to be resolved relatively quickly.  “It appears to be a technical issue,” said Zhang.  “Technical things can usually be overcome,” he added.

This is very possibly a technical issue that is due to our inability to properly manipulate iPSCs to form nerve cells.  However, if the same protocols that drive embryonic stem cells to form nerve cells are used on iPSCs, they only form nerve cells poorly.  There are probably other protocols that can do just this.  We just haven’t found them yet.

Also, it is worth mentioning, that the ability of iPSCs to differentiate into neurons is probably a line-specific property.  Therefore if these lines to not form lines effectively, then perhaps other lines do.

Mesenchymal stem cells form heart muscle

On August 3rd, 2009, the University of Miami Miller School of Medicine released a press piece that reported the results on a study by Joshua M. Hare, who is the director of the Interdisciplinary Stem Cell Institute at the Miller School. This study examined the ability of mesenchymal stem cells to fix ailing hearts.

Mesenchymal stem cells are found in lots of different places in our bodies. They are found in bone marrow stroma, fat, connective tissue, blood vessels, umbilical cord, and lots of other places too. These cells might come from “perivascular” cells, which are cells that hang around blood vessels. Nevertheless, mesenchymal stem cells have the ability to form bone, cartilage, fat, and muscle. They also have a fascinating capacity to hide from the immune system. They have groups of surface proteins that prevent cells from the immune systems from recognizing them as foreign, and therefore, mesenchymal stem cells from one person can be transferred into an unrelated person without fear of transplantation rejection.

Several experiments have shown that mesenchymal stem cells (MSCs) can differentiate into heart muscle if treated with the right chemicals (S. Tomita, et al., Circulation 1999;100:II-247–II-256; Also see H. Okura, et al., Tissue Eng Part C Methods, 2009). Transplanting MSCs into the hearts of laboratory animals that have had heart attacks can also help the fix the heart (D. Wolf, et al., J Am Soc Echocardiogr 2007;20:512-20). However, there is a raging debate over how MSCs help broken hearts get better.

Even though MSCs can form heart muscle in culture, they seem to do so rather poorly (Y. Zhang, et al., Interact Cardiovasc Thorac Surg. 2009 Dec;9(6):943-6). Also, several studies suggest that once MSCs are transplanted into ailing hearts, they do not differentiate into heart muscle with any efficiency worth bragging about and seem to help the heart by means of the chemicals they produce (Ryota Uemura, et al., Circulation Res 98 (2006): 1414-21).

There are, however, some reasons to suspect that this is not the end of the story. Engineering MSCs with various genes or administering MSCs with certain chemicals can push then to form heart muscle at higher rates (Yigang Wang, et al. Am J Physiol Heart Circ Physiol (nov 6, 2009, doi:10.1152/ajpheart.00765.2009). Also, in particular experiments, MSCs clearly form heart muscle (J. Tang, et al., Eur J Cardiothorac Surg 30 (2006): 353-61).

Clinical studies with MSCs for heart problems have been conducted but the data are limited. Initial studies were very encouraging (S. Chen, et al., Am J Cardiol 94 (2004): 92-5 and S. Chen, et al., J. Invasive Cardiol 18 (2006): 552-6). Now a new study has shown that MSCs not only help people who have had a recent heart attack, but that they turn into heart muscle and other heart tissues.  MSCs can also help form blood vessels and the increase of blood flow to the heart also helps an ailing heart.  This seems to be one of the main ways that bone marrow-based stem cells help hearts after a heart attack.  Therefore MSCs might be one of the best ways to treat bum hearts, but certainly more work needs to be done.