Likagen: Growing Lost Limbs

Creating a world without limits!

Limbs help us do so many things: our legs let us get from place to place, they let us dance around in the comfort of our homes, and they let us kick around a soccer ball with our friends. And our arms are awesome too! They let us hug people, grab a snack to feed ourselves, and type things out, much like I’m doing right now with this article.

Can you imagine leading your life without even one of your limbs? We’re so used to using our limbs every single day for seemingly mundane things, but people without limbs are reminded of these things as things.

This is the case for 30 million people who live with limb loss globally. About 185,000 people have an amputation each year. This means that 300 to 500 amputations are performed .

The main causes of limb loss are:

  1. Vascular disease (54% of cases)
  2. Trauma, often war-induced (45% of cases)
  3. Cancer (about 1% of cases)

Now, normally an amputee could go do the doctor and get a prosthetic, and everything would be okay, right? Well, there are more than a few problems with prosthetics:

  • For an amputee who needs to replace their prosthetic every few years or so, costs can range from
  • Prosthetics can often be incredibly uncomfortable and won’t always work the right way either.

Most importantly, most prosthetics don’t have the right electrical signaling that mimics the nerve connection between the peripheral nerves of the limb and the brain — rendering them ineffective.

So now we look to nature for solutions, which brings us to the amphibian of the hour: the salamander.

If salamanders can grow their own limbs, what’s stopping us from doing it?

“Salamanders do have certain genes that are “turned off” in humans”, Gardiner said. “Perhaps those genes enable regeneration or at least help control the process. Something in humans’ evolutionary past selected against expressing those genes the way salamanders do. Nobody knows what that something was.”

According to a study performed by James Godwin and his colleagues, salamanders rely heavily on the use of macrophages, which are immune cells that were proven to be critical in regenerating lost limbs.

Salamanders that were amputated and depleted of all of their macrophages couldn’t grow back their limbs at all, where other amputated salamanders that were depleted of some of their macrophages grew back the lost limbs at a slow rate.

According to the study, macrophage-depleted salamander limb stumps also failed to fully activate expression of TGF-β1, a pleiotropic growth factor and key regulator of embryonic development and mammalian wound healing, as well as its target genes and . Inhibition of TGF-β1 signaling blocks successful limb regeneration in the salamander, and fibronectin forms part of the provisional matrix during scar-free repair that may contribute to a environment that allows for successful regeneration. The stunted activation of MMP9 and MMP3 on macrophage ablation is consistent with an essential role for matrix remodeling in successful limb re-generation and implicates macrophages as a key regulator of matrix degrading enzymes, enzymes which would ultimately prevent the formation of a new limb.

So why can’t humans do the same?

For a human limb to regenerate, you need bone, muscle, blood vessels, nerves and skin. According to David Gardiner, professor of developmental and cell biology at the University of California, Irvine, “To regrow a limb, the cells need to know where they are — are they at the very tip of a limb by the fingers, or are they at the elbow joint? — and they need to build the right structures in the right order.” (Hint: this is where special cells called fibroblasts come in.)

Nanotech & Regrowing Your Limbs!

My research project Likagen is tackling this exact problem.

The most promising solution that we’re working on is using targeted carbon nanotubes to deliver the genes involved in development of blood, bone, muscle, and nerves at the site of amputation. Essentially, I’m leveraging nanotech to mimic the way salamanders regrow their own limbs!

In a nutshell, this is what the process looks like:

  1. 3-D printed Monocryl scaffolds for structuring regrowth
  2. Use target genes to….
  3. Begin bone regeneration with bone stem cells
  4. Blood and muscle growth with mesenchymal stem cells
  5. Nerve and skin growth with induced pluripotent stem cells.

A Closer Look at the Carbon Nanotubes

Carbon nanotubes, specifically multi-walled carbon nanotubes (or MWCNTs), exhibit suitable characteristics as large contact surface, stability, flexibility and nucleic acid binding affinity, which make them optimal for gene delivery. Additionally, non-covalent binding of nucleic acids on the surface of the MWCNTs increases the efficiency of content release in the cell through endocytosis. We plan use to carboxylic multi-walled carbon nanotubes (or COOH-MWCNTS) specifically, due to their high solubility and low toxicity, two barriers which prevent the use of MWNCTs for biomedical purposes.

A study performed by researchers in Brazil showed that COOH-MWCNTs can effectively deliver DNA into the fibroblasts (or developmental cells) of cows; due to these results, we hypothesize the same can be done for humans if the COOH-MWCNTS are targeted properly (the same study indicates that this can be done with relative ease).

In short, MWNCTs are synthesizes through a vapor deposition process that consists of using ferrocene and ethylene as the transition metal and carbon precursors, respectively. They’re then heated to remove any abnormal carbon structures that remain and then filtered using HCl to remove any chemical residues.

Nitric acid oxidation is used to add the carboxyl (-COOH) groups to the surface of the nanotubes. After the cleaning process, the nanotubes are dried at 80 degrees Celsius for 12 hours.

What are the Target Genes?

We broke down the target genes into four essential categories: blood vesssels, bone, muscles and nerves.

Genes for Blood Vessel Development:

  • Nkx2–5, activates a certain gene, and in turn, determines the fate of a group of cells in a developing embryo. They’re essential for blood vessel development specifically.
  • Vascular endothelial growth factor is another important family of genes for blood vessel development.

Genes for Bone Development:

Caption: “Sequence and stages of the osteoblast lineage from a self-renewing, pluripotent mesenchymal stem cell to terminally differentiated osteocyte is diagrammatically illustrated. The characteristic feature of each developmental stage is indicated below the cell morphology. Next row summarizes the key transcription factor and co-regulatory protein involved in genetic control of osteoblast differentiation. Factors that negatively regulate Runx2 activity and osteoblast differentiation are indicated in red. Several physiologic mediators influencing osteoblast development, including transforming growth factor β (TGFβ), the bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs), Wnt/β-catenin signaling and hormones are also indicated. Secretory molecules, receptor and signal transducer that inhibit osteoblast maturation are highlighted in red. Last row summarize phenotypic marker genes expressed at different developmental stages of osteoblast differentiation.”

Target Genes:

  • Sox2, Msx1,2 Bapx1 (osteoblast differentiation)
  • FGF18, GH, FGFr2 (genes that code for proteins that influence osteoblast development)
  • Oct4, e-Myc, Nanog, CD15 (marker genes expressed at different developmental stages of osteoblast differentiation)

Genes Involved with Skin Development:

  • The Human Protein Atlas has a page which describes a family of genes that code for various keratin proteins that are essential for skin development.
  • The genes that code for the p63 and Satb1 proteins, which were found by researchers to also be key in skin development.

Genes Involved in Muscle Development:

  • provides instructions for making a protein called myostatin. . Myostatin is found almost exclusively in muscles used for movement (skeletal muscles), where it is active both before and after birth. This protein normally restrains muscle growth, ensuring that muscles do not grow too large.
  • controls muscle growth with help from the gene, which produces the myostatin protein.

Genes Involved With Nerve Development:

  • The linked study above, performed by researchers at the Heinrich-Heine-University, Dusseldorf, Germany, links a family of genes that are involved with neural development. Genes of interest include: cytosolic phosphoprotein, myelin basic protein, myelin protein zero, myelin proteolipid protein, and myocilin to name a few.

Genes Involved with Artery Development:

  • Engineer stem cells with Notch receptor and and (essential for arterial differentiation). ( pathwaycontrols Notch signaling.
  • Vascular Endothelial Growth Factor A (VEGFA) will promote the development of arteries.

Genes Involved with Vein Development:

  • We’ll still need to make sure to include VEGFA , but those two genes will differentiate the veins from the arteries and regular blood cells.

Other Genes Involved with Limb Development:

  • Hox family of genes as well as genes influencing the SHH pathway and Apical ectodermal ridge (AER) pathway influence hand, finger and toe development.

The treatment will also include the insertion of FGF-10, a gene coding a growth factor protein that has been shown to be essential for limb positioning during development.

Copies of these genes will be taken from the patient’s own genome in order to ensure a growth process that’s as efficient as possible.

Overall, we hypothesize that inserting copies of these target genes from the amputee’s genome makes sense as we are actively trying to culture the arm from the patient’s own body and stem cells.

How Does the System Actually Work?

As shown in the image above, the goal of the system is to tag the DNA of the different target genes to a nanotube to bone stem cells for bone growth, mesenchymal stem cells for blood and muscle growth and induced pluripotent stem cells for nerve and skin growth.

The product itself would be a topical applicant that would be put on the site of amputation. There wouldn’t be a worry of the nanotubes working on just the bone application site, or just for the muscles — proper targeting would take care of that issue.

We then generate the Monocryl scaffolding through 3-D printing for the patient’s limb based on the patients’ body proportions. We hypothesize that providing this scaffolding will help the stem cells proliferate and grow to form a new limb that would be exactly like the original and lost one.

There are minimal safety concerns involved with using Monocryl as well.

A Breakdown of the Growth and Incubation Period

Estimated Time of Incubation: 15 days

Incubation Method: We apply stem cell topical applicant and have it grow inside of an incubation tube to prevent any harm.

How Did We Calculate That:

What is the average rate of mitosis for human epidermis? About 90 min

What is the average rate of mitosis for bones?

  • This paper suggests that the mitotic index (number of cells undergoing mitosis is 8.86 for every 1000 cells in a given day given that mitosis occurs at a constant rate.
  • How many cells does an adult human bone have?
  • 37.2 trillion cells in human body, 15% of which is bone, divided by 206 bones in the adult human body = 27 billion cells per human bone
  • 23 billion will grow in a day if the above mitosis rate is applied
  • Therefore we can expect the bone to fully develop in two days or so.
  • Same can go for cartilage.

What is the average rate of mitosis for blood vessels, arteries and veins?

  • Using the same paper for bones, we can estimate this to be about 5–7 days .

What is the average rate of mitosis for muscle cells?

  • Assume that bone mitotic rate is the same for muscle calls
  • 37.2 trillion cells in human body, 50% of which is muscle, divided by 4 muscles in an adult arm = 45.6 trillion cells per muscle
  • 4.2 trillion will grow in a day with the same mitosis rate
  • So assuming that muscles grow side by side, we can expect two days.

What is the average rate of mitosis for nerves?

  • This study indicates that over a period of 3–7 peripheral nerves grow back after injury almost completely after MMP-9 is inhibited
  • We can likely say nerve development will take at least 8 days.

Assumptions Made:

  • The individual parts grow alongside each other
  • The research used to make these estimates was mostly done on non-human species and that we will likely need to begin by doing our own research to accrue more data and develop better timelines and estimates.

Likagen: A Company That Grows Lost Limbs

I started this project because I knew that there were millions of people who couldn’t maximize their lives due to losing their limbs. I thought “if salamanders can regrow their own limbs, and use them as normally as before,

Likagen’s vision for the future is as simple yet powerful as this: Creating a world without limits. The overall goal is pushing the human body beyond its current limits so mankind can .

The mission is this: Leveraging nanotechnology, gene therapy, and stem cells to regrow lost limbs and empower amputees. These three technologies are going to revolutionize the future, and we want to start using them now to empower the amputees of today.

Finally, we have Likhagen’s core values. Present in the way our employees build relationships with customers and each other in teams, as well as how we handle projects, these values are:

  • : This is our most important company value. Hopefully this is also a no-brainer, as we’re committed to growing limbs (another key thing we look for — a good sense of humor). Everyone has room for improvement, and Likhagen’s no exception. When it comes to doing any experiment or project, we’re focused on viewing results as objectively as possible. We’re asking ourselves: what went right / wrong here and why? What can I do better the next time around?
  • : Everyone’s extremely unique. We make sure that everyone feels comfortable to express themselves freely, which is why we also promote
  • : This one falls right in line with the growth mindset. It’s all about keep the urge to get better alive, and never stopping working towards our goals and visions.
  • : With every big problem that exists in today’s world, there needs to be a big solution. At Likhagen, we push ourselves to think outside the box and focus on how we can make tomorrow’s reality possible today.

Who’s Going to Pay for Likhagen’s Product?

The amputees who want a better chance at life.

I’m also looking for investors who want to use their wealth to support good causes and make a tangible impact on the world.

How Do We Want People to Feel?

Likhagen wants you to have faith and feel empowered: you have the power to change your life for the better, and we’re helping you grow your own limbs!

Cost Breakdown

Implementation and our Long-Term Goals

By 2030, we plan to begin clinical trials with Likhagen’s products and empower amputees across the world. Here’s a breakdown of how we’re getting to that stage, from now until 2030.

2020–2022, Validation of Ideas:

  • Conduct company research on the average mitotic rates for cells for all of the parts of a limb (blood, bone, etc) to generate more realistic estimates.
  • Conduct research using carbon nanotubes to transfect target genes into stem cells and iterate based on the results
  • Conduct further research on genes that differentiate between growing hands and fingers and feet and toes.

2022–2024, Human Limb Generation:

  • We work on physically generating an entire human arm using stem cell treatment which includes the target genes.
  • We ask the following questions are the genes helping the stem cells develop into the right parts of them? Which genes are inhibiting the development of the arm, and which genes would need to replace them in order to ensure optimal development?
  • We then move on to generating a human leg with the same or modified version of the stem cell treatment, asking the same questions.
  • We plan to iterate versions of the treatment from the experimental results as necessary.

2024–2030, Animal Experimentation:

Begin trials on mice:

  • We will conduct at least 100 trials a year with at least 10 mice per trial.
  • We amputate the mouse’s paw and apply our treatment. From these experiments, we will better assess the efficacy of our treatment at the molecular level.
  • Similar questions to the limb generation experiments will be asked: are the genes helping the stem cells develop into the right parts of the limb? Which genes are inhibiting the development of the limb, and which genes would need to replace them in order to ensure optimal limb development? Are they developing proportionally to the rest of the mouse’s body?
  • We plan to iterate on this feedback as necessary
  • Once we have at least a 95% success rate for the mice trials, we move on to…

2030 — Launch Likagen:

  • Begin human clinical trials for amputees across the United States and beyond.

What’s Likagen Up To Now?

I’m currently developing an experiment looking at the creation of fruit fly induced pluripotent stem cells in conjunction with imaginal disc cells to observe developmental processes. The overall goal of this is to understand how this development of a fruit fly leg progress, in order to understand how human limb regeneration methods can be derived similarly.

Looking Towards the Future

We’re devoting to creating a world without any limits, especially the limits of the human body. We’re reshaping the status quo so that amputees don’t have to feel despair for not being able to do the same things with their own limbs. Thanks to Likhagen, a new future is coming: one where you lose a limb today, and get a better one tomorrow.

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Apurva Joshi

Currently conducting independent research in iPSC derivation. Outside of that: 2nd-yr bchm & neuro @ brandeis, alum @ TKS, writer of medium articles