Scientists Create Germ Cell-Supporting Embryonic Sertoli-Like Cells From Skin Cells

Stem cell researchers from the Whitehead Institute in Cambridge, Massachusetts have used a novel, stepwise cell reprogramming protocol to convert skin cells into embryonic Sertoli-like cells.

Sertoli cells are found in the testes of men and they provide vital support, protection, and nutrition to developing sperm cells. Sertoli cells also possess “trophic” properties, which simply mean that they secrete factors that help cells grow and survive. In fact, Sertoli cells have been used to protect and promote the growth, and survival of non-testicular cellular grafts in transplantations. Mature Sertoli cells, however, do not divide, and primary immature Sertoli cells have the unfortunate tendency to degenerate during prolonged culture in the laboratory. Therefore, it is desirable to find some kind of alternative source of Sertoli cells independent of the donor testis cells, but for basic research and clinical applications.

Whitehead Institute Founding Member Rudolf Jaenisch said, “The idea is if you could make Sertoli cells from a skin cell, they’d be accessible for supporting the spermatogenesis process when conducting in vitro fertilization assays or protecting other cell types such as neurons when co-transplanted in vivo. Otherwise, you could get proliferating cells only from fetal testis.”

Researchers in the Jaenisch lab seem to have overcome the supply and lifespan challenges of cultured Sertoli cells by means of using cellular reprogramming to direct one mature cell type, in this case a skin cell, into immature Sertoli cells. The process of cellular reprogramming, otherwise known as trans-differentiation, reprograms a cell directly from one mature cell type to another without first de-differentiating the cell back to an embryonic stem-cell stage. Unlike other reprogramming methods that generate induced pluripotent stem cells (iPSCs), trans-differentiation does not rely on the use of genes that can cause cancer.

The Whitehead Institute scientists trans-differentiated mouse skin cells into embryonic Sertoli-like cells by dividing the trans-differentiation process into two main steps that mimic Sertoli cell development inside the testes. This first step involves the progression transformed skin fibroblasts from their free-moving, unorganized mesenchymal state into an organized, sheet-like epithelial state. For the second step, the cells were induced so that they acquired the ability to attract each so that they formed aggregates that are very similar to those observed in co-cultures of embryonic Sertoli cells and germ cells.

Next, Jaenisch’s lab workers invented a cocktail that consisted of five different transcription factors that specifically activate the developmental program for embryonic Sertoli cells. The cells that resulted from this induction behaved in ways that were reminiscent of embryonic Sertoli cells. They aggregated, formed tubular structures similar to seminiferous tubules found in the testes, and secreted a host of Sertoli-specific factors. When these reprogrammed cells were injected into the testis of fetal mice, the trans-differentiated cells properly migrated to the right location and integrated into the seminiferous tubules. The injected cells behaved exactly like endogenous embryonic Sertoli cells, even though they expressed a few genes differently.

Yossi Buganim, a postdoctoral researcher in the Jaenisch lab and first author of the Cell Stem Cell paper said: “The injected trans-differentiated cells were closely interacting with the native germ cells, which shows [sic] that they definitely do not have any bad effect on the germ cells. Instead, they enable those germ cells to survive.”

Buganim also showed that when their embryonic Sertoli-like cells made from trans-differentiated skin cells were used to sustain other cultured cells in a Petri dish, the cells thrived and lived longer than cells sustained by actual native Sertoli cells. The reprogrammed Sertoli cells supported and nourished the cultured cells and acted like tried and true Sertoli cells.

These encouraging results from their cell culture work have inspired Buganim to investigate if the embryonic Sertoli-like cells retain their enhanced supportive capacity after transplantation into the brain. Once in the brain, the cells could potentially sustain ailing neurons. If these cells truly have this ability, they could have clinical applications. Such applications would include the support and protection of implanted neurons in regenerative therapies for neurodegenerative disorders such as ALS and Parkinson’s disease.

Identifying the Actors Who Play the Part During Reprogramming

A remarkable paper in the journal Nature by Claudia Doege and others (Nature 488, 652-655 (2012)) has revealed the mechanism by which cells are reprogrammed to induced pluripotent stem cells (iPSCs).

Fully differentiated cells have those genes that induce pluripotency (that is, the ability to form any cell type in the adult body) completely shut off (see Takahashi and Yamanaka, Cell 126, 663-676 (2006)). However, if four different genes are introduced into these differentiated cells, namely Oct4, Sox2, c-Myc and Klf4, then the differentiated cell de-differentiates into an iPSC. However, how these genes do this has been rather elusive. However the Doege et al. paper has elucidated our understanding of this process.

To begin, we must understand that gene expression is jointly controlled by two classes of proteins and these include transcription factors, which bind to targets in DNA and activate DNA, and epigenetic regulators that alter the proteins that package DNA (histones). Doege and others have identified two epigenetic regulators called Parp1 and Tet2 that stimulate the expression of the dormant pluripotency genes in differentiated cells that convert them into iPSCs.

What do these proteins do? Parp1 and Tet2 induce the removal of a chemical tag (H3K27me3, for those who are interested) from those histones associated with pluripotency genes and induce the addition of a different chemical tag (H3K4me2, again for the interested). The first chemical tag on the histones shut down gene expression, but the second type of chemical tag induce gene expression.

Doege and his colleagues showed that these epigenetic changes occur before increased expression is detected in two pluripotency genes (Nanog and Esrrb). These epigenetic changes are highly correlated with the binding of the transcription factor Oct4 (also known as POU5F1). Oct4, you see, activates the expression of Parp1, and after the histones are properly modified, Oct4 can bind to the promoter of these genes and activate their expression.

This report shows, for the first time, that epigenetic regulators are equally as important as transcription factors in the status switch from differentiated state to iPSC. According to the accompanying summary of Doege’s article by Kyle Loh and Bing Lim, “reprogramming transcription factors liaise with endogenous epigenetic regulators to execute reprogramming.”

Source – Loh and Lim, Epigenetics: Actors in the cell reprogramming drama. Nature 488,599–600 (30 August 2012); doi:10.1038/488599a

Loh and Lim point out that this work also raises new questions. For example, how do Parp1 and Tet2 specifically activate these pluripotency genes rather than affecting the genome globally? Are there cell-type specific epigenetic regulators? Does this mechanism work for other cell types as well? Does this explain why some cell types become iPSCs so much more efficiently than others? Doege et al. have written an incredible paper that blasts open the door of on our understanding of iPSC formation. This should provide new insights into reprogramming in general.

X (Chromosome) Marks the Plot

In female mammalian embryos, the X chromosome represents a problem. Since mammalian females have two X chromosomes, the embryo contains twice as much of the gene products of the X chromosome as opposed to male mammalian embryos, which only have one copy of the X chromosome. How is this problem solved? X chromosome inactivation (XCI). XCI occurs very early during female mammalian development, and it occurs on a cell-by-cell basis, and occurs randomly. The embryo has some cells that have one copy of the X chromosome inactivated and all the other cells have the other copy of the X chromosome inactivated. This is the reason the bodies of mammalian females are mosaics in which some cells have one copy of the X chromosome inactivated and yet other cells in which the other copy of the X chromosome is inactivated. Thus genetic diseases that map to the X chromosome will affect the entire body of the mammalian male but only a portion of the mammalian female’s body.

What does this mean for stem cells? Quite a bit. Embryonic stem cells are derived from the inner cell mass of the blastocyst-stage embryo. This is precisely the time when the cells of the embryo begin to randomly select a copy of the X chromosome to inactivate. The timing of XCI differs slightly from one species to another. In mice, for example, both copies of the X chromosome are active in mouse embryonic stem cells (ESCs) (Fan and Tran, Hum Genet 130 (2011):217-22; Chaumeil, et al., Cytogenet Genome Res 99 (2002):75-84), and XCI occurs when the cells differentiate (Murakami, et al., Development 138 (2011):197-202). Human ESCs, however, vary tremendously (Dvash and Fan, Epigenetics 4 (2009):19-22), with a few hESC lines showing activation of both copies of the X chromosome and many others showing inactivation of one or the other copy of the X chromosome. Human induced pluripotent stem cells (iPSCs) are derived from adult cells that already have one copy of the X chromosome inactivated. Therefore, de-differentiation of adult cells into iPSCs undoes XCI and activates both copies of the X chromosome (Maherali, et al., Cell Stem Cell 1 (2007):55-70 & Hanna, et al., PNAS 107 (2010):9222-7).

XCI is a process that is linked to pluripotency. The genes necessary for the maintenance of pluripotency (OCT4, Sox2, Nanog) all repress genes necessary for XCI (Xist) and activate genes that repress XCI (Tsix). Therefore, XCI seems to be a factor in the down-regulation of pluripotency in early embryonic cells.

There is a new study that underscores this link between XCI and pluripotency. Researchers at the Gladstone Institutes at the University of California, San Francisco have expanded upon the so-called Kyoto method for making iPSCs. The Kyoto method uses an animal cell line that grows in the culture dish and makes a protein called LIF (leukemia inhibitory factor). LIF activates the growth of cultured iPSCs and allows them to grow and establish an iPSC line.

According to Kiichiro Tomoda from the Gladstone Institute, iPSC derivation on LIF-making feeder cells always produces IPSCs that have two active copies of the X chromosome. However, if iPSCs are derived on feeder cells that do not make LIF, the result is very poor iPSCs derivation and the resultant iPSCs only have one active copy of the X chromosome. Furthermore, by passaging iPSCs that were derived from non-LIF-making feeder cells on LIF-making feeder cells, the inactivated X chromosome became active. This shows that iPSC derivation is highly sensitive to the environment in which the cells are derived. If also shows how to make iPSCs that more closely resemble early embryonic cells.

Unique Drug Responses of Stem Cells from Parkinson’s Patients

Induced pluripotent stem cells (iPSCs) are made from adult cells by means of genetic engineering techniques that introduce specific genes into the adult cell and force it to de-differentiate into an embryonic-like cell. This procedure might provide cells for therapeutic uses some day, but this technology must overcome the mutations introduced into these cells by this procedure and the tumors they can cause. Until then, iPSCs will remain off-limits as therapeutic tools.

That does not disqualify iPSCs as tools for research and even therapeutic investigation. This present paper that comes from a collaborative effort led by Ole Isacson, professor of neurology at McLean Hospital and Harvard Medical School in Boston, uses this very strategy to examine the response of patients with particular forms of Parkinson’s disease to various drugs.

Parkinson’s disease is a progressive, insidious disease that affects a portion of the brain called the midbrain. Within the midbrain is a black body called the substantia nigra, which is Latin for “black stuff.” The substantia nigra is rich in neurons that release a neurotransmitter called “dopamine.”

First of all, to review, neurotransmitters are chemicals that neurons (the cells that make and transmit nerve impulses to other neurons in the brain) use to talk to each other. Neurotransmitters bind to the surfaces of nearby neurons and initiate the production of a nerve impulse. If the neuron receives enough neurotransmitter, it will generate a nerve impulse. Neurons typically can only respond to particular neurotransmitters. The neurotransmitters to which they respond elicit particular responses from them.

Parkinson’s disease results from the death of dopamine-releasing neurons in the midbrain. These neurons connect to cells of the “striatum.” The striatum is responsible for balance, movement control, and walking. Dopamine, produced in the substantia nigra, passes messages between the striatum and the substantia nigra, and when the cells of the substantia nigra deteriorate, which is the case of Parkinson’s disease, there is a corresponding decrease in the amount of dopamine produced between these cells. The decreased levels of dopamine cause the neurons of the striatum to fire uncontrollably, and this prevents the patient from properly controlling their direct motor functions.

Most of the cases of Parkinson’s disease are spontaneous and have no apparent cause. However, there are several types of inherited forms of Parkinson’s and mutations in approximately 17 different genes are associated with inherited forms of Parkinson’s disease.  Of these, only nine have been studied in any detail.  Nevertheless, two genes in particular are important in this paper.

Isacson found two Parkinson’s patients with inherited forms of the disease.  One of then had a mutation in the LRRK2 (Leucine-rich repeat kinase-2) gene, which encodes the Dardarin protein and is intimately involved in the onset of Parkinson’s disease.  The other had a mutation in the PINK1 gene (PTEN induced putative kinase 1), which encodes a protein known to enter mitochondria (the powerhouses of the cell).  Isacson used cells from each patient to make iPSCs.  He also used additional patients, and he had a total of 3 patients with mutations in LRRK2 and two with mutations in PINK1.

Because mutations in LRRK2 and PINK1 are thought to interfere with the function of mitochondria in neurons, Isacson examined the mitochondria of these patient-specific iPSCs.  When compared to mitochondria from volunteers without Parkinson’s disease, Isacson found that the Parkinson’s patient-specific iPSCs were much more susceptible to damage after exposure to toxins.  Thus, the mitochondria of these patient-specific iPSCs were certainly much more fragile than normal mitochondria.

Could this mitochondrial fragility be ameliorated with medicines?  Isacson tested the ability of particular substance to mitigate this condition in the patient-specific iPSCs.  A supplement called Q10, which is known to aid mitochondrial function was administered the to Parkinson’s patient-specific iPSCs, was given to the cells, and all cells were prevented from experiencing mitochondrial damage after exposure to toxins.  However, when a different drug called rapamycin was administered, the results were very different.  Rapamycin diminishes the immune response of an organism, and therefore, it can spare weak cells from being cleared by the immune system.  Rapamycin prevented damage in the cells with mutations in LRRK2, but not those with mutations in PINK1.

This paper shows how iPSC-based research can lead to information that can fashion personal treatments for each patient.  Even though this work focused on Parkinson’s disease, there are many other diseases that could benefit from iPSC-based research.

Proof-of Concept Study: Stem Cell Treatment for Muscular Dystrophy

Recently, I posted here about an experiment that used induced pluripotent stem cells (iPSCs) from muscular dystrophy (MD) patients to treat mice with MD. A similar but different experiment has successfully treated MD mice using stem cells made from a MD patient.

Francesco Saverio Tedesco from University College London and the San Raffaele Scientific Institute in Milan, Italy, has used a stem cell known to hang out around blood vessels called “mesoangioblasts” to initiate this gene therapy/stem cell treatment strategy. Mesoangioblasts are multipotent progenitors of tissues that form from the middle layer of the embryo (mesoderm). More importantly, mesoangioblasts express genes that are normally found in the circulatory system (blood vessels in particular). Therefore, they seem to be able to form blood vessels rather readily, but there are also indications that they also can differentiate into muscle (Cossu G, Bianco P. (2003). Mesoangioblasts–vascular progenitors for extravascular mesodermal tissues. Curr Opin Genet Dev. 13(5):537-42).

Tedesco and his colleagues examined mesoangioblasts of patients with a moderately mild version of muscular dystrophy called limb-girdle muscular dystrophy. Tedesco and his colleagues found that the numbers of mesoangioblasts in these patients was greatly reduced. This is potentially significant, since mesoangioblasts are being strongly considered as candidate stem cell to treat MD patients.

Next, Tedesco’ laboratory isolated fibroblasts and muscle cells (myoblasts) from the MD patients and converted them into iPSCs, using standard lentiviral-based transfection procedures. They then used the patient-derived iPSCs to to make mesoangioblasts, which they grew in culture. The cultured mesoangioblasts were then subjected to genetic engineering techniques that fixed the mutation that caused MD.

Limb-girdle MD (LGD2D) results from a mutation in a gene that encodes a protein called “alpha-sarcoglycan.” This protein, alpha-sarcoglycan is part of a large complex called the dystrophin-associated protein complex. This complex consists of a host of proteins that includes alpha-sarcoglycan, alpha-dystrobrevin, syncoilin, synemin, sarcoglycan, dystroglycan, and sarcospan. These proteins are embedded in the cell membrane of the muscle cell, and they bind to a long, fibrous protein called dystropin inside the cell and a large protein outside the cell called laminin. Collectively, this structure is called a “costamere.” Costameres bind to to the protein dystrophin and thereby physically link the cell membrane of the muscle cell to the contractile proteins inside the muscle. Likewise, costameres bind to an extracellular protein called laminin and physically link the cell membrane of the muscle to the extracellular matrix. Costameres guarantee that the muscle contracts while firmly anchored to a substratum. Mutations in dystrophin tend to cause Duchenne’s muscular dystrophy (DMD), which is the most severe form of MD. However, mutations in any of the genes that encode costamere proteins can cause a form of MD.

Back to Tedesco and his crew. After genetically fixing the mutation in the mesoangioblasts, Tedesco and his collaborators implanted them into the muscles of mice that completely lacked a functional alpha-sarcoglycan gene and had immune systems that did not work properly. Such mice had muscles that worked very poorly and showed some, though not all the characteristics of MD.

The implanted mesoangioblasts went right to work and made muscle fibers that were normal and contained lots of alpha-sarcoglycan. After growing for a time, Tedesco and co-workers put these mice on a treadmill. The mice that had been implanted with mesoangioblasts performed far better than the control mice. This experiment showed that it is at least possible, in principle to treat MD patients with a combination of stem cells and gene therapy.

Tedesco, who was understandably excited by these results. said: “This is a major proof of concept study. We have shown that we can bypass the limited amount of patients’ muscle stem cells using induced pluripotent stem cells and then produce unlimited numbers of genetically corrected progenitor cells.”

Professor Giulio Cossu, another author of this study from University College London said: “This procedure is very promising, but it will need to be strenuously validated before it can be translated into a clinical setting, also considering that clinical safety for these “reprogrammed” stem cells has not yet been demonstrated for any disease.”

Huntington’s Disease Mutation Corrected in Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs)are made from adult cells by means of genetic engineering techniques that introduce transcription factors into the cells that drive the cell into an embryonic state that divides readily and can differentiate into a wide variety of cell adult cell types.

iPSCs are terrific tools for studying human genetic diseases, since iPSCs that bear the genetic mutations that cause the disease can be easily made from patients, those diseases can be studied in great detail. In a recent paper, scientists from the Ellerby lab at the Buck Institute of Aging (San Francisco, CA) used iPSCs made from a patient with Huntington’s disease to correct the genetic mutation that causes Huntington’s disease.

Lisa Ellerby, in whose lab this work was done, commented on her research in this way: “We believe that the ability to make patient-specific genetically corrected iPSCs from HD patients is a critical step for the eventual use of these cells in cell replacement therapy. The genetic correction reversed the signs of disease in these cells – the neural stem cells were no longer susceptible to cell death and the function of their mitochondria was normal.”

The corrected cells were potentially able to populate the regions of the brains of mice afflicted with Huntington’s disease, but there are no clear signs to date that transplantation of such cells would improve the function of the mouse. Therefore, the next step in this research is to transplant the corrected cells into the brains of HD-afflicted mice to determine if the mice show functional improvements.

Ellerby’s lab used a technique called homologous recombination to correct the mutation in the iPSCs derived from the skin of a patient with Huntington’s disease. Because Huntington’s disease consists of expansions of a triplet sequence (CAG), recombination replaces the expanded regions of the Htt gene that have excessive numbers of CAG repeats with those that have normal numbers of CAG repeats.

Ellerby’s groups will transplant neural stem cells made from the corrected iPSCs into the brains of mice that have Huntington’s Disease. The results should be rather telling.

University of Wisconsin Scientists Find a New, Better Way to Turn Stem Cells into Heart Muscle Cells

Stem cell researchers and cardiologists from the University of Wisconsin-Madison have designed a new and improved protocol to turn embryonic and induced pluripotent stem cells into heart muscle cells.

The study leader, Sean Palecek, who is also professor of chemical and biological engineering at the University of Wisconsin-Madison, and his colleagues Timothy Kamp, professor of cardiology at UW School of Medicine and Public Health, and Xiaojun Lian, a UW graduate student, have developed a technique for efficient and abundant production of heart muscle cells. This technique will provide scientists a better and more abundant source of material for drug studies and a better model system to study diseases and heart pathologies.

Heart muscle cells (also known as cardiomyocytes) are essential cells that compose the beating heart. However, it is rather difficult to make large quantities of them. Typically, cultured heart muscle cells only survive or a short period time, which greatly complicates using them for any experiments or drug tests. Now, however, these UW researchers have devised an inexpensive method for developing an abundance of heart muscle cells in the laboratory.

Cardiologist Timothy Kamp explained: “Many forms of heart disease are due to the loss or death of functioning cardiomyocytes, so strategies to replace heart cells in the diseased heart continue to be of interest. For example, in a large heart attack up to 1 billion cardiomyocytes die. The heart has a limited ability to repair itself, so being able to supply large numbers of potentially patient-matched cardiomyocytes could help.”

Why is their method so much more efficient? The UW research team discovered that by changing a signaling pathway called Wnt pathway, they could guide the stem cells to differentiate into heart muscle cells. All they had to do was turn the Wnt pathway on and off at different times by using two small molecules.

The Wnt signaling pathway is an extremely common signaling pathway that exists in virtually all multicellular organisms and is used multiple times during development.  Wnt signaling begins with the secretion of a small protein can a Wnt protein.  Wnt proteins are produced by cells to send signals to nearby cells.  When the cells receiving the signal are bound by the Wnt protein, a series of events are set into motion inside the cell.  The receptor that binds the Wnt protein consists of a protein that is a member of the Frizzled gene family.  Frizzled receptors bind the Wnt protein in combination with another protein called LRP.  The binding of Wnt, LRP and Frizzled brings an internal protein called Disheveled to the membrane.  Once Disheveled come to the membrane, it becomes activated.  How this activation occurs in still unclear, but Disheveled inhibits GSK-3 (glycogen synthase kinase-3).  GSK-3 normally attaches phosphate groups to a protein called beta-catenin.  This phosphate group attachment marks beta-catenin for destruction, but once GSK-3 is inhibited by activated Disheveled, beta-catenin is no longer destroyed and when the levels of beta-catenin build up in the cytoplasm, it goes to the nucleus where it combines with another protein called TCF and regulates gene expression.  Once again we see that a signal transduction pathway begins at the cell surface and results in changes in gene expression.

“Our protocol is more efficient and robust,” said Palecek. “We have been able to reliably generate greater than 80 percent cardiomyocytes in the final population while other methods produce about 30 percent cardiomyocytes with high batch-to-batch variability.”

Palacek continued: “The biggest advantage of our method is that it uses small molecule chemicals to regulate biological signals. It is completely defined, and therefore more reproducible. And the small molecules are much less expensive than protein growth factors.”

Kamp noted that the “fact that turning on and off one master signaling pathway in the cells can orchestrate the complex developmental dance completely is a remarkable finding as there are many other signaling pathways and molecules involved.”

This protocol has the capacity to revolutionized the use of heart muscle cells for drug testing.  Also, because the Wnt signaling pathway is required during heart development, this protocol also has the ability to clarify the exact role of this pathway during heart differentiation.  Finally, if stem cells are eventually used for therapeutic purposes, this protocol or one like it will certainly be employed to convert stem cells into heart muscle cells.