Stem Cell Science Double Feature: Reprogramming Cardiac Fibroblasts – Recap of talk by Dr. Deepak Srivastava

The Talk:

Cardiac Reprogramming: From Developmental Biology to Regeneration

In Plain English:

How to turn the heart-dwelling cells that build connective tissues into replacements for damaged heart muscle cells

The Speaker:

Dr. Deepak Srivastava, MD of UC-SF‘s Gladstone Institutes

The Sponsor:

MIT Biology Colloquium

What it covered:

Dr. Deepak Srivastava is a cardiologist who experiments with using stem cells to create replacement cells for damaged heart muscle tissue. However, while most labs try to grow replacements in petri dishes, Dr. Srivastava’s lab is working on finding a way to transform the cells that build scar tissue in the heart (the cardiac fibroblasts) into fully functional heart muscle cells.

A typical fibroblast’s job is to secrete collagen and other chemicals that help the cells in muscle and skeletal tissues stick together. If they weren’t there, your muscle cells might slide around or come apart whenever you moved. Luckily, muscle cells like to form stringy structures called sarcomeres that can contract and release. When you’re flexing your muscles, you’re actually contracting the sarcomeres and that contractile force is what causes your arm to tense up. Without the fibroblasts, the muscle cells would have a hard time aligning themselves correctly (and if one cell is even slightly out of line with its neighbor cells, the rogue cell can throw everything off), so they’re a pretty important cell group.

Many fibroblasts remain in their dormant fibrocyte stage until they’re needed to help rebuild an injured body part. However, cardiac fibroblasts don’t rebuild complete heart tissue; they build scar tissue that keeps the heart from falling apart. Which is good, but not as good as being able to completely regrow damaged heart muscle.

Dr. Srivastava wondered if there was a way to convince the fibroblasts in damaged hearts to organize themselves into something that more closely resembled cardiomyocytes, the specialized muscle cells that form the heart. However, before he could attempt to create a replacement heart tissue, he had to understand which chemical signals caused the cardiomyocytes to behave differently from their neighbors.

One of the key features of cardiomyocytes is that they bind to each other in very specific ways. All cells have at least a few portals in their membranes that called “gap junctions” that allow electrically charge particles from neighboring cells to enter and/or leave the cell. Cardiomyocytes have a very specific set of gap junctions that allows them to feel the “heart-beat” signal without disrupting normal function in neighboring tissues. The scar tissue cells that typical fibroblasts produce don’t “beat”, so finding a way to convince fibroblasts to develop into “beating cells” was a key priority for Dr. Srivastava’s team.

He spent the mid-2000s investigating how a special type of RNA-transcript called microRNAs affected the formation of heart tissues. MicroRNAs (or “miRNAs” for short) are different from “messenger-RNAs” that take gene transcripts to the ribosome to be converted into proteins; microRNAs are too short too code for proteins. For a long time, scientists were confused by the miRNAs, because what’s the point of building an RNA if it doesn’t help you make a protein?

It turns out that the miRNAs can bind to certain sections unzipped DNA. That means that cells can basically “turn down the volume” on a particular gene by producing miRNAs. They’re also really for scientists who want to figure out which genes are “upregulated” or “downregulated” in specific cell types, because we know how to read RNA base pairs.

Based on miRNA research, Dr. Srivastava’s team developed a cocktail of transcription-controlling chemicals that he hoped would cause the fibroblasts to start acting more like cardiomyocytes.

Perfecting the cocktail was a tricky process. He found that several different chemical signals were needed to get the fibroblasts to respond at all, and even when they did, only a fraction of the treated cells changed their gene expression.

Furthermore, the fibroblasts that did respond only underwent a partial transformation; when Dr. Srivastava’s team analyzed the gene expression levels of the transformed fibroblasts, he found that their expression levels fell somewhere in between the expression levels you’d expect for a typical fibroblast and the ones you’d expect from a cardiomyocyte.

Even a partial transformation is a pretty encouraging result. The transformed fibroblasts were “beating”, so even though they hadn’t transformed perfectly, they were still helping to replace the damaged muscle cells.

The next challenge was to find a way to integrate these reprogrammed cells with the existing tissues of the heart. The replacement cells would have to line up with the existing heart cells correctly in order to form appropriate gap junctions, and they would have to form sarcomeres to be able to contract and release properly. Partially reprogrammed cells that aren’t in exactly the right place would potentially confuse the neighboring cardiomyocytes, and that confusion could lead to all kinds of problems.

He decided to try using a retrovirus (a virus that cuts and pastes its genetic material directly into the host’s nucleus-dwelling DNA.) to transform the fibroblasts* that live in mammalian hearts, figuring that cells who already live in the neighborhood would be less likely to disrupt the local gap junctions than cells grafted in from a petri dish.

Once again, he saw partial transformations in the cardiomyocytes. His team has attempted applying this technique in both mouse and pig hearts, and even though they’re still trying to perfect the transformation-inducing cocktail, the initial results have been promising.

Right now, his team is focusing on two goals: 1) Find a combination of transcription factors that cause human fibroblasts to transform in petri dishes and 2) Work on finding a way to make sure that only the cardiac fibroblasts are infected by working with the pig model. (Pig metabolisms are more similar to human metabolisms than mouse ones in many ways. The downside of this approach is that we don’t know as much about the pig immune system as we do about the mouse immune system, but Dr. Srivastava thinks it’s important to test this idea in several different model organisms before even thinking about attempting to implement this approach in a human.)

Obviously, there are a lot of safety issues that need to be taken into account before applying transcription factors as medical treatment, but it’s an interesting alternative to using pluripotent stem cells to replace damaged tissue.

*Scientists have found that if they suck the viral genetic data out of the virus’s capsid and add whichever genes they want to insert into the cell genome, the virus’s cell-targeting and gene-splicing machinery still work. Cell-targeting and gene-splicing are still really hard for us to do, so it’s much easier to reprogram a virus that targets heart cells than to build a robot that breaks into heart cells and changes their genes. And even if we did build a robot like that, it would probably look almost exactly like a virus. When you hear about scientists “reprogramming” cells with viruses, they are basically using the virus’s body like a very special syringe that can find very specific cell types, and the RNA that causes viral disease is lying at the bottom of a biohazardous waste container, far away from the patient.

My Personal Take:

Most the cell biology classes I’ve taken have focused on cancer and immune cells, which are known for their ability to move around, so I’ve always kind of assumed that the world of skeletal and muscular cells was more fixed (and by extension, less interesting.)

However, as I was listening to Dr. Srivastava’s talk, I began to realized that I’d seriously underestimated the complexity of structural cells’ lives. I also realized that I really didn’t know much of anything about fibroblasts. (I can’t think about connective tissue disorders without getting sad about Jonathan Larson, so when people talk to me about the formation of the aorta, I have a hard time hearing them over the chorus of “Without You” blasting in my head.) I knew vaguely that fibroblasts were in charge of collagen and other substances that help cells stick together, but I hadn’t seriously thought about how they might be affected by chemical and electrical signals.

And then a “stupid question” started brewing in the back of my head: Are fibroblasts stem cells? Because this talk was billed as a stem cell talk, but it seemed like we were shifting away from stem cell grafts and more toward reprogramming cells that are already living in the heart. But if fibroblasts are adult somatic cells, why would they be more susceptible to this type of transformation? I did some googling and found out three things: 1) The first time scientists were able to create pluripotent cells from an ordinary cell, the original cell was a fibroblast. 2) The suffix -blast is frequently used to describe stem cells, but it can also indicate that the cell has “an active metabolism” and 3) Fibroblasts definitely aren’t embryonic cells, but they can still give rise to different types of related cells, depending on what structures they need to build. This ability is especially crucial for their role in healing wounds.

The biological definition of a stem cell hinges on its ability to produce daughter cells that are functionally different from the stem cell. A stem cell is “undifferentiated”, but most cells in our bodies are “differentiated”, meaning that they have a specific function, and when they divide they can only produce daughter cells that do the same thing they do. Differentiated muscle cells can only give rise to more muscle cells and so on.

But it seems like fibroblasts fall into a little bit of a gray area. They aren’t totipotent; the modified fibroblasts that Dr. Srivastava and his team created still retained some fibroblast characteristics. However, fibroblasts can divide and give rise to the cells that form scar tissue, and those scar tissue cells are clearly different from their fibroblast progenitors.

I’m honestly still a bit confused about whether fibroblasts count as stem cells, but thinking about them made me realize that “stem-cell-ish-ness” is a spectrum. All cells can react to their environment and change their gene expression to some degree; it’s just that stem cells can alter their gene expression patterns so rapidly that they can give rise to completely different cells. However, every cell in our body is receiving chemical and electrical signals from all directions that tell it which genes to activate, which means that, in a way, every cell is in a constant process of transforming itself.

That idea really hits home for me, because usually when we talk about applying drugs and medication to treat a disease, we talk about the cells like they’re passive recipients that are transformed by the drugs (or we don’t talk about the cells at all.), but it’s the cell that transforms itself in response to the chemical or electrical cue.

Dr. Srivastava’s idea of convincing the heart’s fibroblasts to transform themselves into cardiomyocyte-like “beating” cells sounds a little bit creepy at first blush. (I couldn’t help but think of all the Stargate episodes where people get infected by retroviruses that turn them into a completely different species. The idea of any transformation like that happening in the space a of a few hours is ludicrous but unnerving). However, I’d argue that it actually makes a lot more sense (and is actually less creepy) than trying to graft outside cells into an existing heart. In this case, the fibroblasts are natives that are transforming themselves in accordance with the chemical instructions they hear in the environment.

Dr. Srivastava’s project is to make sure that we give the cells precise instructions about we need them to do and make sure that the right cells hear those instructions. It’s pretty important stuff.

Biggest Misconception to Avoid:

This isn’t a magic cure that will allow us to completely undo the damage done in a heart-disease-ridden heart. Dr. Srivastava’s research represents the first attempts to devise what could turn out to be a very powerful tool for cardio-regenerative medicine, but we’re still a long, long way from being able to cure chronic heart disease.

Best One-Liner:

Dr. Srivastava on why this approach may be more promising than tissue grafting: The converted fibroblasts transform in vivo with beating cardiomyocytes around them. They express the right gap junctions. They’re likely getting the right cues from their neighbors. It’s like they grew up on the block rather being plunked down into a new environment as an adult cell.

Best Audience Question:

Audience member: It seems like there would be a lot of safety issues associated with implementing this in humans. I’d be worrying about which other cell typess are susceptible to these retroviral infections. And how do you track where the converted fibroblasts are going? How do you know another beating heart or tumor won’t appear somewhere else?

Dr. Srivastava: Yes. We’re also worried about beating fibroblasts falling out of alignment. (Having cells that are “beating” scattered around the body could be a problem, especially neighboring cells may have weird responses to the electrical pulse from the “heartbeat”.)

We’ve tried converting a few different types of cells. Dermal fibroblasts will be converted also, but you need really high levels of certain transcription factors. For some reason, the fibroblasts in the heart are more susceptible. But we’re having a hard enough time converting cells in the heart that we haven’t seen beating elsewhere.

Key Terms:

  • Fibroblast = long, spindly cells that form connective tissues and are responsible for producing substances like collagen. Very active in wound healing. Different types of fibroblast hang out in different regions of the body. Here we’re talking about “cardiac fibroblasts”, which are in charge of making sure the heart maintains its structural integrity.
  • Cardiomyocyte = the cells that make up the heart muscle. They’re set apart by their “heart-beat”-inducing gap junctions and their sarcomere patterns.
  • Gap junction = a connection between two cells that allows electrically charged particles to move from one cell to the next without getting lost in between cells. Like a tunnel. But for calcium ions.
  • Sarcomere = the “stripe-y” patterns formed by muscle cells. By contracting and releasing the sarcomeres, we make our muscles move.
  • Transcription factor = a chemical signal that affects gene expression in cells
  • miRNAs = a family of short RNA-strands that act as transcription factors
  • Progenitor cells = cells that are too specialized to be considered full-blown stem cells but are capable of differentiating into more specific cell types. For instance, immune progenitor cells can differentiate into different types of white blood cells but can’t turn into neurons.
  • Hematopoietic stem cells and mesenchymal stem cells = The two biggest & best known stem cell families. Hematopoietic stem cells give rise to neural and immune progenitor cells, so they get tons of attention. However, fibroblasts are descended from the mesenchymal or “stromal” stem cells that give rise to different types of skeletal, muscular, and connective tissue cells.

Tl;dr: Cardiac fibroblasts aren’t stem cells per se, but they respond to the same chemical cues that cause stem cells to produce muscle cells. So we may be able to reprogram fibroblasts that already live in the heart to act as replacements for the heart muscle cells that get killed by heart disease.

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