Accelerated Reprogramming and Gene Editing Protocol Can Make Fixed Cells Much Faster

Sara Howden and her colleagues at the Morgridge Institute for Research and the Murdoch Children’s Research Institute in Australia have devised a protocol that can significantly decrease the time involved in reprogramming mature adult cells while genetically repairing them at the same time. Such an advance is essential for making future therapies possible.

Howden and others demonstrated that genetically repaired cells can be derived from patient skin cells in as little as two weeks. This is much shorter than the multistep approaches that take more than three months.

How were they able to shorten the time necessary to do this? They combined two integral steps in the procedure. Adult cells were reprogrammed to an embryonic stem cell-like state in order to be differentiated into the cells that we want. Secondly, the cells must undergo gene editing in order to correct the disease-causing mutation.

By in this new protocol developed by Howden and her colleagues, they combined the reprogramming and gene editing steps.

To test their new protocol, Howden and her team used cells isolated from a patient with an inherited retinal degeneration disorder, and an infant with severe immunodeficiency. In both cases, the team not only derived induced pluripotent stem cell lines from the adult cells of these patients, but they were also able to repair the genetic lesion that causes the genetic disease.

This protocol might advance transplant medicine by making gene-correction therapies available to patients in a much timelier fashion and at lower cost.

Presently, making induced pluripotent stem cell lines from a patient’s cells, genetically repairing those cells, expanding them, differentiating them, and then isolating the right cells from transplantation, while checking the cells all along the way and properly characterizing them for safety reasons would take too long and cost too much.

With this new approach, however, Howden and others used the CRISPR/Cas9 technology to edit the damaged genes while reprogramming the cells, greatly reducing the time required to make the cells for transplantation.

Faster reprogramming also decreases the amount of time the cells remain in culture, which minimizes the risks of gene instability or epigenetic changes that can sometimes occur when culturing cells outside the human body.

Howden’s next goal is to adapt her protocol to work with blood cells so that blood samples rather than skin biopsies can be used to secure the cells for reprogramming/gene editing procedure. Blood cells also do not require the expansion that skin cells require, which would even further shorten the time needed to make the desired cell types.

The accelerated pace of the reprogramming procedure could make a genuine difference in those cases where medical interventions are required in as little time as possible. For example, children born with severe combined immunodeficiency usually die within the first few years of life from massive infections.

Howden cautioned, however, that she and her team must first derive a long-term source of blood cells from pluripotent stem cells before such treatments are viable and demonstrate the safety of such treatments as well.

See Stem Cell Reports, 2015: DOI: 10.1016/j.stemcr.2015.10.009.

Skin Cells Converted into Placenta-Generating Cells

Yosef Buganim and his colleagues from Hebrew University of Jerusalem have successfully reprogrammed skin fibroblasts in placenta-generating cells.

The placenta is a marvelously complex, but it is also a vital organ for the unborn baby. It supplies oxygen and nutrients to the growing baby and removes waste products from the baby’s blood. The placenta firmly attaches to the wall of the uterus and the umbilical cord arises from it.

The placenta forms from a population of cells in the blastocyst-stage embryo known as trophoblast cells. These flat, outer cells interact with the endometrial layer of the mother’s uterus to gradually form the placenta, which firmly anchors the embryo to the side of the uterus and produce a structure that serves as an embryonic kidney, endocrine gland, lung, gastrointestinal tract, immune system, and cardiovascular organ.

Trophoblast form after an embryonic event known as “compaction,” which occurs at about the 12-cell stage (around day 3). Compaction binds the cells of the embryo tightly together and distinguishes inner cells from outer cells. The outer cells will express the transcription factor Cdx2 and become trophoblast cells. The inner cells will express the transcription factor Oct4 (among others too), and will become the cells of the inner cell mass, which make the embryo proper.

Fetal growth restriction, which is also known as intrauterine growth restriction, refers to a condition in which a fetus is unable to achieve its genetically determined potential size. It occurs when gas exchange and nutrient delivery to the fetus are not sufficient to allow it to thrive in utero. Fetal growth restriction can lead to mild mental retardation or even fetal death. This disease also cause complications for the mother.

Modeling a disease like fetal growth restriction has proven to be very difficult largely because attempts to isolate and propagate trophoblast cells in culture have been unsuccessful. However, these new findings by Buganim and his colleagues may change that.

Buganim and his coworkers screened mouse embryos for genes that support the development of the placenta. They identified three genes – Gata3, Eomes, and Tfap2c – that, when transfected into skin fibroblasts, could drive the cells to differentiate into stable, fully-functional trophoblast cells. Buganim called these cells “induced trophoblast stem cells” or iTSCs.

In further tests, Hana Benchetrit in Buganim’s laboratory and her colleagues showed that these iTSCs could integrate into a developing placenta and contribute to it.

Buganim and his team are using the same technology to generate fully functional human placenta-generating cells.

If this project succeeds, it might give women who suffer from the curse of recurrent miscarriages or other placenta dysfunctions diseases the chance to have healthy babies. Also, since these iTSCs integrate into the placenta and not the embryo, they pose little risk to the developing baby.

This work was published in Cell Stem Cell 2015; DOI: 10.1016/j.stem.2015.08.006.

Skin Cells Converted into Placenta-Generating Cells

Researchers from the laboratory of Yosef (Yossi) Buganim at Hebrew University of Jerusalem have used genetic engineering techniques to directly reprogram mouse skin cells into stable, and fully functional placenta-generating cells called induced trophoblast stem cells (iTSCs).

The placenta forms a vital link between a mother and her baby. When the placenta does not work as well as it should, the baby will receive less oxygen and nutrients from the mother. Consequently, the baby might show signs of fetal stress (that is the baby’s heart does not work properly), not grow nearly as well, and have a more difficult time during labor. Such a condition is called “placental insufficiency” and it can cause recurrent miscarriages, low birth weight, and birth defects.

Placental dysfunction has also been linked to a condition called fetal growth restriction (AKA Intrauterine growth restriction). Intrauterine growth restriction or IUGR is a condition characterized by poor growth of a baby while in the mother’s womb during pregnancy.

How can scientists study the placenta? Virtually all attempts to grow placental cells in culture have been largely unsuccessful.

Buganim and his colleagues have solved this problem. A screen for genes that support the development of the placenta yielded three genes: GATA3, Eomes, and Tfap2c. Next the Buganim team took mouse skin fibroblasts and forced the expression of these three placenta-specific genes in them. This initiated a cascade of events in the cells that converted them into stable and fully functional placenta-generating cells.

These skin-derived TSCs behave and look like native TSCs and they also function and contribute to developing placenta. The Bugamin laboratory used mouse cells for these experiments, but they want to expand their experiments to include human cells to make human iTSCs.

The success of this study could potentially give women who suffer from recurrent miscarriage and placental dysfunction diseases the ability to have healthy babies. The embryo is not at risk from such cells, since iTSCs integrate into the placenta and not into the embryos itself.

See Cell Stem Cell. 2015 Sep 22. pii: S1934-5909(15)00361-6. doi: 10.1016/j.stem.2015.08.006.

Rebooting Pancreatic Cells Can Normalize Blood Sugar Levels in Diabetic Mice

Type 1 diabetes results from the inability of the endocrine portion of the pancreas to secrete sufficient quantities of the hormone insulin. The cells that make insulin, beta cells, have been destroyed. Consequently, type 1 diabetics must inject themselves with insulin routinely in order to stay alive. Is there a better way?

A new strategy suggests that maybe pancreatic cells can be “rebooted” to produce insulin and that sure reprogramming could potentially help people with type 1 diabetes manage their blood sugar levels without the need for daily injections. This therapeutic approach is simpler and potentially safer than giving people stem cells that have been differentiated into pancreatic beta cells.

Philippe Lysy at the Cliniques Universitaires Saint Luc, which is part of the Catholic University of Louvain in Belgium, and his colleagues have reprogrammed pancreatic duct cells extracted from dead donors who were not diabetic at the time of death. The duct cells do not produce insulin, but they have a natural tendency to grow and differentiate into specific types of cells.

Lysy and his team grew the cells in the laboratory and encouraged them to become insulin-producing cells by exposing them to fatty particles. These fatty particles are absorbed into the cells after which they induce the synthesis of the MAFA transcription factor. MAFA acts as a genetic “switch” that binds to DNA and activates insulin production.

Implantation of these altered cells into diabetic mice showed that the cells were able to secrete insulin in a way that controls blood sugar levels. “The results are encouraging,” says Lysy.

Lysy’s colleague, Elisa Corritore, reported these results at this week’s annual meeting of the European Society for Pediatric Endocrinology in Barcelona, Spain. Lysy and others are preparing to submit their results for publication.

This work, if it continues to pan out, might lead to the harvesting of pancreatic ducts from deceased donors and converted in bulk into insulin-making cells. Such “off-the-shelf” cells could then be transplanted into people with type 1 diabetes to compensate for their inability to make their own insulin.

“We would hope to put the cells in a device under the skin that isolates them from the body’s immune system, so they’re not rejected as foreign,” says Lysy. He says devices like this are already being tested for their ability to house insulin-producing cells derived from stem cells.

Previous attempts to get round this problem have included embedding insulin-producing cells in a seaweed derivative prior to transplantation in order to keep them from being destroyed by the recipient’s immune system.

Lysy thinks that since insulin-producing cells originate from pancreatic tissue, they have an inherently lower risk of becoming cancerous after the transplant. This has always been a worry associated with tissues produced from embryonic stem cells, since these have the capability to form tumors if any are left in their original state in the transplanted tissue.

The basic premise of the work looks solid, says Juan Dominguez-Bendala, director of stem cell development for Translational Research at the University of Miami Miller School of Medicine’s Diabetes Research Institute in Florida. “However, until a peer-reviewed manuscript is published and all the details of the work become available to the scientific community, it is difficult to judge if this advance represents a meaningful leap in the state of the art.”

Lysy expects it will take between three and five years before the technique is ready to be tested in human clinical trials.

Rejuvenation Factor Discovered in Human Eggs

When the egg is fertilized by a sperm, it is transformed into a single-celled embryo or zygote that is metabolically active and driven to divide and develop. The egg, on the other hand, is a rather inert cell from a metabolic perspective. What is it in the egg that allows it to transform into something so remarkably different?

A new study by Swea-Ling Khaw and others in the laboratory of Ng Shyh-Chang at the Genome Institute of Singapore (GIS) has elucidated two main factors that help rejuvenate the egg and might also help reprogram adult cells into induced pluripotent stem cells (iPSCs).

Eggs express large amounts of a protein called Tcl1. Tcl1 suppresses the function of old, potentially malfunctioning mitochondria (the structure in cells that makes the energy for the cell). This suppression prevents damaged mitochondria from adversely affecting the egg’s transformation from into an embryo.

Remember also that if an adult cell is fused to an egg, it can cause the egg to divide and form an early embryo. Therefore, the egg cytoplasm is able to reprogram adult cells as well, and Tcl1 seems to play a role in this reprogramming capability as well.

In a screen for genes that are important to the reprogramming process, Shyh-Chang’s laboratory isolated two genes, Tcl1 and Tcl1b1. Further investigation of these two proteins showed that Tcl1 affects mitochondria by inhibiting a mitochondrial protein called polynucleotide phosphorylase (PNP). By locking PNP in the cytoplasm rather than the mitochondria, the growth and function of the mitochondria are inhibited. Tcl1b1 activates the Akt kinase, which stimulate cell growth, survival, and metabolism.

In a review article in the journal Stem Cells and Development, Anaïs Wanet and others explain that energy production in pluripotent stem cells is largely by means of glycolysis, which occurs in the cytoplasm. Mitochondria in pluripotent stem cells are immature subfunctional. When adult cells are reprogrammed into iPSCs, mitochondria function is shut down and energy production is largely derived from glycolysis. When the cells differentiate, the mitochondria are remodeled and become functional once again. Tcl1 is the protein that help shut down the mitochondria so that the pluripotent state can ensue and Tcf1b1 gears up the pluripotent stem cells to grow and divide at will.

Given this remarkable finding, can Tcf1 help make better iPSCs? Almost certainly, but how does one use this important factor to make better iPSCs?  That awaits further experimentation.  Additionally, this finding might also help aging and infertility issues as well. Hopefully this work by Shyh-Chang and her colleagues will lead to many more fruitful and exciting experiments.

Chemical-Only Cell Reprogramming Cocktails Direct Converts Skin Cells into Neurons

Two Chinese laboratories have independently transformed skin cells into neurons using only a cocktail of chemicals. One laboratory used skins cells from Alzheimer’s patients and the other used healthy laboratory mice, and therefore, the protocols developed by each laboratory differ. However, the success of these protocols suggests that it might be economically possible to use neurons made a patient’s own cells to test drug regiments for clinical purposes.

These two studies reinforce the idea that a purely chemical approach represents a promising way to scale up cell reprogramming research that might avoid the technical challenges and safety concerns associated with the more popular method of using transcription factors.

One of the challenges of reprogramming cells to change their identity is that you may end up with cells that look normal on the outside, but inside, many of their internal workings are quite different from the cell type you want to make. In these two papers, neurons made from chemically reprogrammed cells showed neuron-specific gene expression, action potentials, and synapse formation, which is strong evidence that these protocols produce fully operational neurons.

In both cases, the protocols employed decreased the expression of skin-specific genes and increased the expression of neuron-specific genes. These chemicals promoted neuronal cell fates by coordinating multiple signaling pathways that worked together to commit the cells to a neuronal fate. This direct reprogramming procedure bypasses the so-called “proliferative intermediate stage” that put cells under stress and increases the mutation rates. Therefore direct conversion protocols are inherently safer than other reprogramming protocols.

The paper from the laboratory of Jian Zhao (Cell Stem Cell 2015;17(2):204) designed a purely chemical protocol to convert skin cells from human Alzheimer’s disease patients into neurons. Direct reprogramming protocols are available for converting human skin cells into neurons, but these protocols require that cells be transfected with genes that encode transcription factors. Such manipulation requires that cells be treated with viruses or subjected to potentially stressful transfection conditions. This purely chemical protocol is a potentially welcome alternative that would be both safer and easier. The chemicals used in these procedures are easy to synthesize, stable, and standardization of the procedures would also be much easier.

The paper that uses a purely chemical protocol to directly reprogram mouse skin cells comes from the laboratory of Hongkui Deng (Cell Stem Cell 2015;17(2):195) is the culmination of four years of work. The main hurdle was suppressing skin-specific gene expression. Then Dong identified a compound called I-BET151 that suppressed skin cell-specific gene expression. This allowed Deng and his colleagues to successfully reprogram mouse cells with a purely chemical protocol.

The next step for both of these laboratories is to show that, in principle, these chemically reprogrammed cells can be used for therapeutic purposes. Such a proof-of-principle experiment will put direct reprogramming on the map for regenerative medicine in a powerful way.

Converting Immune Cell into Another Type of Immune Cell

What does it take to directly convert an antibody-producing B cell into a scavenging macrophage? The answer: one gene, according to a report in the July 30th issue of Stem Cell Reports. This directly reprogramming is transformation is possible because a transcription factor called C/EBPa can short-circuit the cells so that they re-express genes reserved for embryonic development.

Over the past 65 years, research teams in laboratories all over the world have shown that many different types of specialized cell types can be forcibly reprogrammed into another, but how this occurs is only recently been realized. These “transdifferentiations,” as they’re called, include reprogramming a skin cell into a muscle cell, or a muscle cell into a brown fat cell with the addition of just one or two transcription factors that bind to a cell’s DNA and induce the expression of other genes.

“For a long time it was unclear whether forcing cell fate decisions by expressing transcription factors in the wrong cell type could teach us something about what happens normally during physiological differentiation,” said senior study author Thomas Graf, Ph.D., group leader at the Centre for Genomic Regulation in Spain. “What we have now found is that the two processes are actually surprisingly similar.”

According to lead author of this study, Chris van Oevelen, Ph.D., B cell transdifferentiation occurs when C/EBPa binds to two regions of DNA that act as gene expression enhancers. One of these regions is typically active in immune cells, but the other is only activated when macrophage precursors are ready to differentiate. Thus, the synergism of these two enhancer pathways can cause the B cell to act like a macrophage precursor, which triggers B cell-to-macrophage transdifferentiation.

“This has taught us a great deal about how a transcription factor can activate a new gene expression program (in our case, that of macrophages) but has left us in the dark about the other part of the equation; namely, how the factor silences the B cell program, something that must happen if transdifferentiation is to work,” Dr. Graf said. “This is one of the questions we are focusing on now.”

Dr. Graf is interested in this pathway because of its potential therapeutic applications. As it turns out, C/EBPa-induced B cell-to-macrophage transdifferentiation can convert both human B cell lymphoma or leukemia cells into functional, non-cancerous macrophages. Graf believes that induced transdifferentiation could become therapeutically relevant if drug researchers can find a molecule that can replace C/EBPa. Additionally, understanding the mechanisms of this process would help labs worldwide who use transdifferentiation approach to generate cells for regenerative purposes.