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.”

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.

Direct Reprogramming Cells with Recombinant Proteins


In my opinion, for what it’s worth, we will probably see direct reprogramming take a prominent place in regenerative medicine in the future. It will not be in the near future, but as direct reprogramming becomes better understood and more feasible, it will probably become a central part of the discussion of regenerative medical strategies.

Direct reprogramming, which is also known as lineage conversion, uses cell type-specific transcription factors to convert a mature, adult cell into a different type of mature, adult cell. The cell does not pass through a pluripotent intermediate, and becomes a wholly different type of cell.

Of course, forcing the expression of lineage-specific transcription factors in cells requires that they be treated with recombinant viruses or other such tools. These genetic manipulations present problems for regenerative medicine, since such viruses can cause mutations or cause the introduced genes to be constantly activated, both of which can cause cells to die to grow uncontrollably. Genetically engineering cells needs to be done in a “kinder and gentler” way (to quote George HW Bush).

To that end Dennis Clegg and his colleagues from the Center for Stem Cell Biology and Engineering at UC Santa Barbara have used specially designed proteins to directly cultured retinal pigmented epithelial cells to neurons.

Newly discovered C-end rule (CendR) cell- and tissue-penetrating peptides have a arginine-rich sequence at the end of proteins that allows them to bind particular cell receptors and be internalized into the cell. These CendR peptides bind to the NRP-1 protein and are internalized. Several laboratories have used CendR peptides to increase the efficacy of anti-cancer drugs in experimental cases (see Alberici L, et al (2013) Cancer Res 73:804–812; Sugahara KN, et al. (2010) Science 328:1031–1035; Sugahara KN, et al. (2009) Cancer Cell 16:510–520; and Roth L,, et al. (2012) Oncogene 31:3754–3763).

By tacking a CendR peptide to the end of the Sox2 protein, Clegg and others were able to convert retinal pigmented epithelial (RPEs) cells to neurons. The Sox2 protein is highly expressed in neural progenitor cells. Other studies have shown that Sox2 can reprogram mouse and human fibroblasts to neural stem cells (Ring KL, et al. (2012) Cell Stem Cell 11:100–109). Thus, Sox2 should do the trick.

Making cultured RPE cells from embryonic stem cells is relatively easy to do. Therefore, Clegg and his coworkers made cultured RPEs and then treated them with viruses that expressed Sox2. The cultured RPEs showed conversion to neurons and the expression of neuron-specific genes.

Since they had established that Sox2 could convert RPEs to neurons, they tried recombinant Sox2 protein with the CendR peptide RPARPAR at the end of the protein. After 60 days in culture, the cells expressed a host of neuron-specific genes, and were capable of taking up a dye that only active neurons can take up (FM1-43).

Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.
Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.

The efficiency for this experiment was lousy (0.3%) as opposed to the efficiency for the use of recombinant viruses (11%). Nevertheless, this experiment shows that it is possible to directly reprogram cells without using recombinant viruses.

Putting Peps in Your Heps


The liver is a special organ that performs a whole host of essential functions. The liver stores iron, vitamins and minerals; it detoxifies alcohol, drugs, and other chemicals that accumulate in our bloodstreams, and it produces bile (used to dissolve fats so that they can be degraded), and blood-based proteins like clotting factors and albumin. The liver also stores sugar in the form of glycogen. All of these tasks are undertaken by a single cell type, the hepatocyte (otherwise known as a liver cell).

human-liver-diagram

When your liver fails, you get really sick. This was greatly illustrated to me by one of my colleagues where I teach whose wife suffered extensive liver damage as a result of her battle with lupus (short for systemic lupus erythematosus, an autoimmune disease). Now that this dear lady has had a liver transplant, she is a new person. What a difference a healthy liver makes.

What can regenerative medicine do for patients with failing livers? Human pluripotent stem cells, either embryonic stem cells or induced pluripotent stem cells, can be directed to differentiate into liver cells in culture, but the liver cells made by these cells are very immature. They express proteins commonly found in fetal liver cells (for example, alpha-fetoprotein) and they also lack key enzymes associated with adult cells (such as cytochrome P450s). Rashid and others in the Journal of Clinical Investigation (2010; 120: 3127-3136) showed this. The development of three-dimensional culture systems have increased the maturity of such cells, but there is still a long way to go (see T Takebe and others, Nature 2013; 499:481-484 and J Shan and others, Nature Chemical Biology 2013; 9: 514-520).

Two papers from the journal Cell Stem Cell might show a way forward to making mature liver cells for regenerative liver treatments without destroying embryos or even using and pluripotent stem cell lines. These papers utilize the procedure known as “direct reprogramming,” otherwise known as “direct lineage conversion.” Direct reprogramming requires the forced overexpression of particular genes that causes the cells to switch their cell types.

In the first of these papers, Pengyu Huang and his colleagues from the Chinese Academy of Sciences in Shanghai, China overexpressed a three-gene combination in mouse embryonic fibroblasts that converted the cells into hepatocytes at an efficiency of 20% after 14 days in culture. This gene combination, known as 3TF (HNF4/HNF1A/FOXA3), converted the mouse embryonic skin cells into mature liver cells that made blood proteins and drug-processing enzymes. The only problem was that these mature cells could not grow in culture because they were mature. Therefore, Huang and others infected these cells with a virus called SV40, which drove the cells to divide. Now these cells could be grow in culture and expanded for further experiments.

When transplanted into the livers of mice with failing livers, the induced liver cells made by Huang and others restored proper liver function and allowed the mice to survive.

A second paper by Yuanyuan Du and others from the Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China, used a large gene combination to make mature liver cells from human skin fibroblasts. This gene combination included eight genes (HNF1A/HNF4A/HNF6/ATF5/PROX1/CEBPA/p53 ShRNA/C-MYC) that converted the human skin cells into liver cells after 30 days in culture at an efficiency of nearly 80%. Again, these cells metabolized drugs as they should, made blood proteins, took up cholesterol, and stored glycogen. Du and others compared the gene expression profile of these human induced hepatocytes or “hiHeps” to the gene expression profile of liver cells taken from liver biopsies. While there were differences in gene expression, there was also significant overlap and a large overall similarity. In fact the authors state, “these results indicate that hiHeps show a similar expression profile to primary human hepatocytes.”

Next, Du and others used three different mouse models of liver failure in all three cases, the hiHeps were capable of colonizing the damaged liver of the mouse and regenerating it. Mind you, the hiHeps did not do as good a job as human primary hepatocytes, but they still worked pretty well. This shows that this direct reprogramming protocol, as good as it is, can still be optimized and improved.

These studies show that the production of highly functional human hepatocyte-like cells using direct reprogramming is feasible and represents an exciting step towards the production of a supply source of cells for drug development, and therapies for liver disease.

Directly Reprogramming Skin Cells into White Blood Cells


Scientists from the Salk Institute have, for the first time, directly converted human skin cells into transplantable white blood cells, which are the soldiers of the immune system that fight infections and invaders. This work could prompt the creation of new therapies that introduce new white blood cells into the body that can attack diseased or cancerous cells or augment immune responses for other conditions.

This work, which shows that only a small amount of genetic manipulation could prompt this direct conversion, was published in the journal Stem Cells.

“The process is quick and safe in mice,” says senior author Juan Carlos Izpisua Belmonte, who holds the Salk’s Roger Guillemin Chair. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

The problems that Izpisua Belmonte mentions, includes the long time (at least two months) numbingly tedious cell culture work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells. Blood cells derived from iPSCs also have other obstacles: they engraft into organs or bone marrow poorly and can cause tumors.

The new method designed by Izpisua Belmonte and his team, however, only takes two weeks, does not produce tumors, and engrafts well.

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells,” says one of the first authors and Salk researcher Ignacio Sancho-Martinez. “Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

This faster reprogramming technique developed by Belmonte’s team utilized a form of reprogramming that does not go through a pluripotency stage. Such techniques are called indirect lineage conversion or direct reprogramming. Belmonte’s group has demonstrated that such approaches can reprogram cells to form the cells that line blood vessels. Thus instead of de-differentiating cells into an embryonic stem cell-type stage, these cells are rewound just enough to instruct them to form the more than 200 cell types that constitute the human body.

Direct reprogramming used in this study uses a molecule called SOX2 to move the cells into a more plastic state. Then, the cells are transfected with a genetic factor called miRNA125b that drives the cells to become white blood cells. Belmonte and his group are presently conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

“It is fair to say that the promise of stem cell transplantation is now closer to realization,” Sancho-Martinez says.

Study co-authors include investigators from the Center of Regenerative Medicine in Barcelona, Spain, and the Centro de Investigacion Biomedica en Red de Enfermedades Raras in Madrid, Spain.

Skin Cells Converted into Blood Cells By Direct Reprogramming


Making tissue-specific progenitor cells that possess the ability to survive, but have not passed through the pluripotency state is a highly desirable goal of regenerative medicine. The technique known as “direct reprogramming” uses various genetic tricks to transdifferentiate mature, adult cells into different cell types that can be used for regenerative treatments.

Juan Carlos Izpisua Belmonte and his colleagues from the Salk Institute for Biological Studies in La Jolla, California and his collaborators from Spain have used direct reprogramming to convert human skin cells into a type of white blood cells.

These experiments began with harvesting skin fibroblasts from human volunteers that were then forced to overexpress a gene called “Sox2.” The Sox2 gene is heavily expressed in mice whose bone marrow stem cells are being reconstituted with an infusion of new stem cells. Thus this gene might play a central role is the differentiation of bone marrow stem cells.

Sox2 overexpression in human skin fibroblasts cause the cells express a cell surface protein called CD34. Now this might seem so boring and unimportant, but it is actually really important because CD34 is expressed of the surfaces of hematopoietic stem cells. Hematopoietic stem cells make all the different types of white and red blood cells in our bodies. Therefore, the expression of these protein is not small potatoes.

In addition to the expression of CD34, other genes found in hematopoietic stem cells were also induced, but not strongly. Thus overexpression of SOX2 seems to induce an incipient hematopoietic stem cell‐like status on these fibroblasts. However, could these cells be pushed further?

Gene profiling of hematopoietic stem cells from Umbilical Cord Blood identified a small regulatory RNA known as miR-125b as a factor that pushes SOX2-generated CD34+ cells towards an immature hematopoietic stem cell-like progenitor cell that can be grafted into a laboratory animal.

When SOX2 and miR-125b were overexpressed in combination, the cells transdifferentiated into monocytic lineage progenitor cells.

What are monocytes? They are a type of white blood cells and are, in fact, the largest of all white blood cells. Monocytes compose 2% to 10% of all white blood cells in the human body. They play multiple roles in immune function, including phagocytosis (gobbling up bacteria and other stuff), antigen presentation (identifying and altering other cells to the presence of foreign substances), and cytokine production (small proteins that regulate the immune response).

Monocytes express a molecule on their cell surfaces called CD14, and when human fibroblasts overexpressed Sox2 and miR-125b, they became CD14-expressing cells that looked and acted like monocytes. These cells were able to gobble up bacteria and other foreign material, and when transplanted into a laboratory animal, these directly reprogrammed cells generated cells that established the monocytic/macrophage lineage.

Cancer patients, and other patients with bone marrow diseases can have trouble making sufficient white blood cells. A technique like this can generate transplantable monocytes (at least in laboratory animals) without many of the drawbacks associated with reprogramming human cells into hematopoietic stem cells that possess true clinical potential. Also because this technique skips the pluipotency stage, it is potentially safer.

Digestive Cells Converted into Insulin-Secreting Cells


By switching off a single gene, Columbia Medical Center scientists have converted cells from the digestive tract into insulin-secreting cells. This suggests that drug treatments might be able to convert gut cells into insulin-secreting cells.

Senior author Domenico Accili said this of this work: “People have been talking about turning one cell into another for a long time, but until now we hadn’t gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target.”

Accili’s work suggests that lost pancreatic beta cells might be replaced by retraining existing cells rather than transplanting new insulin-secreting cells. For nearly two decades, scientists have been trying to differentiate a wide variety of stem cells into pancreatic beta cells to treat type 1 diabetes. In type 1 diabetes, the patient’s insulin-producing beta cells are destroyed, usually by the patient’s own immune system. The patient becomes dependent on insulin shots in order to survive.

Without insulin, cells have no signal to take up sugar and metabolize it. Also muscles and the liver do not take up amino acids and make protein, and the body tends to waste away, ravaged by high blood sugar levels that progressively and relentlessly damage it without the means to repair this damage.

Insulin-producing beta cells can be made in the lab from several different types of stem cells, but the resulting beta cells often do not possess all the properties of naturally occurring beta cells.

This led Accili and others to attempt to transform existing cells into insulin-secreting beta cells. In previous work, Accili and others demonstrated that mouse intestinal cells could be converted into insulin-secreting cells (see Talchai C, et al., Nat Genet. 2012 44(4):406-12), This recent paper demonstrates that a similar technique also works in human intestinal cells.

The gene of interest, FOXO1, is indeed present in human gut endocrine progenitor and serotonin-producing cells. In order to determine in FOXO1 inhibition could induce the formation of insulin-secreting cells, Accili and others used human induced pluripotent stem cells (iPSCs) and small “gut organoids,” which are small balls of gut tissue that grow in culture.

Inhibition of FOXO1 by either introducing a mutant version of the gene that encoded a protein that soaked up all the wild-type protein or by using viruses that forced the expression of a small RNA that prevented the expression of the FOXO1 gene caused loss of FOXO1 activity. FOXO1 inhibition promoted the generation of insulin-positive cells within the gut organoids that express all the genes and proteins normally found in mature pancreatic β-cells. These transdifferentiated cells also released “C-peptide,” which is a byproduct of insulin production, in response to drugs that drive insulin secretion (insulin secretagogues). Furthermore, these cultured insulin-secreting cells and survive when transplanted into mice where they continue to secrete insulin in response to increased blood sugar concentrations.

The findings of Accili and his colleagues provide some evidence that gut-targeted FOXO1 inhibition or transplantation of cultured gut organoids made from iPSCs could serve as a source of insulin-producing cells to treat human diabetes.

This is a remarkable piece of research, but there is one thing that troubles me about it. If the patient’s immune system has been sensitized to beta cells, making new beta cells will simply give the immune system something else to attack. It seems to me that retraining to immune system needs to be done first before replacement of the beta cells can ever hope to succeed.

Mouse Blood Cells Reprogrammed into Blood Cell Stem Cells


Boston Children’s Hospital researchers have directly reprogrammed mature blood cells from mice into blood-forming hematopoietic stem cells by using a cocktail of eight different transcription factors.

These reprogrammed cells have been called induced hematopoietic stem stem cells or iHSCs. These cells have all the hallmarks of mature mouse HSCs and they have the capacity to self-renew and differentiate into all the blood cells that circulate throughout the body.

These findings are highly significant from a clinical perspective because they indicate that it might be entirely possible to directly reprogram a patient’s existing, mature blood cells into a hematopoietic stem cell for transplantation purposes. Such a procedure, known as hematopoietic stem cells transplantation or HSCT, is a common treatment for patients whose bone marrow has suffered irreparable damage due to environmental causes (heavy metals, chloramphenicol, etc) or disease (cancer). The problem with HSCT is finding a proper match for the patient and then procuring clinically significant quantities of high-quality bone marrow for HSCT.

Derrick J. Rossi, an investigator in the Program in Cellular and Molecular Medicine at Boston Children’s Hospital and Assistant Professor in the Department of Stem Cell & Regenerative Biology, explained: “HSCs comprise only about one in every 20,000 cells in the bone marrow. If we could generate autologous (a patient’s own) HSCs from other cells, it could be transformative for transplant medicine and for our ability to model diseases of blood development.”

Rossi and his collaborators have screened genes that are expressed in 40 different types of blood progenitor cells in mice. This screen identified 36 different genes that control the expression of the other genes. These 36 genes encode so-called “transcription factors,” which are proteins that bind to DNA and turn gene express on or shut it off.

Blood cell production tends to go from the stem cells to progeny cells called progenitor cells that can still divide to some limited extent, and to effector cells that are completely mature and, in most cases, do not divide (the exception is lymphocytes, which expand when activated by specific foreign substances called antigens).

Further work by Rossi and others identified six transcription factors (Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, and Prdm5) of these 36 genes, plus two others that were not part of their original screen (N-Myc and Meis1) that could robustly reprogram myeloid progenitor cells or pro/pre B lymphocytes into iHSCs.

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To put these genes into these blood cells, Rossi and others uses souped-up viruses that introduced all either genes under the control of sequences that only allowed expression of these eight genes in the presence of the antibiotic doxycycline. Once these transfected cells were transplanted into mice, the recipient mice were treated with doxycycline, and the implanted cells formed HSCs.

When this experiment utilized mice that were unable to make their own blood cells, because their bone marrow had been wiped out, the implanted iHSCs reconstituted the bone marrow and blood cells of the recipient mice.

To further show that this reconstituted bone marrow was normal, high-quality bone marrow, Rossi and others used these recipient mice as bone marrow donors for sibling mice whose bone marrow had been wiped out. This experiment showed that the mice that had received the iHSCs had bone marrow that completely reconstituted the bone marrow of their siblings. This established that the iHSCs could completely reestablish the bone marrow of another mouse.

Thus Rossi and others had established that iHSCs could in fact created HSCs from progenitor cells, but could they do the same thing with mature blood cells that were not progenitor cells? Make that another yes. When Rossi and others transfected their eight-gene cocktail into mature myeloid cells, these mature cells also made high-quality iHSCs.

Rossi noted that no one has been able to culture HSCs in the laboratory for long periods of time. A few laboratories have managed expand HSCs in culture, but only on a limited basis for short periods of time (see Aggarwal R1, Lu J, Pompili VJ, Das H. Curr Mol Med. 2012 Jan;12(1):34-49).  In these experiments, Rossi used his laboratory mice as living culture systems to expand his HSCs, which overcomes the problems associated with growing these fussy stem cells in culture.

Gene expression studies of his iHSCs established that, from a gene expression perspective, the iHSCs were remarkably similar to HSCs isolated from adult mice.

This is certainly an exciting advance in regenerative medicine, but it is far from being translated into the clinic.  Can human blood progenitor cells also be directly reprogrammed using the same cocktail?  Can mature myeloid cells be successfully reprogrammed?  Will some non-blood cell be a better starting cell for iHSC production in humans?  As you can see there are many questions that have to be satisfactorily answered before this procedure can come to the clinic.

Nevertheless, Rossi and his team has succeeded where others have failed and the results are remarkable.  HSCs can be generated and transplanted with the use of only a few genes.  This is certainly the start of what will hopefully be a fruitful regenerative clinical strategy.

On a personal note, my mother passed about almost a decade ago after a long battle with myelodysplastic syndrome, which is a pre-leukemic condition in which the bone marrow fails to make mature red blood cells.  Instead the bone marrow fills up with immature red blood cells and the patient has to survive on blood transfusions.

The reason for this condition almost certainly stems from defective HSCs that do not make normal progeny.  Therefore the possibility of using a patient’s own cells to make new HSCs that can repopulate the bone marrow is a joyful discovery for me to read about, even though it is some ways from the clinic at this point.

Directly Reprogramming Gut Cells into Beta Cells to Treat Diabetes


Type 1 diabetes mellitus results from destruction of insulin-producing beta cells in the pancreas. Diabetics have to give themselves routine shots of insulin. The hope that stem cells offer is the production of cells that can replace the lost beta cells. “We are looking for ways to make new beta cells for these patients to one day replace daily insulin injections,” says Ben Stanger, MD, PhD, assistant professor of Medicine in the Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania.

Some diabetics have had beta cells from cadavers transplanted into their bodies to replace the missing beta cells. Such a procedure shows that replacement therapy is, in principle possible. Therefore, transplanting islet cells to restore normal blood sugar levels in type 1 diabetics could treat and even cure disease. Unfortunately, transplantable islet cells are in short supply, and stem cell-based approaches have a long way to go before they reach the clinic. However, Stanger and his colleagues have tried a different strategy for treating type 1 diabetes. “It’s a powerful idea that if you have the right combination of transcription factors you can make any cell into any other cell. It’s cellular alchemy,” comments Stanger.

New research from Stanger and a postdoctoral fellow in his laboratory, Yi-Ju Chen that was published in Cell Reports, describes the production of new insulin-making cells in the gut of laboratory animals by introducing three new transcription factors. This experiment raises the prospect of using directly reprogrammed adult cells as a source for new beta cells.

In 2008, Stanger and others in Doug Melton’s laboratory used three beta-cell reprogramming factors (Pdx1, MafA, and Ngn3, collectively called PMN) to convert pancreatic acinar cells (the cells in the pancreas that secrete enzymes rather than hormones) into cells that had many of the features of pancreatic beta cells.

Following this report, the Stanger and his team set out to determine if other cells types could be directly reprogrammed into beta cells. “We expressed PMN in a wide spectrum of tissues in one-to-two-month-old mice,” says Stanger. “Three days later the mice died of hypoglycemia.” It was clear that Stanger and his crew were on to something. Further work showed that some of the mouse cells were making way too much extra insulin and that killed the mice.

When the dead mice were autopsied, “we saw transient expression of the three factors in crypt cells of the intestine near the pancreas,” explained Stanger.

They dubbed these beta-like, transformed cells “neoislet” cells. These neoislet cells express insulin and show outward structural features akin to beta cells. These neoislets also respond to glucose and release insulin when exposed to glucose. The cells were also able to improve hyperglycemia in diabetic mice.

Stanger and his co-workers also figured out how to turn on the expression of PMN in only the intestinal crypt cells to prevent the deadly whole-body hypoglycemia side effect that first killed the mice.

In culture, the expression of PMN in human intestinal ‘‘organoids,’ which are miniature intestinal units grown in culture, also converted intestinal epithelial cells into beta-like cells.

“Our results demonstrate that the intestine could be an accessible and abundant source of functional insulin-producing cells,” says Stanger. “Our ultimate goal is to obtain epithelial cells from diabetes patients who have had endoscopies, expand these cells, add PMN to them to make beta-like cells, and then give them back to the patient as an alternate therapy. There is a long way to go for this to be possible, including improving the functional properties of the cells, so that they more closely resemble beta cells, and figuring out alternate ways of converting intestinal cells to beta-like cells without gene therapy.”

This is hopefully a grand start to what might be a cure for type 1 diabetes.

Transformation of Non-Beating Human Cells into Heart Muscle Cells Lays Foundation for Regenerating Damaged Hearts


After a heart attack, the cells within the damaged part of the heart stop beating and become ensconced in scar tissue. Not only does this region not beat, it does not conduct the signal to beat either and that can not only lead to a slow, sluggish heartbeat, it can also cause irregular heart rates or arrhythmias.

Now, however, scientists have demonstrated that this damage to the heart muscle need not be permanent. Instead there is a way to transform those cells that form the human scar tissue into cells that closely resemble beating heart cells.

Last year, researchers from the laboratory of Deepak Srivastava, MD, the director of Cardiovascular and Stem Cell Research at the Gladstone Institute, transformed scar-forming heart cells (fibroblasts) into beating heart-muscle cells in live mice. Now they report doing the same to human cells in a culture dishes.

“Fibroblasts make up about 50 percent of all cells in the heart and therefore represent a vast pool of cells that could one day be harnessed and reprogrammed to create new muscle,” said Dr. Srivastava, who is also a professor at the University of California, San Francisco. “Our findings here serve as a proof of concept that human fibroblasts can be reprogrammed successfully into beating heart cells.”

In 2012, Srivastava and his team reported that fibroblasts could be reprogrammed into beating heart cells by injecting just three genes (collectively known as GMT, which is short for Gata4, Mef2c, and Tbx5), into the hearts of live mice that had been damaged by a heart attack (Qian L, et al., Nature. 2012 31;485(7400):593-8). From this work, they reasonably concluded that the same three genes could have the same effect on human cells.

“When we injected GMT into each of the three types of human fibroblasts (fetal heart cells, embryonic stem cells and neonatal skin cells) nothing happened—they never transformed—so we went back to the drawing board to look for additional genes that would help initiate the transformation,” said Gladstone staff scientist Ji-dong Fu, Ph.D., the study’s lead author. “We narrowed our search to just 16 potential genes, which we then screened alongside GMT, in the hopes that we could find the right combination.”

The research team began by injecting all candidate genes into the human fibroblasts. They then systematically removed each one to see which were necessary for reprogramming and which were dispensable. In the end, they found that injecting a cocktail of five genes—the 3-gene GMT mix plus the genes ESRRG and MESP1—were sufficient to reprogram the fibroblasts into heart-like cells. They then found that with the addition of two more genes, called MYOCD and ZFPM2, the transformation was even more complete.

To help things along, the team used a growth factor known as Transforming Growth Factor-Beta (TGF-Beta) to induce a signaling pathway during the early stages of reprogramming that further improved reprogramming success rates.

“While almost all the cells in our study exhibited at least a partial transformation, about 20 percent of them were capable of transmitting electrical signals—a key feature of beating heart cells,” said Dr. Fu. “Clearly, there are some yet-to-be-determined barriers preventing a more complete transformation for many of the cells. For example, success rates might be improved by transforming the fibroblasts within living hearts rather than in a dish—something we also observed during our initial experiments in mice.”

The immediate next steps are to test the five-gene cocktail in hearts of larger mammals. Eventually, the team hopes that a combination of small, drug-like molecules could be developed to replace the cocktail, which would offer a safer and easier method of delivery.

This latest study was published online August 22 in Stem Cell Reports.

Reprogramming Skin Cells into Neural Stem Cells By Introducing One Gene


Transforming skin cells into nerve cells that interconnect and send nerve impulses to each other requires an extensive amount of reprogramming. The production of induced pluripotent stem cells is rather labor-intensive and introduces some risks. However, a new procedure designed by Yadong Huang at the Gladstone Institutes has shown that the introduction of a single gene into skin cell can generate nerve cells from skin cells.

This single gene, Sox2, transforms skin cells within days into early-stage brain stem cells known as induced neural stem cells or iNSCs. In culture, iNSCs self-renew and mature into neurons that can connect with each other and then transmit electro-chemical signals between each other. When the iNSCs were cultured for one month, they had already formed a completely new neural network.

An excited Huang made these points: “Many drug candidates, especially those developed for neurodegenerative diseases, fail in clinical trials because current models don’t accurately predict the drug’s effects on the human brain. Human neurons derived from reengineered skin cells could help assess the efficacy and safety of these drugs, thereby reducing risks and resources associated with human trials.”

Huang’s findings build on the work of Japanese research Shinya Yamanaka, who was the first scientist to publish the production of induced pluripotent stem cells. Since that time, other researchers have used genetic engineering techniques to directly reprogram adult cells into other types of adult cells without passing through the embryonic-stem-cell stage. Last year, Sheng Ding managed to use a combination of small molecules and genes to transform skin cells directly into neural stem cells. Huang’s technique now simplifies this technique even more so that only one gene is required to reprogram skin cells into neural stem cells. By avoiding the induced pluripotent stem cell stage, Huang and Ding hope to avoid the risk of tumor formation and the mutations induced by the production of induced pluripotent stem cells.

Karen Ring, a graduate student in Biomedical Sciences at the University of California, San Francisco, who was the lead author on this paper vouched for the safety of the iNSCs: “We wanted to see whether these newly generated neurons could result in tumor growth after transplanting them into mouse brains. Instead, we saw the reprogrammed cells integrate into the mouse’s brain, and not a single tumor developed.”

Huang’s paper also addresses the function Sox2 in the reprogramming of the skin cells. Huang and his research team also want to identify similar regulators that direct the development of specific types of neurons in the brain that tend to degenerate in the case of particular types of neurodegenerative diseases. Huang noted: “If we can pinpoint which genes control the development of each neuron type, we can generate them in the Petri dish from a single sample of human skin cells. We could then test drugs that affect different neuron types, such as those involved in Parkinson’s disease.” Huang added that such a discovery would help drug developers design treatments for neurodegenerative diseases that are much more specific, and the drug design would probably occur much faster.

Alzheimer’s disease still afflicts 5.4 million people in the US alone and this number is thought to triple by 2050. There are still no medications that can reverse the devastation wrought by this disease. Huang’s data might provide the means to test such new drugs.

Neurons from Skin Cells


Can we make nerve cells from skin cells? The answer seems to be yes. Furthermore, it seems to be really easy to do.

Marius Wernig at the Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology at Stanford University School of Medicine had published a remarkable paper. They started from a pool of nineteen candidate genes, but they identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that rapidly and efficiently convert mouse embryonic and adult mouse fibroblasts into functional neurons in vitro. He called these cells induced neuronal (iN) cells. He further showed that they expressed multiple neuron-specific proteins, and were able to generate nerve impulses and form functional connections between other nerve cells (synapses). Because they could make iN cells from non-nerve cells, they might be able to make large quantities studies of neurons for research.

Of even greater importance is the ability to make nerve cells for regenerative medicine. While it is too early to get too excited about this, the ability to form neurons from your own skin cells to repair spinal cord injuries and other neurological disorders without killing embryos is thrilling to say the least.

A recipe for heart cells from amnion


Embryonic stem cells can be made from adult cells. Such cells are called iPSCs or induced embryonic stem cells, and they have all the characteristics of embryonic stem cells made by means of the destruction of embryos.

Lately, scientists have found a way to convert one type of adult cell into another type of adult without going through any embryonic step.

Qi Zhou and his colleagues from the Melton lab at Harvard were able to transform pancreatic enzyme-secreting cells (exocrine cells) into insulin-secreting cells by inserting three transcription factors (Ngn3, also known as Neurog3), Pdx1 and Mafa into the exocrine cells and they reprogrammed themselves into beta-cells (Nature 455, 627–32 (2008)). Also, Yechoor and his colleagues used a similar technique that placed neurogenin into liver cells in a live animal. These animals shows insulin-secreting cells into their livers, which showed that the liver cells had been reprogrammed into beta cells (V. Yechoor et al., Dev. Cell 16, 358–73 (2009)).

This shows that reprogramming is vastly superior and cheaper than making cloned embryos that we subsequently kill and use to make embryonic stem cells. This is the therapeutic way of the future.

Now, Jun Takeuchi and Benoit Bruneau at the Gladstone Institute of Cardiovascular Disease in San Francisco have found that adding cardiac-specific genes to developing mouse embryos can make even some extra-embryonic parts become beating heart cells.  They made the cells from amnion, the thin layer that surrounds the embryo and fetus throughout development.  This is the sac that breaks when we say that a mother’s water breaks.  The amnion is normally medical waste, but can now be used to make heart cells.

See this link for the paper.