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.

Astrocytes
Astrocytes

“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. http://linkinghub.elsevier.com/retrieve/pii/S221112471300171X