Preventing Rejection of Embryonic Stem Cell-Based Tissues


Embryonic stem cells (ESCs) are derived from human embryos. Because they are pluripotent, or have the capacity to make any adult cell type, ESCs are thought to hold great promise for cell therapy as a source of differentiated cell types.

One main drawback to the use of ESCs in regenerative medicine is the rejection of ESC-derived cells by the immune system of the patient. Transplantation of ESC-derived tissues would require the patient to take powerful anti-rejection drugs, which tend to have a boatload of severe side effects.

However, a paper reports a strategy to circumvent rejection of ESC-derived cells. If these strategies prove workable, then they might clear the way to the use of ESCs in regenerative medicine.

The first paper comes from the journal Cell Stem Cell, by Zhili Rong, and others (Volume 14, Issue 1, 121-130, 2 January 2014). In this paper, Rong and his colleagues from the laboratory of Yang Xu at UC San Diego and their Chinese collaborators used mice whose immune systems had been reconstituted with a functional human immune system. These humanized mice mount a robust immune response against ESCs and any cells derived from ESCs.

In their next few experiments, Xu and others genetically engineered human ESCs to routinely express two proteins called CTLA4-Ig and PD-L1. Now this gets a little complicated, but stay with me. The protein known as CTLA4-Ig monkeys with particular cells of the immune system called T cells, and prevents those T cells from mounting an immune response against the cells that display this protein on their surfaces. The second protein, PD-L1, also targets T cells and when T cells bind to cells that have this protein on their surfaces, they are completely prevented from acting.

CTLA-4 mechanism

Think of it this way: T cells are the “detectives” of the immune system. When they find something fishy in the body (immunologically speaking), they get on their “cell phones” and call in the cavalry. However, when these detectives come upon these cells, their cell phones are inactivated, and their memories are wiped. The detectives wander away and then do not remember that they ever came across these cells.

Further experiments showed that any derivatives of these engineered ESCs, (teratomas, fibroblasts, and heart muscle cells) were completely tolerated by the immune system of these humanized mice.

This is a remarkable paper. However, I have a few questions. Genetic engineering of these cells might be potentially dangerous, depending upon how it was done, where in the genome the introduced genes insert, and how they are expressed. Secondly, if cells experience any mutations during the expansion of these cells, these mutations might cause the cells to be detected by the immune system. Third, do these types of immune repression last long-term? Clearly more work will need to be done, but these questions are potentially addressable.

My final concern is that if this procedure is used widespread, it might lead to the wholesale destruction of human embryos. Human embryos, however, are the youngest, weakest, and most vulnerable among us. What does that say about us if we do not value the weakest among us and dismember them for their cells? Would we allow this with toddlers?

Thus my interest and admiration for this paper is tempered by my concerns for human embryos.

Cardiac Muscle Repair with Molecular Beacons


Pure heart muscle cells that are ready for transplantation. This is one of the Holy Grails of regenerative medicine. Of course when working with pluripotent stem cell lines, isolating nothing but beating heart muscle cells is rather difficult. A new technique makes the isolation of pure cultures of beating heart muscle cells that much easier.

Researchers at Emory and Georgia Tech have developed a method that utilizes molecules called “molecular beacons” to isolate heart muscle cells from pluripotent stem cells. Molecular beacons fluoresce when they come into contact with cells that express certain genes. In this case, the beacons target cells that express heart-specific myosin.

Physicians can use these purified cardiac muscle cells to heal damaged areas of the heart in patient that have suffered a heart attack or are suffering heart failure. This molecular beacon technique might also have applications in other fields of regenerative medicine as well.

“Often, we want to generate a particular cell population from stem cells for introduction into patients,” said Young-sup Yoon, professor of medicine and director for stem cell biology at Emory University School of Medicine. “But the desired cells often lack a readily accessible surface marker, or that marker is not specific enough, as is the case for cardiac muscle cells. This technique could allow us to purify almost any type of cell.”

Gang Bao pioneered he use of molecular beacons and was a co-author of this publication. Yoon and is colleagues and collaborators grew mouse and human embryonic stem cells and induced pluripotent stem cells and differentiated them into heart muscle cells (cardiomyocytes). They then used molecular beacons to label only those cells that expressed messenger RNAs with just the right sequences. These molecular beacons hybridized with the mRNAs and fluoresced. Bao and others then used flow cytometry to sort the fluorescent cells from the non- fluorescent cells. The fluorescent cells have differentiated into heart muscle cells and were isolated from all the other cells.

These purified heart muscle cells could engraft into the heart of a mouse that had suffered a heart attack and they improved heart function and formed no tumors. This proof-of-principle experiment shows that this technique is feasible.

“In previous experiments with injected bare cells, investigators at Emory and elsewhere found that a large proportion of the cells are washed away. We need to engineer the cells into compatible biomaterials to enhance engraftment and retention,” said Yoon,

Human Embryonic Stem Cell Communication Network Discovered


Cells use a variety of mechanisms to talk to each other. These signaling pathways are called “signal transduction” pathways, and they vary extensively from one cell type to another.

Therefore, it should be no surprise that human embryonic stem cells signal to each other. The precise signal transduction pathway that human embryonic stem cells use to communicate with each other is the subject of a research project from a laboratory in Singapore.

Human embryonic stem cells or hESCs can differentiate into any adult cell type. The factors that keep hESCs in their pluripotent state are of interest to stem cell scientists because they might allow them to better direct the differentiation of hESCs or even grow them in culture better.

Cell-to-cell communication is vitally important to multicellular organisms. The coordinated development of tissues in the embryo that culminate in the formation of specific organs requires that cells receive signals and respond accordingly. If there are errors in these signals, the cells will respond differently and the embryo will either be grossly abnormal, or the cell might divide uncontrollably to make a tumor.

Human ESCs communicate by means of a signal transduction pathway known as the extracellular regulated kinase or ERK pathway.  The ERK signal transduction pathway begins with the binding of a growth factor receptor by a growth factor.  These growth factors are almost always bound to the extracellular matrix, which is the goo that surrounds cells and provides a structure in which the cells live.  The binding of the receptor causes the receptor to pair with another copy of itself, and that activates the bits of the receptor found inside the cell (tyrosine kinase domain for the interested).  The activated receptor attaches phosphates to itself, which causes particular proteins to find and bind the receptor, which recruits particular proteins to the cell membrane.  One of the recruited proteins is a protein kinase called RAF.  RAF attaches phosphate groups to the protein kinase MEK, and MEK attaches phosphate groups to the protein kinase ERK.  Once ERK has a phosphate attached to it, it can move into the nucleus and regulate transcription factors involved in the control of gene expression.  Thus a phenomenon that began at the cell membrane culminates in a change in gene expression.

ERK_pathway

Stem cell scientists a A*STAR’s Genomic Institute of Singapore and the Max Planck Institute of Molecular Genetics (MPIMG) in Berlin, Germany studied how genetic information is accessed in hESCs. To do this they mapped the kinase interactions across the entire human genome (kinases are enzymes that attach phosphate groups to other molecules) and discovered that ERK2, a protein that belongs to the ERK signal transduction pathway targets important sites such as non-coding genes, and histones, cell cycle, metabolism, and stem cell-specific genes.

The ERK signaling pathway involves an additional protein called ELK1 that interacts with ERK2. However, this research team discovered that ELK1 has a second, totally opposite function. At genomic sites not targeted by ERK signaling, ELK1 silences genetic information, which keeps the cell in its undifferentiated state.

ELK1 Interaction with ERK2

The authors propose a model that integrates this bi-directional control to keep the cell in the stem cell state, in which genes necessary for differentiation are repressed by ELK1 that is not associated with ERK2, and cell-cycle, translation and other pluripotency genes are activated by ELK1 in association with ERK2 or ERK2 plus other transcription factors.

Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.
Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.

First author Jonathan Göke from Stem Cell and Developmental Biology at the GIS said, “The ERK signaling pathway has been known for many years, but this is the first time we are able to see the full spectrum of the response in the genome of stem cells. We have found many biological processes that are associated with this signaling pathway, but we also found new and unexpected patterns such as this dual-mode of ELK1. It will be interesting to see how this communication network changes in other cells, tissues, or in disease.”

A co-author of this study, Martin Vingron said, “A remarkable feature of this study is, how information was extracted by computational means from the data.”

Professor Ng Huck Hui, managing author of this paper, added, “This is an important study because it describes the cell’s signaling network and its integration into the general regulatory network. Understanding the biology of embryonic stem cells is a first step to understanding the capabilities and caveats of stem cells in future medical applications.”

Exporting Tissue Engineering


Professor György K.B. Sándor from the Finland Distinguished Professor Program or FiDiPro believes that tissue engineering has the ability to become a new global export item.

FiDiPro is a joint funding program of the Academy of Finland and Tekes (the Finnish Funding Agency for Technology and Innovation) that enables top researchers to do work in Finland for a fixed period of time.

Sándor is a Canadian professor who specializes in oral and maxillofacial surgery who has participated in FiDIPro. Sandor’s research program examines bone regeneration, hyperbaric oxygen, tissue regeneration, and stem cells. He works at the BioMediTech research institute which is run by the University of Tampere and the Tampere University of Technology. BioMediTech is an innovation center that combines biomedical research with new technologies.

The goal of Sándor’s research program is to produce bone and cartilage by means of tissue engineering techniques that grow tissue-derived stem cells. Some people are missing bone at birth as a result of a developmental disorder, or, in some cases, bone defects from accidents, and various inflammatory diseases can cause bone loss. Particular surgeries that require bone removal can also cause bone loss,

Tissue engineering can produce tailored, living spare parts for human beings. If the protocols and methods of tissue engineering can be up-scaled appropriately, it could become the third alternative form of treatment alongside more traditional forms of treatment for such conditions that include surgery and drug treatments.

“Tissue-derived stem cells can be isolated from the patient’s own tissue. In that way, they will not cause a rejection reaction. Compared to tissue stem cells, human embryonic stem cells have a greater ability to differentiate into different cell types, In practice, that means that all cell types can be used,” Sándor said.

Sandor noted that Finland is a forerunner in developing bone engineering techniques. “At the moment expertise in the field is concentrated in Finland, but it also generated global interest in other medically advanced countries,” said Sándor .

In the near future, large numbers of patients might travel to Finland to receive tissue engineering-based treatments. As such forms of treatment increase and are perfected, expertise in tissue engineering can be exported for use on a larger scale.

“We have proven with more than 20 clinically successful operations that tissue engineering works,” Sándor said.

Sándor considers the research community in Tampere to be unique when it comes to the way it is run and functions. One of the key reasons why Sandor decided to stay and continue his research in Finland even after his experience with FiDiPro came to an end.

“In the field, BioMediTech is a unique concentration of researchers and expertise. In the Pirkanmaa region, also the cooperation between research, industry, and administration works well. That enables efficient decision-making which, in turn, contributes to the creation of new innovations,” he said.

“Cooperation with colleagues is smooth too. That was the determining factor in my decision to stay in Finland. Each day is like a new adventure.”

Tests to Improve Stem Cell Safety


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

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

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

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

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

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

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

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

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

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

An Easy Way to Make Retinal Pigment Epithelium from Pluripotent Stem Cells


Age-related macular degeneration is the leading cause of irreversible vision loss and blindness among the aged in industrialized countries. One of the earliest events associated with age-related macular degeneration (AMD) is damage to the retinal pigmented epithelium (RPE), which lies just behind the photoreceptor cells in the retinal. The RPE serves several roles in visual function, including absorption of stray light, formation of blood retina barrier, transport of nutrients, secretion of growth factors, isomerization of retinol, and daily clearance of shed outer photoreceptor outer segments. RPE cell death and dysfunction is associated with both wet (neovascular) and dry (atrophic) forms of AMD.

How then do we make RPE cells from stem cells in order to treat AMD? In previous experiments, scientists have used RPEs made from human embryonic stem cells to treat two patients with inherited eye diseases. The results from these experiments were underwhelming to say the least. Also, the derivation of RPEs from embryonic stem cells was tedious and laborious. Is there a better way?

Make that a yes. A paper in Stem Cells Translational Medicine from Donald Zack’s laboratory at Johns Hopkins University School of Medicine describes a simple and highly scalable process for deriving RPEs from human pluripotent stem cells.

To begin with, the cells were plated at relatively high densities (20,000 cells / cm square centimeter) in a medium called TeSR1. This medium can support the growth of human pluripotent stem cells and can also keep them undifferentiated without the use of animal feeder cell lines. SInce there are no feeder cells to make, the cultivation of these cells is much simpler than before and the variability from culture to culture decreases.

After five days of growth, the cells grew to a monolayer (the cells had grown and spread throughout the culture dish) and were transferred to a 5% carbon dioxide and 20% oxygen incubator. Three days later, they were transferred to Delbecco’s Modified Eagle Medium with F12 supplement or DMEM/F12. This culture medium supports stem cell differentiation. The cells grew and differentiated, for about 25 days, but RPEs were easily visible because they make loads of dark pigment. Once the dark colonies appeared, the cells were allowed to grow another 25 days. The cells were transferred into Delbecco’s Medium with enzymes to pull the cells apart from each other for four hours, then, after pipetting them vigorously, the cells were centrifuged, and suspended in a cell detachment solution called Accumax.

The separated cells were filtered and plated on specially coated plates, and cultured in “RPE medium.” This is a mixture of several different culture media that favors the survival and growth of RPEs. Because RPE colonies were easily seen with their dark pigments, they were specifically picked and passaged. The result was extremely clean RPE cultures from pluripotent stem cells.

Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.
Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.

The cells were subjected to a battery of tests: flow cytometry, Western blotting, Immunostaining and so on. These cells passed with flying colors and they are clearly RPE cells that express RPE-specific genes, have RPE-specific proteins on their cell surfaces, and even snuggle up to photoreceptors and recycle their terminal segments.  The final functional test came from a transplantation experiment in which human RPEs made from human pluripotent stem cells were transplanted behind the retinas of mice with impaired immune systems.  The cells, as you can see in the figures below, integrated beautifully, and were also highly functional, as indicated by the rhodopsin-positive vesicles in the implanted RPE cells.   No tumors were seen in any of the laboratory animals implanted with the stem cell-derived RPEs.

Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.
Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.

This new procedure is able to make RPEs from pluripotent stem cells in a simple and highly scalable way.  If human induced pluripotent stem cells could be used with this protocol, and there seems little reason that should not be highly possible, then such cells could be easily used for human clinical trials.

Making Pancreatic Beta Cells from Embryonic Stem Cells


Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.

Pancreatic beta cells

The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.

Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).

A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.

Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.

When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”

Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?

Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.

What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.

Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .
Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .

In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.

Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”

Induced Pluripotent Stem Cells Form Layered Retina-Like Structure in Culture


Embryonic stem cells can form several different types of eye-specific cells. In the early years of the 21st century, reproducible and efficient methods for differentiating embryonic stem cells into lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells were developed (Haruta M., Embryonic stem cells: potential source for ocular repair. Semin Ophthalmol. 2005 Jan-Mar;20(1):17-23).

Other experiments showed that embryonic stem cells could be differentiated into neural progenitor cells (NPCs). These NPCs differentiated in culture and some of them even expressed genes characteristic of developing retinal cells. Although it must be noted that this was uncommon and cells expressing markers of mature photoreceptors were not observed. Implantation of these differentiated NPCs into the retinas of laboratory animals allowed them to survive for at least 16 weeks, migrate over large distances, and form photoreceptor-like cells that made blue-absorbing pigments. These cells also integrated into the host retina (Banin E, Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells. Steem Cells. 2006 Feb;24(2):246-57).

These early experiments were followed by several others that showed equally remarkable promise. Workers in Takahashi’s laboratory in Kobe, Japan found that embryonic stem cells could form retinal precursors, but that they rarely formed photoreceptors unless they were treated with extracts from embryonic retinas. However in a follow-up paper in 2008, Takahashi, research group found that specific cocktails of small molecules and/or growth factors could push retinal precursors to form photoreceptors (Osakada, et al., Nat Biotechnol. 2008 Feb;26(2):215-24). Kunisada’s lab in Gifu, Japan used various techniques to differentiate embryonic stem cells in culture so that they would form an elaborate retinal-like structure. When this structure was transplanted into the eyes of rodents with inherited eye diseases, these transplanted cells regenerated the ganglion cells in the retina (Aoki H, et al., Graefes Arch Clin Exp Ophthalmol. 2008 Feb;246(2):255-65). Yu’s lab from Seoul National University, Seoul, South Korea made pure RPE cell cultures from embryonic stem cells and then transplanted them into the eyes of rodents with RPE-based retinal degeneration diseases (Park UC, et al., Clin Exp Reprod Med. 2011 Dec;38(4):216-21). The transplanted cells formed RPEs and integrated into the retinas of the laboratory animals. Sophisticated functional assays definitively showed that the RPEs made from embryonic stem cells gobbled up the old segments from photoreceptors and recycled the components back to the photoreceptors (Carr AJ, et al., Mol Vis. 2009;15:283-95).

Using embryonic stem cells to make retina-like structures in culture can provide a model for testing new drugs and procedures to treat degenerative eye diseased such a macular degeneration. Also, such structures might be used to transplant sections of retina into the eyes of individuals where the retina has died off.

With this goal in mind, researchers at the University of Wisconsin-Madison have succeeded in making made early retina structures that contain growing neuroretinal progenitor cells. The novelty in this experiment is that they did it using induced pluripotent stem (iPS) cells that were derived from human blood cells.

In 2011, the laboratory, of David Gamm lab, pediatric ophthalmologist and senior author of the study whose lab is at the Waisman Center, created structures from the most primitive stage of retinal development using embryonic stem cells and iPS cells derived from human skin. These structures generated the major types of retinal cells, including photoreceptors, they did not possess the layered structure found in more mature retina. Clearly something was missing t form a retinal-like structure.

The iPS cells used in this study were made by scientists at a biotechnology company called Cellular Dynamics International (CDI) of Madison, Wisconsin. CDI pioneered the technique to convert blood cells into iPS cells, and they extracted a type of blood cell called a T-lymphocyte from donor samples. These T-lymphocytes were reprogrammed into iPS cells (full disclosure: CDI was founded by UW-Madison stem cell pioneer James Thomson).

With these iPS cells, Gamm and postdoctoral researcher and lead author Joseph Phillips, used their previously-established protocol to grow retina-like tissue from iPS cells. However, this time, about 16% of the initial retinal structures developed distinct layers, which is the structure observed in a mature retina. The outermost layer primarily contained photoreceptors, whereas the middle and inner layers harbored intermediary retinal neurons and ganglion cells, respectively. This particular arrangement of cells is reminiscent of what is found in the back of the eye.

At 72 days, stem cells derived from human blood formed an early retina structure, with specialized cells resembling photoreceptors (red) and ganglion cells (green) located within the outer and inner layers, respectively. Nuclei of cells within the middle layer are shown in blue. These layers are similar to those present during normal human eye development.

These retinal structures also showed proper connections that could allow the cells to communicate information. In the retina, light-sensitive photoreceptor cells along the back wall of the eye produce impulses that are ultimately transmitted through the optic nerve and then to the brain, and this allows. Because these layered retinal structures not only had the proper cell types, but also the proper connections, these findings suggest that it is possible to assemble human retinal cells into the rather complex retinal tissues found in an adult retina. This is extremely stupefying when one considers that these structures all started from a single blood sample.

There are several applications to which these structures might be subjected. They could be used to test drugs and study degenerative diseases of the retina such as retinitis pigmentosa (a major cause of blindness in children and young adults). Also, it might be possible one day to replace multiple layers of the retina in order to help patients with more widespread retinal damage.

Gamm said, “We don’t know how far this technology will take us, but the fact that we are able to grow a rudimentary retina structure from a patient’s blood cells is encouraging, not only because it confirms our earlier work using human skin cells, but also because blood as a starting source is convenient to obtain. This is a solid step forward.” He also added, “We were fortunate that CDI shared an interest in our work. Combining our lab’s expertise with that of CDI was critical to the success of this study.”

This work was published in the March 12, 2012 online issue of Investigative Ophthalmology & Visual Science. The research is supported by the Foundation Fighting Blindness, the National Institutes of Health, the Retina Research Foundation, the UW Institute for Clinical and Translational Research, the UW Eye Research Institute and the E. Matilda Ziegler Foundation for the Blind, Inc.

Planarian Stem Cell Genes Provide Insight into Human Stem Cells


When I was an undergraduate, I cut up planarians in the laboratory and watched them regenerate. Planarians are a type of free-living flatworm that has an uncanny ability to regenerate. Cut them in half, and the tail half will grow a head and the head half will grow a tail. Cut them in half, and the left half will grow a new right side and the left half will grow a new left side. They are truly remarkable critters. How do these worms do this? It appears that the cells of this worm can de-differentiate and become like embryonic cells that originally made the damaged structures. Essentially, the bit of the worm that needs to regrow, recapitulates the developmental process that made it in the first place.

In this way, the bodies of planarians act like stem cells. Stem cells have the potential to regenerate tissues that have been irreparably damaged. One of the problems with stem cells is how to control them. Yet planarians have a genome full of genes that have human homologs. Therefore, planarians seem a logical choice as a model system to study stem cell behavior. Yet, until now, scientists have been unable to efficiently identify the genes that regulate the planarian stem cell system.

At the Whitehead Institute, Peter Reddien‘s lab has revealed some unique insights into planarian biology. These discoveries might help stem cell scientists deliver on a promising role in regenerative medicine. Published in the journal Cell Stem Cell, Reddien and his co-workers used a novel approach to identify and study those genes that control stem cell behavior in planarians. Perhaps unsurprisingly, at least one class of these genes has a counterpart in human embryonic stem cells.

Once injured, planarians (Schmidtea mediterranea) use stem cells, called cNeoblasts, to regrow missing tissues and organs. Within about a week after being injured, the worms have formed two complete planarians. These cNeoblasts are similar to embryonic stem cells in that they are “pluripotent,” which simply means that they have the capacity to form almost cell type in the body. In order to regrow damaged tissues, researchers want to be able to turn on pluripotency and then turn it off after cells have replaced the damaged or missing adult cells.

Reddien, associate professor of biology at MIT and a Howard Hughes Medical Institute (HHMI) Early Career Scientist, said this about his paper: “This is a huge step forward in establishing planarians as an in vivo system for which the roles of stem cell regulators can be dissected. In the grand scheme of things for understanding stem cell biology, I think this is a beginning foray into seeking general principles that all animals utilize. I’d say we’re at the beginning of that process.”

Dan Wagner, a postdoctoral research fellow in Reddien’s lab, and Reddien constructed a protocol to identify genes that regulate the differentiation and renewal of the stem cell population. After identifying genes active in cNeoblasts, Wagner exposed the planarians to ionizing radiation. This left only one surviving cNeoblast in each planarian. After this treatment, each cNeoblast can divide and form colonies of new cells that will differentiate into distinct cell types and divide at specific rates.

Now Wagner and his colleagues eliminated each of the active genes, one per planarian, and observed to determine the behavior of the surviving cNeoblasts without that missing gene. Because the cNeoblasts divide and differentiate at a reproducible rate, the research group could easily determine the role of each gene in cNeoblast behavior. If a colony cNeoblasts was missing a particular gene and had fewer stem cells than the controls, that gene plays in stem cell renewal. Conversely, if the colony had fewer differentiated cells than normal, then the missing gene played a role in differentiation.

Wagner explained, “Because it’s a quantitative method, we can now precisely measure the role of each gene in different aspects of stem cell function. Being able to measure stem cell activity with a colony is a great improvement over methods that existed before, which were much more indirect.”

This screen identified 10 genes that affect cNeoblast renewal, and two of these genes also play roles in cNeoblast renewal and differentiation. Three of the stem cell renewal genes are rather interesting because they code for proteins that are similar to components of Polycomb Repressive Complex 2 (PRC2). PRC2 is known to regulate stem cell biology in mammalian embryonic stem cells and other types of stem cells as well.

These data suggests that the mechanisms that control stem cells in planarians and mammals certainly share some similarities. This might even extend to the mechanisms by which cNeoblasts and embryonic stem cells maintain their naive developmental state. Such work might lead to more insights into stem cell biology that will allows better control and manipulation of stem cells, which will make their use in regenerative medicine much safer.

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.