Reconstructing Skin from Stem Cells

Karl Koehler, PhD, and Jiyoon Lee, PhD, have engineered the first appendaged human skin inside a petri dish. Their findings could transform the future of reconstructive surgery.

Skin disorders are one of the most common non-fatal conditions in the world. In fact, experts predict one in four people in the United States alone will develop a skin disease. Many of these disorders, such as alopecia or deep scarring from head and neck cancers, are benign. Others, such as third-degree burns and congenital skin blisters, can lead to severe infections. Nonetheless, a disfigured appearance from any of these conditions, no matter how severe, may cause immeasurable psychological and emotional harm.

While skin grafts are a conventional method of reconstructing damaged skin, they also require transplanting healthy, yet sometimes mismatched, tissue from another part of the body. Skin grafts from a donor are another option, but one that risks rejection by the immune system or complications such as hematomas and limited range of motion.

Researchers have turned to engineering artificial skin organoids, or clusters of cells and tissues grown from stem cells, as a more effective way of reconstructing skin back to its natural appearance and function. However, no human skin organoid has ever been engineered with the appendages necessary for retaining fluids or mediating sensation. These appendages, which include nerves, fats, glands and hair follicles, allow skin to perform its essential functions.

“Growing these appendages has been the ‘holy grail’ of the skin biology field for decades,” said Karl Koehler, PhD, Assistant Professor of Otolaryngology–Head and Neck Surgery at Harvard Medical School and Research Associate at the F.M. Kirby Neurobiology Center at Boston Children’s Hospital. “The mind instantly wanders to its applications for facial plastic surgery and using the artificial grafts to rebuild skin sites in pediatric head and neck cancer cases.”

Working in collaboration with Jiyoon Lee, PhD, an Instructor in Surgery at Harvard Medical School and a Research Associate at Boston Children’s Hospital, Dr. Koehler has successfully implemented a novel method of engineering stem cells into the first-ever appendaged human skin organoid. Reported for the first time in Nature, their findings have provided a foundation for studies that could completely change the future of reconstructive and facial plastic surgery.

A proof-of-concept that began in the ear

For more than 50 years, researchers have attempted to grow appendaged human skin organoids in a myriad of ways. One of the most common methods has been growing the top layer of skin, also known as the epidermis, separate from the bottom layer of skin, the dermis. By manipulating stem cells so that each layer is engineered separately, researchers thought they could control how the two layers communicated with one another. In theory, this method would facilitate the development of nerves, fats and hair follicles. However, these attempts have failed.

In 2013, Dr. Koehler, then an Associate Researcher at the Indiana University School of Medicine, was researching the development of hair cells in mice when he attempted to develop a complete inner ear organoid ex vivo. He stumbled upon something unexpected: the epidermis and dermis of an ear canal growing together next to the inner ear.

“We almost thought of it as a weed in our garden,” Dr. Koehler said. “Ironically, we were trying to eliminate this unwanted byproduct and maximize the development of the inner-ear tissue.”

Once hair follicles appeared on the unwanted skin tissue, Dr. Koehler recognized the opportunity at hand and shifted some of his focus away from inner-ear hair cells to characterizing this new method of generating hair-bearing skin.

In a 2018 study published in Cell Reports, Drs. Koehler and Lee reported their step-by-step approach to hair follicle development in mouse organoids. Instead of developing the dermis and epidermis separate from one another, they had induced the mouse organoid so that the two layers of skin would grow together in a 3D design. The subsequent hair follicles developed at around the same time they normally would in pre-natal development, providing a proof-of-concept that could be applied to human skin organoids.

“The approach we’ve stumbled on is that we need to get these cells to develop together,” Dr. Koehler explained. “It’s exactly how they develop in the embryo; juxtaposed right next to each other and going through each stage of development and progression in concert with each other.”

Finding the “holy grail”

Dr. Koehler arrived at Boston Children’s Hospital in 2020 ready to test his new methodology on human stem cells in collaboration with Dr. Lee. They began by reverting human red blood cells back to an embryonic state, creating pluripotent stem cells. After organizing the cells into a sphere, the researchers screened numerous human embryonic growth factors and drugs — such as transforming growth factor β inhibitors and fibroblast growth factors — to grow a dermis and epidermis within the cluster of cells.

After 80 days, not only had the dermis and epidermis grown together inside an organoid, but so had hair follicles and other appendages, including nerves, fats and Merkel cells. Single-cell RNA sequencing and direct comparison to fetal specimens revealed that the skin organoid was equivalent to the facial skin of human fetuses in the second trimester of development.

“It was hard not to get too excited at the sight of these hair follicles,” said Dr. Lee. “It has allowed us to hypothesize that, at some point very early in development, there’s a period when the precursor cells of the epidermis and the precursor cells of the dermis need to be together to develop skin as we know it.”

Taking their study one step further, the team transplanted the appendaged human organoid onto a nude, immunosuppressed mouse. According to their findings published in Nature, Drs. Koehler and Lee witnessed the transplanted graft reconstitute skin on the mouse, providing the first translational evidence that skin organoids could feasibly be used for human skin reconstruction and wound healing.

The future of skin reconstruction

According to the American Academy of Dermatology, the direct health care cost of skin disease in the United States is $75 billion, inclusive of patient visits, medications and procedures used to manage symptoms. Dr. Koehler believes doctors could one day rely on appendaged organoids grown in laboratories to reconstruct damaged skin and eliminate the costs of traditional treatments.

Researchers across the Harvard Department of Otolaryngology–Head and Neck Surgery have already expressed interest in using the appendaged organoids to test gene therapies for rare skin conditions, such as epidermolysis bullosa. Other members of the otolaryngology– head and neck surgery community would like to use the organoids to better understand the evolution of human face anatomy, and how melanoma develops in melanocytes, the skin cells responsible for pigment in the skin.

While the potential of these organoids seems limitless, plenty of barriers exist before they can reach the clinic for reconstructive purposes, which is Dr. Koehler’s ultimate goal. Researchers must first find a way to convert the appendaged human organoids from its spherical shape into planar layers that match the physiology of natural skin, all while ensuring the organoids can withstand immune responses. Researchers must also consider the cost of generating pluripotent stem cells from blood or urine samples, which Dr. Koehler said can be millions of dollars.

“Growing fully-functional skin in a petri dish once sounded like something you’d only read about in a science fiction novel,” Dr. Koehler said. “Now that we know it’s at least a possibility, it’s time to leverage our existing partnerships across the Harvard network and work together to bring these organoids from the bench to the bedside.”