Reinventing assisted reproductive technology.
Giving infertile and same sex couples a chance at having their own family.
We all know someone who has struggled with infertility.
Whether it be the woman who has gone through multiple rounds of IVF, or the couple who loves kids but can’t have their own; infertility carries a huge psychological and physical toll on those affected.
Statistically speaking, 1 in 8 couples struggle to have biological children. This numbers totals to 186 million people worldwide. It’s even worse in developing countries where 1 in 4 couples face this reality.
Check out this article my team member wrote for more information on the causes of infertility.
The two most commonly used assisted reproductive technologies (ART) are IVF (in vitro fertilization) and IUI (intrauterine insemination). IUI has a 10–20% effectiveness rate and IVF is costly, incredibly painful, and only 20–45% effective.
My team has spent the past 5 weeks trying to understand this problem and come up with a solution 10x better than the status quo.
Introducing project Janam, a moonshot with a mission of reinventing assisted reproductive technology by completely bypassing the need to have viable eggs and sperm.
We had two goals in mind:
- Decrease pain of treatment 🤕
- Increase effectiveness of treatment 📈
Our solution? Make babies by reprogramming skin cells.
(or any somatic cell for that matter!)
Yep, you heard that right!
The pipeline consists of 5 key steps.
- Get skin fibroblasts through a skin biopsy
2. Use CRISPRa to turn skin fibroblasts into induced pluripotent stem cells (iPSCs)
3. Reprogram iPSCs into Primordial Germ Cells (pre-cursors to egg and sperm)
4. Maturing the PGCs into eggs and sperm using an artificial tissue engineered environment
5. Combine the egg and sperm to form viable embryos that can be implanted in the uterus
Turning Skin Cells into Induced Pluripotent Stem Cells 🧬
We’re able to reprogram primary skin fibroblasts into IPS cell by targeting 5 endogenous genes: OCT4, SOX2, KLF4, MYC (the 4 Yamanaka factors) and LIN28A promoters using CRISPRa. Over-expressing this genes helps us induce pluripotency.
CRISPRa is a gene-editing technology that does not cut DNA, but instead activates the expression of certain genes without mutating the genome using dCas9VP192 as the gRNA. Through experiments conducted, researchers have seen that along with dCas9 additionally guides were required for gene activation.
The reprogramming efficiency can be improved by targeting Alu-motif enriched near genes which play a role in embryo genome activation (EEA-motif) whose effect is mediated in part by a stronger activation of NANOG and REX1 genes.
The process of electroporation makes cell membranes become more permeable by introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to briefly open the pores.
Electroporation of the skin fibroblasts is used to replicate dCas9VPH plasmid containing TP53 targeting shRNA, EEA motif targeting gRNA plasmid, reprogramming factor targeting gRNA plasmid (OMKSL), and an additional KLF4 and MYC targeting gRNA plasmid (KM), resulting in the emergence of iPSC-like colonies. The resulting colonies could be expanded into iPSC lines demonstrating typical pluripotency markers as well as differentiation into 3 germ layers in vitro and in vivo.
Turning IPSCs into Primordial Germ Cells (pre-cursors to egg and sperm) 🦠
One of the main goals of reproductive medicine right now is to generate eggs and sperm from pluripotent stem cells fully in vitro. In 2013, Hayashi, K & Saitou were able to generate eggs from mouse embryonic stem cells and induced pluripotent stem cells using both in vivo and in vitro methods.
We only use the in vitro portion of the protocol they used.
It goes from iPSCs ➡️ EpiLCs ➡️ PGCLCs.
EpiLC are epiblast cells which later differentiate into three layers: definitive endoderm, mesoderm and ectoderm. Epiblast stem cells, like embryonic cells, are pluripotent. The epiblast will differentiate very early into germ cell progenitors, the primordial germ cells (PGC).
If you want to read the full procedure we intend on using, check out steps 1–53 from “Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells”. This was developed by Hayashi, K., & Saitou, M in 2013 and is extremely comprehensive- it goes through the mediums used for culturing, exact measurements, and anything else one would possibly need to know when doing this in their basement (Just kidding. This is definitely illegal to do at home 😔). The protocol is quite complicated, which is why I condensed the steps into 5 major milestones:
- Maintenance of iPSCs ● 2 hours for coating the culture plate and 30 min for passage
2. EpiLC differentiation ● 30 min for treating cells and 2 days incubation
3. PGCLC differentiation ● 1 hour for treating cells and 3–4 days for incubation
4. Dissociation of PGCLCs for purification ● 1–2 hours
5. FACS sorting of PGCLCs ●2 hours
Once we have PGCLC’s, we want to turn them into germ cells.
Artificial Tissue Engineered Environment for Cell Proliferation: PGCLCs to Germ Cells 🧪
Turning PGCs/PGCLCs into germ cells has been done in vivo in mice, however it is much harder to do outside the body.
Once PGCs or PGCLCs (primordial germ cell like-cells) have been developed from IPSCs, researchers put the pre cursors back into mice’s testes to generate sperm-which they would extract afterwards to continue the IVF process. In this study they actually birthed live pups! 🐁🐁🐁
We know the procedure works the way Hayashi and Saitou, but optimally this wouldn’t be done by putting the PGCLCs back into the body.
My team wanted to mimic this process without going in vivo, but it hasn’t really been done before. This is the step where scientists are currently stuck- they had the PGCs but they could only put them back in the body to produce viable sperm or egg. This is because they need to be able to undergo mitosis and meiosis, which is difficult to make happen in a petri dish environment.
Here’s the moonshot 🚀: placing the PGCLCs in an artificial tissue engineered environment for cell proliferation. The PGCLCs will turn into immature eggs and sperm through their respective tissue environments.
This way, we can even take a male somatic cell and turn it into an egg. We can take a female’s somatic cell and turn it into sperm. Same sex couples would be able to reproduce if we took PGCs from both partners and induced one to become a sperm cell and one to become an egg.
Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts using viable cells in a suitable matrix for regenerative medicine.
Engineered tissues are made by ‘seeding’ cells into a bioengineered scaffold where they reorganize it into a material suitable for use as an artificial tissue, such as egg or sperm.
It’s also been found that a cell population can survive and undergo division in a tissue engineered environment.
Using the light microscopy technique to proliferate the cells, we would also need glutaraldehyde, vincristine, osmium tetroxide (dehydrated alcohol used to multiply cells in environment), Premarin (for oocytes to develop into egg through hormonal growth), testosterone, a bio-reactor, and scaffolds.
Vincristine is quite dangerous however it works as it allows us to interrupt the cell cycle in order for chromosomes to disperse and causes DNA to multiply.
Scaffold Design
Scaffolds are 3d structures whose purpose is to provide a favourable environment for cells adhesion and growth and to give structural support. Studies show it may just be possible to build a suitable scaffold for any tissue however the design of scaffold is important. The architecture must be tissue specific in terms of porosity (pore shape and size), surface topography (shape and roughness). These features are essential to improve cell homing (adhesion, survival, migration, differentiation) and to facilitate the flow of culture medium (in vitro) through the construct in order to ensure the supply of nutrients and oxygenation.
In Vitro Maturation (IVM) 🧫
The egg needs to be matured using IVM. This technique has been around for quite some time, however the old process didn’t produce very healthy mature eggs. It used hormones to encourage eggs to reach maturation.
Lucky for us- scientists have come up with a better procedure. The new method produces 50% more healthy mature eggs using a protein dimer called cumulin which encourages eggs to reach maturation by communicating with the oocyte’s supporting cells.
Cumulin is an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family. It’s a potent activator of Granulosa Cells, improving oocyte quality. When oocyte is combined with cell signalling molecules called cyclic-AMP (c-AMP) modulators, we harness the power of an oocyte protein (growth factor) that promotes maturation.
Now we have a mature egg and the sperm, we need to fertilize and implant the embryos. Janam will send the eggs and sperm to fertility clinics who can then perform in vitro fertilization.
Fertilizing Egg & Implantation of Embryo💉🥚
The process:
- An egg is fertilized by injecting a single sperm into the egg or mixing the egg with sperm in a petri dish
- The fertilized egg (embryo) is transferred into the uterus.
Injecting a single sperm into the egg is known as intracytoplasmic sperm injection (ICSI) to manually fertilize the eggs.
ICSI was developed to help achieve fertilization for couples with severe male factor infertility or couples who have had failure to fertilize in a previous in vitro fertilization (IVF) attempt.
The most important indicator of ICSI success appears to be the fertilization rate of the procedure. The fertilization rate in the UCSF IVF laboratory is exceptional — currently 80 to 85 percent.
The fertilized egg is an embryo ready for implantation in the woman’s uterus. For a gay male couple, they have the option of getting a surrogate or using an artificial womb (this will be commercialized some years in the future).
Where are we at right now? 🔬
Skin ➡️ IPSC works effectively in human cells.
I’ve linked a paper from 2018 here. It talks about iPS cells generated by retroviral transduction of the 4 transcription factors: Oct3/4, Sox2, Klf4, and c-Myc and using CRIPR-Cas9-based gene activation to reprogram primary human skin fibroblasts into pluripotent cells.
Also here’s a paper from 2007 showing successful reprogramming of differentiated human somatic cells into a pluripotent state. The study demonstrates the generation of iPSCs from adult human dermal fibroblasts with the same 4 Yamanaka factors.
IPSC ➡️ PGC in mice has been done successfully in several papers.
In 2016, they were able to obtain fertile offspring starting with mouse embryonic and induced pluripotent cells. Here’s a paper that goes through making gametes (eggs and sperm) from stem cells. Keep in mind PGCs are the precursors of gametes, we are only attempting to complete half the protocol done in that study (iPSC to PGC).
IPSC ➡️ PGC in humans has low success rates, but they’re working on it
It’s challenging to convert iPSCs into PGCLCs to obtain eggs & sperm. The differentiation of iPS cells into PGCLCs in vitro hasn’t been achieved yet.
What still needs to be done?
IPSC ➡️ PGC in humans with high success rates
PGC ➡️ Germ in vitro
This has been done in vivo, but in vitro is more difficult- which is why we proposed an artificial tissue engineered environment.
This study grew porcine aortic wall cells in a porous sponge scaffold to become aortic valve substitutes. Cells were freshly acquired and seeded on identical sponges, grown under culture conditions for a 4 week period. It was done in 2005, which is why it’s important to examine advances made since then.
Biomaterials used for scaffold fabrication have improved 10x, as have fabrication techniques in general.
Here are a list of different fabrication methods developed since 2005: Stimulus-Triggered Approaches, 3-D Printing Based on Particle Bonding, Laser-Based Techniques, Stereolithography, Two Photon Polymerization, Deposition-Based Approaches, Extrusion-Based Techniques: Fuse Deposition Modelling, Droplet-Based Techniques: Multijet Printing, and 3-D Bioprinting Techniques.
This just goes to show that we will be able to intermix different fabrication types to have better results. We just need people to be working on these tissue engineered environments which allow cells to proliferate.
How long until commercial viability?
After reading a billion (or so 🙃) papers and talking to experts in the field, I would roughly estimate:
Figuring out IPSC ➡️ PGC in humans + PGC ➡️ Germ in vitro will probably take 3–5 years. Clinical trials and testing will take 5 years.
186 million people world wide are infertile or unable to have a biological child. These people spend their lives yearning for children of their own for the unmatched joy that comes with bringing another human into the world.
With Janam’s technology everyone has a chance to start a family of their own. There is no hormone injection or ovarian stimulation but best of all; There is no constant pain for you or your partner.
This is the revolution of assisted reproductive technology, and my team and I can’t wait to see the day our moonshot becomes reality around the globe. 🚀
I hope you enjoyed this article, as much as I enjoyed writing it :) Check out my amazing team members who helped make this possible: Manroop Kalsi, Richa Pandya, and Alisha Arora.
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