Shinya Yamanaka's 2006 discovery of induced pluripotent stem cells (iPSCs) ignited a revolution in the field of stem cell biology (1). For the first time, nearly all human somatic tissues could be produced from iPSCs reprogrammed from blood or skin cells, in a process that took only weeks. This advance was particularly crucial for obtaining surrogate tissues from cell types that are otherwise difficult to procure and do not readily expand in vitro, such as cardiac or neural cells. Additionally, many ethical concerns are avoided, because this technology uses a patient's own genetic material to create iPSCs rather than relying on embryonic stem cells. In the aftermath of Yamanaka's discovery, entire biomedical industries have developed around the promise of using human iPSCs (hiPSCs) and their derivatives for in vitro disease modeling, drug screening, and cell therapy (2).

The hiPSC technology has had a particularly notable impact in cardiac regenerative medicine, a field where scientists and clinicians have been working to devise new methods to better understand how cardiovascular disease manifests and how to restore cardiovascular function after disease strikes (3). The heart is limited in its ability to regenerate lost cardiomyocytes (beating heart muscle cells), following an adverse event such as a heart attack (4). Cardiomyocytes derived from hiPSCs (hiPSC-CMs) may represent a potential replacement option for dead cells in such a scenario. However, certain issues remain to be addressed, such as whether hiPSC-CMs can integrate with host myocardial tissue in the long term (5).

While using hiPSC-CMs for in vivo cell therapy may become practical in the future, employing hiPSC-CMs for high-throughput drug discovery and screening is becoming a reality in the present (6). Cardiovascular diseases can be recapitulated in a dish with patient-specific hiPSC-CMs. For example, if a patient exhibits a cardiac arrhythmia caused by a genetic abnormality in a sarcomeric protein or ion channel, that same rhythm problem can be recapitulated in vitro (7). Thanks to advances in hiPSC differentiation protocols, hiPSC-CMs can now be mass-produced to study cardiovascular disease mechanisms in vitro (8).

My graduate thesis in the laboratories of Joseph Wu and Sean Wu at Stanford University focused on in vitro applications of hiPSC-CMs for cardiovascular disease modeling and for high-throughput screening of chemotherapeutic compounds to predict cardiotoxicity. I initially embarked on a project using hiPSC-CMs to model viral myocarditis, a viral infection of the heart, caused by the B3 strain of coxsackievirus (9). I began by demonstrating that hiPSC-CMs express the receptors necessary for viral internalization and subsequently found that hiPSC-CMs were highly susceptible to coxsackievirus infection, exhibiting viral cytopathic effect within hours of infection. I also identified compounds that could alleviate coxsackievirus infection on hiPSC-CMs, a translationally relevant finding, as there remains a shortage of treatments for viral myocarditis.

Using a genetically modified variant of coxsackievirus B3 expressing luciferase, I developed a screening platform for assessing the efficacy of antiviral compounds. Pretreatment with interferon-, ribavirin, or pyrrolidine dithiocarbamate markedly suppressed viral replication on hiPSC-CMs by activating intracellular antiviral response and viral protein clearance pathways. These compounds alleviated viral replication in a dose-dependent fashion at low concentrations without causing cellular toxicity.

I next sought to use hiPSC-CMs to screen anticancer chemotherapeutic compounds for their off-target cardiovascular toxicities (10). Cardiotoxicity represents a major cause of drug withdrawal from the pharmaceutical market, and several chemotherapeutic agents can cause unintended cardiovascular damage (11). Using cultured hiPSC-CMs, I evaluated 21 U.S. Food and Drug Administrationapproved tyrosine kinase inhibitors (TKIs), commonly prescribed anticancer compounds, for their cardiotoxic potential. HiPSC-CMs express the major tyrosine kinase receptor proteins such as the insulin, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors, lending validity to this cellular model.

Initially, human induced pluripotent stem cells (hiPSCs) can be produced by reprogramming skin or blood cells by nonviral or viral reprogramming methods. Cardiac differentiation protocols allow for the creation of cardiomyocytes derived from hiPSCs (hiPSC-CMs) for downstream applications, including in vitro disease modeling, drug screening, and regenerative cell therapy.

With data from a battery of cellular apoptosis, contractility, electrophysiology, and signaling assays, I generated a cardiac safety index to help align in vitro toxicity data to clinical drug safety guidelines (12). From the safety index, I determined that a subclass of VEGF receptor 2/PDGF receptorinhibiting tyrosine kinase inhibitors, some of which exhibit toxicity clinically, also elicited cardiotoxicities in hiPSC-CMs. These manifested as substantial alterations in cellular electrophysiology, contractility, and viability when administered at clinically relevant concentrations. I also discovered that cotreatment with either IGF or insulin partially rescued TKI-induced toxicity by up-regulating antiapoptotic signaling pathways. This work could prove useful for groups aiming to develop effective screening platforms to assess new chemotherapeutic compounds for cardiotoxic side effects.

I also collaborated with the Center for the Advancement of Science in Space (CASIS) to send a sample of hiPSC-CMs to the International Space Station. As humankind ventures beyond our home planet, it is imperative that we better understand how the heart functions for long periods of time in microgravity. Analysis of these hiPSC-CMs revealed microgravity-induced alterations in metabolic gene expression and calcium handling (13).

In recent years, the stem cell field has experienced an explosion of studies using hiPSC-CMs as a model cellular system to study cardiovascular biology. As improvements in hiPSC-CM mass production continue, we will see a rise in studies using these cells for disease modeling and drug screening. Thus, although hiPSC-CM technology is in its infancy, it holds great potential to improve cardiovascular health.

PHOTO: COURTESY OF A. SHARMA

FINALIST

Arun Sharma

Arun Sharma received his undergraduate degree from Duke University and a Ph.D. from Stanford University. Having completed a postdoctoral fellowship at the Harvard Medical School, Sharma is now a senior research fellow jointly appointed at the Smidt Heart Institute and Board of Governors Regenerative Medicine Institute at the Cedars-Sinai Medical Center in Los Angeles. His research seeks to develop in vitro platforms for cardiovascular disease modeling and drug cardiotoxicity assessment. http://www.sciencemag.org/content/367/6483/1206.1

The rest is here:
Stem cells to help the heart - Science Magazine

A 40-year-old HIV patient has been declared cured after a promising treatment has left him with no active virus. The man, Adam Castillejo, was the subject of extensive research in early 2019 after doctors failed to find HIV in his body over an 18-month period after previously being diagnosed in 2003.

Castillejo, known by the nickname London Patient lived with the disease for many years, taking medicine to manage it since 2012. That same year he was diagnosed with Hodgkins Lymphoma and later endured a bone marrow transplant. That operation may have ultimately cured him of HIV and appears to have made him only the second person to ever be cured of the disease that causes AIDS.

As ScienceAlert reports, the bone marrow transplant that doctors performed on Castillejo used cells from a donor with a very special genetic quirk. The cells are thought to work against HIV in the body, but there was no guarantee that the transplant would provide any concrete benefits beyond treating the cancer.

However, it appears as though the decision to treat Castillejo with the unique stem cells worked in more ways than one and last year doctors announced they couldnt find the virus in his body after 18 months. At the time, they were hesitant to declare the London Patient cured, but after a new round of testing returned the same results, they are more confident that the active form of the virus has indeed been defeated.

This is a unique position to be in, a unique and very humbling position, Castillejo told the New York Times. I want to be an ambassador of hope.

While this sounds like incredible news and for Castillejo, it certainly is the treatment is not an option for everyone. With cancer limiting their options, doctors used the stem cell transplant as a last resort to keep him alive. Its a serious operation and one that was only performed because Castillejos condition was so dire.

Castillejo and the other HIV patient who had similar results, known as the Berlin Patient, may be uniquely fortunate. The doctors note that there are others who have had the same transplant performed but did not improve as rapidly as the others. There are obviously many factors at work here and as exciting as it is to see a second person cured of this terrible disease, theres a lot more work to be done before we can say HIV has been truly beaten.

Read more here:
HIV patient appears to be cured after stem cell treatment - New York Post

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the capability to replenish mature osteoblasts and repair the skeleton.

Until now, it has been thought that stem cells for bone lie within the bone marrow and the outer surface of the bone. Many studies have described the existence of a network of vascular channels that helped distribute blood cells out of the bone marrow. However, none of the studies had proved the existence of cells within these channels.

A new study by the scientists from the UConn School of Dental Medicine has discovered the population of stem cells that reside along the vascular channels within the cortical bone and have the ability to generate new bone. These stem cells stretch across the bone and connect the inner and outer parts of the bone.

Lead investigator Dr. Ivo Kalajzic, professor of reconstructive sciences, said, This is a discovery of perivascular cells residing within the bone itself that can generate new bone-forming cells. These cells likely regulate bone formation or participate in bone mass maintenance and repair.

This is the first study that reports the existence of these progenitor cells within the cortical bone that can generate new bone-forming cellsosteoblaststhat can be used to help remodel a bone.

To reach this conclusion, the scientists observed the stem cells within an ex vivo bone transplantation model. These cells migrated out of the transplant and started to reconstruct the bone marrow cavity and form new bone.

However, further study is required to determine the cells potential to regulate bone formation and resorption.

The study is presented in the journal Stem Cells.

Read this article:
These new stem cells have the ability to generate new bone - Tech Explorist

By Stephanie Chow | March 12, 2020

Three Faculty of Medicine researchers Drs. Zachary Laksman, Bruce Verchere and Tim Kieffer have collectively received more than $1.6M from the Stem Cell Network (SCN) to advance their work in stem cell and regenerative medicine research.

The SCN investment, which will advance research collaborations across the country, aims to translate stem cell-based therapies from bench to bedside for the benefit of all Canadians.

Dr. Zachary Laksman, Department of Medicine, Division of Cardiology

UBC Collaborators: Dr. Glen Tibbits, Dr. Liam Brunham, Dr. Francis Lynn, Dr. Shubhayan Sanatani

Project: Pipeline Towards Stem Cell Driven Personalized Medicine for Atrial Fibrillation

Dr. Bruce Verchere, Department of Pathology & Laboratory Medicine

UBC Collaborators: Dr. Francis Lynn, Dr. Megan Levings, Tim Kieffer, Dr. Dina Panagiotopoulos, Dr. Brad Hoffman

Project: Genetic Manipulation of hES-derived Insulin-producing Cells to Improve Graft Outcomes

Dr. Tim Kieffer, Department of Cellular & Physiological Sciences

UBC Collaborators: Dr. James Piret, Dr. Megan Levings

Project: A Bioprinted Insulin-Producing Device for Diabetes

Read this article:
Faculty members receive funding to advance stem cell research - UBC Faculty - UBC Faculty of Medicine

This is a full transcript of Disabled and out of money in North Korea as first broadcast on 13 March and presented by Beth Rose

JITE- I got a few stares of course. I'm bald. I had a beard. I was in a wheelchair. I'm black. The first two that I went to said, "No, no, no, we probably can't do that." I didn't want to do something which was challenging for me only, rather than North Korea. Oh, well that's a tough place to go to.

[jingle: Ouch]

BETH-I've been so excited about bringing you this Ouch podcast. A few months ago I received an email. It said, "Hi Beth, a friend of mine, Jite Ugono has multiple sclerosis, or MS, and uses a wheelchair. He's just about to travel to North Korea. Would you like to talk to him?" "Yes," was my answer, "very much so."

I'm Beth Rose, and you're listening to the BBC Ouch podcast, and for a while Jite has been on my mind. From the day he flew to China to get his visa, to the five days he would spend in the country we know very little about. And finally, he's back. Also, just a quick note to say that this podcast was recorded long before the Corona virus outbreak.

[music]

BETH-Hello.

JITE-Hello, hi.

BETH-So how was the trip?

JITE-Everyone says surreal, but it was surreal. Being inside a communist country and being restricted. Also in a wheelchair, there are no provisions at all for wheelchair access and that kind of stuff. Most of the places I went to were only accessible by stairs, so they carried me, which was nice. And that's one of the good things about having a guide, because I had two guides and a driver.

BETH-So you said you were thinking about this trip a year ago. It's the kind of trip that most people won't even think you can do, so why did you suddenly decide to book your holiday to North Korea?

JITE-Well I've got MS so they said one of the treatments of MS could be stem cell therapy. So stem cell therapy involves chemo and the rest of it. I thought to myself why not do something as rare as stem cell therapy? It was almost like a redefinition of my identity. I didn't really want to be known solely because of MS or the treatment, because everyone's going to ask about the chemo. I wanted to do something else which was kind of equal and opposite.

BETH-It's quite rare, stem cell therapy for multiple sclerosis isn't it?

JITE-It is. I hadn't heard of it. Chemo for cancer, we know all about that, but as soon as she said chemo for this For me it was quite emotional because my mum died the year before of cancer and she went through chemo as well. It was a shock, but it was also some hope. It seems less bleak. What I have is Primary Progressive MS, a steady degradation of mobilities. And they have less treatment for that, so most other treatment comes for Secondary Remitting, when you have attacks and then you can recover.

BETH-So what does the chemo do?

JITE-Chemo reduces your immune system. So what they want to do is kind of knock out the immune system and then reintroduce the stem cells and then restart the immune system.

BETH-That sounds quite an intense treatment.

JITE-I was in hospital for a month. So I went in for chemo, I was in hospital for a week or so, first of all, came back out, did the injections, back into hospital for a month. It was tough going through, but easier when you do it in stages. You think, okay I'm going to do this chemo first, in ten days I'll do the injections. Bite size. So by the end of it it's like oh, I've done it. I think it taught me whatever I go through I have to be a bit more patient.

BETH-How long ago were you diagnosed with MS?

JITE-2009.

BETH-So you were quite young?

JITE-I'm 45 now, so yeah, the symptoms got worse maybe six or seven years ago in terms of difficulty walking. And that's the main thing. The first thing was the eyesight, so the eyes were playing up and I thought maybe I should go to the optician. It didn't really make a difference. So it got progressively worse. I did an MRI scan and then the consultant said, "Well, it could be MS." So I was kind of aware and I kind of knew that it was something quite serious. So when he came back and he said MS. You make a decision about how you're going to deal with it.

For me, it was you're not going to feel sorry for yourself because people go through worse. For me, it's only when I'm faced with stuff you realise you can do it. I didn't just want to survive. Because when you're diagnosed with stuff it's like getting through the day. Everyone says, "Oh, you're so brave. You went to work?" For me it's just one life, you can't spend it getting through the day, you want to do something else.

BETH-So was it when you were having your chemo when you were in hospital, the idea for North Korea?

JITE-It was actually the first consultation when she told me, "You're going to do stem cell therapy." They told me that I was going to be able to maybe walk with sticks and I thought, why waste it?

BETH-I feel like a lot of people would have had similar thoughts but maybe thought South of France would be quite nice?

JITE-It would have been challenging. If anyone said they were going to the South of France, oh okay. I didn't want to do something which was challenging for me only, rather than North Korea, oh well, that's a tough place to go to, regardless of whether you're in a wheelchair. It was important to me to do something which was challenging, not because of MS, not because of the wheelchair, but it was challenging.

BETH-So how do you go about booking a trip? Can you go to a travel agent?

JITE-I mean, that's what I did. So the first two that I went to said, "No, no, no. We can't do that, there's no access." And I was probably more determined. That's another lesson it taught me, it's more important for me that I wanted to do it. And no one was coming back to me to say, "Why don't you go?" So when the third person came back and said, "Actually, we could do that," the normal way of going to North Korea is through a group tour, with my condition anyway. You think about what the problems could be. Getting onto the coach. Holding people up.

So my tour was me on my own. I had two guides and a driver and that was it. They sorted out the visa to China and once you get to China you get the visa to North Korea from China.

BETH-Touching upon the issues of getting onto a bus, what is it like for you with MS? How does it manifest itself?

JITE-My balance is a problem. I can't really use my left leg at all. My eyesight's a problem. Maybe sometimes my memory and my vocabulary. They're difficulties which arose mainly because I did chemo. We know that the drugs are quite aggressive and concentrated, so they give you lots of water to dilute and because you're given that you're given drugs to help you relieve that stuff, so you're peeing like every ten minutes.

So it went down to probably once every hour and that became a problem and that affects your confidence, you're afraid to kind of go out, maybe there won't be toilets around, that's kind of what I was thinking about, going to North Korea.

BETH-Did you even know about that? Is there information about toilets or accessibility?

JITE-Not at all, not at all. It's only when I got there that I realised that the And sorry to go on about toilets, but it was important to me. [laughs] Okay, so in North Korea they had two types of toilets, they had the European toilets and then they had the Korean toilets, ground toilets, so you have to kind of balance, which I didn't even attempt. So everywhere we went to it was okay, "Is it a Korean toilet here or a European toilet?" Even the guides started to realise and started to know after a while.

BETH-I mean, that's such a gamble isn't it, not knowing the accessibility, not knowing what the toilet situation's going to be like. I'm guessing this was all in your mind?

JITE-Every problem has to have a solution. So before I went I'd got it up to you can pass an hour now, because I'd gone to the gym, I'd started doing core stuff, even in the plane, because it was ten and a half hours there. You think about the problems that you could face, it's personal of course, but also there are people around that can give you a hand.

And that was another thing, getting vaccinations was a problem, because when you do chemo and your immune system is low they don't advise that you have vaccinations. So I was intending to go to Korea in September but that was super close to my stem cell.

BETH-When you were flying, initially to China, what was going through our mind?

JITE-It was just getting through that first bit, hoping that someone's going to be there to meet me. The luggage I even took I had to make sure that I could carry. That's one of the solutions with a wheelchair, you're going to have to push the luggage as well so it can't be too big. Two pieces of hand luggage is what I took. That's what I was thinking about, I wasn't thinking about Pyongyang yet, I was thinking about how to get to China.

Beijing was packed, traffic everywhere. It was surprisingly western. The cars were German cars. In North Korea I had the guides, in China I didn't have guides, I had a person to take me from the airport to the hotel and that was it. So I didn't really have the confidence to kind of venture out. I got in a day before, so as soon as I landed in China I had to go and get the visa. As soon as you get the visa is when they give you a briefing, what you should and shouldn't do. The chap apparently had been doing it for 28 years, and no one had ever missed a briefing until me.

BETH-Ah! [laughs]

JITE-I mean, only because the person who picked me up said, "Oh, I can get the visa for you."

BETH-So they were being helpful, but actually

JITE-Yeah, so they went out and got the And I was appreciative, because getting in and out of the car was such a pain. And I am quite lazy naturally. If I can do without it then I won't do it, you know. So when they gave me an opportunity not to, oh okay. The travel agent contact in China was almost panicky on the phone, "No one's ever done this."

BETH-Wow, and I bet your heart was racing at that point.

JITE-To an extent, but I kind of knew what not to do. I mean, I'm not rude, and plus I'd seen stuff on YouTube and the guides tell you as well. So I was quite prepared. I flew into Pyongyang. The airport was a surprise. They only have a few planes that land for the day. They had one from Beijing, one from Shanghai and one from Moscow. There are soldiers everywhere, but the soldiers were, "Oh, look at this guy," I suppose maybe because I was a novelty in a sense. They'd never really seen someone in a wheelchair before. They were super helpful.

I'd met the guides at the airport as well. I got a few stares of course. I'm bald, and they have like five haircuts. I had a beard, I was in a wheelchair. I'm black. So all those things together.

BETH-So did you feel like you stuck out?

JITE-I didn't feel like I could relax, only because you feel like you're always on. I couldn't be anonymous, there's always someone watching, and that's tiring.

BETH-And did you feel like you were being watched by your guides?

JITE-Maybe the brief was to watch, but it is different when you have a relationship with people. So I didn't feel that way. I suppose they were constantly on about how great the leader is and after a while it got a bit tedious. Everyone walked around with badges. And it's difficult to tell because they spoke the language quite a bit. I don't know what they're saying.

BETH-They greeted you at the airport.

JITE-Yes.

BETH-Had they had disabled travellers before?

JITE-I don't think they had. What happens is that when you go on your own there is no camaraderie, I was mostly alone, but the advantage is you could probably get closer to people. There's good and there's bad about it.

BETH-What's it like, Pyongyang?

JITE-For me it was super quiet. I mean here we have adverts and stuff, people are selling you stuff all the time, there is different, you have pictures of the leaders surrounded by flowers and you have to respect that. If there's an image of a leader you can't really take a photo of it and you can't stand in front of it obscuring it. Or you can't crop it. Apparently they check people's phones to see what they've taken.

BETH-Did you take photos?

JITE-I took photos but they didn't check. But everywhere was empty. The place is set up for tourists but there are not many tourists. You go into a restaurant and there are people standing around. The restaurants are empty. It's bizarre.

BETH-So it's not really like a bustling city?

JITE-Not at all. Actually I went during King Il Sung who's the grandad of this present leader, it was his birthday, so there were two days of celebrations. I think there were more people on the street than normal, and then they had volunteers picking up stuff or gardening or I mean, because it's a communist environment they pay for everything but you have to work. They've got big roads, no cars.

BETH-Wow.

JITE-Yeah. The days were quite long. Maybe eight o'clock they'll come for me and then eight o'clock in the evening I'd finish. So there was always something to do and you were always with people. I think they had five channels, that was about it.

BETH-TV channels?

JITE-Five TV channels. On the channels they have the leader, Kim, pointing at stuff. He designed the theme park.

BETH-What's the tourist trail like?

JITE-There is an itinerary, so you would go to the war museum, flower exhibition. I went to their subway, it's the deepest subway in the world. So everything's the best in the world or the tallest in the world.

BETH-How did the subway compare to the tube?

JITE-It was more opulent. I only saw two of them and I think those are the two they show people, so maybe the others are less. There are chandeliers and stuff.

BETH-And the restaurants, you said you went into one, but they've got all the staff just waiting around?

JITE-Yeah, the restaurants seem to be for tourists, and because I was on my own, seven, ten people just standing around looking. I went to a casino, which was strange.

BETH-Oh, okay?

JITE-Yeah. But the casino was in the hotel. I think I was the only one in there. So when I went to North Korea I didn't take enough cash, and that was a problem obviously because no cards. So the guys were like, "You need some money? Go to the casino, you can change your money."

BETH-Oh, I thought you were going to say to like gamble and win.

JITE-At first I went to change money, but they didn't take sterling, they took US dollars and euros, but I didn't have either, so they allowed me to gamble, so I did.

BETH-Did you win? Did you get some money?

JITE-Yeah, I did. I don't want to get used to it. [laughs]

BETH-What game did you play?

JITE-Black Jack. I didn't know what was going on, but people around, they were almost cheering, and I was thinking by the time I won a hundred dollars I thought it's time to go, it's time to go. And everyone's around you willing you on and you don't want to disappoint them but you think okay, I'm going guys.

BETH-Is it expensive then, if you ran out of money and you're having to gamble to boost your-?

JITE-To boost. Okay, so I mean they have their own currency and they don't let you take the currency out.

BETH-I bet your guides quite enjoyed being in the casino.

JITE-The guides said, "Oh, we're not allowed in." Even when they came up to my hotel room I had to have Al Jazeera because that's the only English speaking channel, but they were almost transfixed. They were shaking their heads. Look around the world, look how happy we are type of thing. So you kind of understand why they would let Al Jazeera in, because Al Jazeera can be quite, look what's happening around the world, the protests here, the protests there.

BETH-And did you find people were willing to help you?

JITE-I think it was more because they see you as being vulnerable. "Oh, you're not comfortable, let me move your legs." So you always get somebody helping, which is not necessarily what you want all the time. Because you want to be able to be self-sufficient. Certainly in London people are a bit more patient to offer, "Okay, how can I help?" and then they stand back. In Korea it was, "Oh, we can do that for you." [laughs]

BETH-Did you see any other disabled people out and about?

JITE-No, I didn't.

BETH-No one at all?

JITE-I didn't at all. One of the guides was quite insistent on how great their society is. That's why they stay kind of thing, away from everyone else, and they obviously saw it as a good thing.

BETH-Oh, that's interesting. I was going some research, and there's a lot of reports from the UN and different charities where they say basically they send people away in an out of town community.

JITE-Yeah, they don't expect you to try. So maybe that was part of it, they were almost surprised that this person is doing something on their own.

BETH-And were they quite surprised how you just got on with everything?

JITE-Yeah, I suppose. Maybe they were. So even when I'd be going down the road people would lean over and look. They weren't rude about it. They would look, they were curious, but they weren't intrusive. And sometimes you look and they look away, except the kids, so the kids would be staring. But that's normal though, even in London you'll get kids staring. One of the guides took a video of me being lifted up the stairs, and it was quite tough to watch because you don't really see yourself as being vulnerable, except when you see it.

It's like hearing a recording of yourself and you think oh, do I sound like that? Or do I look like that? Am I really that vulnerable kind of thing? No wonder everyone helps. [laughs] It was tough to see. I didn't really see the footage until I got to the hotel and you kind of think, you know, is that how it is? They were helpful, and it sounds ungrateful almost, but it is what you think about.

It's a lack of confidence to think people only help you because you look so vulnerable. Maybe people are just nice. And that was one of the good things about going to North Korea. People say that Londoners are quite cold and I don't find that, Londoners can be helpful, and especially if you're patient enough. And MS for me does that, it allows you to be patient.

BETH-So what kinds of things is nice to have help for?

JITE-Probably getting in and out of cars. In London not so much, in London you kind of want to get strong. I know that I'm going to have to get in a car, and not everybody gives the same level of help, so you have to be self-sufficient. In North Korea there's no need. And I'm never going to be in North Korea again.

BETH-How did the access pan out? Because that was the big mystery wasn't it really? I mean, you had no idea.

JITE-It was just people lifting me. Only one place, the museum was difficult.

More:
Transcript: Disabled and out of money in North Korea - BBC News

Genes may determine what characteristics are passed down from parent to offspring, but each cell expresses these genes differently based on external epigenetic modifications. Epigenetics dont alter the gene sequence (genotype), but they do influence cell behavior and function (phenotype). The study of epigenetics helps us understand how phenotypic changes lead to disease, stem cell differentiation, and essentially, what drives the fate of every cell in the human body.The epigenome is not consistent between cells, or even between cells of the same type. Individual modifications come and go throughout a cells lifetime. Therefore, scientists are faced with the steep challenge as they try to decipher the role of epigenetics in disease and development.[i] Understanding intercellular heterogeneity is key here. The epigenome must be examined at single-cell resolution.

Now, with the advancement of single-cell sequencing methods like the single-cell assay for transposase accessible chromatin (scATAC-seq), researchers have access to sophisticated techniques to map large cell populations, one cell at a time. The resulting epigenomic information provides unprecedented insight into the different cell types that come together to form organs and organ systems, as well as pathogenic modifications associated with disease.

Every single cell has unique epigenomic instructions that guide how it expresses its genes and these instructions are subject to change. A map locating epigenetic modifications in the genome would help scientists understand how epigenetics drives cellular differentiation. But until recently, epigenetic assays mainly focused on select regions of DNA or gave bulk results across an entire sample of cells.[ii] These assays were not designed to detect epigenetic patterns in individual cells.

Single-cell tools like scATAC-seq help us get a grasp on intracellular heterogeneity, differentiate between cell populations and map the role of epigenetics in the larger context of an organism. By building a collection of scATAC-seq data, scientists have begun generating a cell atlas to provide insight into the role of epigenetics during the intricate biological processes that occur throughout the human lifetime.

During ATAC-seq, a hyperactive transposase mutant, Tn5, binds to open chromatin (euchromatin) regions. Wherever Tn5 binds, it cleaves the DNA and attaches sequencing adapters. Then, after PCR amplification, ATAC fragments are sequenced to identify open chromatin regions. ATAC results indicate where nucleosomes are typically positioned in the cell sample and which regions of the genome are open for transcription factors to bind. As such, scientists use ATAC-Seq as a first-pass screening approach to identify changes in chromatin accessibility between samples.

ATAC-seq has many practical applications, but it cant account for the cell-to-cell variability thats often an important aspect of developmental processes and disease. So, researchers developed a new assay in which microfluidic technology is used to isolate individual cells before ATAC-seq.[iv] This assay provides epigenomic information at single cell resolution, earning it the name scATAC-seq.

The key to the scATAC-seq method is that it isolates genomes of individual cells early on to perform a separate ATAC-seq reaction on each individual cell. Then, open regions of the genome are cleaved by the Tn5 transposase, tagged with sequencing adapters and amplified with barcoded cell-identifying primers. Subsequently, the barcoded libraries of ATAC fragments, (each representing an individual cell) are pooled together and sequenced to reveal open chromatin regions of thousands of individual cells.

The first droplet-based iteration of the scATAC-seq method (dscATAC-seq) uses a single cell isolator to encapsulate thousands of individual nuclei in nanoliter-sized droplets for ATAC sequencing. It uses a custom Tn5 transposase to enhance library complexity and signal resolution. Compared to the original microfluidic method, the new workflow is faster and yields greater biological insight with less time and effort spent on sequencing. To demonstrate its power and potential, this technique has been used to conduct an unbiased analysis of the many different cell types and regulatory elements in a mouse brain. [v]

Figure 1:In scATAC-seq, droplet-based technology partitions thousands of whole cells or nuclei into individual nanoliter-sized droplets, enabling researchers to prepare a library of ATAC fragments for sequencing to reveal open chromatin regions. Credit:Bio-Rad Laboratories.

To capture single cell data on a truly massive scale, combinatorial indexing was next introduced into the dscATAC-seq workflow. This new method, called dsciATAC-seq, enables researchers to assess up to 50,000 cells in a single assay. Assaying a large volume of cells is possible because, in dsciATAC-seq, the hyperactive mutant transposase integrates a first set of barcodes as it cleaves open regions of chromatin in each nucleus. Because every cells DNA already carries a barcode, multiple cells can be loaded into a single droplet. Then, as usual, ATAC fragments are amplified with a second set of barcoded primers. After sequencing these fragments, the two sets of barcodes are used to derive epigenomic profiles for tens of thousands of cells.

Putting the dsciATAC-seq method to the test, researchers have studied immune cell clusters from human bone marrow derived cells to illustrate how the chromatin accessibility landscape in these cells changes according to different stimulants at the single cell level.5Although the number of cells that a single scATAC-seq experiment can evaluate has grown dramatically, it will take a continued concerted effort from scientists across many disciplines to create a comprehensive map of the human epigenome, encompassing data from trillions of cells.[vi] Furthermore, to help decode the patterns we find in the human epigenome, it may be valuable to gather information about the epigenomes of animals commonly used as research models. As each of these maps become increasingly detailed, scientists will gain a more thorough understanding of how biological process work and may apply this knowledge towards developing better treatments for complex diseases.

Reference:

[i] Egger, G., et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004, 429, 457463. doi:10.1038/nature02625[ii] DeAngelis, J. T., Farrington, W. J., & Tollefsbol, T. O. An overview of epigenetic assays. Molecular biotechnology, 2008, 38(2), 179183. doi:10.1007/s12033-007-9010-y[iii] Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods, 2013, 10(12):1213-8. doi: 10.1038/nmeth.2688.[iv] Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature, 2015, 523(7561):486-90. doi: 10.1038/nature14590.[v] Lareau, C.A., Duarte, F.M., Chew, J.G. et al. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature Biotechnology 37, 916924 (2019) doi:10.1038/s41587-019-0147-6.[vi] Bianconi, E., Piovesan, A., Facchin F., Beraudi, A., Casadei. R., Frabetti, F., Vitale, L., Pelleri, M., Tassani. S., Piva, F., Perez-Amodio, S, Strippoli, P. & Canaider, S. An estimation of the number of cells in the human body. Annals of Human Biology, 2013, 40:6, 463-471. doi: 10.3109/03014460.2013.807878.

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Using Single Cells To Get the Whole Picture of the Epigenome - Technology Networks

Stromal Cells May Help to Overcome Resistance to Chemotherapy in Patients with Glioblastoma

The researchers found that GBM causes these stromal cells to act like stem cells, naturally resisting attempts to kill them and promoting tumor growth instead. They also identified the pathway that makes cancer vulnerable in a lab setting.

GBM is an aggressive form of brain cancer and the tumors are usually heterogenous or contain different genetic mutations. This means that treatments focused on 1 target are ineffective or only partially effective.

There have also been few new treatments over the past few decades for GBM, so its clear that we need to find a way to make current treatments more effective for these patients, said study senior author Yi Fan, MD, PhD.

The study focused on overcoming resistance and researchers found that GBM transforms a type of stromal cells, called endothelial cells, so that they act like stem cells. The researchers then found that the resistance is enabled by a signaling pathway called Wnt. The more that Wnt is activated, the more a cell is able to resist treatment like chemotherapy. Previous research has shown that GBM stimulates Wnt activity. Therefore, these findings show the way in which tumors co-opt their environment to survive an attack.

Blocking Wnt signaling may be an effective way to help the cells overcome resistance to treatment. Therefore, the research team used an experimental approach to shut off Wnt signaling in the stromal cells of tumor samples. They found these cancer cells were vulnerable to chemotherapy once the signal was blocked.

The findings support the development of other cells, including cancer. By targeting them, a treatment would effectively get closer to the cause of the tumors ability to survive, which may make therapy more efficient, according to the study authors. Additionally, the findings indicate that treatments will remain effective even as the tumor changes.

Reference

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Stromal Cells May Help to Overcome Resistance to Chemotherapy in Patients with Glioblastoma - Pharmacy Times