The Speed of the Cell Cycle Makes Aging Cells Young Again

When Shinya Yamanaka and his colleagues at the RIKEN Institute discovered a way to reprogram adult cells into embryonic stem cell-like cells, known as induced pluripotent stem cells (iPSCs), they overthrew a core understanding of cell and developmental biology; namely that once cells become committed to a particular cell fate, they irreversibly remain committed to that cell fate.

Most of the work on iPSCs has examined how to increase the efficiency and safety of this reprogramming procedure. The slowness and inefficiency of this process has frustrated stem cell scientists for some time. Even though some progress has been made at increasing the efficiency of the reprogramming process, the “nuts and bolts” of why this procedure is so slow has remained unclear.

However a recent paper from the laboratory of Shangqin Guo at the Yale School of Medicine has revealed a key component of why this procedure is so slow. That component is the speed of the cell cycle or the length of time the cell takes to divide.

Fast-growing cells have lower barriers to keeping the cell committed to a particular cell fate. Thus faster-growing cells are more easily coaxed into being reprogrammed into pluripotency (the ability to differentiate into all adult cell types).

Guo’s research team examined blood cell-forming stem cells in bone marrow. Normally these stem cells are multipotent, which means that they can differentiate into a limited number of adult cell types. The particular type of blood cells that the progeny of these stem cells differentiate into depends on the particular types of growth factors available to the cells.

Guo and others found that these fast growing bone marrow stem cells could be reprogrammed in as little as four cell divisions.  Ultrafast cell cycle is a key feature of these “privileged cells” that can be reprogrammed to efficiently.  Slower-growing stem cells could not be reprogrammed nearly as fast. Thus the length of the cell cycle seemed to be the key to the speed with which cells could be reprogrammed to iPSCs.

This study also has implications for several other applications, besides making individualized iPSCs for patients. Several human diseases are associated with abnormalities in the establishment of proper cell fates and abnormalities in the cell cycle. Therefore, Guo’s paper could provide insights into why certain genetic diseases affect cells the way they do.

An Even Better Way to Make Induced Pluripotent Stem Cells

Researchers from the Centre for Genomic Regulation in Barcelona, Spain, have discovered an even faster and more efficient way to reprogram adult cells to make induced pluripotent stem cells (iPSCs).

This new discovery decreases the time it takes to derived iPSCs from adult cells from a few weeks to a few days. It also elucidated new things about the reprogramming process for iPSCs and their potential for regenerative medical applications.

iPSCs behave similarly to embryonic stem cells, but they can be created from terminally differentiated adult cells. The problem with the earlier protocols for the derivation of iPSCs is that only a very small percentage of cells were successfully reprogrammed (0.1%-2%). Also this reprogramming process takes weeks and is a rather hit-and-miss process.

The Centre for Genomic Regulation (CRG) research team have been able to reprogram adult cells very efficiently and in a very short period of time.

“Our group was using a particular transcription factor (C/EBPalpha) to reprogram one type of blood cells into another (transdifferentiation). We have now discovered that this factor also acts as a catalyst when reprogramming adult cells into iPS,” said Thomas Graf, senior group leader at the CRG and ICREA research professor.

“The work that we’ve just published presents a detailed description of the mechanism for transforming a blood cell into an iPS. We now understand the mechanics used by the cell so we can reprogram it and make it become pluripotent again in a controlled way, successfully and in a short period of time,” said Graf.

Genetic information is compacted into the nucleus like a wadded up ball of yarn. In order to access genes for gene expression, that ball of yarn has to be unwound so that the cell can find the information it needs.

The C/EBPalpha (CCAAT/Enhancer Binding Protein alpha) protein temporarily unwinds that region of DNA that contains the genes necessary for the induction of pluripotency. Thus, when the reprogramming process begin, the right genes are activated and they enable the successful reprogramming all the cells.

“We already knew that C/EBPalpha was related to cell transdifferentiation processes. We now know its role and why it serves as a catalyst in the reprogramming,” said Bruno Di Stefano, a PhD student. “Following the process described by Yamanaka the reprogramming took weeks, had a very small success rate and, in addition, accumulated mutations and errors. If we incorporate C/EBPalpha, the same process takes only a few days, has a much higher success rate and less possibility of errors, said Di Stefano.

This discovery provides a remarkable insight into stem cell-forming molecular mechanisms, and is of great interest for those studies on the early stages of life, during embryonic development. At the same time, the work provides new clues for successfully reprogramming cells in humans and advances in regenerative medicine and its medical applications.

Forming Induced Pluripotent Stem Cells Inside a Living Organism

A team from the Spanish National Cancer Research Centre (CNIO) has become the first research team to convert adult cells that are still within a living organism into cells that show characteristics of embryonic stem cells.

The CNIO researchers also say that these embryonic stem cells, which were obtained directly from inside an organism, have a broader capacity for differentiation than those obtained by means of an in vitro culture system. Specifically, they have the characteristics of totipotent cells, a primitive state never before obtained in a laboratory, according to the CNIO team.

Manuel Serrano, Ph.D., director of CNIO’s Molecular Oncology Program and head of the Tumor Suppression Laboratory, led this study. It was supported by Manuel Manzanares, Ph.D., and his team from the Spanish National Cardiovascular Research Centre.

The CNIO researchers say their work extends that of Nobel Prize winner Shinya Yamanaka, M.D., Ph.D., one step forward. Yamanaka opened a new horizon in regenerative medicine when, in 2006, he demonstrated that stem cells could be created from adult cells by using a cocktail of genes. But while Yamanaka induced his cells in culture in the lab (in vitro), the CNIO team created theirs directly in mice (in vivo). Generating these cells within an organism brings this technology even closer to regenerative medicine, they say.

In a study published online Sept. 11 in the journal Nature, the CNIO research team details how it used genetic manipulation techniques to create mice in which Dr. Yamanaka’s four genes could be activated at will. When these genes were activated, they observed that the adult cells were able to de-differentiate into embryonic stem cells in multiple tissues and organs.

María Abad, Ph.D., lead author of the article and a researcher in Dr. Serrano’s group, said, “This change of direction in development has never been observed in nature. We have demonstrated that we can also obtain embryonic stem cells in adult organisms and not only in the laboratory.”

Dr. Serrano added, “We can now start to think about methods for inducing regeneration locally and in a transitory manner for a particular damaged tissue.” Stem cells obtained in mice also show totipotent characteristics never generated in a laboratory. Totipotent cells can form all the cell types in a body, including the placental cells. Embryonic cells within the first couple of cell divisions after fertilization are the only cells that are totipotent.

The researchers reported that they were also able to induce the formation of pseudo-embryonic structures in the thoracic and abdominal cavities of the mice. These pseudo-embryos displayed the three layers typical of embryos (ectoderm, mesoderm, and endoderm), and extra-embryonic structures such as the vitelline membrane, which surrounds the egg, and even signs of blood cell formation, which first appears in the primary embryonic vesicle (otherwise known as the “yolk sac”).

“This data tell us that our stem cells are much more versatile than Dr. Yamanaka’s in vitro inducted pluripotent stem cells, whose potency generates the different layers of the embryo but never tissues that sustain the development of a new embryo, like the placenta,” the CNIO researcher said.  Below is a figure from their paper.  The pictures look pretty convincing.

a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.
a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.

The researchers emphasize that any possible therapeutic applications of their work are still distant, but they believe that it could mean a change of direction for stem cell research, regenerative medicine and tissue engineering.

“Our stem cells also survive outside of mice in a culture, so we can also manipulate them in a laboratory,” said Dr. Abad. “The next step is studying if these new stem cells are capable of efficiently generating different tissues such as that of the pancreas, liver or kidney.”

This paper is very interesting, but I find it rather unlikely that their approach will take regenerative medicine by storm.  Engineering mice to express these four genes in an inducible manner caused the formation of unusual tumors throughout the mice.  Maybe they can be coaxed to differentiate into kidney or heart muscle or whatever, but learning how to get them to do that will take a fair amount of in vitro work.  This is interesting, but I doubt that it will change the field overnight.

A More Efficient Way to Make Human Induced Pluripotent Stem Cells

Stem cell researchers at the University of California, San Diego have designed a simple, reproducible, RNA-based method of generating human induced pluripotent stem cells (iPSCs). This new technique broad applications for the successful production of iPSCs for use in therapies and human stem cell studies.

Human iPSCs are made from adult cells by genetically engineering adult cells to overexpress four different genes (Oct4, Klf4, Sox2, and c-Myc). This overexpression drives the cells to de-differentiate into pluripotent stem cells that have many of the same characteristics as embryonic stem cells, which are made from embryos. However, because iPSCs are made from the patient’s own cells, the chances that the immune system of the patient will reject the implanted cells is low.

The problem comes with the overexpression of these four genes. Initially, retroviruses have been used to reprogram the adult cells. Unfortunately, retroviruses plop their DNA right into the genome of the host cell, and this change is permanent. If these genes get stuck in the middle of another gene, then that cell has suffered a mutation. Secondly, if these genes are stuck near another highly-expressed gene, then they too might be highly expressed, thus driving the cells to divide uncontrollably.

Several studies have shown that in order to reprogram these cells, these four genes only need to be overexpressed transiently. Therefore, laboratories have developed ways of reprogramming adult cells that do not use retroviruses. Plasmid-based systems have been used, adenovirus and Sendai virus-based systems, which do not integrate into the genome of the host cell, have also been used, and even RNA has been used (see Federico González, Stéphanie Boué & Juan Carlos Izpisúa Belmonte, Nature Reviews Genetics 12, 231-242).

The UC San Diego team led by Steven Dowdy has used Venezuelan equine virus (VEE) that they engineered to express the reprogramming genes required to make iPSCs from adult cells. Because this virus does not integrate into the host genome, and expresses RNA in the host cell only transiently, it seems to be a safe and effective way to make buckets of messenger RNA over a short period of time.

The results were impressive. The use of this souped-up VEE produced good-quality iPSCs very efficiently. Furthermore, it worked on old and young human cells, which is important, since those patients who will need regenerative medicine are more likely to be young patients than old patients. Also, changing the reprogramming factors is rather easy to do as well.

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.

Studying Tough-to-Examine Disease by Using Brain Cells Made from Stem Cells

Diseases that are hard to study, such as Alzheimer’s, schizophrenia, and autism can be examined more safely and effectively thanks to an innovative new method for making mature brain cells from reprogrammed skin cells. Gong Chen, the Verne M. William Chair in Life Sciences and professor of biology at Penn State University and the leader of the research team that designed this method said this: “The most exciting part of this research is that it offers the promise of direct disease modeling, allowing for the creation, in a Petri dish, of mature human neurons that behave a lot like neurons that grow naturally in the human brain.”

Chen’s method could lead to customized treatment for individual patients that are based on their own genetic and cellular profile. Chen explained it this way: “Obviously we do not want to remove someone’s brain to experiment on, so recreating the patient’s brain cells in a Petri dish is the next best thing for research purposes and drug screening.”

In previous work, scientists at the University of Wisconsin in James Thomson’s laboratory and in Shinya Yamanaka’s laboratory at Kyoto University in Kyoto, Japan discovered a way to reprogram adult cells into pluripotent stem cells. Such stem cells are called induced pluripotent stem cells or iPSCs. To make iPSCs, scientists infect adult cells with genetically engineered viruses that introduce four specific genes (OCT4, SOX2, KLF4 and cMYC for those who are interested). These genes encode transcription factors, which are proteins that bind to DNA or to the machinery that directly regulates gene expression.  These transcription factors turn on those genes (e.g., OCT4, NANOG, REX1, DNMT3β and SALL4, and OCT4) that induce pluripotency, which means the ability to form any adult cell type.  Once in the pluripotent state, iPSCs can be cultured and grown life embryonic stem cells and can differentiate into adult cell types and tissues.

As Chen explained, “A pluripotent stem cell is a kind of blank slate.”  Chen continued, “During development, such stem cells differentiate into many diverse specialized cell types, such as a muscle cell, a brain cell, or a blood cell.  So, after generating iPSCs from skin cells, researchers then can culture them to become brain cells, or neurons, which can be studies safely in a Petri dish.”

Chen’s team invented a protocol to differentiate iPSCs into mature human neurons much more effectively than previous protocols.  This generates cells that behave neurons in our own brains and can be used to model the individualized disease of a single patient.

In the brain, neurons rarely work alone, but instead are usually in close proximity to star-shaped cells called astrocytes.  Astrocytes are very abundant cells and they assist neuron function and mediate neuronal survival.  “Because neurons are adjacent to astrocytes in the brain, we predicted that this direct physical contact might be an integral part of neuronal growth and health,” said Chen.  To test this hypothesis, Chen and his colleagues began by culturing iPSCs-derived neural stem cells, which are stem cells that have the potential to become neurons.  These cells were cultured on top of a one-cell-thick layer of astrocytes sop that the two cell types were physically touching each other.


“We found that these neural stem cells cultured on astrocytes differentiated into mature neurons much more effectively,” Chen said.  This contrasts Chen’s method with other neural stem cells that were cultured alone in a Petri dish.  As Chen put it, the astrocytes seems to be “cheering the stem cells on, telling them what to do, and helping them to fulfill their destiny to become neurons.”

While this sounds a little cheesy, it is undeniable that the astrocyte layer increases the efficiency of neuronal differentiation of iPSCs.  Personalized medicine is moving beyond the gene level, to the level of cellular organization and tissue physiology, and iPSCs are showing the way.

Turning Adult Cells into Early Stage Neurons and Bypassing the Pluripotent Cell Stage

Researchers at the University of Wisconsin, Madison have converted skin cells from monkeys and humans into early neural stem cells that can form a wide variety of nervous system-specific cells. This reprogramming did not require converting adult cells into induced pluripotent stem cells or iPSCs. Su-Chun Zhang, professor of neuroscience and neurology at the University of Wisconsin, Madison, served as the senior author of this research. Bypassing the ultraflexible iPSC stage was the key advantage in this research, accord to Zhang.

Zhang added, “IPSC cells [sic] can generate any cell type , which could be a problem for cell-based therapy to repair damage due to disease in the nervous system.” In particular, the absence of iPSCs greatly reduces the risk of tumor formation in the recipient of the stem cell therapy.

There is a second advantage to this procedure. Namely that iPSC generation usually requires the recombinant viruses that deliver genes to the adult cells. These viruses, retroviruses, insert their genes directly into the genomes of the host cell. While there are ways are using such viruses, the use of retroviruses is definitely the most popular strategy for converting adult cells into iPSCs.

Retroviral life cycle
Retroviral life cycle

However, the procedure used in Zhang’s laboratory, utilized recombinant Sendai viruses that do not integrate their genes into the genome of the host cell, but expressed them transiently, after which, the exogenous genes are degraded.

Sendai virus
Sendai virus

Jaingfeng Lu, a postdoctoral researcher in Zhang’s lab, removed skin cells from monkeys and people, and exposed them to recombinant Sendai viruses that contained the four genes normally used to make iPSCs for 24 hours. Then Lu heated the cells to thirty-nine degrees to kill the viruses and prevent the cells from becoming iPSCs. However, 13 days later, Lu found that the cells had become induced neural progenitors or iNPs. When implanted into newborn mice, the iNPs grew normally and differentiated into neural cell types without forming any tumors.

While other researchers have managed to convert adult cells directly into neurons, Zhang admitted that he had a different goal. “our idea was to turn skin cells into neural progenitors, cells that can produce cells relating to the neural tissue. These progenitors can be propagated in large numbers.”

the research overcomes limitations of previous efforts, according to Zhang. The Sendai, which produces little more than a cold, is not a severe pathogen, does not integrate its genes into the genome of the host cell, does not cause tumors, and is considered safe, since it can be killed by heat within 24 hours. This illustrates how fevers in our bodies can kill off cold viruses. Secondly, the iNPs have a greater ability to grow in culture. Third, iNPs are far enough along in their differentiation so that they can only form nervous system-specific cell types. They cannot form muscle or live. However, the iNPs can form many more specialized cells.

Interestingly, the neurons produced from the iNPs had the characteristics of neurons normally formed in the back part of the brain, something that is potentially helpful. As Zhang noted, “For therapeutic use, it is essential to use specific types of neural progenitors. We need region-specific and function-specific neuronal types for specific neurological diseases.”

Progenitor cells grown from the skin of ALS or spinal muscular atrophy patients can be used to make a whole host of neural cells in order to model each disease and allow rapid drug screening. Such cells could also be used to treat patients with neurological disease too.

“These transplantation experiments confirmed that the reprogrammed cells indeed belong to ells of the intended brain regions and the progenitors produced the three major classes of neural cells: neurons, astrocytes, and oligodendrocytes. This proof-of-principle study highlights the possibility to generate [sic] many specialized neural progenitors for specific neurological disorders.”

Neural progenitors
Neural progenitors

Lu, Jianfeng, Liu, Huisheng, Huang, Cindy Tzu-Ling, Chen, Hong, Du, Zhongwei, Liu, Yan, Sherafat, Mohammad Amin, Zhang, Su-Chun.  Generation of Integration-free and Region-Specific Neural Progenitors from Primate Fibroblasts.  2013/05/02. Cell Reports 2211-1247.

A New Automated Protocol to Prepare and Purify Induced Pluripotent Stem Cell Lines

Induced pluripotent stem cells (iPSCs) come from adult cells and not embryos. By genetically engineering adult cells to express a cadre of genes that are normally found in early embryonic cells, scientists can de-differentiate the adult cells into cells that resemble embryonic stem cells in many (although not all) ways.

Generating iPSCs from human adult cells is tedious and not terribly efficient, but there are ways to increase the efficiency of iPSC generation (see here). Additionally, iPSCs can show a substantial tendency to form tumors, but this tendency is cell line-specific (see here and here). Furthermore, there are ways to screen iPSC lines for tumorgenicity.

Because iPSCs are directly from the patient’s cells, the chances of rejection by the immune system are less likely (see here). Therefore, many stem cells scientists believe that iPSCs may represent one of the best future possibilities for regenerative medicine. However, a hurdle in iPSC development is the ability to generate and evaluation iPSC lines in a rapid, but reliable manner. Once adult cells are induced to become iPSCs, the iPSC cultures are a mixed bag of iPSCs, undifferentiated adult cells that failed to make the transition to iPSCs, and partially reprogrammed cells. Selecting the iPSCs by merely eye-balling the cells through the microscope is tricky and fraught with errors. If the scientist wants to select iPSCs for toxicity studies and not partially differentiated cells, selecting the wrong cells for the experiment can be fatal to the experiment itself.

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute have developed a protocol for iPSC generation and evaluation is automated and efficient, and may bring us closer to the goal of using iPSCs in the clinic some day. This protocol is the culmination of three and a half years of work. This protocol uses a technology called “fluorescence activated cell sorting” or FACS to identify fully reprogrammed cells. FACS sorts the cells according to their expression of two specific cell surface molecules and the absence of another cell surface molecule. This negative selection for a cell surface molecule found in partially reprogrammed cells but not iPSCs is a very powerful technique for purifying iPSCs.

David Kahler, the NYSCF director of laboratory automation, said, “To date, this protocol has enabled our group to derive (and characterize over) 228 individual iPS cell lines, representing one of the largest collections derived in a single lab.” Kahler continued: “This standardized method means that these iPS cells can be compared to one another, an essential step for the use in drug screens and the development of cell therapies.”

This particular cell selection technique provides the basis for a new technology developed by NYSCF, the Global Stem Cell Array, which is a fully automated, robotic platform to generate cell lines in parallel.

Underway at the NYSCF Laboratory, the Array reprograms thousands of adult cells from kin and blood samples taken from healthy donors and diseased patients into iPSC lines. Sorting and characterizing cells at an early stage of reprogramming allows efficient development of iPSC clones and derivation of adult cell types.

“We are excited about the promise this protocol holds to the field. As stem cells move towards the clinic, Kahler’s work is a critical step to ensure safe, effective treatments for everyone.” said Susan L. Solomon, who is the Chief Executive Officer of NYSCF.

Decoding the Mechanisms Behind Stem Cell Reprogramming

Kenneth Zaret is the associate director of the Penn Institute for Regenerative Medicine and is also Professor of Cell and Developmental Biology at the University of Pennsylvania. Zaret’s laboratory has examined the process by which adult cells are reprogrammed to make induced pluripotent stem cells (IPSCs). Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs, and iPSCs seem to offer many of the advantages of embryonic stem without the moral messiness of destroying human embryos to make them.

The production of iPSCs required genetic engineering techniques that introduce genes into cells. Introducing four genes – Oct4, Sox2, Klf4, and c-Myc – into adult cells drives them to de-differentiate at become iPSCs, but this process is very slow – it can take up to a month – and is quite inefficient – one in 1,000 cells becomes an iPSC. Also, even though iPSCs share many characteristics with embryonic stem cells, they are not exactly the same and differ in some ways.

Oct4 in Mammalian ESC Pluripotency

See this site for this figure.

This study, which was published in the journal Cell, attempted to define the reasons why iPSC production takes so long and is so inefficient. Zaret and his group examined the genomes of adult cells, 48 hours after the introduction of the four genes, and compared them to the genomes of the starting adult cells, fully reprogrammed iPSCs, cells near the end of the re-programming process (pre-iPSCs), and embryonic stem cells.

At 48 hours, Zaret and others found that of the transcription factors that had been introduced and overexpressed in the adult cells, three of them, Oct4, Klf4, and Sox2, tended to bind to the “distal enhancer elements” of genes. The phrase “distal enhancer elements” refers to regions of genes that help control when the gene is turned on. Most genes consist of a sequence of DNA known as the “coding region” that contains the sequences that are used to make the messenger RNA that will be translated into protein. However, genes also have other sequences that tell the cell when and where to make the messenger RNA. These control sequences are called “enhancers.” The Oct4, Klf4, and Sox2 proteins bind to enhancers in target genes and influence their expression. Because these proteins do this early heavy lifting, Zaret called them “pioneer factors.”

Now, there is a problem. The DNA of the cell is not an open book, but is bound up into a compact structure called chromatin. DNA tightly wound into chromatin does not allow access to proteins. Therefore, the pioneer factors for cell reprogramming are ready to bind to enhancers, but the DNA of the genome is too tightly wound to permit their binding. What is the cell to do? This is the job of the c-Myc protein, which enhances the binding of the other pluripotency factors to chromatin.


There is another problem, however, and this is the genuinely remarkable finding of Zaret’s lab: 48 hours after the initiation of reprogramming, large sections of the genome of the cell are refractory to the binding of the pioneering factors. In Zaret’s own words: “Basically, large chunks of the human genome were physically resisting these factors from entering. That provided some understanding that you’ve got to overcome the binding requirement to get these factors to their final destination.”

What caused these chunks of the genome to be off-limits to the pioneer factors? Chromatin results from the assembly of DNA with very positively-charged proteins called histones. Histones act as miniature spools around which the DNA is wound and packaged. Chemical modification of the histones can influence the tightness of the chromatin. For example, the attachment of acetate groups tends to make chromating rather loose, and gene expression can readily occur, but the attachment of methyl (CH3-) groups tends to cinch the chromatin down so tightly that little gene expression can occur.

Histone modification

In the case of the off-limits portions of the genome in adult cells undergoing reprogramming, a histone modification called “H3K9me3,” which is a short hand for saying lysine residue number 9 on histone #3 had three methyl groups attached to it, blocks the pioneer factors from accessing the DNA under its structural compaction. However, if Zaret and his workers treated cells with an inhibitor that prevents the enzymes from modifying histones in this manner, they found that the reprogramming process was significantly accelerated.

Zaret thinks that these findings might not only tell us about the roadblocks to reprogramming, but also give us clues as to a way to work around the difficulties to reprogramming. His lab has uncovered a normal mechanism by which cells protect themselves from being reprogrammed under normal circumstances. In his own words: “We went into this thinking that we were going to learn something about the mechanism of conversion to pluripotency, but at the end of the day we ended up discovering new ways that cells control gene expression by shutting down parts of their genome.”

The importance of this work is difficult to overstate. In the words of Susan Haynes, from the National Institutes of Health General Medical Sciences division, which funded Zaret’s work, “These studies provide detailed insights into how reprogramming factors interact with the chromatin of differentiated cells and start them down the path toward becoming stem cells. Dr. Zaret’s work also identified a major structural roadblock in the chromatin that the factors must overcome in order to bind DNA. This knowledge will help improve the efficiency of reprogramming, which is important for any future therapeutic applications.”

Induced Pluripotent Stem Cell Mutations Do Not Cluster in Protein-Coding Genes

Induced pluripotent stem cells (iPSCs) are made by introducing genes into adult cells that push the adult cell into an embryonic-like state. The earliest iPSCs were made with engineered retroviruses that actually inserted their genomes into the chromosomes of the host cell. The use of such tools to form iPSCs produces cells with large segments of DNA inserted into their chromosomes, which can cause mutations. This is not a desirable trait if such are to be used in a clinical setting. Additionally, detailed genetic examinations of iPSCs have shown that the very process of reprogramming adult cells to form cells with embryonic characteristics causes mutations (Gore, et al., Nature 471 (2011): 63–67).

Thus, iPSCs tend to harbor a variety of mutations that range from base sequence changes in their DNA to changes in the number of copies of various genes. These types of mutations can make iPSCs rather dangerous to use clinically. In fact one study suggest that the mutations generated by making iPSCs can potentially illicitly activate the expression of particular genes.  These inappropriately activated genes can induce the immune system of the person who donated the adult cells from which the iPSCs ere made to attack and reject the iPSCs (Zhao, et al., Nature 474, (2011): 212–215).

One issue that has not been properly addressed to date is the status of iPSCs made by alternative methods.  The Gore paper examined 22 iPSC lines and three of them were derived by methods that do not use viruses that insert themselves into the genome of the host cell.  Their data suggested that these iPSC lines also had higher numbers of mutations than the cells from which they were derived, but the tables in the Gore paper tend to show that the mRNA-derived iPSCs had lower numbers of mutations than those derived from more traditional means. Another issue is that the human genome has a tremendous amount of empty space.  Mutations that do not occur within the coding region of a gene is likely to not cause a problem.

Into the fray comes a paper from the laboratory of Linzhao Cheng at the Johns Hopkins Institute for Cell Engineering.  Cheng and his co-workers made iPSCs from bone marrow stem cells and discovered that while they possessed more mutations than the cells from which they were made, those mutations were typically not in genes that will affect the function of the cells. Cheng and his colleagues also used techniques to make iPSCs that did not utilize viruses that insert into the genomes of the host cell.

Cheng’s group took the bone marrow-derived iPSCs and differentiated them into mesenchymal stem cells, and then sequenced their genomes to determine the new mutations that were caused by the reprogramming.  They discovered that there were 1,000 to 1,800 new mutations in each cell line, but the mutations rarely occurred in protein coding regions.

On the average, each iPSC had six mutations that occurred in coding regions, but each mesenchymal stem cell made from the iPSC lines had about 12 mutations per cell in coding regions.  While this sounds awful, we must remember that some mutations are very consequential for the proteins that are encoded by genes, but many are not.  For example, the sickle-cell disease is due to one mutation in the hemoglobin gene that causes the hemoglobin protein to form chains that deform the red blood cell.  However, there are many other mutations in the hemoglobin gene that do not affect its function in the least.

When Cheng and his colleagues examined where the mutations occurred, they found that none of the mutations in protein coding regions that they had detected were in genes that would cause the iPSCs to grow uncontrollably or predispose the cells to form cancerous tumors.

Based on his findings, Cheng thinks that iPSCs form a smaller risk than was previously thought.  His results are published in Cell Stem Cell.

University of Georgia Lab Generates Blueprint for Stem Cell Responses to Signaling Molecules

What makes a stem cell a stem cell? This is not a trivial question, but an answer to this question is essential in order to understand how to make adult cells stem cells and how to find, and manipulate other stem cells in the body to amplify their healing properties.

Fortunately a great deal of work has been done in this area – genes expressed by stem cells under particular conditions. However, data from different labs tends to conflict with each other. What is a stem cell scientist to do?

From this morass of cacophony comes a very satisfying study from the University of Georgia at Athens, GA. This study, which comes from the laboratory of Stephen Dalton, professor of cellular biology, has generated a wiring diagram of sorts that describes how stem cells respond to external signaling molecules. In one paper, Dalton and his band of intrepid scientists have managed to reconcile several conflicting observations from many different labs.

This paper, which appeared in the March 2 edition of the journal Cell Stem Cell, can potentially provide stem cell scientists with the ability to control precisely the differentiation of particular stem cells into specific cell types. Dalton offered this assessment of his publication: ‘We can use the information from this study as an instruction book to control the behavior of stem cells. “We’ll be able to allow them to differentiate into therapeutic cell types much more efficiently and in a far more controlled manner.”

Many researchers have tended to view signaling in stem cells in an atomistic way. In other words, a single type of signaling molecule sets in motion a specific signal transduction pathway that culminates in maintaining or changing the fate of the stem cell. This, however, appears to be far too simplistic. In the Dalton paper, evidence is presented that several signaling molecules work together in complex ways to control a variety of molecular switches that specified is a stem cell continues to divide and renew itself, or becomes a specific cell type, such as a neuron, heart muscle or skin cell.

To paint of picture of our understanding of stem cell signaling before the publication of the Dalton paper, let us take the “Wnt” signaling molecule as an example. Approximately half the published studies presented evidence that Wnt signaling molecules drove stem cells to renew themselves and not differentiate, but remain in the naïve development state. For example:
1. Cai C, Zhu X. The Wnt/β-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol Med Report. 2012 Feb 21. doi: 10.3892/mmr.2012.802.
2. Miki T, Yasuda SY, Kahn M. Wnt/β-catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming. Stem Cell Rev. 2011 Nov;7(4):836-46.
3. Bisson I, Prowse DM. WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics. Cell Res. 2009 Jun;19(6):683-97.
4. Shimizu T, Kagawa T, Inoue T, Nonaka A, Takada S, Aburatani H, Taga T. Stabilized beta-catenin functions through TCF/LEF proteins and the Notch/RBP-Jkappa complex to promote proliferation and suppress differentiation of neural precursor cells. Mol Cell Biol. 2008 Dec;28(24):7427-41.

However, several other papers argued just the opposite. Instead Wnt drove stem cells to differentiate and not stay in the developmentally naïve state:
1. Davidson KC, et al., Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A. 2012 Mar 5.
2. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol. 2008 Sep 10;291(1-2):116-24.
3. Munji RN, Choe Y, Li G, Siegenthaler JA, Pleasure SJ. Wnt signaling regulates neuronal differentiation of cortical intermediate progenitors. J Neurosci. 2011 Feb 2;31(5):1676-87.
4. Kirton JP, Crofts NJ, George SJ, Brennan K, Canfield AE. Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease? Circ Res. 2007 Sep 14;101(6):581-9

Could Wnt molecules drive cells to do both differentiate and remain in the naive state? According to Dalton the answer is yes and there is a simple reason why. Dalton’s research team showed that at low concentrations, Wnt signaling keeps the stem cell in its naive, developmental, pluripotent state. However, at higher concentrations, Wnt signaling does just the opposite and drives the stem cell to stop dividing and differentiate.

However, we must avoid viewing Wnt signaling in a linear fashion because Wnt does not work alone. Other signaling molecules, such as fibroblast growth factor (FGF2), Activin A, and insulin-like growth factor (IGF), work with Wnt to modify stem cell behavior. If that doesn’t make things complicated enough, these signaling pathways can amplify or inhibit each other to cause what would be a two-fold increase under one set of conditions to become a 10-fold increase under another distinct set of conditions. The timing of cell signaling (when the cells are given the signaling molecule) also plays a crucial role with respect to the outcome.

Dalton remarked on his findings: “One of the things that surprised us was how all of the pathways ‘talk’ to each other. You can’t do anything to the IGF pathway without affecting the FGF2 pathway, and you can’t do anything to FGF2 without affecting Wnt. It’s like a house of cards; everything is totally interconnected.”

In another example, when activated, the PI3K/Akt signaling pathway maintains stem cell self-renewal, and it does so by inhibiting Raf/Mek/Erk and Wnt signal transduction pathways. The PI3K/Akt pathway also drives another signal transduction pathways called the “Activin A/Smad2,3” pathway to promote self-renewal, and this is mediated by stimulating the expression of a gene long known to be essential for stem cell self-renewal called Nanog. However, at low levels of PI3K/Akt signaling, the Wnt pathway is activated an, in combination with the Smad2,3 pathway, promotes differentiation.

Why is it that the Smad2,3 signaling proteins promote stem cell self-renewal and differentiation? When PI3K/Akt signaling decreases, the Wnt signal transduction pathway teams up with the Raf/Mek/Erk signal transduction pathway, which was suppressed by PI3K/Akt. Together, these two pathways target the protein kinase Gsk3β, which drives cells to differentiate. Thus, the signal to self-renew or differentiate revolves around Smad2,3 and the state of this signaling pathway determines if the stem cell differentiates of continues in its naïve developmental state, self-renewing with abandon.

This paper is the result of five years of generating hypotheses, testing them, and then revising the hypotheses in light of new data. This painstaking process was continued until the discrepancies were properly resolved. Fortunately, these data can provide scientists with a better grasp of that first step that stem cells might take as they differentiate. Furthermore, Dalton is quite confident that the same approach can be used to dissect and elucidate the molecular events that underlie other developmental steps that occur as the cells in an embryo divide and differentiate into more specific cell types.

Dalton sounded a hopeful note: “Hopefully this type of approach will give us a greater understanding of cells and how they can be manipulated so that we can progress much more rapidly toward the routine use of stem cells in therapeutic settings.” Dalton said.

Marion Zatz, who is chief of the Developmental and Cellular Processes Branch in the Division of Genetics and Developmental Biology at the National Institutes of Health (NIH), oversees stem cell biology grants awarded by the NIH (which partially supported Dalton’s work). Zatz made this comment about Dalton’s paper: “This work addresses one of the biggest challenges in stem cell research—figuring out how to direct a stem cell toward becoming a specific cell type. In this paper, Dr. Dalton puts together several pieces of the puzzle and offers a model for understanding how multiple signaling pathways coordinate to steer a stem cell toward differentiating into a particular type of cell. This framework ultimately should not only advance a fundamental understanding of embryonic development, but facilitate the use of stem cells in regenerative medicine.”

Dalton’s paper is truly a remarkable achievement that will allow a deeper and more accurate understanding of stem cell biology and development.