Haploid embryonic stem cells

Dieter Egli


video: https://www.youtube.com/watch?v=rfz4k7HGsOw&index=4&list=PLHpV_30XFQ8R2Kpcc1pwwXsFnJOCQVndt&t=19m53s

I am going to talk about a project we conducted that I believe has about half the work to write the human genome. That's great. We already did half, so why not do the rest of it?

Eukaryotic genomes exist in various forms. Haploid genome of yeast to make a synthetic chromosome-- if you want to write a genome, you would rather do it once, rather than 8 times like in strawberry. So I think haploid has a great advantage. It's not just less DNA in it. But also it greatly facilitates the analysis of the genome. There is great value in doing this in human cells. If we want to understand the human genome, we need to be able to change it as much as we can and to gain the most functional insights.

Haploid cells have an absence of complementation by other alleles, so whatever you do to the cell should be functionally expressed. Haploid cells are often just in germ cells. The question really was, can a haploid human genome divide and differentiate? If a cell is just haploid, can it also differentiate?

At my lab in New York, we took human oocytes. When they are at mitosis, they have replicated a geonme. They segregate those. The oocyte is already haploid. Then you have a single unreplicated genome, and the mitotic division, then you start developing as a haploid cell, and occassionally the cell does not divide so you have two nuclei in one cell which generates a diploid cell. So you have a mixture of cells, some haploid cells and some diploid cells.

So what we had to do, in cooperation with another lab, is to isolate those cells. This can be done by staining those cells and then analyzing in a FACS and sorting them. You can see here that the more intense the staining the more DNA it has. The less intense it is the less DNA. So the unreplicated one in g1, there is no diploid cell, ..... this way, we can generate highly pure cells that are haploid.

What these cells do is-- over time, cells do diploidize. When cells fail, even if it's just a fraction of the culture, because there's no reversion to haploidy, so the culture becomes gradually more diploidy. So the sorting needs to be done perhaps every 3 to 4 weeks.

The karyotypes are stable. There are easy ways to check for ploidy. One that I really like is simply using unistaining and checking the number of centomere spots. It works in Che cells. If it's around 40 or 46, then it's diploid. This is a common technique. Also sorting is not hard either.

There's very few differences between haploid cells and diploid cells, surprisingly. Gene expression... and they have one X chromosome. Human embryonic stem cells inactivate one X, which means X to other, which shows here, in gene expression, is one to two. Haploid cells have an X chromosome so it's a 1-to-1 ratio. So this somehow doesn't seem to effect those cells in remarkable ways. Also, they have smaller volumes and less total RNA.

We know they can be inspected, they can be used for genetic modification, and genetic screening and they are ameniable to genetic modification. Can they differentiate into various cell types? Various eukaryotic organisms can exist as haploids. For human cells, what are they capable of? We differentiated those cells into various lineages. Here you see neurons, FACS sorted and centomere staining, and the same for cardiomyocytes and the same for endoderm cells, we also made some beta cells. There's a trend to more cells becoming diploid, although a significant fraction remain haploid.

Here you can see a teratomas, which is in vivo differentiation of stem cells, there's a surprisingly high number of haploid and FACS analysis. Huma ncells as haploids have the capability to give to differentiate to various cell types.

What's the strength of haploid embryonic stem cell systems? There are stringent molecular and functional testing of genetic changes. It can be differentiated in any cell.

My research has been the target of many ethical studies over the years. Often what happens is that there's misinformation, at least restriction on research and strong regulation, whereas it's not used in actual application. I think the focus should be on what's done with it, and not holding back on what research is pursued. We should determine if there is value to this. This is a law that as of today still effects stem cell research. NIH cannot fund this type of work right now. These cells you just saw have no potential for making a human being, but they fall under the jurisdiction of that law. I asked the NIH if they would consider including my cells beyond the mandate of the law, and they said no.

Some of my collaborators include Gloryn Chia, Lina Sui, Mark Sauer, Nissim Benvenisty, Tamar Golav-Lev, Ofra Yanuka, Uri Weissbein. Thank you.

Q: I was wondering if you had tried to fuse two haploid cells together.

A: If you fuse them, you're going to have a diploid, you'd be done. We haven't done that yet. I think the focus here is on keeping them haploid. There are many things that could be done with those cells.

Q: I was wondering practical question.. sorting them every so often. What's the mutagenic impact of one round of sort on-- how many mutations would you get every time you do this cell sorting?

A: Good question. We don't know the answer yet. There's a stain. There's a low dose of... I suspect there's some impact. We haven't quantified it yet. It looks, on the level of using those cells, it certainly hasn't impacted their function or their ability to grow and differentiate. It seems to be low toxicity. Yes, we have to address that question.

Q: ...

A: Don't know yet. I suspect it's simple lack of .. in a fraction of the cells. If you grow diploid embryonic stem cell culture, you will have.. these cells don't become diploid anymore, but they have ... so they divide normally. So you don't really.. culture from diploid to tetraploid. In this case, if you culture for a year without sorting, you would probably lose most haploid cells.