Personalized Stem Cells for Curing Parkinson’s Disease


Stem cell treatments for curing Parkinson’s disease have been one of the dreams of stem cell scientists ever since the first embryonic stem cells were derived from mouse embryos in 1981. Unfortunately, this proved to be one of the harder therapeutic nuts to crack. Several experiments have shown that while feasible, getting the recipe right has required a fair amount of tweaking.

brain-labels

Parkinson’s disease (PD) results from the progressive death of neurons in the midbrain that release a neurotransmitter called dopamine, To review briefly, the brain consists of the forebrain, midbrain and hindbrain. The forebrain consists of the two large cerebral hemispheres that occupy the vast majority of the space within your skull. In addition to the left and right cerebral hemispheres is the diencephalon that consists of the thalamus, subthalamus, hypothalamus, and epithalamus. The thalamus serves as a relay station for a whole variety of nerve fiber tracts, the hypothalamus regulates visceral activities by way of other brain regions and the autonomic nervous system. and the epithalamus connects the limbic system to the rest of the brain. The midbrain, which lies below the diencephalon, is part of the brain stem and dopamine produced in two regions of the midbrain, the substantia nigra and ventral tegmental area play roles in motivation and habituation, and refinement of the control of voluntary movement, The hindbrain consists of the metencephalon and the myelencephalon, both of which contain mutiple fiber tracts and nuclei for vital functions.

Midbrain 2

The death of dopamine-producing neurons in the pars compacta region of the substantia nigra region of the midbrain causes PD. The par compacta sends nerve fibers to the cerebral hemispheres, in particular to cluster of neurons called the basal ganglia. The basal ganglia do not initiate movement, but they refine movement and stabilize the limbs and other body parts while moving. Thus the basal ganglia normally exert a constant inhibitory influence on a wide range of movements. preventing movement at inappropriate times. When someone decides to move, this inhibition is reduced for the required motor system, thereby releasing it for activation. Dopamine releases this inhibition, and therefore high levels of dopamine tend to promote movement and low levels of dopamine demand greater exertion to generate any given movement. Thus the net effect of dopamine depletion is to produce hypokinesia, or less overall movement.

Basal ganglia

Now that we have some knowledge of PD and what causes it, we can examine how to cure it. Since the death of dopamine-secreting neurons causes PD, replacing death or moribund neurons should be possible. Several preclinical studies in laboratory animals and clinical studies with human patients has shown that this is possible.

Rodents can contract a synthetic form of PD if they are treated with a drug called 6-hydroxydopamine. This drug kills off their dopamine-secreting neurons and creates a PD-like disease. Embryonic stem cells can be differentiated in the laboratory into dopamine-secreting neurons, which can then be transplanted into the midbrain. In PD rats, this strategy has reversed the symptoms of PD, but tumor growth has been a nagging problem. The biggest problem is that isolating fully differentiated dopamine-secreting cells has proven difficult because of a lack of good, solid indicators that say to the scientists, “This one is a dopamine-secreting neuron and this one is not.” Thus, isolating nice, clean cultures of only dopamine-secreting cells has been kind of tough to do.

Fortunately, Doi and others in the Takahashi lab at the University of Kyoto showed that prolonged maturation culture system (42 days long) can eliminate most of the tumor-making cells. However, this culture system is laboriously long. Now, Takahashi and Doi and others have struck again in a paper published in Stem Cell Reports in which they used induced pluripotent stem cells to derive dopamine-secreting neurons to treat PD rats.  Because induced pluripotent stem cells are made from a patient’s own adult cells and are converted into embryonic-like stem cells by means of a combination of genetic engineering and cell culture techniques, they are patient-specific and do not require the dismembering of human embryos.

The novelty of this paper is that Doi and others used a protein that acts as an earmark for dopamine-secreting midbrain neurons and this protein is called CORIN. CORIN is a protease, which simply means that it clips other proteins into small pieces. Nevertheless, by using the CORIN protein, Takahashi, Doi and others successfully and efficiently isolated dopamine-secreting midbrain neurons from other cells in their cultures.  Additionally, Doi and the gang were able to differentiate the induced pluripotent stem cells into dopamine-secreting progenitor cells.  This means that the cells were poised to differentiate into dopamine-secreting neurons, but were not quite there yet.  This way, the cells would grow in culture, but upon transplantation, they would differentiate into dopamine-secreting neurons rather than form tumors.  High numbers of cells are required for clinical purposes and this technique allows the for production of large number of cells.

The technique used in this paper also produced the cells under conditions that were safe, scalable and potentially usable for clinical use. These high-quality cells never produced any tumors and produced definitive behavioral improvements in the implanted laboratory animals. The problems that remain are one of scale. The grafts of dopamine-secreting cells that survived in the midbrains of these mice were relatively small (about 1 square millimeter in size or the thickness of a dime).  This is probably due to the fact that the cells differentiate when transplanted rather than growing.  Therefore, this technique will need to be adapted to somehow increase the size of the graphs of dopamine-secreting neurons.  In some PD patients such small graphs will probably work just fine, but in others, probably not.  The other issue is that these implanted cells might be subjected to the same bad intracerebral environment as the original cells and die off quickly, thus abrogating any positive clinical effect they might have.  This is another issue that will need to be examined.

The work goes on, without the need to destroy any embryos.

See Daisuke Doi at al., Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation. Stem Cell Reports 2014, 2: 337-350.

Using Stem Cells for Muscle Repair


Stem cell treatments for muscular dystrophy and other degenerative diseases of muscle might be a realistic possibility, since scientists have discovered protocols to make muscle cells from human pluripotent stem cells.

Tiziano Barberi, Ph.D., chief investigator in the Australian Regenerative Medicine Institute (ARMI) at Monash University in Clayton, Victoria, and Bianca Borchin, a graduate student in the Barberi laboratory, have developed techniques to generate skeletal muscle cells. Barberi and Borchin isolated muscle precursor cells from human pluripotent stem cells (hPSCs), after which they applied a purification technique that allows these cells to differentiate further into muscle cells.

Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the ability to become any cell in the human body, including skeletal muscles, which control movement. Once the stem cells begin to differentiate, controlling that process is very challenging, but essential in order to produce only the desired cells. Barberi and Borchin used a technique known as fluorescence activated cell sorting (FACS) to identify those cells that contained the precise combination of protein markers that are expressed in muscle precursor cells. FACS also enabled them to successfully isolate those muscle precursor cells.

“There is an urgent need to find a source of muscle cells that could be used to replace the defective muscle fibers in degenerative disease. Pluripotent stem cells could be the source of these muscle cells,” Dr. Barberi said. “Beyond obtaining muscle from hPSCs, we also found a way to isolate the muscle precursor cells we generated, which is a prerequisite for their use in regenerative medicine.”

Borchin said there were existing clinical trials based on the use of specialized cells derived from hPSCs in the treatment of some degenerative diseases, but deriving muscle cells from pluripotent stem cells proved to be challenging. “These results are extremely promising because they mark a significant step towards the use of hPSCs for muscle repair,” she said.

“The production of a large number of pure muscle precursor cells does not only have potential therapeutic applications, but also provides a platform for large-scale screening of new drugs against muscle disease,” Dr. Barberi added.

This study was published early online Nov. 27 in Stem Cell Reports.  This study does not address the immune response against dystrophin that has plagued gene therapy and stem cell-based muscular dystrophy clinical trials that has been noted in previous posts.  The use of embryonic stem cells, in particular, would create muscles that are not tissue matched to the patient and would generate robust inflammation against the implanted muscles.   Thus embryonic stem cells would generate a “cure” that would be much worse than the disease itself.  Nevertheless, adapting the Barberi-Borchin protocol to induced pluripotent stem cells would produce skeletal muscle cells that are tissue matched to the patient.

Human Fat Contains Multilineage Differentiating Stress Enduring Cells With Great Potential for Regenerative Medicine


A collaboration between American and Japanese scientists has discovered and characterized a new stem cell population from human fat that do not cause tumors and can differentiate into derivatives from ectoderm, mesoderm, and endoderm.

Multilineage Differentiating Stress-Enduring or Muse cells are found in bone marrow and the lower layers of the skin (dermis). Muse cells are a subpopulation of mesenchymal stem cells, and even express a few mesenchymal stem cell-specific genes (e.g., CD105, a cell-surface protein specific to mesenchymal stem cells). However, Muse cells also express cell surface proteins normally found in embryonic stem cells (e.g., stage-specific embryonic antigen-3, SSEA-3). Additionally, Muse cells have the ability to self-renew, and differentiate into cell types from all three embryonic germ layers, ectoderm (which forms skin and brain), mesoderm, (which forms muscle, bone, kidneys, gonads, heart, blood vessels, adrenal glands, and connective tissue), and endoderm (which forms the gastrointestinal tract and its associated tissues). Finally, Muse cells can home to damaged sites and spontaneously differentiate into tissue-specific cells as dictated by the microenvironment in which the cells find themselves.

A new publication by Fumitaka Ogura and others from Tohoku University Graduate School of Medicine in Sendai, Japan and Saleh Heneidi from the Medical College of Georgia (Augusta, Georgia), and Gregorio Chazenbalk from the David Geffen School of Medicine at UCLA has shown that Muse cells also exist in human fat.

The source of cells came from two places: commercially available fat tissue and freshly collected fat from human subjects, collected by means of liposuction. After growing these cells in culture, the mesenchymal stem cells and Muse cells grew steadily over the 3 weeks. Then the Dezawa research group used fluorescence-activated cell sorting (FACS) to isolate from all these cells those cells that express SSEA-3 on their cell surfaces.

FACS uses antibodies conjugated to dyes that can bind to specific cell proteins. Once the antibodies bind to cells, the cells are sluiced through a small orifice while they are illuminated by the laser. The laser activates the dyes if the cell fluoresces, one door opens and the other closes. The cell goes to one test tube. If the cell does not fluoresce, then the door stay shut and another door opens and the cell goes into a different test tube.  In this way, cells with a particular cell-surface protein are isolated from other cells that do not have that cell-surface protein.

Fluorescent-Activated Cell Sorting
Fluorescent-Activated Cell Sorting

In addition to expression SSEA-3, the fat-based Muse cells expressed other mesenchymal stem cell-specific cell-surface proteins (CD29, CD90), but they did not express proteins usually thought to be diagnostic for fat-based mesenchymal stem cells (MSCs) such as CD34 and CD146.  Muse cells also expressed pluripotency genes (Nanog, Oct3/4, PAR4, Sox2, and Tra-1-81).  The Muse cells grew in small clusters and some cell expressed ectodermal-specific genes (neurofilament, MAP2), others expressed mesodermal-specific genes (smooth muscle actin, NKX2) and endodermal-specific genes (alpha-fetoprotein, GATA6).  These data suggested that the cultured Muse cells were poised to form either ectoderm, mesodermal, or endodermal derivatives.

When transplanted into mice with non-functional immune systems, the Muse cells never formed any tumors or disrupted the normal structure of the nearly tissues.  When placed in differentiating media, fat-derived Muse cells differentiated into cells with neuron-like morphology that expressed neuron-specific genes (Tuj-1), liver cells, and fat.  When compared with Muse cells from bone marrow or skin, the fat-derived Muse cells were better at making bone, fat, and muscle, but not as good as bone marrow Muse cells at making neuronal cell types, but not as good at making glial cells.  Many of these assays were based on gene expression experiments and not more rigorous tests.  Therefore, the results of these experiments might be doubtful until they are corroborated by more rigorous experiments.

These cells are expandable and apparently rather safe to use.  More work needs to be done in order to fully understand the full regenerative capacity of these cells and protocols for handling them must also be developed.  However, hopefully pre-clinical experiments in rodents will give way to larger animal experiments.  If these are successful, then maybe human trials come next.  Here’s to hoping.

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.”

Clinical-Scale NK Cells for Cancer Therapy Made from Pluripotent Stem Cells


Dan Kaufman’s laboratory has done it again. The Kaufman laboratory at the University of Minnesota in collaboration with scientists from MD Anderson Cancer Center in Houston, Texas have designed a protocol to make natural killer cells from embryonic stem and induced pluripotent stem cells.

Natural killer cells provide a very important contribution to the innate immune response. These cells produce molecules called cytokines and they also kill virally infected cells and malignant cells. NK cells are unique among the cells of the immune system in that they have the ability to recognize foreign, infected or stressed cells in the absence of antibodies and Major Histocompatibility Complex proteins (the cell surface proteins that act as bar codes used by the immune system uses to determine if a cell is yours or not yours). Therefore, NKs typically work faster than the rest of the immune system.

Natural killer cells or NK cells have been used to treat patients with refractory cancers. Unfortunately, a major problem with using NK cells is growing a sufficient quantity of cells for therapy. Using pluripotent stem cells to make NK cells is an intriguing possibility, but the protocols for differentiating NK cells from embryonic stem cells (ESCs) is tedious and inefficient. However, the Kaufman laboratory has provided a much more efficient and straight-forward way to derive NK cells, thus allowing for the production of clinical scale quantities of NK cells.

The Kaufman lab protocol involves first deriving embryoid bodies from ESCs or induced pluripotent stem cells (iPSCs), which are made from adult cells through genetic engineering techniques that causes the cells to de-differentiate into ESC-like cells known as iPSCs. Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that assume a kind of spherical shape and have a variety of cells differentiating into a wide range of cell types. Embryoid bodies can contain beating heart muscle, neural-type cells, blood progenitors cells, and even muscle or bone cells in their interiors in a haphazard arrangement. Forming embryoid bodies or EBs from cultured ESCs or iPSCs is rather easy, but controlling the differentiation of the cells in the EBs is quite another matter.

embryoid bodies
embryoid bodies

Kaufman and others discovered that if the EBs were incubated with artificial antigen-presenting cells that expressed a surface-bound version of the protein IL21 (interleukin 21) plus a cocktail of cytokines, these pluripotent stem cells could efficiently form NK cells.

Functional assays of the NK cells differentiated from ESCs and iPSCs easily showed that the NK cells for functional in every way and expressed all the cell surface molecules characteristic of NK cells. Furthermore, all ESC and iPSC lines examined were able to make NK cells, but the efficiency with which they made them different rather widely.

In conclusion, Kaufman and others state in their paper, “our ability to now produce large numbers of cytotoxic NK cells means that prospect hESC- and iPSC-derived hematopoietic products for diverse clinical therapies can be realized in the not-too-distant future.” For some cancer patients, that day cannot come soon enough.