Induced Pluripotent Stem Cells Slow to Grow Tumors in Monkeys


One of the major concerns that dogs the use of pluripotent stem cells in human clinical trials is the risk of tumor formation. Embryonic stem cells and induced pluripotent stem cells have an inherent ability to form special tumors known as teratomas. Teratomas are a rather strange group of tumors that develop from cells early in the developmental program of cells, before they have become committed to mature, adult cell types. Therefore, they contain a mixture of cell types organized to a greater or lesser extent into recognizable structures such as muscles or nerve tissue. In bizarre cases partial teeth may be found.

Embryonic stem cells and their derivatives have a distinct disadvantage in that they are rejected by the immune system of the patient. However, induced pluripotent stem cells (iPSCs), which are made from the patient’s own mature, adult cells, possess the same array of cell surface proteins as the patient’s own cells. Therefore, they are not rejected by the patient’s immune systems. Unfortunately, iPSCs can harbor cancer-causing mutations that were induced during the reprogramming process, and these mutations can seriously compromise their clinical usefulness and safety. Having, not all iPSC lines are the same. Some appear to be safer than others and screening methods that have been developed by stem cell scientists seem to be able to detect unsafe iPSC lines over others.

Now, a new study has shown that it takes a lot of effort to get iPSCs to form tumors after transplantation into a monkey. These findings will bolster the prospects of one day using iPSCs human patients.

Making iPSCs from an animal’s own skin cells and then transplanting them back into the creature also does not trigger an inflammatory response as long as the cells have first been differentiated into a more mature, specialized cell type.

“It’s important because the field is very controversial right now,” says Ashleigh Boyd, a stem-cell researcher at University College London, who was not involved in the work. “It is showing that the weight of evidence is pointing towards the fact that the cells won’t be rejected.”

Pluripotent stem cells have the ability to differentiate into many specialized cell types in culture. Therefore, they have been held out as potential sources of treatments for regenerative therapies for diseases such as Parkinson’s and some forms of diabetes and blindness. iPSCs, which are made by reprogramming adult cells, have an extra advantage because transplants made from them could be genetically matched to the recipient. Also, iPSC derivation is cheaper than cloning procedures and does not destroy a young embryo.

Globally, stem cells researchers are pursuing a variety of iPSCs-based therapies. For example, a group in Japan began enrolling patients for an iPSC-based human clinical trial last year. Experiments in mice from 2011 suggested that even genetically matched iPSCs can elicit an immune response, and pluripotent stem cells can also form slow-growing tumors. Both of these results have elicited deep safety concerns.

A stem-cell scientist from the National Institutes of Health in Bethesda, Maryland, named Cynthia Dunbar led this new study. She decided to evaluate both of these above-mentioned concerns in healthy rhesus macaques. The ability of pluripotent stem cells to form teratomas in laboratory mice is normally a test of their pluripotency. However, to prevent the immune systems of the mice from attacking and destroying these implanted stem cells, mice that lack the cell-mediated arm of the immune response are used. Such mice are called “nude” mice because they do have any hair.

Dunbar said, “We really wanted to set up a model that was closer to human. It was somewhat reassuring that in a normal monkey with a normal immune system you had to give a whole lot of immature cells to get any kind of tumor to grow, and they were very slow-growing.”

Dunbar and her team made iPSCs from skin and white blood cells from two rhesus macaques, and transplanted them back into the monkeys. She and her coworkers were careful to make sure that each monkey was injected with those iPSCs that had been derived from their own cells. For example, if monkey A provided cells that were used to derive iPSC cell line A1, then monkey A was only injected with iPSC line A1 cells and so on. Dunbar and others found that tumor formation required 20 times as many iPSCs as those needed for form a tumor in a nude mouse. These data are invaluable for safety assessments of potential iPSC-dependent therapies. Additionally, even though the injected iPSCs did trigger a mild immune response (white blood cells were attracted to the site of injection, which caused local but not systemic inflammation), when iPSCs were differentiated to a more mature cell types caused no such response.

Dunbar’s study is the first to examine the effects of transplanting undifferentiated iPSCs into the monkey they came from. However it is not the first primate study what happens when cells differentiated from iPSCs are transplanted into non-human primates. Scientists at Kyoto University in Japan transplanted monkey iPSCs that had been differentiated into dopaminergic neurons (the type of neuron that dies in Parkinson’s disease) into the brains of other monkeys and notes that these cells survived for months without forming tumors. Researchers at RIKEN in Kobe, Japan, observed similar results when they transplanted iPSCs that had been differentiated into retinal pigment epithelial cells, which support the photoreceptors at the back of the eye. In neither study did the implanted cells form tumors nor were they immunologically rejected when animals received their own cells. However, in both cases, the transplantation sites that were chosen tend to have a weak capacity to trigger immune responses.

In contrast, Dunbar differentiated iPSCs into bone precursor cells and placed them into small scaffolds just under the skin. Such a location can potentially elicit a robust immune response. However, the transplants did not cause irritation or inflammation, since the differentiated cells do not express embryonic proteins that are normally absent in mature tissues. By eight weeks, new bone had formed, and almost a year later no tumors had formed, and bone formation persisted.

The caveat to these studies is that some work has suggested that bone precursor cells can suppress the immune response against them. To circumvent this problem, Dunbar hopes to repeat these studies using iPSCs that have been differentiated into heart and liver cells.

Safe and Efficient Cell Reprogramming Inside a Living Animal


Research groups at the University of Manchester, and University College, London, UK, have developed a new technique for reprogramming adult cells into induced pluripotent stem cells that greatly reduces the risk of tumor formation.

Kostas Kostarelos, who is the principal investigator of the Nanomedicine Lab at the University of Manchester said that he and his colleagues have discovered a safe protocol for reprogramming adult cells into induced pluripotent stem cells (iPSCs). Because of their similarities to embryonic stem cells, many scientist hope that iPSCs are a viable to embryonic stem cells.

How did they do it? According to Kostarelos, “We have induced somatic cells within the liver of adult mice to transient behave as pluripotent stem cells,” said Kostarelos. “This was done by transfer for four specific gene, previously described by the Nobel-prize winning Shinya Yamanaka, without the use of viruses but simply plasmid DNA, a small circular, double-stranded piece of DNA used for manipulating gene expression in a cell.”

This technique does not use viruses, which was the technique of choice in Yamanaka’s research to get genes into cells. Viruses like the kind used by Yamanaka, can cause mutations in the cells. Kostarelos’ technique uses no viruses, and therefore, the mutagenic properties of viruses are not an issue.

Kostarelos continued, “One of the central dogmas of this emerging field is that in vivo implantation of (these stem) cells will lead to their uncontrolled differentiation and the formation of a tumor-like mass.”

However, Kostarelos and his team have determined that the technique they designed does not show this risk, unlike the virus-based methods.

“[This is the ] only experimental technique to report the in vivo reprogramming of adult somatic cells to plurpotentcy using nonviral, transient, rapid and safe methods,” said Kostarelos.

Since this approach uses circular plasmid DNA, the tumor risk is quite low, since plasmid DNA is rather short-lived under these conditions. Therefore, the risk of uncontrolled growth is rather low. While large volumes of plasmid DNA are required to reprogram these cells, the technique appears to be rather safe in laboratory animals.

Also, after a burst of expression of the reprogramming factors, the expression of these genes decreased after several days. Furthermore, the cells that were reprogrammed differentiated into the surrounding tissues (in this case, liver cells). There were no signs in any of the laboratory animals of tumors or liver dysfunction.

This is a remarkable proof-of-principle experiment that shows that reprogramming cells in a living body is fast and efficient and safe.

A great deal more work is necessary in order to show that such a technique can use useful for regenerative medicine, but it is certainly a glorious start.

 

Also involved in this paper were r, , and .

Pure Heart Muscle Cells from Induced Pluripotent Stem Cells With Molecular Beacons


Using induced pluripotent stem cells to have heart muscle cells is one of the goals of regenerative medicine. Successful cultivation of heart muscle cells from a patient’s own cells would provide material to replace dead heart muscle, and could potentially extend the life of a heart-sick patient.

Unfortunately, induced pluripotent stem cells, which are made by applying genetic engineering techniques to a patient’s own adult cells, like embryonic stem cells, will cause tumors when implanted into a living organism. To beat the problem of tumor formation, scientists must be able to efficiently isolate the cells that have properly differentiated from those cells that have not differentiated.

A new paper from a laboratory the Emory University School of Medicine in Atlanta, Georgia, have used “molecular beacons” to purify heart muscle cells from induced pluripotent stem cells, thus bringing us one step closer to a protocol that isolates pure heart muscle cells from induced pluripotent stem cells made from a patient’s own cells.

Molecular beacons are nanoscale probes that fluoresce when they bind to a cell-specific messenger RNA molecule. Because heart muscle cells express several genes that are only found in heart muscle cells, Kiwon Ban in the laboratory of Young-Sup Yoon designed heart muscle-specific molecular beacons and used them to purify heart muscle cells from cultured induced pluripotent stem cells from both mice and humans.

The molecular beacons made by this team successfully isolated heart muscle cells from an established heart muscle cell line called HL-1. Then Ban and co-workers applied these heart-specific molecular beacons to successfully isolate heart muscle cells that were made from human embryonic stem cells and human induced pluripotent stem cells. The purity of their isolated heart muscle cells topped 99% purity.

Finally, Ban and others implanted these heart muscle cells into the hearts of laboratory mice that had suffered heart attacks. When heart muscle cells that had not been purified were used, tumors resulted. However, when heart muscle cells that had been purified with their molecular beacons were transplanted, no tumors were observed and the heart function of the mice that received them steadily increased.

Because the molecular beacons are not toxic to the cells, they are an ideal way to isolate cells that have fully differentiated to the desired cell fate away from potentially tumor-causing undifferentiated cells. in the words of Ban and his colleagues, “This purification technique in combination with cardiomyocytes (heart muscle cells) generated from patient-specific hiPSCs will be of great value for drug screening and disease modeling, as well as cell therapy.”

Pluripotent Stem Cells Derived From Mouse and Human White Mature Fat Cells


Several studies have shown that adult white fat cells can differentiate into other cell types by first dedifferentiating into a less committed cell type and then differentiating into heart, bone, cartilage, fat or other cell types. These dedifferentiated fat cells, which are also called DFAT cells, do not have any of the characteristics of the stem cell population normally found in fat (fat-based mesenchymal stromal cells).

No one has studied DFAT cells in much detail. One study of rat DFAT cells showed that a very low percentage of cultured rat DFAT cells (0.4% – 1.2%) expressed embryonic stem cell-specific genes after 2 weeks. Beyond that, there is little known about DFAT cells. Could they be a potential source of pluripotent cells?

A new study by Medet Jumabay and colleagues at the David Geffen School of Medicine at UCLA have isolated DFAT cells from adult white fat of mice and humans and characterized them. The results are fascinating and potentially useful for regenerative medicine.

This paper utilizes a new way to isolate fat cells that guarantees their initial purity and a culture system that encourages isolated of DFAT cells. After the fat cells were isolated from liposuction the fat cells showed the characteristics of pluripotent stem cells for five to seven days in culture. In culture, DFAT cells spontaneously clumped into clusters that expressed several stem cell-specific genes. Once these stem cell-specific genes faded, genes associated with specific cell types, such as liver or nerves, or muscle, were expressed. Interestingly, when DFAT cells were implanted into mice with non-functional immune systems, they did not form tumors.

Thus, fat-derived DFAT cells represent a highly plastic stem cell population for pluripotent cell research that is very responsive to changes in culture conditions and may benefit the development of cell-based therapies.

Grafted Stem Cell Derivatives Restore Normal Heart Rhythms in Mice


American researchers, in collaboration with technicians from Fujifilm VisualSonics, Inc., have used advanced ultrasonic software to document microscopic, regenerative improvements to heart muscle that has suffered from previous damage.

High-frequency ultrasound and special cardiac-assessment software was developed by FujiFilm VisualSonics, Inc of Toronto, Canada. Scientists from Mayo Clinic implanted engineered cells into the damaged hearts of mice and then used the special software and ultrasound imaging to observe the regeneration of the heart so that it began to contract with normal cardiac rhythms.

After a heart attack, dead heart tissue is replaced with a cardiac scar that consists of scar tissue that neither contracts nor conducts the signals to contract. Depending of the size of the heart scar, the heart can beat abnormally. An abnormal heart beat is known as arrhythmia. Arrhthymias come in three different categories: a heart that beats too fast (tachycardia), a heart that beats too slowly (bradycardia), and a heart that beats erratically. Arrhythmias after a heart attack can be life-threatening, and restoring normal heart rhythm to the heart after a heart attack is very important.

In this experiment, mice were given heart attacks, and then undifferentiated induced pluripotent stem cells (iPSCs) were implanted into these hearts. Those mice that received induced pluripotent stem cells gradually normalized, their heart beat. The resynchronization of the heart beat of these mice was imaged with high-resolution ultrasound.

Satsuki Yamada, first author of this paper, said, “A high-resolution ultrasound revealed harmonized pumping [of the heart] where iPS cells were introduced to be the previously damaged heart tissue.” Yamada also noted that Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, and achieves resynchronization of the heart beat after a heart attack.

This experiment shows, for the first time that undifferentiated iPSCs have the potential to stabilize a patient’s heart after a heart attack. The healing of the heart was documented by ultrasound imaging and by “speckle-tracking echocardiogram.,” Speckle-tracking echocardiography was designed by VevoStrain Advanced Cardiac Analysis Software, which was manufactured by VisualSonics.

This software package provides advanced imaging and quantification capabilities for studying sensitive movements in heart muscles and it is also the only commercial cardiac-strain package optimized for assessing cardiovascular function preclinical rodent studies.

Yamada and her co-researchers utilized this software during the implantation and observation of the iPSCs within the hearts of mice. This software package the motion of the heart wall both at the regional and global levels and from several different perspectives, measurements of these movements, the changes in dimension in the left ventricle during the heart cycle.

The software definitely showed that homogeneous wall movement was restored in those mice that had received implants of iPSCs.

When iPSCs were implanted into mice that had dysfunctional immune systems, they produced tumors, but in mice with normal immune systems, the implanted iPSCs did not produce tumors. What became of those cells is uncertain, but they clearly helped heal the heart and did not cause tumors.

Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.
Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.

This paper is interesting and suggests that undifferentiated cells can also exert healing effects on the heart.

Foregut Stem Cells


Scientists from Cambridge University have designed a new protocol that will convert pluripotent stem cells into primitive gut stem cells that have the capacity to differentiate into liver, pancreas, or some other gastrointestinal structure.

Nicholas Hannan and his colleagues at the University of Cambridge Welcome Trust MRC Stem Cell Institute have developed a technique that allows researchers to grow a pure, self-renewing population of stem cells that are specific to the human foregut, which is the upper section of the human digestive system. These types of stem cells are known as “foregut stem cells” and they can be used to make liver, pancreas, stomach, esophagus, or even parts of the small intestine. Making these types of gastrointestinal tissues can provide material for research into gastrointestinal abnormalities, but might also serve as a source of material to treat type 1 diabetes, liver disease, esophageal and stomach cancer, and other types of severe gastrointestinal diseases.

“We have developed a cell culture system which allows us to specifically isolate foregut stem cells in the lab,” said Hannan. “These cells have huge implications for regenerative medicine, because they are the precursors to the thyroid upper airways, lungs, liver, pancreas, stomach, and biliary systems.”

Hannan did this work in the laboratory of Ludovic Vallier, and they think that their technique will provide the means to analyze the precise embryonic development of the foregut in greater detail. “We now have a platform from which we can study the early patterning events that occur during human development to produce intestines, liver, lungs, and pancreas,” said Hannan.

To make foregut stem cells, Hannan begins with a pluripotent stem cell line; either an embryonic stem cell line or an induced pluripotent stem cell line. Then he differentiated them into definitive endoderm by treating them with CDM-PVA and activin-A (100 ng/ml), BMP4 (10 ng/ml), bFGF (20 ng/ml), and LY294002 (10 mM) for 3 days. Once they differentiated into endoderm, the endodermal cells were grown in RPMI+B27 medium with activin-A (50 ng/ml) for 3-4 days in order to generate foregut stem cells.

(A) GFP-expressing hPSCs were differentiated into hFSCs. (B) Single GFP-positive hFSCs were seeded onto a layer of non-GFP hFSCs and then expanded for five passages. The resulting population was then split into culture conditions inductive for liver or pancreatic differentiation. (C and D) GFP-hFSCs differentiated for 25 days were found to respectively generate cells expressing liver markers (ALB, LDL-uptake) and pancreatic markers (PDX1, C-peptide) from both hESC-derived (C) and hIPSC-derived (D) hFSCs.
(A) GFP-expressing hPSCs were differentiated into hFSCs.  (B) Single GFP-positive hFSCs were seeded onto a layer of non-GFP hFSCs and then expanded for five passages. The resulting population was then split into culture conditions inductive for liver or pancreatic differentiation.  (C and D) GFP-hFSCs differentiated for 25 days were found to respectively generate cells expressing liver markers (ALB, LDL-uptake) and pancreatic markers (PDX1, C-peptide) from both hESC-derived (C) and hIPSC-derived (D) hFSCs.

These foregut stem cells (FSCs) can self-renew, and can also differentiate into any part of the foregut. Thus, FSCs can grow robustly in culture, and they can also differentiate into foregut derivatives. However, these cells also do not form tumors. When injected into mice, they failed to form tumors.

(A) Large cystic hFSC outgrowth under the kidney capsule of a NOD-SCID mouse. (B) Cryosection of a hFSC outgrowth showing large cystic structures lined with epithelial cells. (C) Immunocytochemistry showing foregut outgrowths expressing EpCAM, PDX1, AFP, and NKX2.1. Scale bars, 100 μm or 50 μm as shown. See also Figure S4.
(A) Large cystic hFSC outgrowth under the kidney capsule of a NOD-SCID mouse.  (B) Cryosection of a hFSC outgrowth showing large cystic structures lined with epithelial cells.  (C) Immunocytochemistry showing foregut outgrowths expressing EpCAM, PDX1, AFP, and NKX2.1.  Scale bars, 100 μm or 50 μm as shown. See also Figure S4.

What are the advantages to FSCs as opposed to making pancreatic cells or liver cells from pluripotent stem cells? These types of experiments always create cultures that are impure. Such cultures are difficult to use because not all the cells have the same growth requirements and they would be dangerous for therapeutic purposes because they might contain undifferentiated cells that might grow uncontrollably and cause a tumor. Therefore, FSCs provide a better starting point to make pure cultures of pancreatic tissues, liver tissues, stomach tissues and so on.

Ludovic Vallier, the senior author of this paper said this of his FSCs, “What we have now is a better starting point – a sustainable platform for producing liver and pancreatic cells. It will improve the quality of the cells that we produce and it will allow us to produce the large number of uncontaminated cells we need for the clinical applications of stem cell therapy.”

Vallier’s groups is presently examining the mechanisms that govern the differentiation of FSCs into specific gastrointestinal cell types in order to improve the production of these cells for regenerative medicine.

Making Induced Pluripotent Stem Cells With Small Molecules


A Journal article in the August 9th edition of Science Magazine features work from the laboratories of Yang Zhao and Hongkui Deng, both of whom are from the College of Life Sciences and Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China. Zhao and Deng and colleagues used small molecules to transform adult cells into induced pluripotent stem cells.

To review, induced pluripotent stem cells are derived from adult cells by genetically engineering the adult cells to express a cocktail of four genes (OCT4, Klf4, Sox2, and c-Myc). To introduce these genes into cells, viruses are normally used, but other techniques are also available. The resultant cells look and act like embryonic stem cells, but they do not require the death of embryos.

In this paper, Deng and colleagues took mouse embryonic fibroblasts (skin cells cultured from mouse embryos) and used them to screen over 10,000 small molecules for their ability to substitute for the OCT4 gene in the production of iPSCs. If this sounds labor intensive, that’s because it is. To conduct the screen, they used mouse embryonic fibroblasts that were infected with viruses that expressed Sox2, Klf4, and c-Myc. These genes are not enough to convert adult cells into iPSCs. However, with these chemicals, these three genes could produce iPSCs from mouse embryonic fibroblasts (MEFs). They identified at least three molecules; Forskolin, 2-methyl-5-hydroxytryptamine and a synthetic molecule called D4476, that could substitute for OCT4.

Thus, by using chemicals, they could get away from using one of the genes required to de-differentiate adult cells into iPSCs. Could they whittle down the number of genes even further? Previously, Deng and Zhao published a paper in which a chemical cocktail was used to substitute for the other three genes so that conversion into iPSCs was achieved by introducing only the OCT4 gene into cells (Li, YQ et al., CELL RESEARCH 21(1): 196-204. They called this cocktail “VC6T.” Therefore, they used VC6T and Forskolin, on their MEFs and the beginnings of de-differentiation occurred, but not much else.

Could chemicals be identified that would take the cells the rest of the way to iPSCs? Another chemical screen examined this possibility. In this test, the MEFs were rigged so that they expressed OCT4 when the cells were treated with the antibiotic doxycycline. By giving the cells doxycycline for 4-8 days, and then testing chemicals to take the cells the rest of the way, they identified a slew of compounds that, when given to the OCT4-expressing MEFs, they became iPSCs.

Then came the real test – make iPSCs with just chemicals and no introduced genes. Could it be done? When they gave the MEFs some of the chemicals identified in the last screen (they called it DZNep), plus VC6T, the expression of OCT4 went up, but the cells simply did not look like iPSCs. So, they changed the culture medium to a “2i” culture system that inhibits some key regulatory proteins in the cells. When they used this same chemical cocktail in a 2i culture system, it worked and iPSCs were produced. Deng and Zhao called these stem cells “chemically induced pluripotent stem cells” or CiPSCs.

(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.
(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.

Next, they optimized the dosages of these chemicals in order to increase the efficiency of iPSC production. They were able to increase the efficiency of iPSC production to 5% (1 of every 20 colonies of cells), which is respectable. They also identified yet another small molecule that beefed up iPSC production by another 40-fold. Also, this chemical cocktail was able to make iPSCs from mouse adult fibroblasts, fat-derived stem cells, and fibroblasts from newly born mice.

When the CiPSC lines were characterized, they made all the right genes to be designated as pluripotent stem cells, and they had normal numbers of normal-looking chromosomes all the way through 13 passages.

When injected into mice with dysfunctional immune systems, the CiPSCs made tumors that were mixtures of tissues of all over the body. When they were transferred into early mouse embryos, they could contribute to the bodies of developing mice, and they could even contribute to the production of eggs and sperm, When baby mice were completely made from CiPSCs, those mice were fertile and had babies of their own. This is the ultimate test of pluripotency and the CiPSCs passed it with flying colors.

A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.
A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.

Other experiments in this paper examined why these chemicals induced pluripotency in adult cells, but these experiments, though interesting, are lost in the fact that this research group has generated iPSCs without using any viruses, or genetic engineering technology. These CiPSCs are true pluripotent stem cells and they were generated without killing any embryos or introducing genes that might drive cells to become abnormal.

If this can be replicated with human cells, it would be earth-shattering for regenerative medicine.

Tests to Improve Stem Cell Safety


Stem cell scientists from the Commonwealth Scientific and Industrial Research Organisation or CSIRO (the Australian version of the NIH) have developed a test to identify unsafe pluripotent stem cells that can potentially cause tumors. This test is one of the first tests specifically designed for human induced pluripotent stem cells or iPSCs.

The development of this test marks a significant breakthrough in improving the quality of iPSCs and identifying unwanted stem cells that can form tumors. The test also directly assesses the stability of iPSCs when they are grown in the lab.

Andrew Laslett and his team have spent the last five years working on this research project and perfecting their test.

Laslett explained: “The test we have developed allows us to easily identify unsafe iPSC cells. Ensuring the safety of these cell lines is paramount and we hope this test will become a routine screen as part of developing safe and effective iPS-based cell therapies.”

Laslett’s research focused on comparing different types of iPS cells with human embryonic stem cells. Induced pluripotent stem cells are, at this time, the most commonly used type of pluripotent stem cell in research.

Laslett’s method has established that iPSCs made in certain ways are inherently less stable and riskier than those made by alternative means. For example, the classical way of making iPSCs, with genetically engineered retroviruses that insert their genes into the chromosomes of the cells they infect, can cause insertional mutations and are inherently more likely to cause tumors. In comparison, iPSCs made with viruses that do not integrate into the host cell’s DNA (that is, with genetically engineered adenoviruses), or made with plasmid DNA, mRNA or modified proteins, do not form tumors.

Laslett hopes the study and the new test method will help to raise the awareness and the importance of stem cell safety. He also predicts that tests like his will promote a kind of quality control over the production of iPSC lines.

“It is widely accepted that iPS cells made using viruses should not be used for human treatment, but they can also be used in research to understand diseases and identify new drugs. Having the assurance of safe and stable cells in all situations should be a priority,” said Laslett.

This test utilizes laser technology that activates fluorescent dyes attached to antibodies that are bound to specific cell surface proteins.  If the cell has the cell surface protein bound by the antibody, the cell and its surface proteins fluoresce, and it is sent into the positive test tube.  If it does not fluoresce, it is sent to the negative test tube.  This technique is called fluorescence activated cell sorting or FACS.  In order to identify proteins found the surfaces of iPSCs, Laslett’s team used dye-conjugated antibodies that bound to surface proteins TG30 (CD9) and GCTM-2.  The presence of these specific cell-surface proteins provides a means to separate cells into safe and unsafe cell lines.  Very early-stage differentiated stem cells that expressed TG30 (CD9) and GCTM-2 on their cell surfaces tend to dedifferentiate into pluripotent cells after differentiation and cause tumors, whereas those very early-stage differentiation stem cell lines that do not express TG30 (CD9) and GCTM-2 on their cell surfaces do not cause tumors.  After separation of the stem cell lines by FACS, the iPSC lines were further monitored as they grew in culture.  Unsafe iPS cell lines that form tumors usual clump together to make recognizable clusters of cells.  However, the safe iPS cell lines do no such thing. This test can also be applied to somatic cell nuclear transfer human embryonic stem cells.

Professor Martin Pera, the Program Leader of Stem Cells, Australia said, “Although cell transplantation therapies based on iPS cells are being fast tracked for testing in humans, there is still much debate in the scientific community over the potential hazards of this new technology.”

Transplantable Hematopoietic Stem Cells Made From Induced Pluripotent Stem Cells


Induced pluripotent stem cells (iPSCs) are made from adult cells by genetic manipulation. In short, four different genes, all of which encode DNA-binding proteins that direct gene expression, are introduced into adult cells. The four proteins direct a gene expression program that dedifferentiates a small proportion of the cells to become stem cells that greatly resemble embryonic stem cells.

These iPSCs have the capacity to differentiate into any cell type in the adult body, but there are particular cell types that have proven difficult for iPSCs to make. One of these is the blood cell-making stem cell that normally resides in bone marrow. This stem cells, the hematopoietic stem cell or HSC. Several different types of blood cells have been made from iPSCs, but, again, making HSCs from iPSCs has proven elusive.

A paper from the laboratories of Leslie Silberstein and Daniel Tenen at the Harvard Stem Cell Institute and Harvard Medical School has used a new approach to make HSCs from iPSCs. In this paper, Giovanni Amabile and colleagues injected undifferentiated HSCs into mice whose immune systems were compromised to prevent them from rejecting the implanted cells. The iPSCs formed tumors known as teratomas that contained a wide variety of cells types that included HSCs. Isolation of these HSCs from the teratomas produced pure cultures of HSCs that could be used to reconstitute the immune system of mice.

Isolation of HSCs from teratomas is actually rather easy, since very high-affinity antibodies can bind to the surfaces of HSCs and facilitate their isolation. Once isolated, Amabile and others used them to reconstitute the immune system of imunodeficient mice. This demonstrates that HSCs isolated in this manner are transplantable.

Embryonic stem cells can be converted to HSCs by co-culturing them with OP9 cells, a special mouse bone marrow-derived cell line. If iPSCs were injected into mice with OP9 cells, the number of HSCs they made in culture greatly increased.

OP9 cells
OP9 cells

The cells produced by the HSCs were evaluated for functionality, and the white blood cells made all the right molecules, ate bacteria like they should and also moved like white blood cells. Antibody making cells all made antibodies and T cells responded just as they should and made all the right molecules in response to stimulation. Thus, these HSCs were normal HSCs and produced blood cells that were completely normal from a functional perspective.

This technique could provide a way to make HSCs for human antibody production, drug screening, and, possibly, transplanation. Unfortunately, if these cells have been passed through an animal, there is no way they can be used for human treatments, since they might have picked up animal viruses and animal sugars on their surfaces. If these procedure could be refined to eliminate passing the iPSCs through an animal , then this technique could certainly be used to make transplantable HSCs for the treatment of human diseases of the blood.

See Amabile et al., Blood 121(8):1255-1264.

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.

The eyes have it.


Amber Dance at Nature Reports Stem Cells has a very interesting article on the use of stem cell treatments to cure blindness.

Hundreds of people have had limbal stem cell transplants to treat chemical burns or diseases that scar the cornea.  Unfortunately this therapy is not commercially available to date, since acquiring data on the efficacy of such treatments is slow.  However, 60-70% of patients who have these procedures have improved vision.

This therapy is an “adult” stem cell treatment, but treatments for other types of blindness might require a more creative strategy.

Once the light passes through the transparent cornea and is bent by the lens, it hits the retina at the back of the eye.  The retina is composed of an inner neural retina that consists of photoreceptors, bipolar cells and ganglion cells that extend axons to form the optic nerve, and an outer pigmented retina into which the photoreceptors extend.  The pigmented retina secretes growth factors and clean up cell fragments from spent photoreceptor cells.  If the photoreceptors break down, then no reception of light is possible in that portion of the retina, but if the pigmented retina breaks down, then the photoreceptors will also die, since the tissue that maintains them has died.

Source: http://www.glaucoma.org/uploads/eye-anatomy-2012_650.gif
Source: http://www.glaucoma.org/uploads/eye-anatomy-2012_650.gif

Age-related macular degeneration is the third-most common cause of blindness in the world, and it results from the death of the photoreceptors in the macula – that part of the retina where the concentration of photoreceptors are the highest and the resolution of the vision is the best.  In animals, scientists have been able to differentiate embryonic stem cells into retinal epithelial cells and transplant them into the retinas.  In rats that tend to suffer from sight degeneration, transplantation of retinal epithelial cells made from embryonic stem cells greatly slows loss of sight (R. Lund, et al., Cloning Stem Cells (2006) 8, 189-199).

Can such a treatment work?  Clinical studies suggest that it can.  In one study where ten patients were treated with fetal retinal cells, none of them experienced rejection (N. Radke, et al., Am. J. Ophthalmol. (2008) 146, 172-182).   The eye, you see, is sealed from the immune system, and there is no need to match tissue types before transplants.  However, injuries to the eye could sensitize the immune system to transplanted tissues, and a possibility might be using induced pluripotent stem cells (iPSCs).  As it turns out, differentiating embryonic stem cells into retinal epithelial cells is rather easy.  Therefore, the use of iPSCs might be quite easy.

There is reason for caution, however, because in animals the transplantation of neural stem cells into animal eyes can cause tumors (S. Arnhold, et al., Invest. Ophthalmol. Vis., (2004), 45, 4251-4255).  However, transplanted retinal epithelial cells made from embryonic stem cells have never formed teratomas.  Therefore, this cell type might not cause tumors at a high rate, and treatments with such cells might actually be feasible.