Stem Cells in Ophthalmology Update 7: Research Studies with Induced Pluripotent Stem Cells Suggest Opposite Results

Two research studies were published this week, about the use of induced pluripotent stem cells (iPSCs) in treating retinal problems, but with opposing results.

In one study, published in Nature, Dr.Yang Xu and his colleagues at the University of California, San Diego, found that iPSCs made from mouse skin cells were rejected by genetically identical mice. This study was just published in Nature. (Similar studies with iPSCs, also published in Nature earlier this year, also showed problems, including genetic and epigenectic abnormalities. See Stem Cells in Ophthalmology Update 5: Gene Defects Common in Induced Stem Cells.)

In the second study, Dr. Budd Tucker and his colleagues at the Schepens Eye Research Institute used iPSCs derived from skin to regenerate large areas of damaged retinas and improve visual function in specially  grown degenerative mice. This study was just published in PloS ONE.

The difference may be in the way the stem cells were used in the experiments. In the first case, the iPSCs (along with embryonic stem cells (hESCs)) were directly introduced into the mice of the same strain from which they were created and the implanted iPSCs encountered an immune response, but not the implanted hESCs. In the Schepens study, the harvested iPSCs were forced to express transcription factors, and with additional chemical coaxing, developed into precursors of retinal cells. These latter cells were introduced into the mouse model eyes with retina degenerative disease. Within four to six weeks, the researchers observed that the transplanted cells had taken up residence in the appropriate retinal area (the photoreceptor layer) and had begun to integrate and assemble into healthy looking retinal tissue.

Another difference, as pointed out to me by an expert in the field in a private communication, is the extreme difference in transplant sites between the two studies. The eye, used in the Schepen’s study, is considered an immune privileged body, whereas the flank, where the teratomas were formed in the UC-SD study, is highly immunogenic. As for the immune rejection seen in the Nature paper it was far from complete, i.e., see the specific details of teratoma formation percentages.  Nevertheless, the true importance of the Nature paper comes from the fact that everyone in the field just assumed that iPSCs would be unseen by the immune system, and that was not the case.

Here are highlights of both studies:

Immunogenicity of induced pluripotent stem cells

Tongbiao Zhao, Zhen-Ning Zhang, Zhili Rong & Yang Xu
University of California, San Diego

Nature (2011) doi:10.1038/nature10135
Received: 07 July 2010, Accepted 19 April 2011, Published online 13 May 2011

Abstract:

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration.

In contrast to B6 ESCs, teratomas (tumors containing a chaotic jumble of cell types) formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice.

These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

Basically, what the researchers found was that the immune system of one mouse could not recognize the cells derived from embryonic stem cells of the same strain of mice. But the experiments also showed that the immune system rejected cells derived from iPSCs reprogrammed from fibroblasts of the same strain of mice, mimicking the situation whereby a patient would be treated with cells derived from iPSCs reprogrammed from the patient's own cells. The scientists also found that the abnormal gene expression during the differentiation of iPSCs causes the immune responses.

As noted in his writeup about this study in the New York Times, Andrew Pollack said, “The initial creation of human iPS cells in 2007 (actually it was in 2006 by Shinya Yamanaka at Kyoto University in Japan) electrified scientists because the cells seemed to have two big advantages over embryonic stem cells. They were not controversial, because their creation did not entail the destruction of human embryos, and since the stem cells could be made from a particular patient's skin cells, they could be used to make tissues that presumably would not be rejected by that patient's immune system.

But that latter assumption was never really tested, until now. When Yang Xu, a biologist at the University of California, San Diego, and colleagues did so, they found that iPS cells made from mouse skin cells were nonetheless rejected by genetically identical mice.”

Dr. Xu, whose research was paid for by the National Institutes of Health and by California's stem cell program, created both embryonic stem cells and iPS cells from an inbred strain of mice and implanted those stem cells into other mice of the same strain. The mice did not have an immune response to the implanted embryonic stem cells. But their immune systems attacked the implanted iPS cells, and in some cases, were completely destroyed by the mice’s immune system.

Further experiments suggested that the reaction was caused by the abnormal activation of certain genes in the iPS cells, resulting in the production of proteins that seemed foreign to the immune systems of the mice. The degree of immune response depended on how the iPS cells were made. The strongest response was to cells made by incorporating genes for certain growth factors into the DNA of the skin cells. Cells made that way are not likely to be used for medical treatments anyway because at least one of the inserted genes can cause cancer.

As noted in a story about the study in The Guardian, Paul Fairchild, director of the Oxford Stem Cell Institute, said nobody would have anticipated the immune rejection problem, but added it was too soon to know the implications for medical therapies based on iPS cells.

"It does beg an important question as to whether the same would happen in humans, but it's premature to suggest it casts a cloud over the whole field. We have no idea if human cells would respond in the same way," he said.

Writing in the same issue of Nature, in the News Section, Erika Check Hayden wrote, “But Xu's study is not necessarily the dire news for the iPS field that it might seem. Researchers working with iPS-derived cells that have matured to an adult fate - for instance, neurons or heart cells - have been able to transplant them into mice without rejection, but these experiments have mostly looked at mice without functional immune systems. And scientists designing therapies are mostly proposing to transplant only one type of differentiated cell at a time made from patients' own skin cells back into their bodies, rather than the jumble of differentiated cells found in a teratoma.”

“Xu and other researchers don't yet know whether purified differentiated iPS-derived cells would be rejected, or whether the problem is specific to undifferentiated cells.”

Xu agrees. His group's next steps will be to examine which specific cells in the teratomas trigger immune rejection, and under what conditions. The team used two different methods to make the iPS cells, and they showed slightly different propensities to trigger immune rejection, so it may be that reprogramming methods can be fine-tuned to avoid the problem altogether.

"We propose that the technology to generate iPS cells needs to be improved in order to minimize the difference between iPS and embryonic stem cells, so that iPS cells can be more useful in human therapies," says Xu.


Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

Budd A. Tucker, In-Hyun Park, Sara D. Qi, Henry J. Klassen, Caihui Jiang, Jing Yao, Stephen Redenti, George Q. Daley, Michael J. Young

PLoS ONE 6(4): e18992. doi:10.1371/journal.pone.0018992
Received: October 22, 2010; Accepted: March 23, 2011; Published: April 29, 2011

Abstract

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog.

Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters.

Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

As noted in the press release from the Schepens Eye Research Institute, scientists from Schepens are the first to regenerate large areas of damaged retinas and improve visual function using iPS cells (induced pluripotent stem cells) derived from skin. The results of their study, which is published in PLoS ONE this month, holds great promise for future treatments and cures for diseases such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy and other retinal diseases that affect millions worldwide.

“We are very excited about these results,” said Dr. Budd A. Tucker, the study’s prime author. “While other researchers have been successful in converting skin cells into induced pluripotent stem cells (iPSCs) and subsequently into retinal neurons, we believe that this is the first time that this degree of retinal reconstruction and restoration of visual function has been detected.” Tucker, who is currently an Assistant Professor of Ophthalmology at the University of Iowa, Carver College of Medicine, completed the study at Schepens Eye Research Institute in collaboration with Dr. Michael J. Young, the principle investigator of the study, who heads the Institute’s regenerative medicine center.

While Tucker, Young and other scientists were beginning to tap the potential of embryonic and adult stem cells early in this decade, the discovery that skin cells could be transformed into “pluripotent” cells, nearly identical to embryonic cells, stirred excitement in the vision research community. Since 2006 when researchers in Japan first used a set of four “transcription factors” to signal skin cells to become iPSCs, vision scientists have been exploring ways to use this new technology. Like embryonic stem cells, iPSCs have the ability to become any other cell in the body, but are not fraught with the ethical, emotional and political issues associated with the use of tissue from human embryos.

Tucker and Young harvested skin cells from the tails of red fluorescent mice. They used red mice, because the red tissue would be easy to track when transplanted into the eyes of non-fluorescent diseased mice. By forcing these cells to express the four Yamanaka transcription factors (named for their discoverer) the group generated red fluorescent iPSCs, and, with additional chemical coaxing, precursors of retinal cells. Precursor cells are immature photoreceptors that only mature in their natural habitat – the eye.

Within 33 days the cells were ready to be transplanted and were introduced into the eyes of a mouse model with retinal degenerative disease. Due to a genetic mutation, the retinas of these recipient mice quickly degenerate, the photoreceptor cells die and at the time of transplant electrical activity, as detected by ERG (electroretinography), was absent.

Within four to six weeks, the researchers observed that the transplanted “red cells” had taken up residence in the appropriate retinal area (photoreceptor layer) of the eye and had begun to integrate and assemble into healthily looking retinal tissue.

The team then retested the mice with ERG and found a significant increase in electrical activity in the newly reconstructed retinal tissue. In fact, the amount of electrical activity was approximately half of what would be expected in a normal retina.  They also conducted a dark adaption test to see if connections were being made between the new photoreceptor cells and the rest of the retina.  In brief, the group found that by stimulating the newly integrated photoreceptor cells with light they could detect a signal in the downstream neurons, which was absent in the other untreated eye.

Based on the results of this study, Tucker and Young believe that harvesting skin cells for use in retinal regeneration is and will continue to be a promising resource for the future. The two scientists say their next step will be to take this technology into large animal models of retinal degenerative disease and eventually toward human clinical trials.