New Study of Stem Cell Treatments for Knee Problems

Christopher Centeno, who performs stem cells treatments for patients who are considering knee replacement, has submitted another paper that examines the outcomes of his techniques. His former publication examined 227 patients who were followed for between 3 months and 2-3 years. This paper also utilized data from 50-60 ultra-high field MRIs of the re-implant sites. The results were quite encouraging.

Their new paper has 339 patients followed for up to 3-4 years with 210 MRIs of the places where cells were re-implanted. This paper also includes knee outcome data for the “Regenexx-C” procedure. The data included in this work shows that the Regenexx-C procedure is dramatically safer than the knee replacement surgeries it helped many patients avoid. The paper is still in the galley proof stage, and therefore, it will be a few months before it makes it into press.

Centeno and his colleagues also have other publications on the way.  There is a paper that focuses on knee/hip patient outcome, which will be published sometime this year or early next (the paper is winding it’s way through the scientific review process).  Stem cell orthopedic treatments come in all sorts of shapes and sizes, but many of them are subjected to peer-review.  Make sure the specific therapy you’re being offered has data showing it works.

Human artifical livers transplanted into mice

Artificial organs are made by using artificial scaffolds to which stem cells and other supporting cells are added.  However, by making smaller versions of human organs, scientists are making small versions of these organs and then implanting them into mice so that they can test the effects of various drugs on them.  Researchers at the Massachusetts Institute of Technology (MIT) have developed artificial humanized mouse livers and implanted them into mice. These manufactured livers responded to drugs in ways that are very similar to the way a human liver does, paving the way for safer and more efficient testing of drugs.

According to Alice Chen and her colleagues, in a paper published in the Proceedings of the National Academy of Sciences (PNAS), her team, led by MIT biomedical engineer Sangeeta Bhatia, engineered an artificial liver by growing a triculture of human liver cells (hepatocytes), mouse fibroblasts, and human liver endothelial cells in a three-dimensional polymeric scaffold in a Petri dish.  Because primary hepatocytes do not grow well in culture on their own, the fibroblasts and endothelial cells are necessary to stabilize and help the hepatocytes survive.

After about a week the artificial livers resemble a contact lens in shape and texture.  By implanting these small structures into the abdomen of a mouse, the livers will recruit blood vessels and successfully integrate with their host’s circulatory systems.  The livers will go on to produce human proteins that circulate throughout the mouse’s blood.  Furthermore, these artificial livers continued to function for weeks after implantation.

Human Artificial Liver
Human Artificial Liver

When the researchers treated the animals with drugs known to be metabolized differently by mice and humans, the mice produced drug breakdown products characteristic of human metabolism.  These livers could be useful for studying the immune response to infectious pathogens, such as the hepatitis B and C viruses and malaria, which only infect humans and other primates.

In an interview, Chen said, “We’re stabilizing cells on the bench top first, then putting them into mice, in a way where integration and engraftment occurs nearly 100% of the time.”  Chen is a former graduate student in the MIT-Harvard Division of Health Sciences and Technology.

The polymer scaffold protects the artificial liver from the host’s immune system, so the devices are not rejected and can be implanted into any mouse strain, including those whose immune systems work normally.

Dimiter Bissig, professor of molecular and cellular biology at Baylor College of Medicine, recently made a chimeric mouse whose livers are almost 95% human.  (see Bissig, K.D., S.F. Wieland, P. Tran, M. Isogawa, T.T. Le, F.V. Chisari, I.M. Verma. 2010. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J. Clin. Invest. 120:924-93). Bissig said, “I personally admire this marriage between top-notch engineering and biology.”

Previous humanized livers have been made by injecting human liver cells into an immunodeficient mouse with a severely damaged liver.  The human cells repopulate and regenerate the injured organ, yielding a chimeric mouse.  According to Chen, this technique takes months and can produce results that are unpredictable and difficult to reproduce.  “The field hasn’t reached the point where it’s a very robust method,” said Chen.

Chimeric animals, however, can produce many more hepatocytes and much more human liver function than the MIT team’s implantable devices can at this time, Bissig points out.  The levels of human albumin in Bissig’s chimeric animals’ serum are measured in the milligram-per-milliliter range, whereas the levels in Chen’s models measure at several orders of magnitude lower. Despite these differences, Bissig believes that one model doesn’t necessarily exclude the other, and that each model is useful for different types of applications.  “We’re working on the same problem, but coming at it from different angles,” said Bissig.

In the future, Bissig would like to see artificial livers that can actually replace the function of the endogenous liver, rather than just operating alongside it, as in the new model.  He imagines that such a device could temporarily help patients in need of an urgent liver transplant, but in situations where suitable donor organs aren’t immediately available.

Note that no embryonic stem cells were used in this procedure.

The problem with tumors

Stem cell treatments are haunted by the possibility of tumor formation. While these risks are certainly low when it comes to treatment with somatic stem cells (also known as adult stem cells), the risks are much higher when it comes to embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and fetal stem cells (FSCs). For example, so-called “stem cell tourism” whereby travelers in search of untested and unapproved cures travel to countries where less regulation of the art of medicine allows them to participate in stem cell transplants. China has been one of the most prominent places for stem cell transplants, and FSC transplants for neurological conditions have been the treatment of choice. Unfortunately, the results of these treatments has been disastrous, resulting in meningitis, tumors (Ninette Amariglio, et al. Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient. PLoS Medicine 2009;6(2):e1000029), and few real cures (see Olle  and Insoo Hyun, Medical Innovation Versus Stem Cell Tourism. Science 2009;324(5935):1664-5). Having said that, while tumor formation for ESCs and iPSCs remains a concern, there have been some advances on this front that might make an important clinical difference in the future.

When ESC or iPSC cultures differentiate, they become a morass of different cell types.  Some of the cells remain in the embryonic state, while others become committed to one or another cell type.  Differentiation during development sometimes requires cues from other cells that either contact the other cells or provide tissue-specific instructions for cells to become this or that or do this or that.  For example, the an organ called the thymus results from outpouchings of the endoderm that lines the lower part of the throat.  These outpouchings behave in a manner that depends upon the tissue in which they find themselves and the surrounding cues that they encounter.  These types of conditions are excruciatingly difficult to recapitulate in a culture dish, and for that reason, no ESC culture has ever formed thymus in the culture dish, although they will form it when placed inside an animal (A Isotani, et al. Formation of a thymus from rat ES cells in xenogeneic nude mouse/rat chimeras. Genes to Cells 2011;16(4):397-405).

Therefore, when ESC or iPSC cultures are differentiated into a particular cells type, it is exceedingly difficult to isolate cells that are purely one cell type unless there is a specific way to select for just that one cell type.  For example, if you want to make neurons from ESCs and transplant them into mice that have a particular neurological problem, you must try to ensure that you inject neurons and not neurons contaminated with cells that are also embryonic in nature, because those embryonic cells will go on to cause a tumor.  In some cases the isolation techniques have gratly improved, and tumor formation has been brought to zero.  For example, Geron Corporation has a cell line called GRNOPC1, which is an “Oligodendrocytes Progenitor Cell” (OPC) line.  This cell line was derived from an embryonic stem cell line called H1, which was originally made by James Thomson in 1998.  Hans Keirstead at UC Irvine took this ESC line and differentiated it into OPCs, purified them, and then cultured them.  OPCs are able to form the conductive myelin sheath that surrounds the extensions of some neurons in the central nervous system.  Keirstead has implanted many rodents with this cell line and he has NEVER observed tumors.  See the following papers to document this:

  1. Keirstead HS, et al. Human embryonic stem cell-derived oligodendrocyte profenitor cell transplants remyelinate and restore locomotion after spinal injury.  J Neurosci. 2005;25(19):4694-705.
  2. Cloutier F, et al. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm. Regenerative Medicine 2006;1(4):469-79.

There are other examples of this as well.  For example, Qiuxia Lin and colleagues transplanted three different types of cells into the heart of mice that had experienced heart attacks:  ESCs, ESCs that had been converted into heart muscle cells, and heart muscle cells had from ESCs, but had been purified by Percoll density gradient separation.  They saw tumors in the mice that had been
implanted with the ESCs and the heart muscle cells made from ESCs.  However not a single mouse that was implanted
with the heart muscle cells that were made from ESCs and then purified by Percoll density gradient separation showed any evidence of tumors (See Qiuxia Lin, et al. Tumourigenesis in the infarcted rat heart is eliminated through differentiation and enrichment of the transplanted embryonic stem cells. Eur J Heart Fail. 2010;12(11):1179-85). Therefore, with proper purification of the differentiated cells away from the embryonic cells, the risk of tumor formation decreases greatly.

Having said that there are plenty of concerns about tumorigenicity of ESCs and iPSCs.  For example, this paper: Ahmed RP, et al. Cardiac tumorigenic potential of induced pluripotent stem cells in an immunocompetent host with myocardial infarction. Regen Med. 2011;6(2):171-8.  In this publication, Ahmed and colleagues used iPSCs to make skeletal muscle stem cells, but they did not try to purify the derivatives in any rigorous manner.  In 6 of the 16 mice that received implantations of these cells, tumors formed.  Likewise, Eva Hedlund and her colleagues in Rudolf Jaenisch’s lab at the Whitehead Institute used the expression of genes that are found in differentiated midbrain neurons to screen for differentiated iPSCs.  Unfortunately, undifferentiated cells still expressed these genes, and when they examined their cultures, they still contained embryonic cells that could cause tumors.  In a follow-up paper, Marius Wernig and his colleagues implanted these cells after efficiently differenting them into the brains of rats with Parkinson’s disease.  They found integration of the neurons they made and improvement of behavior, but tumors still formed.  When they used a surface protein that is only found in embryonic cells (SSEA1) to remove the embryonic cells, the grafts caused no tumors een though they were smaller.  See Eva Hedlund, et al. Stem Cells 2007;25:1126-35, and Marius Wernig, et al, PNAS 2008;105:5856-61.  There are also several papers of very hopeful therapies that caused tumors in the laboratory animals and had to be sent back to the drawing board.

Tumors are a problem, but there are ways around them.  It is incumbent on the researchers to convince us that the cells they have made are safe enough for us to trust that they can be placed in our bodies.