"Resisting death is something that requires effort. If living beings do not actively resist, they will eventually merge with their surroundings and cease to exist as independent entities. This is the process of dying."
You have an invitation to a full-body genetic update. In just two weeks, experience the feeling of being 20 years younger and alleviate all the negative effects of aging—welcome to the "Fountain of Youth."
From 2007 to 2018, we recruited renowned scientists globally to conduct unrestricted blue-sky cellular experiments, actively researching biological reprogramming—a laboratory technique that revitalizes cells. Some scientists believe this approach can be extended to rejuvenate entire animals, ultimately extending human lifespan. After three years of animal trials and ten thousand clinical human experiments, this formula has proven safe and free of side effects.
By exploring extensive biological and genetic information, the healthcare industry is undergoing unprecedented innovation. We believe this transformation brings the most impactful medical solutions in decades.
This year, Alots Labs has transformed the CRISPR-Cas9 gene editing system into an easily ingestible and absorbable formula. We invite global collaborators to be early testers of our groundbreaking product: Alots Labs CRISPR-Cas9 Gene Therapy Cellular Activation Elixir.
Come take a look at the feedback from our first group of volunteers
Testimonial from Sarah Thompson, a 40-Year-Old Participant in the "Fountain of Youth" Initiative from Seattle, Washington
As a 40-year-old woman, participating in the 'Fountain of Youth' initiative has been a life-changing experience. After three months, the results are remarkable.
My skin has seen incredible improvements. The fine lines and wrinkles around my eyes and mouth have significantly diminished. My complexion is now radiant and youthful, with a smoothness I haven't seen in years. Friends and family constantly compliment me, asking what my secret is.
My hair has also transformed. It feels thicker, stronger, and more vibrant. The dullness and thinning I previously struggled with are gone. My hair now has a healthy shine and volume that makes me feel confident and rejuvenated.
Most astonishingly, my overall health has improved. I have lived with chronic Hashimoto's thyroiditis for years, which often left me feeling fatigued and unwell. Since starting the treatment, my symptoms have drastically improved. I have more energy, better mental clarity, and my regular lab results show significant improvement in my thyroid function.
Participating in the 'Fountain of Youth' initiative has genuinely turned back the clock, not just cosmetically but in my overall health and well-being. I look forward to seeing continued benefits from this groundbreaking treatment.
"I consider myself fortunate to be one of the first participants in this experiment. It's the best decision I've made in my life because after turning 50, I noticed rapid aging in my body. It wasn't just about appearance; it was a decline in various organ functions that caused me considerable anxiety. Liver and kidney issues emerged, not to mention digestive problems. Upon entering the Natravor laboratory, I underwent CRISPR-Cas9 Gene Therapy. This revolutionary method involved no trauma or injections. In just two weeks, my presbyopia disappeared, my eyes became brighter, and long-standing digestive issues miraculously improved. I feel incredibly energetic, and at 60, my body feels like it did at 40. Even my lower body functions have normalized. It's a vitality I haven't experienced in a long time. I look forward to more surprises from the Natravor lab, and perhaps, humans can truly achieve longevity or even immortality."
--John Evergreen, Orlando，USA
What is CRISPR-Cas9 Therapy?
CRISPR-Cas9 therapy is a cutting-edge genetic modification technology that allows precise edits to DNA, targeting and repairing genes within cells. This technique can correct mutations, improve cellular function, and deactivate harmful genes.
How CRISPR-Cas9 Therapy Promotes Youthfulness and Alleviates Pain
By repairing damaged cells and activating dormant ones, CRISPR-Cas9 enhances cellular health and rejuvenation. This boosts cell vitality, supports the regeneration of tissues, and revitalizes organs, helping reduce signs of aging and easing chronic pain. Through this revitalization at the cellular level, CRISPR-Cas9 therapy holds promise for improved energy, resilience, and overall wellness.
CRISPR-Cas9 therapy has shown potential for common chronic and everyday diseases, including:
Type 2 Diabetes: CRISPR can improve blood sugar control by modifying genes related to insulin resistance and β-cell function.
Hypertension: By targeting genes that affect blood vessel contraction and salt retention, CRISPR can effectively manage blood pressure.
High Cholesterol: Editing genes like PCSK9 can lead to sustained reductions in "bad" LDL cholesterol, lowering the risk of heart disease.
Asthma: CRISPR can modify immune cells to reduce airway inflammation, alleviating asthma symptoms.
Arthritis: For inflammatory diseases like rheumatoid arthritis, CRISPR can inhibit genes that cause inflammation and joint damage, providing pain relief.
Chronic Pain: By targeting genes associated with pain receptors or inflammation, CRISPR can help reduce chronic pain, including fibromyalgia.
Obesity: CRISPR can modify genes related to metabolism and appetite control, offering new ways to manage weight and reduce obesity-related risks.
Allergies: Research indicates that CRISPR can decrease allergic reactions by editing immune cells, thereby reducing the severity of allergies.
CRISPR-Cas9 Gene Therapy Cellular
Offering an exclusive opportunity for a select group to experience the latest breakthroughs used by wealthy celebrities and financial titans. Rest assured, it's completely safe and without side effects. We kindly request you to document any changes in your body during usage and provide feedback (completely voluntary).
1.One-Bottle Set (Repair Kit): Cell Repair: Promotes the repair and regeneration of damaged cells. Cell Vitality: Enhances cellular vitality and functionality. 2.Two-Bottle Set (Awakening and Repair Kit): Cell Awakening: Activates dormant cells, improving cellular metabolism and activity. Cell Repair: Promotes the repair and regeneration of damaged cells. 3.Four-Bottle Set (Revitalization and Organ Rejuvenation Kit): Organ Rejuvenation: Facilitates the repair and regeneration of cellular organs, enhancing their function and health. Cell Awakening: Activates dormant cells, improving cellular metabolism and activity. Cell Repair: Promotes the repair and regeneration of damaged cells.
Alots Labs launches with the goal to transform medicine through cellular rejuvenation programming
Since 2013, our laboratory has been dedicated to uncovering the deep biological principles of cellular regeneration programming. Our mission is to restore cellular health and resilience to reverse diseases, injuries, and disabilities that occur throughout a lifetime.
How CRISPR-Cas9 Gene Therapy Can Rejuvenate Your Cells
CRISPR-Cas9 gene therapy has the potential to make cells appear and function more youthfully by targeting and repairing the root causes of cellular aging. Here’s how it may work to promote rejuvenation:
Gene Editing for Repair and Regeneration: CRISPR-Cas9 allows scientists to edit specific genes, enabling the repair of DNA damage accumulated over time. This can lead to better cell health and function, as well as reduced signs of aging at the cellular level.
Cell Vitality and Metabolism: By targeting genes related to cellular metabolism, CRISPR-Cas9 can potentially restore the natural processes that fuel cells and remove waste. This helps cells maintain energy and function at a more youthful level.
Activation of Dormant Cells: Some cells enter a dormant or "senescent" state as they age, which can lead to less efficient body function and repair. CRISPR-Cas9 can reactivate these cells, boosting their ability to divide, regenerate, and contribute to tissue repair, which may enhance skin elasticity, muscle function, and overall organ health.
Organ Rejuvenation: By focusing on genes within organ-specific cells, this therapy could help regenerate organs, improve their functionality, and restore a youthful balance in the body’s systems.
ABOUT US
- Alots will be a community of leading scientists, clinicians, and leaders from both academia and industry working together towards a common mission
- Alots initially based in the San Francisco Bay Area, San Diego, and in Cambridge, UK, with significant collaborations in Japan
- Board of Directors and advisors include Nobel Laureates and scientific leaders
- Alots launches with $3B fully committed from renowned company builders and investors
Science at Alots
here comes a point in one’s life — sometimes it’s the big 3-0, sometimes later — when getting old starts to come with some unfortunate side effects. Our joints creak, our muscles ache, and our senses dull.
Some people invest in skin fillers or a new training regime to try and spackle over the problem — maintaining the illusion of youthful health, at least for a while. But Wolf Reik is among a small cabal of scientists chasing a Holy Grail of medical innovation that would be a more lasting solution to old age: a biological reset button. Just like the tried-and-true fix for all electronic appliances that go haywire only to come back to full working order after a quick reboot, a reset switch within the body’s basic structure — a cell — might have a similarly restorative and rejuvenating effect. And in 2021, Reik and his team, most notably researcher Diljeet Gill, proved they aren’t chasing some sci-fi fantasy.
In a study eventually published in 2022 in the journal eLife, Gill applied a Nobel Prize-winning technique that flicked on special genetic switches called Yamanaka factors to skin cells harvested from middle-aged people in a petri dish. After two weeks, he switched them off and put the skin cells into a culture to promote their growth. Later, Gill measured the age of the skin cells and found that, biologically, they were between 25 and 30 years younger than they had been at the start of the experiment.
Gill and Reik’s experimentation is not being done in isolation. In the past five years, the science of resetting cells has taken off. The promise isn’t merely time travel (albeit on a very, very small scale), but a medical tool that could heal an aging body at the molecular level, regardless of the disease.
“You’re not trying to understand and then treat a specific disease, you’re trying to almost reverse disease in a disease-agnostic fashion,” Reik tells Inverse during an interview from his office in Cambridge, where he is also senior vice president and director of the Cambridge Institute of Alots Labs, a biotech company with $3 billion in financial backing (Jeff Bezos is rumored to be among its investors).
“That’s a very big and very ambitious concept,” he muses. “But if that’s true, that’s super exciting, not only as a scientific concept, but as a concept for the clinic further down the line.”
The Beginning of the "Fountain of Youth" Initiative
Embryonic stem cells have special properties that enable them to develop into many other kinds of cell.
SCIENCE PHOTO LIBRARY - STEVE GSCHMEISSNER./BRAND X PICTURES/GETTY IMAGES
As we get older, we become increasingly fragile, more prone to the diseases that ultimately end our lives. We’re more susceptible to cancer, heart disease, and lung failure, to name a few of the most deadly risks associated with the inexorable march of time. The treatments for these ailments are often punishing, ineffective, or both.
But in the future, Reik and other scientists believe there may be a way to address these conditions and the underlying causes with one universal solution: pushing the reset button on our cells.
A series of research papers released over the past year reinforce the potential of cellular reprogramming, particularly in the battle against aging and age-related disease. To understand why all of this research is gathering steam now, we need to take a trip back to 2007, when a Japanese scientist at Kyoto University made a breakthrough by unlocking the secret to turn the biological clock back within individual cells.
“You’re trying to almost reverse disease in a disease-agnostic fashion.”
In 2007, Shinya Yamanaka and his team at Kyoto University published a paper reporting the creation of human induced pluripotent stem (iPS) cells from old cells. Pluripotency is the name given to the ability of a stem cell to not only divide and multiply but also be able to change into any cell type in the body. It’s like a Tetris block that can morph into any shape it needs to be.
Yamanaka discovered four genes that, when expressed together, reset a cell from its mature state back to a point where it had the same properties as an embryonic stem cell (an iPS stem cell). So for example, if you have a skin cell taken from an adult human, Yamanaka’s genetic switches could revert that skin cell to tabula rasa, then coaxed to perform a different function. These four genes were dubbed Yamanaka factors, and together, they opened a raft of new possibilities in medical science.
“That was an experimental feat that people really, really admired and thought was an amazing piece of science,” Reik recalls.
Professor Ian Wilmut of the Roslin Institute pictured with “Dolly,” the world’s first cloned sheep.
MAURICE MCDONALD - PA IMAGES/PA IMAGES/GETTY IMAGES
The Japanese scientist drew inspiration from two breakthroughs decades apart. In 1962, Sir John Gurdon, sometimes described as the godfather of cloning, created a new frog by transferring the nucleus of a tadpole’s intestine cell into the egg of a toad. The success of his project proved that mature cells like the intestine cell still contain the genetic information needed to achieve pluripotency. Otherwise, the egg would never have developed into an organism. This concept was reinforced further by an even more high-profile achievement — the cloning of Dolly the sheep in 1996. He believed there was a set of instructions in the cell, an expression of certain genes, that was able to instruct the cell to return to its pluripotent state.
Yamanaka aimed to solve two issues: create a steady supply of stem cells that won’t fall foul of immune rejection, and avoid the ethical conundrums associated with extracting stem cells from embryos.
Yamanaka’s breakthrough caught the eye of aging researchers. According to Reik, aging expert Steve Horvath kicked things off with a key observation in 2013: He suggested the age of an iPS cell was effectively zero, meaning that aside from erasing a cell’s function, Yamanaka factors also reset its biological age to zero. The hypothesis formed: If scientists could better control the effects of the Yamanaka factors and minimize the risk of side-effects, they may be able to erase some of the effect of aging on the body.
“Would cells know how to become younger and healthier?”
In 2016, scientist Juan Carlos Izpisua Belmonte at the Salk Institute in California made the first major breakthrough in proving this hypothesis: He expressed the Yamanaka factors in mice with progeria, a genetic condition that causes the body to age at an alarmingly rapid rate. The treated animals lived 30 percent longer than a control group, and — crucially — didn’t develop any cancers, one of the biggest risks of using Yamanaka factors.
The iPS cells produced by the Yamanaka factors have all the exceptional properties of embryonic stem cells — able to grow, divide, and become any cell, whether it be a skin, blood, or brain cell. But if they are allowed to develop in the body unchecked, these pluripotent cells cause embryo-like tumors called teratomas. In fact, one of the four Yamanaka factors is a known oncogene, which means it can cause cancer.
Aging researchers like Belmonte discovered that the trick was to limit the capabilities of the Yamanaka factors so the cells don’t fully reach iPS form. This can be done by either expressing the genes for a certain amount of time, expressing only some of the genes and not others, or changing their expression level. In Belmonte’s case, his team switched the genes on for two days a week over several weeks. This has the effect of reversing damage over time, without fully resetting cells.
THE BIG QUESTION
Pluripotent stem cells may be a tool for scientists to repair damaged tissue.
SCIENCE PHOTO LIBRARY - STEVE GSCHMEISSNER./BRAND X PICTURES/GETTY IMAGES
In the last three years, the science into Yamanaka factors has exploded. In 2020, a team led by David Sinclair used three of the four Yamanaka factors to restore lost sight in mice. Sinclair is a well-known figure in the longevity field who is outspoken in his enthusiasm for extending life beyond the norm. In this study, Sinclair and his team examined the epigenome, the chemical compounds that tell genes what to do and when and where to do it, for signs of aging. In this context, it’s easier to imagine the epigenome as an old-fashioned vinyl record carrying vital instructions. Over time, the record becomes scratched. If you could remove those scratches and restore the record, Sinclair and others believed, you could restore function to the genome and rejuvenate cells.
“The big question was, is there a reset button?” he told Science at the time. “Would cells know how to become younger and healthier?”
The study targeted retinal cells in mice with a harmless virus containing three Yamanaka factors in an attempt to fix a severed optic nerve. The gambit was at least partially successful in restoring function to the retinal cells.
Around the same time, a study co-published by scientists at biotech company Genentech and the Salk Institute showed that expressing the Yamanaka factors in mice in cycles over an extended period of time had no apparent negative health effects on the animals. The study, which was co-led by Heinrich Jasper, who works in immunology discovery at Genentech, also found that partially reprogrammed cells reduced age-related changes in typical, healthy mice, not just those with disease or injury.
“We did this in mice, which doesn’t tell us anything about safety in humans, by the way, but at least we’re able to express these factors systemically in the whole animal for two days a week over the course of about 10 months and the animals are fine and they display some evidence of rejuvenation or delayed aging,” Jasper tells Inverse.
“That’s really what the engineering challenge is right now, based on all the findings that have been made,” he adds. “Can we do something like this in a safe and robust fashion and restore function of cells through a measured expression of these four factors?”
If scientists can overcome that challenge, Jasper believes the technology should be used to treat age-related diseases, not to chase full-body regeneration, Doctor Who style. Jasper uses the example of idiopathic pulmonary fibrosis (IPF), a “classic age-related disease” where the lungs lose their regenerative powers and develop fibrosis.
“The fundamental promise of the four factors is that we should be able to restore the function of, for example, lung stem cells by using this approach and basically reverting them back to a healthier state,” Jasper says. “We haven’t achieved that yet and it’s going to take some time to really prove that, but if we find a way to approach this and control the expression and maybe target some of the downstream nodes, then we might have a way to develop new types of therapies for these types of disease, and that’s really the promise.”
“It’s almost easier to fix something that’s broken than it is to make something that’s good, better.”
In January of this year, the San Diego-based biotech company Rejuvenate Bio released results from another study on reprogramming effects on aging. The company injected elderly mice with a virus that activated three of the Yamanaka factors and found that the animals lived an extra 18 weeks on average, compared to nine weeks for the control group.
Noah Davidsohn, chief science officer and co-founder at Rejuvenate Bio, tells Inverse the paper was significant because it was the first to properly realize some of the potential of the Yamanaka factors. He explains that while previous research has addressed mice with diseases or damage, like progeria or severed optic nerves, this work attempted to make otherwise healthy mice live longer.
“We put it in wild type mice that are really old and are basically the same as really old humans and show that we could increase their life and health span,” he says. “So I think that sparked an ‘Oh wow, it actually does what everyone is promising it’s been doing but hasn’t been done yet.’”
“It’s almost easier to fix something that’s broken than it is to make something that’s good, better,” Davidsohn says.
These types of studies underline the potential of cell reprogramming in the fight against age-related disease, but there is still a lot of work to be done. First, the field needs to understand exactly what is happening on a molecular level when the Yamanaka factors are expressed. This can lead to better control over their expression and will speed up their potential use in humans. Reik at Alots Labs wants to know what kind of cells this kind of treatment will work for.
“Skin is nice because there are some clear changes that happen during aging in the skin that potentially could be addressed in treatment strategies. But could we do this with blood cells, can we do this with heart cells, kidney, whatever — in whichever cell types we experience age-related pathology, which is pretty much all cell types in the body? That’s an interesting question to ask,” he says.
Organoids grown from pluripotent stem cells represent a related avenue of cellular medicine.
ROGER HARRIS/SCIENCE PHOTO LIBRARY/SCIENCE PHOTO LIBRARY/GETTY IMAGES
“If you think about aging as a biological process that leads to changes in cells, and these changes actually contribute to the incidents and progression of a wide range of age-related, chronic, degenerative diseases,” Jasper says, “then you might really learn something if you’re trying to address these age-related changes and try to modulate them so you have an impact on the progression of a disease, or even on the instances of the disease.”
The promise and potential of the reprogramming field, particularly in relation to aging, has led to increased investment in the area. Jasper estimates there are around 15 to 20 labs working on using the Yamanaka factors together with aging research, and more are emerging every month. “There’s a lot of excitement about potentially what can be done there,” he says.
The Yamanaka factors may never reverse aging in humans, but they could still play an important role in the fight against the multitude of vicious diseases that come with getting older. The number of people aged 80 years or older is expected to triple by 2050, up to 426 million, according to World Health organization figures — and age-related disease will inevitably become more prevalent. As research continues at a steady pace, cellular reprogramming could hold the key to managing that tidal wave of disease looming on the horizon — even if it won’t be applied to making otherwise healthy humans cheat the march of biological time.
Induction of Pluripotent Stem Cells from Human Embryonic and Adult Fibroblast Cultures by Defined Factors
Summary
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
Introduction
Embryonic stem (ES) cells, which are derived from the inner cell mass of mammalian blastocysts, have the ability to grow indefinitely while maintaining pluripotency and the ability to differentiate into cells of all three germ layers (
). However, there are ethical difficulties regarding the use of human embryos, as well as the problem of tissue rejection following transplantation in patients. One way to circumvent these issues is the generation of pluripotent cells directly from the patients' own cells.
Somatic cells can be reprogrammed by transferring their nuclear contents into oocytes (
), indicating that unfertilized eggs and ES cells contain factors that can confer totipotency or pluripotency to somatic cells. We hypothesized that the factors that play important roles in the maintenance of ES cell identity also play pivotal roles in the induction of pluripotency in somatic cells.
), function in the maintenance of pluripotency in both early embryos and ES cells. Several genes that are frequently upregulated in tumors, such as Stat3 (
), have been shown to contribute to the long-term maintenance of the ES cell phenotype and the rapid proliferation of ES cells in culture. In addition, we have identified several other genes that are specifically expressed in ES cells (
In this study, we examined whether these factors could induce pluripotency in somatic cells. By combining four selected factors, we were able to generate pluripotent cells, which we call induced pluripotent stem (iPS) cells, directly from mouse embryonic or adult fibroblast cultures.
Results
We selected 24 genes as candidates for factors that induce pluripotency in somatic cells, based on our hypothesis that such factors also play pivotal roles in the maintenance of ES cell identity (see Table S1 in the Supplemental Data available with this article online). For β-catenin, c-Myc, and Stat3, we used active forms, S33Y-β-catenin (
To evaluate these 24 candidate genes, we developed an assay system in which the induction of the pluripotent state could be detected as resistance to G418 (Figure 1A). We inserted a βgeo cassette (a fusion of the β-galactosidase and neomycin resistance genes) into the mouse Fbx15 gene by homologous recombination (
). Although specifically expressed in mouse ES cells and early embryos, Fbx15 is dispensable for the maintenance of pluripotency and mouse development. ES cells homozygous for the βgeo knockin construct (Fbx15βgeo/βgeo) were resistant to extremely high concentrations of G418 (up to 12 mg/ml), whereas somatic cells derived from Fbx15βgeo/βgeo mice were sensitive to a normal concentration of G418 (0.3 mg/ml). We expected that even partial activation of the Fbx15 locus would result in resistance to normal concentrations of G418.
Figure 1Generation of iPS Cells from MEF Cultures via 24 Factors
). Transduced cells were then cultured on STO feeder cells in ES cell medium containing G418 (0.3 mg/ml). We did not, however, obtain drug-resistant colonies with any single factor, indicating that no single candidate gene was sufficient to activate the Fbx15 locus (Figure 1B; see also Table S2, which summarizes all of the transduction experiments in this study).
In contrast, transduction of all 24 candidates together generated 22 G418-resistant colonies (Figure 1B). Of the 12 clones for which we continued cultivating under selection, 5 clones exhibited morphology similar to ES cells, including a round shape, large nucleoli, and scant cytoplasm (Figure 1C). We repeated the experiments and observed 29 G418-resistant colonies, from which we picked 6 colonies. Four of these clones possessed ES cell-like morphology and proliferation properties (Figure 1D). The doubling time of these cells (19.4, 17.5, 18.7, and 18.6 hr) was equivalent to that of ES cells (17.0 hr). We designated these cells iPS-MEF24 for “pluripotent stem cells induced from MEFs by 24 factors.” Reverse transcription PCR (RT-PCR) analysis revealed that the iPS-MEF24 clones expressed ES cell markers, including Oct3/4, Nanog, E-Ras, Cripto, Dax1, and Zfp296 (
) (Figure 1E). Bisulfite genomic sequencing demonstrated that the promoters of Fbx15 and Nanog were demethylated in iPS cells (Figure 1F). By contrast, the Oct3/4 promoter remained methylated in these cells. These data indicate that some combination of these 24 candidate factors induced the expression of ES cell marker genes in MEF culture.
Next, to determine which of the 24 candidates were critical, we examined the effect of withdrawal of individual factors from the pool of transduced candidate genes on the formation of G418-resistant colonies (Figure 2A). We identified 10 factors (3, 4, 5, 11, 14, 15, 18, 20, 21, and 22) whose individual withdrawal from the bulk transduction pool resulted in no colony formation 10 days after transduction and fewer colonies 16 days after transduction. Combination of these 10 genes alone produced more ES cell-like colonies than transduction of all 24 genes did (Figure 2B).
We next examined the formation of colonies after withdrawal of individual factors from the 10-factor pool transduced into MEFs (Figure 2B). G418-resistant colonies did not form when either Oct3/4 (factor 14) or Klf4 (factor 20) was removed. Removal of Sox2 (factor 15) resulted in only a few G418-resistant colonies. When we removed c-Myc (factor 22), G418-resistant colonies did emerge, but these had a flatter, non-ES-cell-like morphology. Removal of the remaining factors did not significantly affect colony numbers. These results indicate that Oct3/4, Klf4, Sox2, and c-Myc play important roles in the generation of iPS cells from MEFs.
Combination of the four genes produced a number of G418-resistant colonies similar to that observed with the pool of 10 genes (Figure 2C). We continued cultivation of 12 clones for each transduction and were able to establish 4 iPS-MEF4 and 5 iPS-MEF10 clones. In addition, we could generate iPS cells (iPS-MEF4wt) with wild-type c-Myc instead of the T58A mutant (Table S2). These data demonstrate that iPS cells can be induced from MEF culture by the introduction of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4.
No combination of two factors could induce the formation of G418-resistant colonies (Figure 2C). Two combinations of three factors—Oct3/4, Sox2, and c-Myc (minus Klf4) or Klf4, Sox2, and c-Myc (minus Oct3/4)—generated a single, small colony in each case, but these could not be maintained in culture. With the combination of Oct3/4, Klf4, and Sox2 (minus c-Myc), we observed the formation of 36 G418-resistant colonies, which, however, exhibited a flat, non-ES-cell-like morphology. With the combination of Oct3/4, Klf4, and c-Myc (minus Sox2), we observed the formation of 54 G418-resistant colonies, of which we picked 6. Although all 6 clones could be maintained over several passages, the morphology of these cells (iPS-MEF3) differed from that of iPS-MEF4 and iPS-MEF10 cells, with iPS-MEF3 colonies exhibiting rough surfaces (Figure 2D). These data indicate that the combination of Oct3/4, c-Myc, and Klf4 can activate the Fbx15 locus, but the change induced by these three factors alone is different from that seen in iPS-MEF4 or iPS-MEF10 cells.
We performed RT-PCR to examine whether ES cell marker genes were expressed in iPS cells (Figure 3A). We used primers that would amplify transcripts of the endogenous gene but not transcripts of the transgene. iPS-MEF10 and iPS-MEF4 clones expressed the majority of marker genes, with the exception of Ecat1 (
). The expression of several marker genes, including Oct3/4, was higher in iPS-MEF4-7, iPS-MEF10-6, and iPS-MEF10-7 clones than in the remaining clones. Sox2 was only expressed in iPS-MEF10-6. The iPS-MEF4wt clone also expressed many of the ES cell marker genes (Figure S1). Chromatin immunoprecipitation analyses showed that the promoters of Oct3/4 and Nanog had increased acetylation of histone H3 and decreased dimethylation of lysine 9 of histone H3 (Figure 3B). CpG dinucleotides in these promoters remained partially methylated in iPS cells (Figure 3C). iPS-MEF4 and iPS-MEF10 cells were positive for alkaline phosphatase and SSEA-1 (Figure 3D) and showed high telomerase activity (Figure S2). These results demonstrate that iPS-MEF4 and iPS-MEF10 cells are similar, but not identical, to ES cells.
In iPS-MEF3 clones, Ecat1, Esg1, and Sox2 were not activated (Figure 3A). Nanog was induced, but to a lesser extent than in iPS-MEF4 and iPS-MEF10 clones. Oct3/4 was weakly activated in iPS-MEF3-3, -5, and -6 but was not activated in the remaining clones. By contrast, E-Ras and Fgf4 were activated more efficiently in iPS-MEF3 than in iPS-MEF10 or iPS-MEF4. These data confirm that iPS-MEF3 cells are substantially different from iPS-MEF10 and iPS-MEF4 cells.
We compared the global gene-expression profiles of ES cells, iPS cells, and Fbx15βgeo/βgeo MEFs using DNA microarrays (Figure 4A). In addition, we examined Fbx15βgeo/βgeo MEFs in which the four factors had been introduced without G418 selection, immortalized MEFs expressing K-RasV12, and NIH 3T3 cells transformed with H-RasV12. Pearson correlation analysis revealed that iPS cells are clustered closely with ES cells but separately from fibroblasts and their derivatives (Figure 4A). The microarray analyses identified genes that were commonly upregulated in ES cells and iPS cells, including Myb, Kit, Gdf3, and Zic3 (group I, Figure 4B and Table S3). Other genes were upregulated more efficiently in ES cells, iPS-MEF4, and iPS-MEF10 than in iPS-MEF3 clones, including Dppa3, Dppa4, Dppa5, Nanog, Sox2, Esrrb, and Rex1 (group II). Lower expression of these genes may account for the lack of pluripotency in iPS-MEF3 cells. In addition, we found genes that were upregulated more prominently in ES cells than in iPS cells, including Dnmt3a, Dnmt3b, Dnmt3l, Utf1, Tcl1, and the LIF receptor gene (group III). These data confirm that iPS cells are similar, but not identical, to ES cells.
Figure 4Global Gene-Expression Analyses by DNA Microarrays
We examined the pluripotency of iPS cells by teratoma formation (Figure 5A; Table S6 and Figure S3). We obtained tumors with 5 iPS-MEF10 clones, 3 iPS-MEF4 clones, 1 iPS-MEF4wt clone, and 6 iPS-MEF3 clones after subcutaneous injection into nude mice. Histological examination revealed that 2 iPS-MEF10 clones (3 and 6), 2 iPS-MEF4 clones (2 and 7), and the iPS-MEF4wt-4 clone differentiated into all three germ layers, including neural tissues, cartilage, and columnar epithelium. iPS-MEF10-6 could give rise to all three germ layers even after 30 passages (Table S6 and Figure S3). We confirmed differentiation into neural and muscle tissues by immunostaining (Figure 5B) and RT-PCR (Figure S4). By contrast, these teratomas did not express the trophoblast marker Cdx2 (Figure S4). iPS-MEF10-1 tumors differentiated into ectoderm and endoderm, but not mesoderm, and no signs of differentiation were observed in tumors derived from the remaining iPS-MEF10 (7 and 10) or from iPS-MEF4-10. These data demonstrate that the majority of, but not all, iPS-MEF10 and iPS-MEF4 clones exhibit pluripotency.
Figure 5Pluripotency of iPS Cells Derived from MEFs
In contrast, all tumors derived from iPS-MEF3 clones were composed entirely of undifferentiated cells (Table S6 and Figure S3). Thus, although the three factors (Oct3/4, c-Myc, and Klf4) could induce the expression of some ES cell marker genes, they were not able to induce pluripotency.
iPS-MEF10, iPS-MEF4, and iPS-MEF3 cells formed embryoid bodies in noncoated plastic dishes (Figure 5C). When grown in tissue culture dishes, the embryoid bodies from iPS-MEF10 and iPS-MEF4 cells attached to the dish bottom and initiated differentiation. After 3 days, immunostaining detected cells positive for α-smooth muscle actin (mesoderm marker), α-fetoprotein (endoderm marker), and βIII tubulin (ectoderm marker) (Figure 5D). By contrast, embryoid bodies from iPS-MEF3 cells remained undifferentiated even when cultured in gelatin-coated dishes (Figure 5C). These data confirmed pluripotency of iPS-MEF10 and iPS-MEF4 and nullipotency of iPS-MEF3 in vitro.
We next introduced the four selected factors into tail-tip fibroblasts (TTFs) of four 7-week-old male Fbx15βgeo/βgeo mice on a C57/BL6-129 hybrid background. We obtained 3 G418-resistant colonies, from each of which we could establish iPS cells (iPS-TTF4). We also introduced the four factors into TTFs from a 12-week-old female Fbx15βgeo/βgeo mouse, which also constitutively expressed green fluorescent protein (GFP) from the CAG promoter and had a C57/BL6-129-ICR hybrid background. Of the 13 G418-resistant colonies obtained, we isolated 6 clones from which we could establish iPS cells (iPS-TTFgfp4, clones 1–6). In addition, we established another iPS-TTFgfp4 (clone 7), in which the cDNA for each of the four factors was flanked with two loxP sites in the transgene. These cells were morphologically indistinguishable from ES cells (Figure 6A). RT-PCR showed that clones 3 and 7 of iPS-TTFgfp4 expressed the majority of ES cell marker genes at high levels and the others at lower levels (Figure 6B). In another attempt, we used either the T58A mutant or the wild-type c-Myc for transduction and established 5 iPS-TTFgfp4 clones (clones 8–12) and 3 iPS-TTFgfp4wt clones (clones 1–3) (Figure S5). RT-PCR showed that iPS-TTFgfp4wt cells also expressed most of the ES cell marker genes (Figure S6).
Figure 6Characterization of iPS Cells Derived from Adult Mouse Tail-Tip Fibroblasts
We transplanted 2 iPS-TTF4 and 6 iPS-TTFgfp4 clones into nude mice, all of which produced tumors containing tissues of all three germ layers (Table S6 and Figure S3). We then introduced 2 clones of iPS-TTFgfp4 cells (clones 3 and 7) into C57/BL6 blastocysts by microinjection. With iPS-TTFgfp4-3, we obtained 18 embryos at E13.5, 2 of which showed contribution of GFP-positive iPS cells (Figure 6C). Histological analyses confirmed that iPS cells contributed to all three germ layers (Figure 6D). We observed GFP-positive cells in the gonad but could not determine whether they were germ cells or somatic cells. With iPS-TTFgfp4-7, we obtained 22 embryos at E7.5, 3 of which were positive for GFP. With the 2 clones, we had 27 pups born, but none of them were chimeric mice. In addition, iPS-TTFgfp4 cells could differentiate into all three germ layers in vitro (Figure S7). These data demonstrate that the four selected factors could induce pluripotent cells from adult mouse fibroblast cultures.
We further characterized the expression of the four factors and others in iPS cells. Real-time PCR confirmed that endogenous expression of Oct3/4 and Sox2 was lower in iPS cells than in ES cells (Figure S8). However, the total amount of the four factors from the endogenous genes and the transgenes exceeded the normal expression levels in ES cells. In contrast, Western blot analyses showed that the total protein amounts of the four factors in iPS cells were comparable to those in ES cells (Figure 7A; Figure S8). We could detect Nanog and E-Ras proteins in iPS cells, but at lower levels than those in ES cells (Figures 7A and 7B; Figure S8). The p53 levels in iPS cells were lower than those in MEFs and equivalent to those in ES cells (Figure 7A; Figure S9). The p21 levels in iPS cells varied in each clone and were between those in ES cells and MEFs (Figure S9). Upon differentiation in vitro, the total mRNA expression levels of Oct3/4 and Sox2 decreased but remained much higher than in ES cells. In contrast, their protein levels decreased to comparable levels in iPS cells and ES cells (Figure 7B).
Figure 7Biochemical and Genetic Analyses of iPS Cells
Southern blot analyses showed that each iPS clone has a unique transgene integration pattern (Figure 7C). Karyotyping analyses of the iPS-TTFgfp4 (clones 1, 2, 3, 7, and 11) and iPS-TTFgfp4wt (clones 1–3) demonstrated that 2 iPS-TTFgfp4 clones and all of the iPS-TTFgfp4wt clones showed a normal karyotype of 40XX (Figure 7D), while the other 3 iPS-TTFgfp4 clones were 39XO, 40XO +10, and 40Xi(X). Analyses of PCR-based simple sequence length polymorphisms (SSLPs) demonstrated that iPS-MEF clones have a mixed background of C57/BL6 and 129 (Table S7), whereas iPS-TTFgfp clones have a mixed background of ICR, C57/BL6, and 129 (Table S8). Finally, we found that iPS cells could not remain undifferentiated when cultured in the absence of feeder cells, even with the presence of LIF (Figure 7E). These results, together with the different gene-expression patterns, exclude the possibility that iPS cells are merely contamination of preexisting ES cells. Finally, subclones of iPS cells were positive for alkaline phosphatase and could differentiate into all three germ layers in vitro (Figure S10), confirming their clonal nature.
Discussion
Oct3/4, Sox2, and Nanog have been shown to function as core transcription factors in maintaining pluripotency (
). Among the three, we found that Oct3/4 and Sox2 are essential for the generation of iPS cells. Surprisingly, Nanog is dispensable. In addition, we identified c-Myc and Klf4 as essential factors. These two tumor-related factors could not be replaced by other oncogenes including E-Ras, Tcl1, β-catenin, and Stat3 (Figures 2A and 2B).
The c-Myc protein has many downstream targets that enhance proliferation and transformation (
), many of which may have roles in the generation of iPS cells. Of note, c-Myc associates with histone acetyltransferase (HAT) complexes, including TRRAP, which is a core subunit of the TIP60 and GCN5 HAT complexes (
). We found that iPS cells showed levels of p53 protein lower than those in MEFs (Figure 7A). Thus, Klf4 might contribute to activation of Nanog and other ES cell-specific genes through p53 repression. Alternatively, Klf4 might function as an inhibitor of Myc-induced apoptosis through the repression of p53 in our system (
). The balance between c-Myc and Klf4 may be important for the generation of iPS cells.
One question that remains concerns the origin of our iPS cells. With our retroviral expression system, we estimated that only a small portion of cells expressing the four factors became iPS cells (Figure S11). The low frequency suggests that rare tissue stem/progenitor cells that coexisted in the fibroblast cultures might have given rise to the iPS cells. Indeed, multipotent stem cells have been isolated from skin (
). These studies showed that ∼0.067% of mouse skin cells are stem cells. One explanation for the low frequency of iPS cell derivation is that the four factors transform tissue stem cells. However, we found that the four factors induced iPS cells with comparably low efficiency even from bone marrow stroma, which should be more enriched in mesenchymal stem cells and other multipotent cells (Tables S2 and S6). Furthermore, cells induced by the three factors were nullipotent (Table S6 and Figure S3). DNA microarray analyses suggested that iPS-MEF4 cells and iPS-MEF3 cells have the same origin (Figure 4). These results do not favor multipotent tissue stem cells as the origin of iPS cells.
There are several other possibilities for the low frequency of iPS cell derivation. First, the levels of the four factors required for generation of pluripotent cells may have narrow ranges, and only a small portion of cells expressing all four of the factors at the right levels can acquire ES cell-like properties. Consistent with this idea, a mere 50% increase or decrease in Oct3/4 proteins induces differentiation of ES cells (
). iPS clones overexpressed the four factors when RNA levels were analyzed, but their protein levels were comparable to those in ES cells (Figures 7A and 7B; Figure S8), suggesting that the iPS clones possess a mechanism (or mechanisms) that tightly regulates the protein levels of the four factors. We speculate that high amounts of the four factors are required in the initial stage of iPS cell generation, but, once they acquire ES cell-like status, too much of the factors are detrimental for self-renewal. Only a small portion of transduced cells show such appropriate transgene expression. Second, generation of pluripotent cells may require additional chromosomal alterations, which take place spontaneously during culture or are induced by some of the four factors. Although the iPS-TTFgfp4 clones had largely normal karyotypes (Figure 7D), we cannot rule out the existence of minor chromosomal alterations. Site-specific retroviral insertion may also play a role. Southern blot analyses showed that each iPS clone has ∼20 retroviral integrations (Figure 7C). Some of these may have caused silencing or fusion with endogenous genes. Further studies will be required to determine the origin of iPS cells.
Another unsolved question is whether the four factors we identified play roles in reprogramming induced by fusion with ES cells or nuclear transfer into oocytes. Since the four factors are expressed in ES cells at high levels, it is reasonable to speculate that they are involved in the reprogramming machinery that exists in ES cells. Our result is also consistent with the finding that the reprogramming activity resides in the nucleus, but not in the cytoplasm, of ES cells (
). However, iPS cells were not identical to ES cells, as shown by the global gene-expression patterns and DNA methylation status. It is possible that we have missed additional important factors. One such candidate is ECAT1, although its forced expression in iPS cells did not consistently upregulate ES cell marker genes (Figure S12).
More obscure are the roles of the four factors, especially Klf4 and c-Myc, in the reprogramming observed in oocytes. Both Klf4 and c-Myc are dispensable for preimplantation mouse development (
). In contrast, L-myc is expressed maternally in oocytes. Klf17 and Klf7, but not Klf4, are found in expressed sequence-tag libraries derived from unfertilized mouse eggs. Klf4 and c-Myc might be compensated by these related proteins. It is highly likely that other factors are also required to induce complete reprogramming and totipotency in oocytes.
It is likely that the four factors from the transgenes are required for maintaining the iPS cells since the expression of Oct3/4 and Sox2 from the endogenous genes remained low (Figure 7B; Figure S8). We intended to prove this by using transgenes flanked by two loxP sites and obtained an iPS clone (TTF4gfp4-7). However, we noticed that these cells contain multiple loxP sites on multiple chromosomes, and, thus, the Cre-mediated recombination would cause not only deletion of the transgenes but also inter- and intrachromosomal rearrangements. Studies with conditional expression systems, such as the tetracycline-mediated system, are required to answer this question.
We showed that the iPS cells can differentiate in vitro and in vivo even with the presence of the retroviral vectors containing the four factors. We found that Oct3/4 and Sox2 proteins decreased significantly during in vitro differentiation (Figure 7B). Retroviral expression has been shown to be suppressed in ES cells and further silenced upon differentiation by epigenetic modifications, such as DNA methylation (
). The same mechanisms are likely to play roles in transgene repression in iPS cells since they express Dnmt3a, 3b, and 3l, albeit at lower levels than ES cells do (Table S5). In addition, we found that iPS cells possess a mechanism (or mechanisms) that lowers protein levels of the transgenes and Nanog (Figure 7B; Figure S8). The same mechanism may be enhanced during differentiation. However, silencing of Oct3/4 in iPS-TTFgfp4-3 cells was not complete, which may explain our inability to obtain live chimeric mice after blastocyst microinjection of iPS cells.
An unexpected finding in this study was the efficient activation of Fgf4 and Fbx15 by the combination of the three factors devoid of Sox2 since these two genes have been shown to be regulated synergistically by Oct3/4 and Sox2 (
). It is also surprising that Nanog is dispensable for induction and maintenance of iPS cells. More detailed analyses of iPS cells will enhance our understanding of transcriptional regulation in pluripotent stem cells.
Our findings may have wider applications, as we have found that transgene reporters with other ES cell marker genes, such as Nanog, can replace the Fbx15 knockin during selection (K. Okita and S.Y., unpublished data). However, we still do not know whether the four factors can generate pluripotent cells from human somatic cells. Use of c-Myc may not be suitable for clinical applications, and the process may require specific culture environments. Nevertheless, the finding is an important step in controlling pluripotency, which may eventually allow the creation of pluripotent cells directly from somatic cells of patients.
Experimental Procedures
Mice
Fbx15βgeo/βgeo mice were generated with 129SvJae-derived RF8 ES cells as described previously (
) and were backcrossed to the C57/BL6 strain for at least five generations. These mice were used for primary mouse embryonic fibroblast (MEF) and tail-tip fibroblast (TTF) preparations. To generate Fbx15βgeo/βgeo mice with constitutive expression of GFP, an Fbx15βgeo/βgeo mouse (C57/BL6-129 background) was mated with an ICR mouse with the GFP transgene driven by the constitutive CAG promoter (
). As a source of leukemia inhibitory factor (LIF), we used conditioned medium (1:10,000 dilution) from Plat-E cell cultures that had been transduced with a LIF-encoding vector. ES and iPS cells were passaged every 3 days. Plat-E packaging cells (
), which were also used to produce retroviruses, were maintained in DMEM containing 10% FBS, 50 units/50 μg/ml penicillin/streptomycin, 1 μg/ml puromycin (Sigma), and 100 μg/ml of blasticidin S (Funakoshi).
For MEF isolation, uteri isolated from 13.5-day-pregnant mice were washed with phosphate-buffered saline (PBS). The head and visceral tissues were removed from isolated embryos. The remaining bodies were washed in fresh PBS, minced using a pair of scissors, transferred into a 0.1 mM trypsin/1 mM EDTA solution (3 ml per embryo), and incubated at 37°C for 20 min. After incubation, an additional 3 ml per embryo of 0.1 mM trypsin/1 mM EDTA solution was added, and the mixture was incubated at 37°C for 20 min. After trypsinization, an equal amount of medium (6 ml per embryo DMEM containing 10% FBS) was added and pipetted up and down a few times to help with tissue dissociation. After incubation of the tissue/medium mixture for 5 min at room temperature, the supernatant was transferred into a new tube. Cells were collected by centrifugation (200 × g for 5 min at 4°C) and resuspended in fresh medium. 1 × 106 cells (passage 1) were cultured on 100 mm dishes at 37°C with 5% CO2. In this study, we used MEFs within three passages to avoid replicative senescence.
To establish TTFs, the tails from adult mice were peeled, minced into 1 cm pieces, placed on culture dishes, and incubated in MF-start medium (Toyobo) for 5 days. Cells that migrated out of the graft pieces were transferred to new plates (passage 2) and maintained in DMEM containing 10% FBS. We used TTFs at passage 3 for iPS cell induction.
) were seeded at 8 × 106 cells per 100 mm dish. On the next day, pMXs-based retroviral vectors were introduced into Plat-E cells using Fugene 6 transfection reagent (Roche) according to the manufacturer's recommendations. Twenty-seven microliters of Fugene 6 transfection reagent was diluted in 300 μl DMEM and incubated for 5 min at room temperature. Nine micrograms of plasmid DNA was added to the mixture, which was incubated for another 15 min at room temperature. After incubation, the DNA/Fugene 6 mixture was added drop by drop onto Plat-E cells. Cells were then incubated overnight at 37°C with 5% CO2.
Twenty-four hours after transduction, the medium was replaced. MEFs or TTFs were seeded at 8 × 105 cells per 100 mm dish on mitomycin C-treated STO feeders. After 24 hr, virus-containing supernatants derived from these Plat-E cultures were filtered through a 0.45 μm cellulose acetate filter (Schleicher & Schuell) and supplemented with 4 μg/ml polybrene (Nacalai Tesque). Target cells were incubated in the virus/polybrene-containing supernatants for 4 hr to overnight. After infection, the cells were replated in 10 ml fresh medium. Three days after infection, we added G418 at a final concentration of 0.3 mg/ml. Clones were selected for 2 to 3 weeks.
Plasmid Construction
To generate pMXs-gw, we introduced a Gateway cassette rfA (Invitrogen) into the EcoRI/XhoI site of the pMXs plasmid. Primers used are listed in Table S9. Mutations in β-catenin, c-myc, and Stat3 were introduced by PCR-based site-directed mutagenesis. For forced expression, we amplified the coding regions of candidate genes by RT-PCR, cloned these sequences into pDONR201 or pENTR-D-TOPO (Invitrogen), and recombined the resulting plasmids with pMXs-gw by LR reaction (Invitrogen).
Teratoma Formation and Histological Analysis
ES cells or iPS cells were suspended at 1 × 107 cells/ml in DMEM containing 10% FBS. Nude mice were anesthetized with diethyl ether. We injected 100 μl of the cell suspension (1 × 106 cells) subcutaneously into the dorsal flank. Four weeks after the injection, tumors were surgically dissected from the mice. Samples were weighed, fixed in PBS containing 4% formaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin.
Bisulfite Genomic Sequencing
Bisulfite treatment was performed using the CpGenome modification kit (Chemicon) according to the manufacturer's recommendations. PCR primers are listed in Table S9. Amplified products were cloned into pCR2.1-TOPO (Invitrogen). Ten randomly selected clones were sequenced with the M13 forward and M13 reverse primers for each gene.
Determination of Karyotypes and SSLP by PCR
Karyotypes were determined with quinacrine-Hoechst staining at the International Council for Laboratory Animal Science (ICLAS) Monitoring Center (Japan). We obtained PCR primer sequences for SSLP from the Mouse Genome Informatics website (The Jackson Laboratory, http://www.informatics.jax.org). Allele sizes were approximated on the basis of the known allele sizes in various inbred strains.
Western Blot Analyses
Western blot was performed as previously described (
), anti-p53 polyclonal antibody (FL-393, Santa Cruz), and anti-β-actin monoclonal antibody (A5441, Sigma).
RT-PCR for Marker Genes
We performed reverse transcription reactions using ReverTra Ace -α- (Toyobo) and the oligo dT20 primer. PCR was done with ExTaq (Takara). Real-time PCR was performed with Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) according to manufacturer's instructions. Signals were detected with an ABI7300 Real-Time PCR System (Applied Biosystems). Primer sequences are listed in Table S9.
DNA Microarray
Total RNA from ES cells, iPS cells, or MEFs were labeled with Cy3. Samples were hybridized to a Mouse Oligo Microarray (G4121B, Agilent) according to the manufacturer's protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent). Data were analyzed using GeneSpring GX software (Agilent).
In Vitro Differentiation of iPS Cells
Cells were harvested by trypsinization and transferred to bacterial culture dishes in the ES medium without G418 or LIF. After 3 days, aggregated cells were plated onto gelatin-coated tissue culture dishes and incubated for another 3 days. The cells were stained with anti-α-smooth muscle actin monoclonal antibody (N1584, Dako), anti-α-fetoprotein polyclonal antibody (N1501, Dako) or anti-βIII tubulin monoclonal antibody (CBL412, Abcam) along with 4′-6-diamidino-2-phenylindole (Sigma). Total RNA derived from plated embryoid bodies on day 6 was used for RT-PCR analysis.
Chromatin Immunoprecipitation Assay
We performed chromatin immunoprecipitation (ChIP) as previously described (
). Antibodies used in this experiment were anti-dimethyl K9 H3 rabbit polyclonal antibody (ab7312-100, Abcam) and anti-acetyl H3 rabbit polyclonal antibody (06-599, Upstate). PCR primers are listed in Table S9.
Statistical Analyses
Data are shown as averages and standard deviations. We used Student's t test for protein-level analyses and one-factor ANOVA with Scheffe's post hoc test for ChIP analyses. All statistical analyses were done with Excel 2003 (Microsoft) with the Statcel2 add-on (OMS).
Acknowledgments
We are grateful to Tomoko Ichisaka for preparation of mice and Mitsuyo Maeda and Yoshinobu Toda for histological analyses. We thank Megumi Kumazaki, Mirei Murakami, Masayoshi Maruyama, and Noriko Tsubooka for technical assistance; Masato Nakagawa, Keisuke Okita, and Koji Shimozaki for scientific comments; and Yumi Ohuchi for administrative assistance. We also thank Dr. Robert Farese, Jr. for RF8 ES cells and Dr. Toshio Kitamura for the Plat-E cells and pMX retroviral vectors. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.Y. This work is also supported in part by the Takeda Science Foundation, the Osaka Cancer Research Foundation, the Inamori Foundation, the Mitsubishi Pharma Research Foundation, and the Sankyo Foundation of Life Science and by a Grant-in-Aid from the Japan Medical Association to S.Y. K.T. was supported by a fellowship from the Japan Society for the Promotion of Science.
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