Synthetic version of Oct4 robustly supports induced pluripotent stem cell formation


A recent paper by members of the Danish Stem Cell Centre in Copenhagen, the MRC Centre for Regenerative Medicine in Edinburgh, and Memorial Sloan Kettering Cancer Center in New York has used a synthetic version of the Oct4 protein to dissect the precise role of this protein in stem cells, and to more effectively generate induced pluripotent stem cells.
Oct4 is a member of the “class V POU” transcription factors. POU stands for Pit-Oct-Unc, which are the founding members of this group of transcription factors. Transcription factors are proteins that bind to specific sequences of DNA and turn on gene expression. The POU family of transcription factors was originally defined on the basis of a common region of ~150–160 amino acids that was identified in the transcription factors Pit-1, Oct-1, and Oct-2, which were known from mammals, and the nematode factor Unc-86. This common POU protein domain is the DNA binding region that consists of two subdomains joined by a common linker.

Embryonic development in mammals is controlled by regulatory genes, many of which regulate the expression of other genes. These regulators activate or repress patterns of gene expression that mediate the changes characteristic of development. Oct4, like other members of the POU family of transcription factors, activates the expression of their target genes by binding an eight-base sequence motif that usually has some similarity to this sequence: AGTCAAAT. During embryonic development, Oct4 is expressed initially in all the cells of the embryo, but eventually becomes restricted to the Inner Cell Mass (ICM) and Oct4 expression fades in the outer cells (known collectively as the trophectoderm). At maturity, Oct4 expression is confined exclusively to the developing germ cells. Disruption of Oct4 in mice produces embryos without a pluripotent ICM. This suggests that Oct4 is required for maintaining pluripotency.

Given the importance of Oct4 in early development, it is no surprise that it plays an important role in embryonic stem cell maintenance. Oct4 also plays an essential role reprogramming adult cells from their mature state to the embryonic state. In the absence of Oct4, embryonic stem cells differentiate. Oct4 plays a powerful role in regulating stem cell genes. However, while large quantities of Oct4 are needed, too much of it can hamstring the properties of stem cells.

Given these data, does Oct4 maintain pluripotency by activating the expression of particular genes or by repressing those genes necessary for differentiation? These scientists, whose work is published in the journal Cell Reports, made fusions of Oct4 with proteins that are known to activate gene expression or fusions with proteins known to repress gene expression. Then they accessed the ability of these fused versions of Oct4 to support pluripotency in embryonic stem cells or induce pluripotency in adult cells.

The synthetic version of Oct4 fused to known activator of gene expression were much more efficient in turning on genes that instruct cells how to be stem cells.  Cells also did not require as much of this synthetic Oct4; stem cells required less of the synthetic Oct4 to remain stem cells and adult cells required less to become reprogrammed as stem cells.  Those synthetic versions of Oct4 that were fused to known transcriptional repressors caused cells to differentiate, and such synthetic versions of Oct4 could not replace endogenous Oct4 in stem cells.

Further tests with the activating synthetic Oct4 showed that it could support stem cells under conditions that are usually not conducive to their growth.  This provides a way to generate stem cells in the laboratory when growth conditions are less than optimal.  Because the activator version of synthetic Oct4 could replace endogenous Oct4 and not the repressor version of synthetic Oct4, Oct4 must work primarily as an activator of gene expression rather than a repressor of gene expression.

Professor Joshua Brickman, who is affiliated with The Danish Stem Cell Center (DanStem), University of Copenhagen and Medical Research Council Centre for Regenerative Medicine at the University of Edinburgh. said “Our discovery is an important step towards generating and maintaining stem cells much more effectively.  Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. But to be able to use them medically, we need to be able to maintain them in a pure state, until they’re needed. When we want to turn a stem cell into a specific cell, such as insulin producing beta cell, or a nerve cell in the brain, we’d like this process to occur accurately and efficiently. This will not be possible if we don’t understand how to maintain stem cells as stem cells. As well as maintaining embryonic stem cells in their pure state more effectively, the artificially created Oct4 was also more effective at reprogramming adult cells into so-called induced Pluripotent Stem cells (iPSCs), which have many of the same traits and characteristics as embryonic stem cells but can derived from the patients to both help study degenerative disease and eventually treat them..”

Exploitation of such technology could improve the efficiency of protocols to generate iPSCs in the laboratory and the clinic.  Such cells could be used to produce individualized cells for developing individualized therapies for degenerative diseases such as type 1 diabetes and neuro-degenerative diseases.

Skin Cells from Alzheimer’s Disease Were Turned into Cultured Neurons Using iPSC Technology


Scientists from the University of California, San Diego School of Medicine, have created stem cell-derived, in vitro models of sporadic and hereditary Alzheimer’s, using induced pluripotent stem cells from patients with the neurodegenerative disorder. This experiment provides the ability to study the precise abnormalities present in neurons that cause the pathology of this neurodegenerative disease.

Senior study author Lawrence Goldstein, PhD, professor in the Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute investigator and director of the UC San Diego Stem Cell Program, noted that the production of highly purified, functional human Alzheimer’s neurons in culture has never been done before. Goldstein said: “It’s a first step. These aren’t perfect models. They’re proof of concept. But now we know how to make them.”

This experiment represents a new method for studying the causes of Alzheimer’s disease. These living cells provide a tool for developing and testing drugs to treat the disorder. According to Goldstein, “We’re dealing with the human brain. You can’t just do a biopsy on living patients. Instead, researchers have had to work around, mimicking some aspects of the disease in non-neuronal human cells or using limited animal models. Neither approach is really satisfactory.”

Goldstein and colleagues extracted skin cells called fibroblasts from skin tissues from two patients with familial Alzheimer’s disease. They also used fibroblasts from two patients with sporadic Alzheimer’s disease, and two persons with no known neurological problems. They reprogrammed the fibroblasts into induced pluripotent stem cells (iPSCs) that then differentiated them into working neurons. These iPSC-derived neurons from the Alzheimer’s patients exhibited normal electrophysiological activity, formed functional synaptic contacts and displayed tell-tale indicators of Alzheimer’s disease. Also, they possessed higher-than-normal levels of proteins associated with Alzheimer’s disease.

With cultured neurons from Alzheimer’s patients, scientists can more deeply investigate how Alzheimer’s disease begins and chart the biochemical processes that eventually destroy brain cells and produce degeneration of elemental cognitive functions like memory. Currently, Alzheimer’s research depends heavily upon autopsies performed after the patient has died and the damage has been done. Goldstein added, “The differences between a healthy neuron and an Alzheimer’s neuron are subtle. It basically comes down to low-level mischief accumulating over a very long time, with catastrophic results.”

Neurons derived from one of the two patients with sporadic Alzheimer’s disease showed biochemical changes possibly linked to the disease. Thus there may be sub-categories of the disorder and, in the future, potential therapies might be targeted to specific groups of Alzheimer’s patients.

French Lab Finds Genetic Abnormalities in Embryonic Stem Cell-Derived Neuronal Derivatives


Human pluripotent stem cells represent a tremendous potential for human treatment, but the mutations introduced into these cells during their derivation renders the safety of these cells questionable. Some French researchers have even generated some cautionary data that suggests that additional quality controls are needed to ensure that neural derivatives of human pluripotent stem cells are not genetically unstable. Such cells are currently being tested in clinical trials, and there is a need to ensure that they are genetically sound.

Human stem cells capable of giving rise to any fetal or adult cell type are known as pluripotent stem cells. It is hoped that such cells, the most well-known being human embryonic stem cells (hESCs), can be used to generate cell populations that can be used in therapeutic regiments. Presently, neural derivatives of embryonic stem cells are being tested in clinical trials.

Nathalie Lefort and colleagues at the Institute for Stem cell Therapy and Exploration of Monogenic Diseases (France) have shown that neural derivatives of human embryonic stem cells frequently acquire extra material from the long arm of chromosome 1 (1q). This particular chromosomal defect is sometimes seen in some blood cell cancers and pediatric brain tumors that have a rather poor clinical prognosis. Fortunately, when Lefort and her colleagues implanted these abnormal neural cells into mice, they were unable to form tumors in mice.

Neil Harrison of the University of Sheffield (U.K.) has commented on Lefort’s work in an accompanying article that these data raise safety issues relevant for the therapeutic use of embryonic stem cell derivatives. The fact that the same chromosome was affected in all cases suggests that it should be possible to design a screen that can effectively detect and remove genetically abnormal cells.

Stanford study finds Induced pluripotent stem cells match embryonic stem cells in modeling human disease


Investigators from Stanford University School of Medicine have shown that induced Pluripotent Stem cells (iPSCs), which are made from adult cells through genetic engineering techniques, are a possible alternative to human embryonic stem cells when it comes to modeling those defects caused by a particular genetic condition. The example used in this study was Marfan syndrome, and in this study, iPSCs modeled the disease as well as embryonic stem cells (ESCs). Thus, iPSCs could be used to examine the molecular aspects of Marfan on a personalized basis. Embryonic stem cells, on the other hand, can’t do this because their genetic contents are those of the donated embryo are not the same as the patient’s.

Marfan syndrome is an inherited connective-tissue disorder that occurs in one in 10,000 to one in 20,000 individuals. It results from a large number of defects in one gene called “fibrillarin.” People with Marfan syndrome tend to be very tall and thin, and also tend to suffer from osteopenia, or poor bone mineralization. Medical experts have speculated that Abraham Lincoln, for example, suffered from this disorder. Marfan can also profoundly affect the eyes and cardiovascular system.

This proof-of-principle study, with regards to the utility of iPSCs also has more universal significance; it advances the credibility of using iPSCs to model a broad range of human diseases. iPSCs, unlike ESCs, are easily obtained from virtually anyone and possess a genetic background identical to the patient from which they were derived. Moreover, they carry none of the ethical controversy associated with the necessity of destroying embryos.

“Our in vitro findings strongly point to the underlying mechanisms that may explain the clinical manifestations of Marfan syndrome,” said Michael Longaker, MD, professor of surgery and senior author of the study, which will be published online Dec. 12 in Proceedings of the National Academy of Sciences. Longaker is the Dean P. and Louise Mitchell Professor in the School of Medicine and co-director of the school’s Institute for Stem Cell Biology and Regenerative Medicine. The study’s first author is Natalina Quarto, PhD, a senior research scientist in Longaker’s laboratory.

In this study, both iPSCs and ESCs, and embryonic stem cells that carried a mutation that causes Marfan syndrome showed impaired ability to form bone, and all too readily formed cartilage. These aberrations mirror the most prominent clinical manifestation of the disease.

iPSCs were discovered in 2006, and are derived from fully differentiated tissues such as the skin. However, they harbor the same capacity as embryonic stem cells; namely to differentiate into all the tissues of the body, and replicate for indefinite periods in a cell culture dish. Because iPSCs offer an ethically uncomplicated alternative to ESCs, IPSCs have fueled the hope that they can replace ESCs in scientists’ efforts to analyze, in a dish, those cellular defects ultimately responsible for diseases ranging from diabetes to Parkinson’s and even such complex conditions as cardiovascular disease and autism.

One hope for iPSCs is to be able to differentiate them in a dish into tissues of interest and then study these cells and their characteristics. This would help scientists better understand diseases in a patient-specific way, which would be impossible to do with ESCs unless ESCs were made from donated human eggs that were modified by cloning procedures. Cloning human embryos to the blastocyst stage has yet to occur, which makes this option technically impossible at the present time.

While scientists want to us iPSCs to develop therapeutic applications for regenerative medicine. This strategy, however, is technically more difficult, since scientists will have to develop the capacity first to repair genetic defects within cells before they can be used for regenerative medicine. iPSCs in theory might be a better bet because they are derived from patients’ own cells and, therefore, are less likely to provoke graft rejection than similar tissues produced using a donor embryo’s ESCs.

Unfortunately, several studies have reported subtle differences between iPSCs and ESCs, and these differences imply that the two cell types may not be equivalent. Stem cell experts have wondered whether these differences may render iPSCs inadequate substitutes for ESCs in modeling disease states, but this Stanford study suggests otherwise.