Transdifferentiating Skin Cells into Heart Muscle and Neural Stem Cells With Nothing But Chemicals


A research effort led by Dr. Sheng Ding from the Gladstone Institute and scientists from the Roddenberry Center for Stem Cell Biology and Medicine has successfully transformed skin cells into heart cells and brain cells using little more than a cocktail of chemicals. Previous work that sought to transdifferentiate mature, adult cells into another cell type used gene vectors (such as viruses) that genetically engineered the cells to express new genes at high levels. Because this new protocol uses no genetic engineering techniques, these results are nothing short of unprecedented. This work lays the foundation for, hopefully, being able to regenerate lost or damaged cells with pharmaceutical agents.

In two publications that appeared in the journals Science and Cell Stem Cell, Ding and his collaborators utilized chemical cocktails to drive skin cells to differentiate into organ-specific stem cell-like cells and, then into terminally differentiated heart or brain cells. These results were achieved without genetically engineering cells.

Ding, who was the senior author on both studies, said: “This method brings us closer to being able to generate new cells at the site of injury in patients. Our hope is to one day treat diseases like heart failure or Parkinson’s disease with drugs that help the heart and brain regenerate damaged areas from their own existing tissue cells. This process is much closer to the natural regeneration that happens in animals like newts and salamanders, which has long fascinated us.”

Mature heart muscle cells have very little regenerative ability. Once a patient has suffered a heart attack, the cells that have died are, for the most part, not replaced. Therefore, stem cell scientists have left no stone unturned to find a way to replace dead and dying heart muscle cells. Several clinical trials have transplanted mature adult heart cells or various types of stem cells into the damaged heart. However, such procedures have either not improved heart function or have only modestly improved heart function (with a few exceptions). Typically, transplanted cells do not survive in the hostile environment of the heart after a heart attack and even those cells that do survive fail to properly integrate into the heart. Also, the ability of transplanted cells to differentiate into heart cells is not stellar. Alternatively, Deepak Srivastava, director of cardiovascular and stem cell research at the Gladstone Institute, and his team pioneered a distinctly novel approach in which scar-forming cells in the heart of animals were genetically engineered to differentiate into heart new muscle that greatly improved heart function. Genetic engineering brings its own safety issues to the table and, for these reasons, chemical reprogramming protocols that can do the same thing might provide an easier way to drive heart muscle to regenerate local lesions.

In the Science study, Dr. Nan Cao (a postdoctoral research fellow at Gladstone, and others applied a cocktail of nine chemicals to reprogram human skin cells into beating heart cells. By using a kind of trial-and-error strategy, they discovered the best combination of chemicals to transdifferentiate skin cells into multipotent stem cells. Multipotent stem cells have the ability to differentiate into several distinct cell types from several different types of organs. A second-growth factor/small molecule cocktail drove the multipotent stem cells to differentiate into heart muscle cells.

Perhaps the most surprising result of this protocol is its efficiency. Typically, chemically-induced differentiation is relatively inefficient, but with Ding’s method, over 97% of the cells began beating. These chemically-derived heart muscle cells also responded appropriately to hormones, and they also molecularly resembled heart muscle cells (and not skin cells). Upon transplantation into a mouse heart, these cells developed into healthy-looking heart muscle cells within the heart of the laboratory animal.

“The ultimate goal in treating heart failure is a robust, reliable way for the heart to create new muscle cells,” said Srivastava, co-senior author on the Science paper. “Reprogramming a patient’s own cells could provide the safest and most efficient way to regenerate dying or diseased heart muscle.”

In the second study, published in Cell Stem Cell, which was authored by Gladstone postdoctoral scholar Dr. Mingliang Zhang, PhD, the Gladstone team created neural stem cells from mouse skin cells using a similar approach.

Once again, the chemical cocktail that transdifferentiated skin cells into neural stem cells contained nine different chemicals. Some of the molecules used in the neural stem cell experiment overlapped with those employed in the heart muscle study. Treatment of the skin cells for about ten days with the cocktail transdifferentiated the skins cells into neural-like cells. Virtually all the skin cell-specific genes were shut off and the neural stem cell-specific genes were gradually activated. When these chemical-differentiated cells were transplanted into mice, the cells spontaneously differentiated into neurons, oligodendrocytes, and astrocytes (three basic nerve cells). The neural stem cells were also able to self-replicate, which makes them ideal for treating neurodegenerative diseases or brain injury.

“With their improved safety, these neural stem cells could one day be used for cell replacement therapy in neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease,” said co-senior author Dr. Yadong Huang, who is a senior investigator at Gladstone. “In the future, we could even imagine treating patients with a drug cocktail that acts on the brain or spinal cord, rejuvenating cells in the brain in real-time.”

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Adult Directly Reprogrammed With Proteins into Cardiac Progenitor Cells Heal Heart After a Heart Attack and Make New Heart Muscle


Jianjun Wang from Wayne State School of Medicine in Detroit, Michigan and Xi-Yong Yu from Guangzhou Medical University and a host of graduate students and postdoctoral research fellows in their two laboratories have teamed up to make human cardiac progenitor cells (CPCs) from human skin fibroblasts through direct reprogramming. Direct reprogramming does not go through a pluripotent intermediate, and, therefore, produces cells that have a low chance of generating tumors.

To begin their study, Wang, and Yu and their colleagues isolated fibroblasts from the lower regions of the skin (dermis) and grew them in culture. Then they reprogrammed these cells in a relatively novel manner. This is a little complicated, but I will try to keep it simple.

Reprogramming cells usually requires scientists to infect cells with recombinant viruses that have been genetically engineered to express particular genes in cells or force cells to take up large foreign DNA. Both of these techniques can work relatively well in the laboratory, but you are left with cells that are filled with foreign DNA or recombinant viruses. It turns out that directly reprogramming cells only requires transient expression of specific genes, and once the cells have recommitted to a different cell fate, the expression of the genes used to get them there can be diminished.

To that end, some enterprising scientists have discovered that inducing cells to up modified proteins can also reprogram cells. Recently a new reagent called the QQ-reagent system can escort proteins across the cell membrane. The QQ-reagent has been patented and can sweep proteins into mammalian cells with high-efficiency and low toxicity (see Li Q, et al (2008) Methods Cell Biol 90:287–325).

Wang and Yu and their coworkers used genetically engineered bacteria to overexpress large quantities of four different proteins: Gata4, Hand2, Mef2c, and Tbx5. Then they mixed these proteins with their cultured human fibroblasts in the presence of the QQ reagent. This reagent drew the proteins into the cells and the fibroblasts were reprogrammed into cardiac progenitor cells (CPCs). Appropriate control experiments showed that cells that were treated with QQ reagent without these proteins were not reprogrammed. Wang and Yu and they research groups also exposed the cells to three growth factors, BMP4 and activin A, to drive the cells to become heart-specific cells, and basic fibroblast growth factor to turn the cells towards a progenitor cell fate.

The next set of experiment was intended to show that their newly reprogrammed were of a cardiac nature. First, the cells clearly expressed heart-specific genes. Flk-1 and Isl-1 are genes that earmark cardiac progenitor cells, and by the eighth day of induction, the vast majority of cells expressed both these genes.

 

Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.
Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.

Second, cardiac cells can differentiate into three different cell types: heart muscle cells, blood vessels cells, and smooth muscle cells that surround the blood vessels. In mesoderm progenitors made from embryonic stem cells, inhibition of the Wnt signaling pathway can drive such cells to become heart muscle cells (see Chen, et al Nat Chem Biol 5:100–107; Willems E, et al Circ Res 109:360–364; Hudson J, et al Stem Cells Dev 21:1513–1523). However, Wang, Yu and company showed that treating the cells with a small molecule called IWR-1 that inhibits Wnt signaling drove their cells to differentiate into, not only heart muscle cells, but also endothelial (blood vessel) cells and smooth muscle cells when the cells were grown on gelatin coated dishes. When left to differentiate in culture, the cells beat synchronously and released calcium in a wave-like fashion that spread from one cell to another, suggesting that some cells were acting as pacemakers and setting the beat.

 

Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.
Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.

Then these cells were transplanted into the heart of mice that had suffered heart attacks. When compared to control hearts that received fluid, but no cells, the hearts of the animals that received protein-induced CPCs showed decreased scarring by 4 weeks after the transplantations. They also showed the growth of new heart muscle. A variety of staining experiments established that the engrafted protein-induced CPCs positive for heart muscle- and endothelial-specific cell markers. These experiments showed that transplantation of cardiac progenitor cells can not only help attenuate remodeling of the left ventricular after a heart attack, but that the protein-induced CPCs (piCPCs) can develop into cells of the cardiac lineage.

In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.
In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.

These are exciting results. It shows that direct reprogramming can occur without introducing genes into cells by means that can complicate the safety of the implanted cells. Also, because the cells are differentiated into progenitor cells, they still have the ability to proliferate and expand their numbers, which is essential for proper regeneration of a damaged tissue.

After a heart attack, the ventricle wall scars over and can become thin. However, piCPCs that have been directly reprogrammed from mature, adult cells can be used to replace dead heart muscle in a living animal.

Despite these exciting advances, further questions remain. For example, are the physiological properties of cells made from piCPCs similar enough to match the functional parameters of the heart into which they are inserting themselves? More work is necessary to answer that question. Functional equivalence is important, since a heart that does not function similarly from one end to the other can become arrhythmic, which is clinically dangerous. Further work is also required to precisely determine how well cells derived from piCPCs mature and coupling with neighboring cells. Therefore, larger animal studies and further studies in culture dishes will be necessary before this technique can come to the clinic. Nevertheless, this is a tremendous start to what will hopefully be a powerful and fruitful technique for healing damaged hearts.

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.

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.

McMaster Scientists Convert Blood into Neural Cells With One Gene


McMaster University stem cell scientists have discovered a way to adult sensory neurons from human patients simply by having them roll up their sleeve and provide a blood sample. The McMaster scientists directly converted adult human blood cells to both central nervous system (brain and spinal cord) neurons and peripheral nervous system (rest of the body) neurons responsible for pain, temperature and itch perception. This means that how a person’s nervous system cells react and respond to stimuli can be determined from their blood.

This breakthrough was published online recently and was also featured on the cover of the journal Cell Reports. The leader of this research, Mick Bhatia, serves as the director of the McMaster and Cancer Research Institute and holds the Canada Research Chair in Human stem Cell Biology and is a professor in the Department of Biochemistry and Biomedical Sciences in the Michael G. DeGroote School of Medicine.

Scientists do not have a robust understanding of pain and how to treat it. Neurons in the peripheral nervous system is composed of different types of nerves that detect mechanical forces like pressure or touch, and others and detect temperature, such as heat. Pain is perceived by the brain when signals are sent by peripheral pain receptors.

“The problem is that unlike blood, a skin sample or even a tissue biopsy, you can’t take a piece of a patient’s neural system. It runs like complex wiring throughout the body and portions cannot be sampled for study,” said Bhatia.

“Now we can take easy to obtain blood samples, and make the main cell types of neurological systems — the central nervous system and the peripheral nervous system — in a dish that is specialized for each patient,” said Bhatia. “Nobody has ever done this with adult blood. Ever.

“We can actually take a patient’s blood sample, as routinely performed in a doctor’s office, and with it we can produce one million sensory neurons, that make up the peripheral nerves in short order with this new approach. We can also make central nervous system cells, as the blood to neural conversion technology we developed creates neural stem cells during the process of conversion.”

This new protocol uses a gene called “Oct4” to directly reprogram blood cells. Additionally, if two proteins (SMAD and GSK-3) are inhibited with small molecules while the cells are transfected with the Oct4 gene, then the resultant cells transdifferentiate into blood-derived induced neural progenitor cells (BD-iNPCs). Now the direct conversion of skin cells called fibroblasts into neural progenitor cells that look a great like neural crest cells. However, these BD-iNPCs have the ability to differentiate into glial cells (support cells in the nervous system, multiple central nervous system neurons, and pain receptors, which are normally found in the peripheral nervous system.

image description
Using OCT-4-induced direct reprogramming, Lee et al. convert human blood to neural progenitors with both CNS and PNS developmental capacity. This fate alternation is distinct from fibroblasts that are primed for neural potential. Furthermore, human sensory neurons derived from blood phenocopy chemo-induced neuropathy in formats suitable for drug screening.

This new, revolutionary protocol that directly converts white blood cells into neurons with one gene has not only been patented, but has “broad and immediate applications,” according to Bhatia. He also added that it allows researchers to start asking questions about understanding disease and improving treatments. These cells could be used to determine why certain people feel pain instead of numbness, or whether or not the degree to which people perceive pain is genetically determines, or whether or not diabetic neuropathy ca be mimicked in a culture dish? Bhatia’s new protocol also provides a slick, new model system to find new pain drugs that don’t just numb the perception of pain, but completely block it.

“If I was a patient and I was feeling pain or experiencing neuropathy, the prized pain drug for me would target the peripheral nervous system neurons, but do nothing to the central nervous system, thus avoiding non-addictive drug side effects,” said Bhatia. “You don’t want to feel sleepy or unaware, you just want your pain to go away. But, up until now, no one’s had the ability and required technology to actually test different drugs to find something that targets the peripheral nervous system and not the central nervous system in a patient specific, or personalized manner.”

Bhatia’s team successfully tested their protocol by using fresh blood and frozen blood. This is an important piece of research since blood samples are usually taken and frozen. Freezing blood samples allows scientists or even physicians to create a kind of “time machine” that can show the evolution of a patient’s response to pain over a period of time.

For future studies, Bhatia and his colleagues would like to examine patients with Type 2 Diabetes to determine if his technique can help predict whether they will experience neuropathy by running tests in the lab using their own neural cells derived from their blood sample.

“This bench to bedside research is very exciting and will have a major impact on the management of neurological diseases, particularly neuropathic pain,” said Akbar Panju, medical director of the Michael G. DeGroote Institute for Pain Research and Care, a clinician and professor of medicine.

“This research will help us understand the response of cells to different drugs and different stimulation responses, and allow us to provide individualized or personalized medical therapy for patients suffering with neuropathic pain.”

New Technology Reprograms Skin Fibroblasts


Fibroblasts are one of the main components of connective tissue, which is the main reason scientists typically exploit them for experiments. A collaborative team of scientists from the University of Pennsylvania, Boston University, and the New Jersey Institute of Technology have invented a way to reprogram fibroblasts without going through a pluripotent stage.

The senior author of this study, Xiaowei Xu, associate professor of pathology and laboratory medicine at the University of Pennsylvania School of Medicine, said, “Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes . So these cells do not have tumorigenicity” (the ability to cause tumors).

Melanocytes are found in the skin and they are responsible for the pigment in our skin. They are in the uppermost layer of the skin, known as the epidermis, and produce melanin, a brown pigment that helps screen against the harmful effects of UV light.

Turning a fibroblast into a melanocytes might seem trivial for a stem cell scientist; just reprogram the fibroblast into an induced pluripotent stem cells and then differentiate it into a melanocytes. However, this procedure utilized direct reprogramming, in which the fibroblast was converted into a melanocytes without traversing through the pluripotent stage. The difficultly with direct reprogramming is finding the right cocktail of genes and/or growth factors that will accomplish the deed.

Xu and his colleagues began their search by examining the genes that are specific to melanocytes. They found 10 different transcription factors that are important for melanocytes development. Next they screened these ten genes for their ability to convert a fibroblast into a melanocytes. They found that of the ten melanocytes-specific genes, three of them, Sox10, MITF, and PAX3 could do the job effectively. They called this gene combination “SMP3.”

When Xu and others tested SMP3 on mouse embryonic fibroblasts, they quickly expressed melanocytes-specific genes. When Xu’s group used SMP3 on human fetal dermal cells, once again, the cells rapidly differentiated into melanocytes. Xu and his team referred to these cells as hi-Mel, which is short for human, induced melanocytes.

When hi-Mel were grown in culture they produced melanin a plenty. When they were implanted into the skin of pigmentless mice, once again they rose to the challenge and made a great deal of pigment. Thus hi-Mel express the same genes as melanocytes and they behave for all intents and purposes as melanocytes.

Xu and his colleagues think that their procedure might be able to treat human patients with a condition called vitiligo in which the skin has patches that are devoid of pigment.

Another potential use of this technology is a way to effectively study melanoma, one of the most dangerous skin cancers known to human medicine. My good friend and SAU colleague died over a year ago from melanoma and having better ways to treat this monster would have been marvelous for Charlie, and his family, who miss him dearly. By generating melanocytes from the fibroblasts of melanoma patients, they can “screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening.”.

Also, because so the body contains so many fibroblasts in the first place, this reprogramming technique is well-suited for other cell-based treatments.