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.”

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Long-term Tumorgenicity of Induced Pluripotent Stem Cells


A paper from the Okano laboratory has shown that implantation of neural stem cells made from induced pluripotent stem cells can still form tumors ever after a long period of time.

This paper is an important contribution to the safety issues surrounding induced pluripotent stem cells (iPSCs). As noted in previous posts, iPSCs are made from adult cells by means of genetic engineering and cell culture techniques. In short, by introducing four different genes into adult cells and then culturing them in a special culture medium, a fraction of these cells will de-differentiate into cells that resemble embryonic stem cells in many ways, but are not exactly like them.

The Okano laboratory made iPSCs using viruses that integrate into the genome of the host cell, which is not the safest option. However, because in the four-gene cocktail that is normally used to reprogram these cells (Oct-4, Klf-4, Sox2, and c-Myc), the c-Myc gene is often thought to be the main cause of tumor formation. Okano and his collaborators made their iPSCs without the c-Myc gene, but only used the three-gene cocktail of Oct-4, Klf-4, and Sox2. Such a cocktail is much less efficient that the four-gene cocktail, but it supposed to make iPSCs that are altogether safer.

These iPSCs were differentiated into neural stem cells that grew as tiny spheres of cells, and these “neurospheres” were transplanted into the spinal cords of mice that had suffered a spinal cord injury. The implanted cells differentiated into neurons and glial cells and restored some neural function to these mice. However, the mice were observed for a long period of time after the implantations to assess the long-term safety of these implanted cells.

After 105 days, the implanted mice began to show deterioration of their neural function and their spinal cords showed tumors. It is clear that the Oct-4 gene that was used in the reprogramming procedure was the reason for the tumor transformation.

Graphical Abstract 20141213

This experiment, once again, calls into question the safety of any method for iPSC generation that leaves the transfected genes in the reprogrammed cells. I reported in a previous post that skin cells made from iPSCs that had their transgenes left in them were good at causing tumors and not as good as forming skin cells, but iPSCs without their reprogramming transgenes were safer and more effective tools for regenerative medicine.  This experiment also shows that c-Myc is not the only concern with iPSCs.  Any of the transgenes used for reprogramming can cause problems, and they must be removed if iPSCs are going to produce safe, differentiated cells.  Finally, this experiment pretty much shows that the use of retrovirus tools to introduce genes into cells for the sake of reprogramming is a bad idea if those cells are going to be used for regenerative medicine.  Non-integrating tools are much safer and preferable in these cases.

The Okano paper appeared in Stem Cell Reports.

Patient-Specific Stem Cells Made More Easily?


A Michigan State University research team uncovered the function of an already characterized gene that could be linchpin in the derivation of patient-specific stem cells that might be able to save millions of lives by differentiating into practically any cell in the body.

The gene is known as ASF1A, and even though it was not discovered by the team, ASF1A is one of the genes responsible for the mechanism of cellular reprogramming. Cellular reprogramming de-differentiates adult cells into less mature stem cells that have the capacity to differentiate into any cell type in the adult body.

This work was published in the journal Science. In this paper, the MSU team analyzed more than 5,000 genes from a human egg (oocyte) and determines that ASF1A in combination with another gene known as OCT4 and another molecule were primarily responsible for reprogramming.

Human oocytes
Human oocytes

“This has the potential to be a major breakthrough in the way we look at how stem cells are developed,” said Elena Gonzalez-Munoz, a former MSU post-doctoral researcher and first author of the paper. “Researchers are just now figuring out how adult somatic cells such as skin cells can be turned into embryonic stem cells. Hopefully this will be the way to understand more about how that mechanism works.”

An MSU team identified the thousands of genes expressed in oocytes in 2006. From this list of genes, the genes responsible for cellular reprogramming were then identified.

In 2007, a Japanese research team led by Shinya Yamanaka found that by introducing four other genes into adult cells, they could derive embryonic-like stem cells without the use of a human egg. These cells are called induced pluripotent stem cells, or iPSCs. “This is important because the iPSCs are derived directly from adult tissue and can be a perfect genetic match for a patient,” said Jose Cibelli, an MSU professor of animal science and a member of the team.

Apparently, ASF1A and OCT4 work in together in combination with a hormone-like substance that also is produced in the oocyte called GDF9 to facilitate the reprogramming process. “We believe that ASF1A and GDF9 are two players among many others that remain to be discovered which are part of the cellular-reprogramming process,” Cibelli said.

“We hope that in the near future, with what we have learned here, we will be able to test new hypotheses that will reveal more secrets the oocyte is hiding from us,” he said. “In turn, we will be able to develop new and safer cell-therapy strategies.”

Repopulation of Damaged Livers With Skin-Derived Stem Cells


Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

Physical Cues Push Mature Cells into Induced Pluripotent Stem Cells


Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.

Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.

Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.

retrovirus_life_cycle

Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).

The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).

“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”

a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p<0.05 (two-tailed, unpaired t-test) compared with the control flat surface. Error bars represent one standard deviation. g, Immunostaining of a stable iPSC line expanded from colonies generated on 10 μm grooves. These cells express mESC-specific markers Oct4, Sox2, Nanog and SSEA-1 (scale bar, 100 μm). h, The expanded iPSCs in g were transplanted into SCID mice to demonstrate the formation of teratomas in vivo (scale bar, 50 μm).
a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p

To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.

“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”

This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.

a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p<0.05 (two-tailed, unpaired t-test) compared with the control surface with random nanofibres. e, Fibroblasts were micropatterned into single-cell islands of 2,000 μm2 area with a CSI value of 1 (round) or 0.1 (elongated). After 24 h, cells were immunostained for AcH3, H3K4me2 or H3K4me3 (in green). Phalloidin staining (red) identifies the cell cytoskeleton for cell shape accuracy. The white arrowhead indicates the location of the nucleus (scale bars, 20 μm). f, Quantification of fluorescence intensity in e (n = 34, 20 and 34 for AcH3, H3K4me2 and H3K4me3, respectively). *p<0.05 (two-tailed, unpaired t-test) compared with the circular micropatterned cells (CSI = 1). Error bars represent one standard deviation.
a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p

“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”

Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.

“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.

Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).