In 1997 Dolly the Sheep became the most famous cloned animal. DNA was removed from an unfertilized donor sheep egg cell, and then was replaced with DNA from a mature cell removed from Dolly's parent. An unfertilized egg has only half of the DNA needed to grow into an organism. The other half comes from the sperm cell. But Dolly's egg received a full set of DNA from a mature cell. The egg was fooled into thinking it was fertilized, and that it should grow into an organism. The egg cell grew into an embryo, and eventually into a sheep that was an exact genetic copy of Dolly's parent, since it had the exact same DNA. This was the first time a large mammal was grown from DNA taken from a mature cell, using that cell's entire DNA blueprint to make a new organism. The process is much more involved than this summary describes, and there were many failures along the way. But if we just want some hair follicles, cloning an entire living organism from a donor cell is a bit excessive. While a fascinating genetic exercise, the Dolly the Sheep method is too complicated and costly for commercial hair follicle cloning. Another method, which is inadvertently used in almost every micrograft hair transplant procedure, is a lot easier.

The Splitting Hairs Method of Cloning:
Hair restoration surgeons have long known about the "splitting hairs" method of cloning hair follicles, and it is a much less complicated process than the Dolly the Sheep method. Surgeons have observed that when a hair follicle is accidentally cut in half, one piece, and sometimes both pieces of the follicle, will survive and grow a new hair if the follicle is transplanted back into the donor. Micrograft surgeons routinely place cut follicles back into the patient's scalp, although they don't generally "count" the cut follicles as "grafts". Each fractional follicle is an exact DNA clone of the original, so if the original follicle is DHT-resistant, the clones will also be DHT-resistant.

Cloningfollicles by "splitting hairs".

click on the image to enlarge

If a follicle is cut in half to form top and bottom pieces, and both pieces are placed back in the scalp, the bottom half (with the hair bulb) will sometimes survive and grow a hair. The top half (with the bulge) will sometimes survive and grow a new bottom half containing a new hair bulb, and that half also eventually grows a new hair. This process is similar to the hair follicle creation that occurs at the beginning of the normal hair follicle growth phase. If the follicle is cut lengthwise, two surviving follicles can result, although they may produce finer hairs than the original full-size follicle.

Survival rates of transected hair follicles is lower than follicles that are not cut in half, so hair restoration surgeons continue to take great care to avoid cutting follicles. The reason the splitting hairs methods works when the cut follicle pieces are placed back into the scalp, is that other nearby hair follicle stem cells probably help with the cell regeneration process. But with improved methods to signal cell regeneration, and ultimately improved transected follicle survival rates, this crude cloning technique has some promise for the immediate future.

Mature Cell Cloning:
Commercially cloning of undifferentiated stem cells for research purposes has been around for a few years. Recently the commercialization of cloning mature skin cells has been accomplished. On June 20, 2000 the US FDA approved Organogenesis Incorporated's Apligraf, a skin patch product containing living human skin cells. To make an Apligraf patch, human skin cells derived from infant foreskins are cultured in a growth medium and are allowed to differentiate into some of the types of cells that form mature human skin. Apligraf skin does not contain pigment cells, blood vessels, sweat glands, or hair follicles, but it does have the types of cells that are typically found in the upper and lower layers of skin. It is approved for use as a wound dressing for slow healing skin ulcers.

Replicating skin cells is much less complicated than replicating entire hair follicles, however the approval and commercialization of this mature cell cloning process is very encouraging for future hair follicle cloning. Because hair follicles are miniature organs composed of several different types of specialized cells, they are difficult to clone and even more difficult to assemble properly. The trick to hair follicle cloning will be signaling hair follicle stem cells to make and assemble all the cells needed for a hair follicle.

1993, United Kingdom researchers Jahoda and Reynolds published in "The Journal of Investigative Dermatology" a paper describing their success in growing a rat whisker in a live rat from cultured rat dermal papilla cells. The dermal papilla cells are found in the hair matrix in the bulb of the hair shaft, and they are the cells that grow hairs. For many years it was believed that the hair follicle bulb contained the stem cells necessary for hair follicle growth. The researchers removed dermal papilla cells from a rat whisker hair follicle bulb, cultured the cells in a growth medium, and created thousands of dermal papilla cell clones. After they had enough dermal papilla cells, they implanted the cultured cells back into the rat, but they placed the cells in the skin forming the rat's ear (so they could better see any results). The cloned dermal papilla cells interacted with neighboring skin cells and made a hair follicle that eventually grew a rat whisker. The researchers patented this procedure of growing hair follicles in a living organism with cloned dermal papilla cells. (Jahoda and Reynolds, Journal of Investigative Dermatology (101:634-638, 1993) (also 1992 115:587-593)

In 1994 the same researchers, Jahoda and Reynolds, published in "The Journal of Dermatological Science" a paper describing their success in growing in a cell culture medium, a crude but functioning rat hair follicle from cultured adult hair follicle cells. The researchers cultured four different types of cells found in rat hair follicles, and using a framework of other living cells, assembled them into somewhat unusual hair bulb structures that grew irregular but recognizable hair shafts. This appears to be the first example of growing "test tube" hair follicles, outside of a living organism.

In 1996, hair restoration surgeon Jerry Cooley, MD successfully grew human hair follicles from cloned dermal papilla cells in a human. He cultured dermal papilla cells from hair follicle bulbs surgically removed from his own scalp, and after multiplying the cells in a culture medium, implanted the cloned cells back into his own forearm, and a new hair follicle formed and grew a scalp hair.

In 1998 Dr. Conradus Ghosal Gho of the Netherlands patented with the World Intellectual Property Organization, a "Method for the Propagation of Hair", in which he describes a method of plucking hairs in the anagen (growth) phase, and culturing the dermal papilla cells from the portion of the hair bulb at the end of the plucked hair. He describes using commercial cell culture media along with various beneficial additives such as amino acids, vitamins, trace elements, growth hormones, and antibiotics, however his patent seems to imply that the best results are achieved by also adding cloned CD34+ progenitor cells, a type of stem cell, to the culture. Presumably these would be obtained by surgically removing a tissue sample from the donor, and culturing the stem cells separately, so they could be added to the dermal papilla cell culture. Alternatively, the CD34+ cells may be obtained commercially. After the dermal papilla cells and CD34+ cell mixture has multiplied and differentiated into the different types of cells that make up hair follicle, Dr Gho injects packets of the cloned cell mixture into the donor's scalp, where they develop into new hair follicles and grow new hairs. Dr. Gho also describes using an electronic repeating injection metering apparatus such as an insulin pen to rapidly and precisely insert the cloned cells into the scalp. Currently Dr. Gho is working to refine and improve his method.

So it seems that it won't be long before cloning thousands of whole human hair follicles in "test tubes" becomes a commercial reality. Or will it? There are still plenty of obstacles to overcome before commercial hair follicle cloning is a reality. The method of cloning dermal papilla cells from hair bulbs and injecting them back into the donor's scalp has not been perfected. Do the hair follicles created from cultured dermal papilla cells cycle through the normal growth cycles, and continue to grow new hairs for a lifetime? What are the chemical signals in living skin that tell the dermal papilla cells to grow into a hair follicle in the first place? If we knew more about how the hair follicle growth cycle functioned, maybe we could improve the results of injecting cloned cells to make hair follicles. Or perhaps we could clone cells into entire hair follicles in "test tubes" and achieve better results than injecting packets of cloned cells.

More answers to the future of hair follicle cloning appear in work done over the last decade by Robert Lavker, MD and his associate George Cotsarelis, MD both of the Department of Dermatology at he University of Pennsylvania School of Medicine.

In a paper published in 1990 in the journal "Cell", Cotsarelis showed that hair follicle stem cells were located in the bulge area of the follicle, midway up the hair shaft, rather than in the bulb area at the base of the hair shaft. The bulge area is located near the middle of the hair follicle, below the sebaceous (oil) gland and near the attachment point for the arrector pili muscle (the tiny muscle that allows hairs to "stand on end").

On behalf of the Trustees of the University of Pennsylvania, Lavker has achieved several patents for methods for regulating hair growth. In a 1996 patent filed with the World Intellectual Property Organization (and cited by Dr. Gho in his patent two years later), Lavker describes a hair follicle bulge activation theory, in which the dermal papilla cells activate the stem cells in the bulge in the late telogen (resting) stage of hair growth. This is the point at which the dermal papilla cells move upwards and next to the bulge area of the follicle, after the follicle has shed the old hair and has shrunk in size. Once the stem cells are activated, a new hair follicle growth cycle begins, the follicle grows to full size, and a new hair bulb forms to grow a new hair.

Lavker states that the hair matrix cells are in fact transient amplifying cells; intermediate cells created by stem cells to rapidly grow other specialized cells, in this case those that make up a hair shaft. The 1996 patent describes growth-modulating molecules that are created by hair follicle cells and which undergo hair-cycle-dependent concentration changes in the hair follicle. One such molecule is a protein called glia-derived nexin 1, which has also been shown to play important roles in regulating a variety of types of cellular growth and differentiation.

Lavker's 1996 patent claims applications in modulating hair growth, as well as for growing hair follicle cells outside of a body with a selected growth-modulating molecule, and combining the expanded cells with selected dermal papilla cells to grow hair.

In a year 2000 paper published in "Dermatology Focus", Cotsarelis reveals that he is working on cloning hair follicle stem cells in cultures, and that once the molecular events responsible for the cycle of hair follicle growth is unraveled, cloning entire hair follicles will be closer to reality.

This research suggests that the most likely method for commercial hair follicle cloning is not cloning dermal papilla cells, but the cloning of hair follicle stem cells collected from the bulge area of DHT-resistant hair follicles. These stem cells will be combined with proteins that provide the right molecular signals to tell the stem cells to generate transient amplifying cells in the form of a hair bulb, which in turn will grow a new hair.

Once these details are figured out, thousands of full-size "test tube" grown hair follicles can be self-assemble from cloned DHT resistant stem cells. The cloned hair follicles could be grown in a standard size and shape, perhaps in a tray that can be directly loaded into an automatic graft placement device. Automated graft placement devices, such as Choi Transplanter developed by Yung Chul Choi, MD in Korea in 1990, use a hollow needle to create a tiny recipient site of the proper depth for the graft, and then automatically insert the graft into the opening.

There will still be some problems associated with cloning. One is an increased risk of cancer. The growth-inducing chemicals such as tetradecanoylphorbol acetate (TPA) used to stimulate cellular proliferation for cloning frequently promote tumors and cancer. While cancer risk is low for most cloned cell products, cloning hair follicle stem cells is riskier.

Dermatologists have observed that many basal cell carcinomas, a type of skin cancer that is the most common human tumor, seem to originate from the hair follicle. Cotsarelis notes in the 2000 paper presented in "Dermatology Focus": "…the fact that that basal cell carcinomas are slow-growing tumors composed of poorly differentiated cells that have the ability to differentiate into various adnexal structures strongly suggests that the cell of origin is a pluripotent, slowly cycling stem cell with a highly proliferative potential." What he's saying is that molecular and genetic evidence points to the hair follicle stem cell as the source for this common form of cancer. Cloning hair follicle stem cells with cancer-inducing chemicals will require many years of study to assure the safety of the cloned follicles that result.

Gene Therapy:
An even more advanced technique for solving inherited hair loss in the future is gene therapy. Gene therapy is the process of changing genes of existing cells in the body, and thereby altering cell function. It is a medical treatment still in its infancy, and there have only been a few recent examples of gene therapy working. But it is a potential future baldness treatment method worth exploring.

Gene therapy requires learning how an inherited medical condition occurs at the DNA molecule level, and then going in and fixing it. With gene therapy, the hair follicles with DHT-sensitive cells could be changed into follicles with DHT-resistant cells, and the hair follicles would continue to grow new hairs for a lifetime. But gene therapy involves several very difficult to achieve steps. The first step is figuring out which of the tens of thousands of genes on strands of DNA are involved in the characteristic to be altered, and the second step is figuring out how exactly the target genes are to be changed, so that they give instructions for making the slightly different proteins that will achieve the desired effect. The third step is getting the target cells in the living organism to incorporate the new and improved genes as replacements for the old undesirable genes.

Gene Identification:
Figuring out which genes are involved in the genetic condition to be changed is not an easy task. Despite all the advances in mapping genes in recent years, we are still very far away from knowing what most of these genes do. We certainly do not have a good understanding of all of the genes that affect the cycle of hair growth, and especially which genes are responsible for inherited hair loss. It is most likely that several genes are responsible for making proteins that cause certain hair follicles to be DHT-sensitive.

Future studies will likely involve comparing the genes and resulting proteins in different follicles from a single individual. In a given individual with androgenetic alopecia (pattern hair loss), some hair follicle cells will express the characteristic of DHT-resistance (the follicles at the back of the scalp), while other hair follicles on the same person will express the characteristic of DHT-sensitivity (at the hairline, for example). Both follicles contain cells with identical DNA, but they express different characteristics. So identifying the responsible genes will be tricky. And even after we identify these genes, we have to figure out how to change them ever so slightly so they will make proteins that create DHT-resistant hair follicles. But scientists have been making progress in gene identification.

Modifying Genes:
In a paper presented in the January 30, 1998 issue of the journal "Science", researchers led by Angela Christiano, PhD identified a defect in a single gene responsible for a rare type of inherited baldness called generalized atrichia observed in a Pakistani family, in which affected individuals are born with infant hair that falls out and never grows back. Shortly after birth, affected individuals are completely hairless. The gene, called hairless, was mapped in humans to chromosome 8p21, and was the first example of a single gene defect being identified as a hair loss cause. Christiano was careful to point out that this was just a first step towards identifying genes that affect hair loss. (Science, January 30,1998, vol 279, No. 5351)

Later in the same year, in a paper presented in the September 11, 1998 issue of the "American Journal of Human Genetics", Christiano's team reported on members of a family of Irish Travelers who also exhibited congenital atrichia, in which affected individuals are born with infant hair that falls out and never grows back. Genetic analysis of the Irish Travelers revealed that a mutation of the hairless gene was again responsible for the hair loss condition, however the mutation was different from the one that resulted in hair loss in the Pakistani family.

In a paper reported in the November 25 1998 issue of "Cell", researchers led by Elaine Fuchs, PhD induced the formation of new hair follicles in mice that were genetically engineered to constantly produce a stabilized form of a protein called beta-catenin. Beta-catenin is a multi-functional protein, which signals a variety of cellular functions, but is normally quickly degraded within a cell after being produced. Researchers altered the mouse gene that contains instructions for making the beta-catenin protein in such a way that the beta-catenin produced was resistant to being broken down. The resulting accumulation of beta-catenin caused a massive growth of new hair follicles to grow in normal mouse skin, until there were hair follicles branching from existing hair follicles. Eventually the mice also developed hair follicle tumors as a result of over-expressing beta-catenin. (Fuchs, university of Chicago 1998) (Gat, Dermatology Focus Vol. 19, No. 2, Summer 2000).

In the October 1999 issue of "The Journal of Clinical Investigation", researchers led by Ronald Crystal MD forced resting hair follicles of mice into the growth phase by exposing cells to larger than normal quantities of a protein produced by the Sonic Hedgehog Gene (abbreviated Shh).

The papers presented by these three groups of genetic researchers reveal the complexity of the task of understanding the genetic basis for inherited hair loss, and reveal the monumental task of figuring out how to correct the condition at the molecular level. In the first case, Angela Christiano's team identified a gene that can cause total hair loss when mutated in either of two different ways. In the second example, the team led by Elaine Fuchs mutated a gene in such a way that it coded for the creation of a slightly different protein that caused massive new hair follicle creation. And the third example showed that increasing the exposure to a naturally occurring protein could signal hair follicles to shift from the resting phase into the growth phase. And while all of these genes and their respective proteins appear to play some role in hair follicles, they are also known to affect other cells and systems in the body. Genetics is very complicated.

But suppose that at some point in the future we develop an adequately complete understanding of how all the genes, and their respective proteins, affect inherited hair loss. And suppose that we can also determine how exactly we want to alter the genes so that the proteins they make result in hair follicles that are DHT-resistant, rather than DHT sensitive, but without causing unwanted side effects.

Changing Genes in Living Cells and Living Organisms:
The third challenge of gene therapy is delivering the new-and-improved genes to the target cells, and then to have those cells use the new genes to make the corresponding new proteins, and then to have the altered cells express the desired characteristic.

The correct target cells are critical to successful gene therapy. If mature cells are altered, the benefits of the gene therapy go away after those cells wear out and are replaced with new cells having the original DNA. For a long-lasting effect, stem cells are targeted. When successful, the altered stem cells will then create altered transient amplifying cells, which in turn will create altered specialized cells that will express the desired characteristics.

The most common altered gene-delivery method involves using crippled viruses to insert desired genes into the target cells. Outside of the laboratory, viruses are tiny organisms that infect cells by replacing some of the cell's DNA with virus DNA. After infection by a virus, a cell begin to make the proteins the virus DNA tells it to make, causing the expression of various diseases. Scientists use the virus infection mechanism to deliver desirable DNA.

First, they cripple the virus DNA so that it cannot reproduce or cause harmful effects, but is still able to insert new DNA into target cells. The desired genes are spliced onto to the virus DNA, and the viruses insert the new DNA into the target cells. The viruses can be injected directly to the location where the stem cells are, or the stem cells may be cultured in a laboratory, altered by viruses containing the new DNA, and then the altered stem cells can be placed back into the organism.

There are many areas of gene therapy that need refinement. Identifying genes, determining exactly how to change them to code for the desired proteins, avoiding an immune response when the viruses are injected directly into the organism, getting an adequate quantity of target cells to take up the altered DNA regardless of how it is delivered, and getting the cells to express the characteristics coded by the altered genes, once the new DNA is inserted, all need more work. But forward progress is being made.

In summary, the future of hair loss treatment shows great promise, from new medications such as dutasteride to advances in cloning and gene therapy. But many of these treatments are years, and maybe decades away from commercial use. Current treatment methods, including cosmetic products, drugs such as Propecia, and surgical procedures such as follicular unit micrografts are available right now, if you really want to do something about your hair loss. Your first step should be scheduling an examination with a dermatologist knowledgeable about hair loss treatment.

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