The Use of Stem Cells in Drug Development


Why is it that one person can have surgery and wake up, eat a full lunch and show no ill effects while others are sick for several days after receiving general anesthesia?

The fact is that we all process drugs differently, and these differences are a function of the genetic diversity between all of us. These differences stem from 1) different targets; 2) different liver enzyme activities; and 3) different levels of absorption, excretion and distribution.

A few examples might be illustrative. It is fairly well established that a particular type of blood pressure medicines called “ACE inhibitors” do not work terribly well in African-Americans (see Park IU, Taylor AL. Ann Fam Med. 2007 Sep-Oct;5(5):444-52). The reason for this is that the target of ACE inhibitors, the enzyme angiotensin converting enzyme, which is mercifully abbreviated ACE, works on a substrate that already exists at low concentrations in most African-American patients. Thus a target difference causes differential responses to particular blood pressure medicines.

As a second example, two liver enzymes that degrade drugs, Cyp2C19 and Cyp2D6 are encoded by genes that are subject to genetic variation. In 3-10% of whites, the Cyp2D6 enzyme does not completely function and the drugs processed by this enzyme, a blood pressure medicine called debrisoquine and a heart medicine called sparteine, show impaired degradation. Thus these patients are in danger of overdosing on these drugs at normal dosages, since they are degraded and excreted at such low rates. Other people, however, have a version of Cyp2D6 that is hyperactive. This variant is most commonly found in Ethiopians and Saudi Arabians that consequently, drugs degraded by this enzyme, such as tricyclic antidepressants (e.g., nortriptyline) must be dosed at two the three times the normal concentration. Also, some drugs are given as prodrugs, which are inactive until the liver activates them. In individuals with the overactive Cyp2D6 enzyme variant, a prodrug, such as codeine is overactivated and at normal doses, causes severe side effects (stomach pains). Thus a distinct enzyme difference causes different clinical outcomes with the same drugs (see JK Hicks, et al., Clin Pharmacol Ther. 2013 May;93(5):402-8).

So then, how do we test for drug safety and efficacy given these variations in drug metabolism?

Stem cell technology has the ability to improve drug testing in a multitude of ways. Drug safety can be tested with stem cells as can drug efficacy without feeding them to human volunteers.

Now scientists from the University of Edinburgh have shown that stem cell-based drug tests are almost ready for the prime time. David Hay from the Medical Research Centre for Regenerative Medicine at the University at the University of Edinburgh and his colleagues have generated cell in the laboratory that reach the gold standard required by the pharmaceutical industry to test drug safety.

In this study, the Hay laboratory made liver cells from H9 human embryonic stem cells and from 33D6 human induced pluripotent stem cells. Since is the liver is the main organ that biochemically processes drugs in our bodies (a phenomenon known as biotransformation), testing drug safety in cultured liver cells makes good sense.

Next, Hay and his colleagues found that these pluripotent-derived liver cells were equally effective in drug safety tests as frozen human liver tissue extracted from cadavers. Such livers are in short supply and the results researchers derive from them varies wildly according to the genetic make-up of the donor. Thus frozen liver tissue is not optimal for such drug testing protocols.

However, these drug-testing protocols that use stem cell-based protocols can provide reproducible drug safety results and can also be adapted for individuals with particular genetic compositions who process drugs differently from other people.

David Hay explained it this way: “Differing genetic information plays a key role in how patients’ livers process drugs. We are now able to efficiently produce human liver cells in the laboratory from different people model the functional differences in human genetics.”

Hay and others hope to generate liver cells that contain distinct DNA sequences that will reflect the genetic variations in metabolism found in the population. These cultured liver cells from human pluripotent stem cells can be used to identify differences in drug biotransformation.

These laboratory-generation liver cells could also be used to screen certain drugs that need close monitoring in order to optimize the efficacy of patient treatment, and the safety of these treatment regimes.

Hay and his colleagues are working with Edinburgh BioQuarter in order to form a spin-off company that will commercialize this research and its clinical ramifications.

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.

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.

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.