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.”

Improving Cartilage Production By Stem Cells


To repair cartilage, surgeons typically take a piece of cartilage from another part of the injured joint and patch the damaged area, this procedure depends on damaging otherwise healthy cartilage. Also, such autotransplantation procedures are little protection against age-dependent cartilage degeneration.

There must be a better way. Bioengineers want to discover more innovative ways to grow cartilage from patient’s own stem cells. A new study from the University of Pennsylvania might make such a wish come true.

This research, comes from the laboratories of Associate professors Jason Burdick and Robert Mauck.

“The broad picture is trying to develop new therapies to replace cartilage tissue, starting with focal defects – things like sports injuries – and then hopefully moving toward surface replacement for cartilage degradation that comes with aging. Here, we’re trying to figure the right environment for adult stem cells to produce the best cartilage,” said Burdick.

Why use stem cells to make cartilage? Mauck explained, “As we age, the health and vitality of cartilage cells declines so the efficacy of any repair with adult chondrocytes is actually quite low. Stem cells, which retain this vital capacity, are therefore ideal.”

Burdick and his colleagues have long studied mesenchymal stem cells (MSCs), a type of adult stem cell found in bone marrow and many other tissues as well that can differentiate into bone, cartilage and fat. Burdick’s laboratory has been investigating the microenvironmental signals that direct MSCs to differentiate into chondrocytes (cartilage-making cells).

chondrocytes
chondrocytes

A recent paper from Burdick’s group investigated the right conditions for inducing fat cell or bone cell differentiation of MSCs while encapsulated in hydrogels, which are polymer networks that simulate some of the environmental conditions as which stem cells naturally grow (see Guvendiren M, Burdick JA. Curr Opin Biotechnol. 2013 Mar 29. pii: S0958-1669(13)00066-9. doi: 10.1016/j.copbio.2013.03.009). The first step in growing new cartilage is initiating cartilage production or chondrogenesis. To do this, you must convince the MSCs to differentiate into chondrocytes, the cells that make cartilage. Chondrocytes secrete the spongy matrix of collagen and acidic sugars that cushion joints. One challenge in promoting MSC differentiation into chondrocytes is that chondrocyte density in adult tissue is rather low. However, cartilage production requires that the chondrocytes be in rather close proximity.

Burdick explained: “In typical hydrogels used in cartilage tissue engineering, we’re spacing cells apart so they’re losing that initial signal and interaction. That’s when we started thinking about cadherins, which are molecules that these cells used to interact with each other, particularly at the point they first become chondrocytes.”

Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.
Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.

In order to simulate this microenvironment, Burdick and his collaborators and colleagues used a peptide sequence that mimics these cadherin interactions and bound them to the hydrogels that were then used to encapsulate the MSCs.

According to Mauck, “While the direct link between cadherins and chondrogenesis is not completely understood, what’s known is that if you enhance these interactions early during tissue formation, you can make more cartilage, and, if you block them, you get very poor cartilage formation. What this gel does is trick the cells into think it’s got friends nearby.”

See L Bian, et al., PNAS 2013; DOI:10.1073/pnas.1214100110.

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.

The HPV Vaccines Work


I have blogged before on the Human Papillomavirus (HPV) vaccines, in particular Gardasil. After reviewing the data, I came to the conclusion that this vaccine is essentially safe and does what Merck advertises what it does. The epidemiological data is pretty hard to argue with, and the safety of the vaccine also seems pretty well established. Some readers did not like my conclusions, but that what the data leads me to conclude.

I am not for mandating the vaccine. HPV is acquired by having sex, and young girls can decide for themselves if they are going to have sex and if they should get vaccinated. Health care professionals should definitely encourage sexually active men and women to be vaccinated.

Now a new study provides further evidence that HPV vaccines are effective. A new paper in the Journal of Infectious Diseases by Lauri E. Markowitz, Susan Hariri, Carol Lin, Eileen F. Dunne, Martin Steinau, Geraldine McQuillan, and Elizabeth R. Unger reports that the prevalence of four strains of HPV that can cause cervical cancer, has decreased more than 50% among females aged 14-19 since the introduction of the vaccine in 2006. This is strongly suggests that the vaccine is effective and should result in a reduction in cervical cancer deaths in the long run.

In this study, Markowitz and others analyzed HPV prevalence data from two periods of time: the vaccine era (2007–2010) and the prevaccine era (2003–2006). These data came from National Health and Nutrition Examination Surveys. The prevalence of HPV was determined by detecting HPV in vaginal swab samples from females aged 14–59 years; there were 4150 provided samples in 2003–2006, and 4253 provided samples in 2007–2010.

The results of these surveys showed that among females aged 14–19 years, the prevalence of those HPV strains against which the vaccine was made (HPV-6, -11, -16, or -18) decreased from 11.5% in 2003–2006 to 5.1% in 2007–2010. This is a decline of 56%, and statistically speaking, the confidence intervals for these findings were very high, indicating that these data are quite trustworthy.

Markowitz and her group concluded, “Within 4 years of vaccine introduction, the vaccine-type HPV prevalence decreased among females aged 14–19 years despite low vaccine uptake. The estimated vaccine effectiveness was high.”

Is HPV a problem? Clearly it is. Consider the following data: Approximately 79 million Americans, most in their late teens and early 20s, are infected with HPV, and every year about 14 million people become newly infected.

“This report shows that HPV vaccine works well, and the report should be a wake-up call to our nation to protect the next generation by increasing HPV vaccination rates,” said CDC Director Tom Frieden, M.D., M.P.H. “Unfortunately only one-third of girls aged 13-17 have been fully vaccinated with HPV vaccine. Countries such as Rwanda have vaccinated more than 80 percent of their teen girls. Our low vaccination rates represent 50,000 preventable tragedies – 50,000 girls alive today will develop cervical cancer over their lifetime that would have been prevented if we reach 80 percent vaccination rates. For every year we delay in doing so, another 4,400 girls will develop cervical cancer in their lifetimes.”

According to CDC, each year in the United States, about 19,000 cancers caused by HPV occur in women, and cervical cancer is the most common. About 8,000 cancers caused by HPV occur each year in men in the United States, and oropharyngeal (throat) cancers are the most common.

Clearly HPV is a health problem, and the fact that there is a vaccine available that works is a good thing.

Some news reports quote experts who are troubled that “only” 49% of females aged 13-17 have received a dose of the vaccine, and “only” 32% have received all three doses recommended by the manufacturer. However, the same survey found that only 50% of females aged 14-19 have had sex. Therefore, it is probable that these data suggest that the vaccine is reaching exactly the people who need it and not those who do not.

The words of the Family Research Council seem rather prescient in this regard: “Not every female “needs” the HPV vaccine — those who practice sexual abstinence until marriage and fidelity within marriage have a negligible risk of infection. Those women (and men) who abstain are, at the same time, protecting themselves from other strains of HPV not covered by the vaccine, other STDs, unintended pregnancy, and a range of emotional and relationship problems.”

The HPV vaccine works. If you need it, get it. If you don’t, then don’t. That’s my take.

Beta Blockers and Cardiac Progenitor Cells


The heart receives nerve input from several nerves. Some of these inputs come from the branches of the autonomic nervous system. If that sounds cryptic, just think of the word “automatic.” In other words, the things your body does without you consciously thinking about it are largely directed by the autonomic nervous system: digestion, breathing, the beating of your heart, and so on are all things that our body does without us consciously thinking about it.

The autonomic nervous system consists of two branches, the sympathetic and the parasympathetic branches of the autonomic nervous system. With respect to the heart, the sympathetic nerve inputs to the heart accelerate the heart beat and the force of the heart’s contractions. The parasympathetic inputs to the heart slow the heartbeat, but do not have any direct effect on the force of the heart’s contractions.

autonomic innervation of the heart

The sympathetic nerves that connect to the heart release the neurotransmitters epinephrine and norepinephrine. These neurotransmitters bind to receptors on the surface of heart muscle cells in order to elicit their stimulatory responses. The receptors that bind epinephrine and norepinephrine are called “adrenergic” receptors because they bind epinephrine, which used to be called “adrenaline.” When pharmacists talk about “adrenergic” stimulation, they mean receptors that bind to epinephrine and norepinephrine (for the sake of brevity, I am going to abbreviate these two molecules as Epi/NE).

Activation of Beta2 resize bronchial tubes

Now if all this seems confusing, I am sorry, but it is going to get worse. You see there are different flavors of adrenergic receptors. There are alpha and beta adrenergic receptors. Both alpha and beta adrenergic receptors bind Ep/NE, but the specific responses they elicit can differ, depending on the cell and the machinery it has to respond to the bound receptor. A quick example might help make this clear. If you get an asthma attack, you can breathe in a product called Primatene Mist, which is simply aerosolized epinephrine. Epi, in your lungs, causes the smooth muscles that surround your breathing passages to relax and your breathing passages dilate. This allows you to breath much more easily. However, that same molecule, Epi, will cause your heart to beat faster and harder. The same molecule – Epi – elicits two completely distinct responses from two tissues. This is due to the fact that the heart has one type of adrenergic receptor on the surfaces of its cells (so-called beta1 adrenergic receptors), and the bronchial smooth muscle has a distinct beta adrenergic receptor the on the surfaces of its cells (so-called beta2 adrenergic receptors).

I realize that this is a very long introduction, but it is necessary in order to talk about the paper that I found. In this paper, scientists in Mark Sussman’s laboratory at the San Diego Heart Research Institute have examined cardiac progenitor cells (CPCs) from male mice and their response to beta adrenergic stimulation. You see, once we are born, adrenergic stimulation causes the heart to grow and mature. However, once the heart muscle cells mature, this stimulation no longer causes the heart to enlarge in the same way that heart normally does shortly after birth, although the heart is still capable of remodeling in response to constant aerobic exercise. However, after a heart attack, the secretion of Epi/Ne tends to drive deterioration of the heart. Therefore, a common drug strategy to treat heart attack patients is to prescribe a class of drugs called “beta blockers,” which protect the heart from the deleterious effects of adrenergic stimulation after a heart attack. However, the effects of adrenergic stimulation on CPCs is unknown, and Sussman’s laboratory used cultured CPCs to determine the effects of adrenergic stimulation on CPCs.

CPCs are a stem cell population that resides in the heart. A respectable corpus of literature has shown that CPCs can differentiate into various heart-specific cell types and replace dying heart muscle. Our hearts do not recover properly after a heart attack because the CPCs healing capacities are overwhelmed after a heart attack (See Leri A, Kajstura J, and Anversa P, Circulation Research 109 (2011) 941-61 for an excellent summary of the physiological tasks performed by CPCs).

In the Sussman paper, cultured CPCs from mice and humans were cultured in the laboratory.  It was quickly discovered that CPCs do NOT express beta1 adrenergic receptors on their surfaces, but beta2 adrenergic receptors.  You might smirk and this and say “so what?”  However this is significant for the following reason:  Early in their lives, heart muscle cells expression beta2 adrenergic receptors, but they later switch to exclusive expression of beta1 adrenergic receptors.  They express beta2 adrenergic receptors during that time when they can rapidly divide and respond to the needs of the heart.  CPCs express beta 2 adrenergic receptors only when they are in their undifferentiated state.  Once they differentiate, they switch to beta1 adrenergic receptors.

Secondly, Sussman and his crew discovered that stimulation of the beta2 adrenergic receptors on the surfaces of CPCs caused them to divide.  Sussman and others used a molecule called fenoterol, which binds very tightly to beta2 adrenergic receptors and activates them.

Third, once the CPCs were differentiated into heart muscle cells, they no longer expressed beta2 adrenergic receptors, but expressed beta 1 adrenergic receptors.  Did this change the response of the cells to adrenergic stimulation?  YES.  Instead of dividing in response to adrenergic stimulation, the cells were much more sensitive to dying.  To make sure that this result was not a fluke, Sussman and others engineered CPCs to express beta1 adrenergic receptors, and, sure enough, those cells were also sensitized to cell death upon expression of beta1 adrenergic receptor.

This is all fine and dandy for a culture dish, but can this make a difference in a living animal?  Sussman used a specific mouse strain called TOT.  These mice have a special pathology in that their hearts enlarge and start to not work very well once they are exposed to large quantities of Epi/NE.  Can beta blockers prevent this enlargement of the heart in TOT mice?  It definitely can.  However, Sussman wanted to know what happened to the CPCs.  Therefore, they broke the mice into three groups.  Two groups received metoprolol and the third did not.  Then four weeks later, one TOT mouse group that had received metoprolol and another that had not received transplantations of marked CPCs into their hearts (the CPCs glowed).  Then they examined the CPCs two weeks after the implantation.  The CPCs in non-metoprolol-treated TOT mice took a beating.  However, in the metoprolol-treated mice, the CPCs were three times more prevalent and showed overall lower levels of programmed cell death.  There was less DNA synthesis in the hearts of metoprolol-treated animals, indicating that there was less of a need for replacement of dead cells.

These results indicate that beta blockers do more than protect the heart from excessive Epi/NE after a heart attack.  They also protect the CPCs in the heart, and that could be an even more significant contribution to the life of the heart after a heart attack.  It is might be possible to direct or even augment the activity of CPCs in the heart after a heart attack to accelerate cardiac healing.  That would be a tremendous step in cardiac healing.

BMP-2 Treatment Limits Infarct Size in After a Heart Attack in Mice


Bone Morphogen Protein 2 (BMP2) is a powerful signaling molecule that is made during development, healing, and other significant physiological events. During the development of the heart, BMP2 modulates the activation of cardiac genes. In culture, BMP2 can protect heart muscle cells from dying during serum starvation. Can BMP2 affect hearts that have just experienced a heart attack?

Scientists from the laboratories of Karl Werdan and Thomas Braun at the Max Planck Institute or Heart and Lung Research in Bad Nauheim, Germany have addressed this question in a publication in the journal Shock.

In this paper, Henning Ebelt and his colleagues Gave intravenous BMP2 to mice after a heart attack. CD-1 mice were subjected to LAD-ligation to induce a heart attack (LAD stands for left anterior descending coronary artery, which is tied shut to deprive the heart muscle of oxygen). 1 hour after the heart attack, mice were given 80 microgram / gram of body weight of intravenous recombinant BMP2. The hearts of some animals were removed 5-7 days after the heart attack, but others were examined 21 days after the heart attack to determine the physiological performance of the hearts. Control animals were given intravenous phosphate buffered saline.

Coronary arteries

The extirpated hearts were analyzed for cell death, and the size of their heart scars. Also, protein expression analyses showed the different proteins expressed in the heart muscle cells as a result of BMP2 treatment. Also, the effects of BMP2 on cultured heart muscle cells was ascertained.

The results showed that BMP2 could protect cultured heart muscle cells from dying in culture if they when they were exposed to hydrogen peroxide. Hydrogen peroxide mimics stressful conditions and under normal circumstances, cultured heart muscle cells pack up and die in the presence of hydrogen peroxide (200 micromolar for those who are interested). However, if cultured with 80 ng / mL BMP2, the survival of cultured heart muscle cells greatly increased.

When it came to the hearts of mice that were administered iv BMP2, the BMP2-administered mice survived better and had a smaller infarct size (almost 50% of the heart in the controls and less than 40% in the BMP2-administered hearts). When the degree of cell death was measured in the mouse hearts, those hearts from mice that were administered BMP2 showed less cell death (as determined by the TUNEL assay). BMP2 also increased the beat frequency and contractile performance of isolated heart muscle cells.

FInally, the physiological parameters of the BMP2-treated animals were slightly better than in the control animals. The improvements were consistent, but not overwhelming.

Interestingly, when the proteins made by the hearts of BMP2- and PBS-administered animals were analyzed, there were some definite surprises. BMP2 normally signals to cells by binding a two-part receptor that sticks phosphates on itself, and in doing so, recruits “SMAD” proteins to it that end up getting attached to them. The SMAD proteins with phosphates on them stick together and go to the nucleus where they activate gene expression.

BMP signaling

However, the heart muscle cells of the BMP2-administered mice did not contain heavily phosphorylated SMAD2, even though they did show phosphorylated SMAD1, 5, & 8.  I realize that this may sound like Greek to you, but it means this:  Different members of the BMP superfamily signal to cells by utilizing different combinations of phosphorylated SMADs.  The related signaling molecule, TGF-beta (transforming growth factor-beta), increases scar formation in the heart after a heart attack.  TGF-beta signals through SMAD2.  BMP2 does not signal through SMAD2, and therefore, elicits a distinct biological response than TGF-beta.

These results show that BMP2 administration after a heart attack decreases cell death and decreases the size of the heart scar.  There might be a clinical use for BMP2 administration after a heart attack.

See Henning Ebelt, et al., Shock 2013 Apr;39(4):353-60.

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.