Assembling an Egg

On this episode of the Bio Eats World Journal Club, we explore the very compelling question of whether we can use our understanding of developmental biology to create oocytes (aka eggs or female gametes) from stem cells in the lab. If possible, this could be on par with the development of in vitro fertilization in terms of extending fertility. But creating an oocyte from a stem cell has some unique and high-stakes challenges. Host Lauren Richardson is joined by a16z general partner Vineeta Agarwala and deal partners Judy Savitskaya and Justin Larkin to discuss the research article “Reconstitution of the oocyte transcriptional network with transcription factors” by Nobuhiko Hamazaki, Hirohisa Kyogoku, Hiromitsu Araki, Fumihito Miura, Chisako Horikawa, Norio Hamada, So Shimamoto, Orie Hikabe, Kinichi Nakashima, Tomoya S. Kitajima, Takashi Ito, Harry G. Leitch and Katsuhiko Hayashi, published in Nature, which makes a big step towards this goal. The conversation covers which aspects of oocyte biology the authors were able to replicate, which they were not, and where we think this field might be heading.

Show Notes

• A brief discussion of the biology [3:24] and terminology [5:27]
• Potential uses for oocyte biology [7:34], what it would take to convert cell types [10:36], and the development of the oocyte [12:36]
• Limitations of this study [16:59] and the future of oocyte biology [19:25]

Transcript

Hanne: Hi, I’m Hanne.

Lauren: And I’m Lauren, and this is the Bio Eats World Journal Club, where we discuss breakthrough scientific research, the new opportunities it presents, and how to take it from paper to practice.

Hanne: So, Lauren, you’ve titled this episode “Assembling an Egg,” but I’m going to guess we aren’t discussing your favorite breakfast recipes today.

Lauren: Hanne, you know me well, nope. Today, we are exploring the very compelling question of whether we can use our understanding of developmental biology to create oocytes, AKA eggs — you know, the female counterpart of sperm — from stem cells in the lab.

Hanne: Okay. So, refresh my memory. Do we have stem cells in our adult human bodies that could be used to turn into eggs?

Lauren: Yes and no. Adults have stem cells, but their ability to turn into other types of cells is limited. For example, we have stem cells in our bone marrow, but they can only produce the different types of blood cells, but scientists now know how to turn some of our cells back into stem cells, which are called induced pluripotent stem cells, or iPS cells. If possible, this adult cell to iPS cell to egg cell transformation could be on par with the development of in vitro fertilization in terms of extending fertility, but creating an oocyte by this path is tricky. So, I’ve gathered some of our colleagues, a16z general partner Vineeta Agarwala, and deal partners Judy Savitskaya and Justin Larkin, to discuss a recent research article published in Nature, by Hamazaki et al., that makes a big step towards this goal. We discuss what aspects of oocyte biology the authors were able to replicate, which they were not, and where we think this field might be heading. We start with Vineeta describing the state of innovation in fertility. 

Vineeta: Fertility, even just within the U.S., is approaching a $10 billion industry, and the majority of innovation that we see happening in the space is related to care delivery. How can we expand access to fertility treatments to couples who need them? That’s an important problem, but at the same time, it makes us wonder whether there are biological breakthroughs that could change the face of the industry even more than care delivery technology might.

Justin: Yeah. For so many aspects of reproductive therapies and infertility challenges, the egg or the oocyte is often the critical limiting step, both from an ability to actually get to a healthy pregnancy, but also in the process of the patient journey. Having seen numerous people go through the IVF process and other kinds of iterations of IVF, it’s often just so hard from a patient journey perspective — literally being painful, takes lots of time, it’s expensive. And so, the thought of being able to fundamentally change access to oocytes, and eggs that potentially could be grown in vitro in ways that cut the process down and make it more affordable, has the potential, I think, to not only open up reproductive technology and therapies to a broader population, but to make that care experience just fundamentally different for patients. And so, seeing the early innings of that potentially be suggested in this paper was fascinating to me.

Judy: Vineeta and I were actually pretty surprised the other day when we realized that the most expensive part of IVF, the single biggest line item, is the drugs that are required for egg retrieval. So, this is a way to just get around that entire process.

Overview of oocyte biology

Lauren:  Right. Could you unpack that? How is this solving the problem?

Vineeta: Women are born with a couple of million potential eggs, and as we age, we lose those eggs, and we don’t make more. And this paper provides this suggestion of a way to engineer, from pluripotent stem cells, oocytes that can potentially be fertilized and give rise to a new being, without being dependent on that very constrained, constantly dwindling supply of oocytes that we’re born with as women.

Lauren: Right. I think that is such an interesting idea, that when your baby is still in you, it has its full complement of eggs that it’s ever going to have. Females don’t make additional eggs after birth, so you have to work with what you have. As Judy said, it’s very expensive to retrieve eggs from a woman. So, this paper asks a really tantalizing question of — can we make new eggs from cells after birth, from your own cells after you’ve been born?

Vineeta: I would think of it as a replacement for egg freezing, is one way to contextualize where this could, hypothetically, fit into the industry. Today, women are freezing eggs in their 20s so that they may access them in their 30s and 40s or beyond, and this would provide an alternative to having to undertake that process of retrieval, freezing, and hoping that the eggs are usable.

Justin: Often in those retrievals, you’re only able to get — you know, let’s call it 1, 2, 3, sometimes if you’re lucky, more than that — but a limited number of oocytes, and so you have a limited number of shots on goal on potentially having a successful pregnancy. Where I think what got me excited about this paper is this idea of potentially having more opportunities [for] a successful pregnancy that aren’t as constrained by the cost and kind of care experience today that drives the typical IVF process that we alluded to earlier.

Lauren: We’ve thrown a couple different terms around. We’ve thrown around oocyte, we’ve talked about eggs. Let’s define this. What is an oocyte, exactly?

Justin: Yeah. An oocyte is not a static thing, and it goes through a number of different developmental phases, starting from an undifferentiated cell, ultimately to being a true oocyte, which is what we would call a gamete or female sex cell. 

The journey to get there is quite a fascinating one. As you alluded to, it starts, actually, in utero. Then as it gets further along in its development, it starts out as what we call a primary oocyte. And as a primary oocyte, it still has the deployed genetic material. Then it starts meiosis, which is the process of going from being deployed and having two copies of chromosomes, to being haploid or just having a single copy. And in that process, it actually arrests and will stay in that arrested state until that female hits puberty, and then that full mitotic process isn’t completed until after ovulation and even with fertilization. 

So, it’s highly dynamic, and I think the critical piece here is that there’s a lot of gene regulation that’s going on throughout, which opens up the opportunity for research studies like this to really define what are the regulators of that process. Can we recapitulate that in vitro setting to potentially have some of the applications that we talked about earlier?

Lauren: Yeah. I definitely was doing my background reading in preparation for this conversation, and really appreciating all those different steps that happen during — in utero. What happens before puberty, what happens during puberty, what happens during ovulation, what happens at fertilization — there’s all of these different steps, all these different developmental changes that all of them seem to carry the name oocyte.

Justin: One other nuance that I think is important, too, is just that the oocyte in isolation isn’t necessarily enough, right? There has to be what they call a follicle, which is a set of cells that surrounds it. It’s very hormonally active, a lot of cell signaling and transcription factors are produced in that process. And we start to see early recapitulations of that in this study. But in vivo, inside of the female, there’s a lot of other, kind of, cellular activity that goes on around the oocyte cell itself that has a lot of hormonal impact downstream.

Potential uses in healthcare

Lauren: Yeah. That’s a really good point, that it’s very dependent and has a very complex interaction with the ovary and the cells of the ovary. So, on previous episodes of “Journal Club,” we have talked about converting one cell type to another. For example, we’ve talked about converting stem cells into the cells of the pancreas that produce insulin. But what are some of the particular hurdles in creating an oocyte that you wouldn’t have to deal with in just converting a stem cell into, say, a pancreatic cell?

Vineeta: One way to think about the second-order challenge here is that you have to not only get to a primary oocyte, which has grown and has the right differentiation to at least have started on the path of egg generation, so to speak. But beyond being a primary oocyte, you have to get to a secondary oocyte. And the difference is that you have to undergo the whole process of meiosis, which is how we generate genetic diversity. 

And so, that secondary oocyte is a pretty complicated thing to make. Not only because its genome has to have halved in a very unique way, by a very unique process, but a lot of epigenetic signaling that then goes on to determine gene expression networks in the subsequent fertilized embryo are thought to be driven by a program set in that oocyte. So, some genes are expressed off of DNA you got from your mom, some genes are expressed off of DNA you got from your dad, and not vice versa. A lot of that epigenetic programming is thought to stem from a pattern that’s encoded in the secondary oocyte.

Judy: Another major difference between this and other cell types that you could potentially reprogram is just the morphology. So, oocytes are the largest cell that we have in humanity. The size and shape is just really different from what you would expect for other cell types.

Lauren: Right. So, in my previous example of turning a stem cell into a pancreatic cell, there might be morphological differences between those two cell types, but they aren’t necessarily at the scale that you would see in a stem cell to an oocyte. And, fundamentally, you aren’t changing the genome when you’re doing this transition from a stem cell to a pancreatic cell — the genome stays exactly the same. You’re just changing what genes are expressed. 

When you’re creating an oocyte, you’re halving the number of chromosomes that you have in a cell. So, a mature oocyte only has half the number of chromosomes that a normal cell does, and that’s because it meets with a sperm that has half the number of chromosomes that a normal cell does. And now, when they fuse and fertilize, now you get the full complement. So, having to do both of those steps is far more challenging, and represents an additional hurdle that you have when creating these iPS or stem cell-derived oocytes. 

Lauren: So, let’s talk about how you convert one cell type into another. What are, kind of, the — how do we think about how cell fate is controlled?

Judy: So, the way that we think about cell types is often which transcription factors are present. And those transcription factors are proteins that will bind to the DNA and basically cause the production of RNA from a given genomic locus, so from a given gene in the DNA. So, transcription factors are used as the sort of master regulators of a cell type, where one transcription factor might be relevant. 

Just to make it super simple, let’s say a certain transcription factor is relevant for neurons, and another one is relevant for hepatocytes. You would expect that all of the genes related to hepatocyte function and development are going to be controlled by that hepatocyte transcription factor. And in the neuron cell, that transcription factor is not present, so none of those relevant genes are made. That’s an oversimplification, but that’s kind of the idea of how these genetic regulatory networks work.

Lauren: Right. And so, when we’re thinking about in the lab, in the clinic, if we want to convert one cell type into another, we can express these transcription factors, which then leads to the expression of all their downstream genes. And then that, kind of, reprograms the cell and says, “We’re a hepatocyte now, we’re a neuron now, we have this transcription factor that’s driving this — let’s call [it] the gene regulatory network.” And that guides the cell to a specific identity. 

So, in this paper, what they’re doing is they’re trying to identify the transcription factors that govern this development of the oocyte so that they can take those transcription factors, express them in a stem cell, and then encourage that stem cell to become an oocyte. So, with that in mind, let’s start with how the authors identified these key genes — these transcription factors that are involved in oocyte development.

The development of the oocyte

Vineeta: My understanding is that they did whole transcriptome profiling of cells at different stages in mouse oocyte development. So, basically, compared the differential expression of lots and lots of genes at each of those different time points, and constructed a network analysis to nominate specifically not just genes, but regulatory genes. And they used a bioinformatic GO search to get to the subset of transcription factors that they believed were driving the evolution of the transcriptome through the differentiation process.

Judy: Yeah. I think a lot of those genes were already known from studies that had done in vivo work, so I think it’s important to make that distinction. You can do this work in vivo, which means taking oocytes at different stages of development in a mouse and actually measuring the transcriptome there. Or, they created a sort of — an organoid model, for lack of a better word — where they put cells into an environment that is similar to what they would experience during oogenesis, and measured the transcriptome at different points in that process. 

And so, I think the purpose of this was partially to find the genes that are relevant, but also partially to identify the period of time within this model system that should map onto the period of time in in vivo oogenesis. To be able to, basically, run their experiments, and know the right time period to look at the cells.

Lauren: I think that’s a very good point. It was both a — can we identify the correct transcription factors? But can we also validate this very handy in vitro system that we can then use to do further study?

Vineeta: And another way to think about it is — in biology, we talk a lot about necessity and sufficiency, and I actually think they did a better job with necessity than sufficiency. They proved that if you knock out any of these top-nominated transcription factors, that you can’t get differentiation past a certain stage. And you really need this set of factors to be expressed in order to get to a primary oocyte. Sufficiency is a much higher bar, right? You have to prove that the thing you got, the primary oocyte you got, can then go on to do all of the things that you expect it to do. And I would say it’s almost impossible to prove until you’ve generated the end state of, like, a mouse baby. So, they make some progress towards sufficiency, but less.

Judy: There’s also a sort of curious result that I would like to, I don’t know — talk to the authors about and maybe understand a little bit more. Which is that they find these eight genes that they collectively called PPT 8, and they do all of their experiments with these eight genes. But there’s a paragraph in the paper that talks about how they found a subset of four that is sufficient to get the same phenotype you see. But if you have some subset of five, six, or seven that contains that subset of four, it doesn’t necessarily mean that that’s going to work. 

So, there’s sort of this — they are sufficient, but then some other part of those eight — that set of eight — has some interaction with that set of four, such that they didn’t trust that the set of four was truly sufficient. So, there’s some complexity going on there that they’ve definitely moved on from by just using the set of eight.

Lauren: Yeah. There does seem to be maybe a higher level of regulation that hasn’t been elucidated yet.

Justin: A couple other things I thought were interesting, too, is that they had to also expose them to just some somatic cells from the ovary to get the development to take place. And so, I think it, again, reiterates the point that while these transcription factors are likely necessary, there’s also some dynamic signaling that’s happening from somatic cells that surround, as opposed to just being purely driven by those transcription factors. 

One other thing that also caught my attention is the timeline that this all took place in. If you look at what’s happening in vivo, this is usually happening over seven or eight days inside the mouse model. But within the in vitro model, they saw this happen over a couple of days. And so, this is likely a necessary set of transcription factors and regulators, but there are likely other regulators — potentially checkpoints or others — that are absent in the system that’s allowing it to run through this process on a much more accelerated timeline. And potentially could explain some of the other issues that they see downstream with having the morphological appearance of an oocyte, but a lot of the functional aspects of it didn’t quite get that.

Limitations of the current research

Lauren: So, what elements of a functional oocyte were they able to recapitulate with these oocyte-like cells, and what’s missing?

Judy: So, what they did successfully show was growth, the morphology. They also showed a couple of expressions of certain factors that we associate with oocytes, but what they didn’t show is that there’s the right dynamics surrounding the DNA. For example, meiosis didn’t occur, which — it’s also not something that they were aiming for, so I don’t think that’s the bar that we should hold them to, but there was no meiosis. So, what you end up with in the end is a cell with a lot of extra DNA in it. And then the other really important piece is that the methylation pattern is incorrect. It’s completely different from what you would expect in an oocyte.

Lauren: The methylation — that’s one of the key epigenetic modifications. So, that governs how the chromosomes are packaged, and that leads to how accessible certain genes are to be turned off and on at particular rates.

Vineeta: The maternal and paternal imprinting, the mechanism by which is most commonly methylation of the DNA, is really important for health and disease. We know now of many different disease states that are actually attributable to incorrect maternal or paternal imprinting. And so, it’s not a minor issue that methylation wasn’t solved, and one that we’d have to pay a lot more attention to as this research advances.

Lauren: Yes, that’s a really good point. So, the real test — the final test of whether you got an oocyte or not — would be to fertilize it with a sperm and to grow a new being — in this case, a mouse pup — up from that. In this paper, they tried that, but it didn’t quite work. What was the result of this experiment?

Justin: So, what they saw — and somewhat not surprisingly — is that when they did fertilize it, a very small percentage of the cells actually went on to cleave at all, and even of those that did, very few made it beyond the two or four-cell division. Which, a lot of the early cleavage in cells is dependent on having that haploid one single set of chromosomes. And so, in this scenario where they weren’t able to achieve meiosis, which means that they weren’t able to go in with a haploid cell, it’s unsurprising that when the cell was fertilized, it resulted ultimately in non-viable embryos — given that the chromosomal count mix is not consistent with traditional fertilization.

The future of oocyte biology

Lauren: Yeah. So, the paper makes some really great advances in our understanding of how an oocyte develops, what the gene networks are, what the transcription factors are that are regulating these — but there are still a lot of mysteries, and still a lot left to study in this process. When you think about these unanswered questions at the end of the paper, what are the questions that interest you? Where are you interested in seeing this work go next?

Justin: I think for me, when I look at this paper, there are two, kind of, key avenues you could take. One is to look at this as, “Can we use the oocyte as a structural scaffold for other scientific applications?” And we see this happening already today, where enucleated oocytes, or rather the oocyte without the nucleus of the DNA material, are used in applications for mitochondrial disease to other potential therapeutic applications. Those are relatively limited. 

And so, for me, the biggest question that this tees up is, “What are the next steps that need to be taken to really understand how we get the nuclear — how we get the meiosis portion of this correct? Because if we’re eventually going to reach, kind of, the vision that we outlined at the beginning — of having this be a critical asset and enabling greater access to fertility treatments — it’s really an absolute necessity and table stake in order for this to progress.

Judy: Yeah, I totally agree with Justin. At the end, the authors say that this is a potential — a potential use case here is somatic cell nuclear transfer, which is another way of saying cloning. I think that there’s not that much need for this kind of a solution for those applications. I think we should really see this as a stepping stone toward [an] entirely ex vivo generation of an oocyte for the purpose of in vitro fertilization.

Vineeta: Yeah. I think one of the things that’s hardest about this particular — the fertility use case of stem cell research is that, presumably, the parents of a prospective child want to see their genome represented in the progeny. A lot of other applications of stem cell research don’t actually have that requirement, especially if you can design creative ways to avoid immunosuppression and to create immune cloaking of a stem cell-derived therapy, and so on. You might actually envision in a lot of other fields an off-the-shelf stem cell-derived or iPSC-derived cell therapy that can be really therapeutic for a lot of patients with different diseases. 

Here, we can’t have that, or at least it doesn’t solve some part of the core fertility challenge. And so, because you’re so dependent on actually running this process on a case-by-case basis with each couple, I think we just have an even further way to go on this. It’s not like you could create a bank at some point, and then differentiate them from that every time you need to spin up a new embryo. You really have to get the whole process right end-to-end from the point of a patient-specific iPSC cell line. And that’s really hard.

Lauren: There’s not to be an allogenic option.

Vineeta: Exactly. There’s no allo-embryo to be had here.

Justin: And the bottom line for me is — in thinking of the ultimate translation of this, we’re obviously in the earliest of early innings in terms of this actually translating to being, kind of, that holy grail for fertility and the fertility treatments that we talked about earlier. I think this does clarify a lot of our understanding about what it takes to create a structurally similar cell to an oocyte. But ultimately, to reach the broader vision that we want, there’s still a lot of work that needs to be done.

Judy: This is actually a really important step for developmental biology as well, because I think it’s one thing to do descriptive research, where you understand an existing system and you characterize all the pieces of it, and it’s an entirely different level of understanding when you can actually rebuild it. So, I think there’s the phrase Feynman says: “You don’t really understand something until you can build it,” or some variation on that. And so, this is a perfect example of a paper that’s using building to get to an understanding that is deeper than what we had before.

Lauren: I think that’s a perfect note to end on. Justin, Judy, Vineeta, thank you for joining me on “Journal Club” today.

Vineeta: Thank you, Lauren.

Judy: Thanks, Lauren.

Justin: Thanks, Lauren. Thanks, Judy. Thanks, Vineeta. This was fun.

Lauren: And that’s it for “Journal Club” this week. If you enjoyed this episode, please subscribe, rate, and review wherever you listen to the podcast. And to learn more about how biology is technology, subscribe to our newsletter at a16z.com/newsletters.