Harvard and Duke University create the first fully human ovarian organoid

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Harvard and Duke University create the first fully human ovarian organoid

Harvard and Duke University create the first fully human ovarian organoid, which is expected to open up new treatments for cancer.

“The fully human ovarian organoids developed by us develop several times faster than the existing human/mouse hybrid method, and also replicate many key functions of human ovarian organs. This marks our first step in the field of female reproductive health research. An important step forward. In the future, similar techniques could also be used to treat infertility.” – Co-first author Dr. Merrick Pierson Smela, Wyss Institute and Harvard Medical School.

Human life initially conceives from an egg cell within a mother’s ovary; however, the ovary, an important reproductive organ in humans, is surprisingly poorly studied.

Scientists have been working to create in vitro models of human ovaries in order to gain a deeper understanding of ovarian diseases and explore related treatments.

But existing models, mostly a combination of human and mouse cells, don’t faithfully replicate human ovary function; moreover, they take a lot of time to mature in the lab.

Recently, researchers from Harvard University’s Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, and Duke University collaborated with Gameto, a biotechnology company dedicated to improving the treatment of female reproductive diseases, to create a fully human ovarian organoid , the organoid is capable of supporting egg cell maturation, enabling follicular development and secretion of sex hormones.

Without having to harvest tissue from patients, the model, called an “ovaroid,” supports research on the biology of the human ovary and could lead to the development of new treatments for infertility, ovarian cancer and other diseases.

The technology is currently licensed to Gameto; the “ovaroid” model is described in detail in a paper published by the research team in the journal eLife.

Harvard and Duke University create the first fully human ovarian organoid
https://prod–journal.elifesciences.org/articles/83291

Whole Human Ovarian Organoids

The developing ovary contains germ cells (which later grow into eggs) and somatic cells (which support germ cell growth).

Current laboratory models of the ovary use human germ cells and mouse somatic cells; therefore, Dr. Smela and team members wanted to see if they could induce human stem cells to grow into a functional, fully human ovary (meaning the fully human ovary). Human ovarian organoids will have human germ cells and somatic cells).

The research team decided to target granulosa cells—ovarian body cells that support the development of unfertilized eggs within follicles and secrete the sex hormones estradiol and progesterone.

There was no method to efficiently generate granulosa cells from human induced pluripotent stem cells (iPSCs), so the research team decided to create their own method.

Experimental workflow flow chart:

Harvard and Duke University create the first fully human ovarian organoid

The emerging field of iPSC technology is based on the discovery that proteins called transcription factors (TFs), which bind directly to DNA and control whether certain genes are turned ‘on’ or ‘off’, are introduced into human induced pluripotent stem cells ( iPSCs), these transcription factors will guide the differentiation of human iPSCs into different types of cells such as neurons and fibroblasts.

The research team chose to employ this strategy to generate human granulosa cells. Through the “piggyBac transposition” technology, the researchers inserted 35 candidate TFs into the iPSCs genome.

After these TFs were expressed in iPSCs, the researchers screened the cells to see which ones produced a protein called FOXL2, a known marker of granulosa cells. Six top TFs associated with FOXL2 expression were identified: NR5A1, RUNX1/RUNX2, TCF21, GATA4, KLF2 and NR2F2. Next, we tested different combinations of these top candidate TFs and found that both combinations, NR5A1 and RUNX1 or NR5A1 and RUNX2, consistently upregulated FOXL2. The two combinations also drove the expression of two proteins, AMHR2 and CD82, which are surface markers found on granulosa cells.

The researchers then looked at the full transcriptome of these new cells and found that they expressed many other genes known to be active in granulosa cells.

When the researchers compared data from these new cells with data from other studies of human fetal ovary cells, they found that the new cells were most similar in gene expression to granulosa cells in human ovaries at 12 weeks of gestation. Remarkably, the new method took just five days.

To ensure that these new granulosa-like cells also replicated normal granulosa cell function, the researchers treated the granulosa-like cells with androstenedione and then added follicle-stimulating hormone (FSH).

These cells successfully produced estradiol from androstenedione without the addition of FSH; their production increased when FSH was added.

These granulosa cells also produced progesterone—the hormone that normal human granulosa cells secrete after ovulation.

Harvard and Duke University create the first fully human ovarian organoid

Follicle formation
(A) Day 35 human egg (F66/N.R1.G #7 + hPGCLC) section stained for FOXL2, OCT4 and AMHR2. Scale bar is 40 um. Follicle-like structures are marked with yellow = horns.
(B) Whole oval view of a human follicle-like structure (F66/NR1G#7). Scale bar is 1 mm.
(C) Humanoid (F66/N.R1.G.F #4 + hPGCLC) at day 70 in culture, stained for FOXL2, NR2F2 and AMHR2, showing multiple small follicles (yellow triangles) consisting of a monolayer of FOXL2 + AMHR2 + cells . NR2F2+ cells interspersed among these cells. Scale bar is 100 pm.
(D) Section of human egg (F2/N.R1 #70 +hPGCLC) at day 66 culture, stained for FOXL2, NR2F2, and AMHR2, showing antral follicles composed of FOXL2+AMHR2+granular cells arranged in several layers around the central lumen . NR2F2 staining is seen outside the follicle (labeled “stroma”). Scale bar is 100 μm.

Achieved 100% humanization in 16% of the time

The new granule-like cells function so much like real cells that the researchers co-cultured them with human primordial germ cell-like cells (hPGCLCs) to form ovarian organoids, or “ovaroids,” that include both germ cells and somatic cells.

Four days after the granulosa-like cells were co-cultured with hPGCLCs, they began to produce a protein called DAZL (a marker that heralds the beginning of germ cell maturation journey).

In contrast, ovaries made from mouse somatic cells did not express DAZL until day 32.

Human germ cells do not live long enough to further develop into eggs, but the ovary-like cells begin to form empty follicle-like structures composed of granulosa-like cells after about 16 days – despite the fact that no eggs are present.

By day 70, many follicles of different sizes had formed within the ovary-like, some of which had developed multiple layers characteristic of mature follicles to support the eggs.

“This is a feat in itself—the human ovaroids efficiently replicate the hormone signaling, germ cell maturation, and follicle formation seen in human ovaries,” said Church, senior author of the paper and professor of genetics at Harvard Medical School. more noteworthy, these can be completed in as little as five days; human/mouse hybrid ovarian organoids take a month to complete. This will undoubtedly greatly speed up research on women’s health and reproductive issues .”

The Wyss team is continuing to develop human ovarian organoid models and plans to integrate other ovarian cell types — including hormone-producing theca cells — to more fully replicate the complex functions of the human ovary.

They also hope to improve their culture system to allow germ cells to fully develop into eggs and to determine the optimal dosage of different TFs.

Meanwhile, Gameto has started preclinical research with some leading clinical institutions to explore the derived co-culture system required for human egg maturation.

Don Ingber, founding director of Wyss and professor at Harvard Medical School and Boston Children’s Hospital, said: “Half the population is women, but historically women’s health has not been given the same attention and investment as men.

This is an important step forward for the laboratory to study the human ovary, and we look forward to the breakthrough insights this model can provide for female reproductive health and disease.”

References:

https://www.news-medical.net/news/20230221/First-fully-human-ovarian-organoids-provide-insights-about-female-reproductive-health-and-disease.aspx

Harvard and Duke University create the first fully human ovarian organoid, which is expected to open up new treatments for cancer.

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