Micro-Grooved Surfaces Influence Stem Cell Differentiation

Martin Knight and his colleagues from the Queen Mary’s School of Engineering and Materials Science and the Institute of Bioengineering in London, UK have shown that growing adult stem cells on micro-grooved surfaces disrupts a particular biochemical pathway that specified the length of a cellular structure called the “primary cilium.” Disruption of the primary cilium ultimately controls the subsequent behavior of these stem cells.

Primary cilia are about one thousand times narrower than a human hair. They are found in most cells and even though they were thought to be irrelevant at one time, this is clearly not the case.

Primary Cilium

The primary cilium acts as a sensory structure that responds to mechanical and chemical stimuli in the environment, and then communicates that external signal to the interior of the cell.  Most of the basic research on this structure was done using a single-celled alga called Chlamydomonas.

Martin Knight and his team, however, are certain that primary cilia in adult stem cells play a definite role in controlling cell differentiation.  Knight said, “Our research shows that they [primary cilia] play a key role in stem cell differentiation.  We found it’s possible to control stem cell specialization by manipulating primary cilia elongation, and that this occurs when stem cells are grown on these special grooved surfaces.”

When mesenchymal stromal cells were grown on grooved surfaces, the tension inside the cells was altered, and this remodeled the cytoskeleton of the cells.  Cytoskeleton refers to a rigid group of protein inside of cells that act as “rebar.” for the cell.  If you have ever worked with concrete, you will know that structural use of concrete requires the use of reinforcing metal bars to prevent the concrete from crumbling under the force of its own weight.  In the same way, cytoskeletal proteins reinforce the cell, give it shape, help it move, and help it resist shear forces.  Remodeling of the cytoskeleton can greatly change the behavior of the cell.

The primary cilium is important for stem cell differentiation.  Growing mesenchymal stromal cells on micro-grooved surfaces disrupts the primary cilium and prevents stem cell differentiation.  This simple culture technique can help maintain stem cells in an undifferentiated state until they have expanded enough for therapeutic purposes.

Once again we that there are ways to milk adult stem cells for all they are worth.  Destroying embryos is simply not necessary to save the lives of patients.

Priming Cocktail for Cardiac Stem Cell Grafts

Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.

In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”

To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells.  Here is a picture of bone marrow stroma to give you an idea of what it looks like:

Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.

Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.

Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.

“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.

Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”

Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.

“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”

Embryonic Stem Cell Lines Accumulate Cancer-Causing Mutations

Embryonic stem cells have an incredible ability to grow in culture. Their ability to fill a culture dish in a short period of time makes them attractive candidates for regenerative medicine. However, embryonic stem cells bring a caveat to the table as well. They can sometimes form tumors. Many times these tumors are not aggressive, but sometimes they are. If embryonic stem cells are differentiated into tissues, their ability to grow and form tumors decreases, but does not completely disappear. There are plenty of cases where cells made from embryonic stem cells do not produce tumors when transplanted into animal hosts, but there are also several cases where even cells differentiated from embryonic stem cells can produce tumors.

Because scientists want to grow embryonic stem cell lines in the laboratory, they will grow them in cultures for long periods of time. However, growing human embryonic stem cells for long periods of time can cause the cell line to show chromosomal instability while being cultured continuously (Hanson C, Caisander G. Human embryonic stem cells and chromosome stability. APMIS. 2005 Nov-Dec; 113 (11-12): 751-5). Long-term growth of human embryonic stem (hES) cells in the laboratory can cause them to gain or lose large sections of chromosomes. According to several reports in Nature Biotechnology, this instability can lessen the reproducibility and reliability of experimental results, and can raise the specter of cancer, which can hinder the clinical application of embryonic stem cells.

Anselme Perrier and his colleagues of The Institute for Stem Cell Therapy in Evry, France discovered that long-term culture of five hES cell lines resulted in a the amplification of a portion of a the 20th chromosome called 20q.11.21 locus in four cases of the five cases (Nature Biotechnology 26, 1364 – 1366, 2008). This portion of the human genome contains 23 genes, many of which have roles in proliferation and cell survival.  Therefore, this amplification may give cells a selective advantage and therefore become more prevalent over time.

In a complementary study, Dr Claudia Spits of Vrije Universiteit Brussel in Belgium examined 17 different hES cell lines with her colleagues and showed the same amplification in five cases (Nature Biotechnology 26, 1361 – 1363, 2008).  A part of chromosome 18 was amplified in three cell lines and had several trisomies (three copies of a chromosome) and monosomies (one copy of a chromosome) as well.  The deletion of part of chromosome 18 led to rapid increase of cell growth, indicating that there may be a tumour suppressor in that area. “It’s still an early stage” says Spits, who intends to look further at chromosome 18. “The potentially oncogenic genes that lie in areas that are amplified or duplicated are not well characterized yet, but they have been found in a number of cancers.”

What are we to make of this?  Simply put, if embryonic stem cells are going to be used in a clinical setting, then they should be made and used within a short period of time.  Culturing them for long periods of time should be avoided, since this selects for cells that grow uncontrollably.  This might not be practical, but I think that there is enough evidence to suggest that making lines and culturing them for long periods should be taboo for clinically used lines.